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Giulia GRISENDI

Ricercatore t.d. art. 24 c. 3 lett. A
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

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Pubblicazioni

- METHOD FOR PRODUCTION OF ANTI-TUMOR TRAIL PROTEIN [Brevetto]
Dominici, Massimo; Bussolari, Rita; Grisendi, Giulia; Conte, Pierfranco
abstract

The method for production of anti-tumor TRAIL comprises: inserting a TRAIL molecule, encoded by a viral vector irreversibly derived from a cell line, into a carrier cell, thereby obtaining a stably TRAIL-producing carrier cell, said TRAIL molecule comprising a soluble molecule.

2023 - Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model [Articolo su rivista]
Bianchi, Elisa; Rontauroli, Sebastiano; Tavernari, Lara; Mirabile, Margherita; Pedrazzi, Francesca; Genovese, Elena; Sartini, Stefano; Dall'Ora, Massimiliano; Grisendi, Giulia; Fabbiani, Luca; Maccaferri, Monica; Carretta, Chiara; Parenti, Sandra; Fantini, Sebastian; Bartalucci, Niccolò; Calabresi, Laura; Balliu, Manjola; Guglielmelli, Paola; Potenza, Leonardo; Tagliafico, Enrico; Losi, Lorena; Dominici, Massimo; Luppi, Mario; Vannucchi, Alessandro Maria; Manfredini, Rossella
abstract

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity.

2022 - A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors [Articolo su rivista]
Golinelli, G.; Talami, R.; Frabetti, S.; Candini, O.; Grisendi, G.; Spano, C.; Chiavelli, C.; Arnaud, G. F.; Mari, G.; Dominici, M.
abstract

We here investigated the dynamic cell-to-cell interactions between tumor and mesenchymal stromal/stem cells (MSCs) by the novel VITVOⓇ 3D bioreactor that was customized to develop in vivo-like metastatic nodules of Ewing’s sarcoma (ES). MSCs are known to contribute to tumor microenvironment as cancer associated fibroblast (CAF) precursors and, for this reason, they have also been used as anti-cancer tools. Using dynamic conditions, the process of tissue colonization and formation of metastatic niches was recreated through tumor cell migration aiming to mimic ES development in patients. ES is an aggressive tumor representing the second most common malignant bone cancer in children and young adults. An urgent and unmet need exists for the development of novel treatment strategies to improve the outcomes of metastatic ES. The tumor-tropic ability of MSCs offers an alternative approach, in which these cells can be used as vehicles for the delivery of antitumor molecules, such as the proapoptotic TNF-related apoptosis inducing ligand (TRAIL). However, the therapeutic targeting of metastases remains challenging and the interaction occurring between tumor cells and MSCs has not yet been deeply investigated. Setting up in vitro and in vivo models to study this interaction is a prerequisite for novel approaches where MSCs affinity for tumor is optimized to ultimately increase their therapeutic efficacy. Here, VITVOⓇ integrating a customized scaffold with an increased inter-fiber distance (VITVO50) was used to develop a dynamic model where MSCs and tumor nodules were evaluated under flow conditions. Colonization and interaction between cell populations were explored by droplet digital PCR (ddPCR). VITVO50 findings were then applied in vivo. An ES metastatic model was established in NSG mice and biodistribution of TRAIL-expressing MSCs in mice organs affected by metastases was investigated using a 4-plex ddPCR assay. VITVOⓇ proved to be an easy handling and versatile bioreactor to develop in vivo-like tumor nodules and investigate dynamic cell-to-cell interactions with MSCs. The proposed fluidic system promises to facilitate the understanding of tumor-stroma interaction for the development of novel tumor targeting strategies, simplifying the analysis of in vivo data, and ultimately accelerating the progress towards the early clinical phase.

2022 - Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma [Articolo su rivista]
Golinelli, G.; Grisendi, G.; Dall'Ora, M.; Casari, G.; Spano, C.; Talami, R.; Banchelli, F.; Prapa, M.; Chiavelli, C.; Rossignoli, F.; Candini, O.; D'Amico, R.; Nasi, M.; Cossarizza, A.; Casarini, L.; Dominici, M.
abstract

Background: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES. Methods: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario. Findings: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver. Interpretation: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies.

2022 - Autologous Marrow Mesenchymal Stem Cell Driving Bone Regeneration in a Rabbit Model of Femoral Head Osteonecrosis [Articolo su rivista]
Mastrolia, I.; Giorgini, A.; Murgia, A.; Loschi, P.; Petrachi, T.; Rasini, V.; Pinelli, M.; Pinto, V.; Lolli, F.; Chiavelli, C.; Grisendi, G.; Baschieri, M. C.; Santis, G. D.; Catani, F.; Dominici, M.; Veronesi, E.
abstract

Osteonecrosis of the femoral head (ONFH) is a progressive degenerative disease that ultimately requires a total hip replacement. Mesenchymal stromal/stem cells (MSCs), particularly the ones isolated from bone marrow (BM), could be promising tools to restore bone tissue in ONFH. Here, we established a rabbit model to mimic the pathogenic features of human ONFH and to challenge an autologous MSC-based treatment. ON has been originally induced by the synergic combination of surgery and steroid administration. Autologous BM-MSCs were then implanted in the FH, aiming to restore the damaged tissue. Histological analyses confirmed bone formation in the BM-MSC treated rabbit femurs but not in the controls. In addition, the model also allowed investigations on BM-MSCs isolated before (ON-BM-MSCs) and after (ON+BM-MSCs) ON induction to dissect the impact of ON damage on MSC behavior in an affected microenvironment, accounting for those clinical approaches foreseeing MSCs generally isolated from affected patients. BM-MSCs, isolated before and after ON induction, revealed similar growth rates, immunophenotypic profiles, and differentiation abilities regardless of the ON. Our data support the use of ON+BM-MSCs as a promising autologous therapeutic tool to treat ON, paving the way for a more consolidated use into the clinical settings.

2022 - CRISPR-mediated T cell engineering against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Ferrari, Tommaso; Masciale, Valentina; Grisendi, Giulia; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2022 - Cancer Stem Cells and Cell Cycle Genes as Independent Predictors of Relapse in Non-small Cell Lung Cancer: Secondary Analysis of a Prospective Study [Articolo su rivista]
Masciale, V.; Banchelli, F.; Grisendi, G.; D'Amico, R.; Maiorana, A.; Stefani, A.; Morandi, U.; Stella, F.; Dominici, M.; Aramini, B.
abstract

PURPOSE: Cancer stem cells (CSCs) are described as resistant to chemotherapy and radiotherapy. It has been shown that CSCs influence disease-free survival in patients undergoing surgery for lung cancer (NCT04634630). We recently described an overexpression of CSCs recurrence-related genes (RG) in lung cancer. This study aims to investigate CSC frequency and RG expression as predictors of disease-free survival in lung cancer. EXPERIMENTAL DESIGN: This secondary analysis of a prospective cohort study involved 22 surgical tumor specimens from 22 patients harboring early (I-II) and locally advanced (IIIA) stages ACL and SCCL. Cell population frequency analysis of ALDHhigh (CSCs) and ALDHlow (cancer cells) was performed on each tumor specimen. In addition, RG expression was assessed for 31 target genes separately in ALDHhigh and ALDHlow populations. CSCs frequency and RG expression were assessed as predictors of disease-free survival by Cox analysis. RESULTS: CSCs frequency and RG expression were independent predictors of disease-free survival. CSC frequency was not related to disease-free survival in early-stage patients (HR = 0.84, 95%CI = 0.53-1.33, P = .454), whereas it was a risk factor for locally advanced-stage patients (HR = 1.22, 95%CI = 1.09-1.35, P = .000). RG expression-if measured in CSCs-was related to a higher risk of recurrence (HR = 1.19, 95%CI = 1.03-1.39, P = .021). The effect of RG expression measured in cancer cells on disease-free survival was lower and was not statistically significant (HR = 1.12, 95%CI = 0.94-1.33, P = .196). CONCLUSIONS: CSCs frequency and RG expression are independent predictors of relapse in lung cancer. Considering these results, CSCs and RG may be considered for both target therapy and prognosis.

2022 - Dissecting Tumor Growth: The Role of Cancer Stem Cells in Drug Resistance and Recurrence [Articolo su rivista]
Aramini, B.; Masciale, V.; Grisendi, G.; Bertolini, F.; Mauer, M.; Guaitoli, G.; Chrystel, I.; Morandi, U.; Stella, F.; Dominici, M.; Haider, K. H.
abstract

Emerging evidence suggests that a small subpopulation of cancer stem cells (CSCs) is responsible for initiation, progression, and metastasis cascade in tumors. CSCs share characteristics with normal stem cells, i.e., self-renewal and differentiation potential, suggesting that they can drive cancer progression. Consequently, targeting CSCs to prevent tumor growth or regrowth might offer a chance to lead the fight against cancer. CSCs create their niche, a specific area within tissue with a unique microenvironment that sustains their vital functions. Interactions between CSCs and their niches play a critical role in regulating CSCs’ self-renewal and tumorigenesis. Differences observed in the frequency of CSCs, due to the phenotypic plasticity of many cancer cells, remain a challenge in cancer therapeutics, since CSCs can modulate their transcriptional activities into a more stem-like state to protect themselves from destruction. This plasticity represents an essential step for future therapeutic approaches. Regarding self-renewal, CSCs are modulated by the same molecular pathways found in normal stem cells, such as Wnt/β-catenin signaling, Notch signaling, and Hedgehog signaling. Another key characteristic of CSCs is their resistance to standard chemotherapy and radiotherapy treatments, due to their capacity to rest in a quiescent state. This review will analyze the primary mechanisms involved in CSC tumorigenesis, with particular attention to the roles of CSCs in tumor progression in benign and malignant diseases; and will examine future perspectives on the identification of new markers to better control tumorigenesis, as well as dissecting the metastasis process.

2022 - Human Adipose Mesenchymal Stromal/Stem Cells Improve Fat Transplantation Performance [Articolo su rivista]
Piccinno, M. S.; Petrachi, T.; Pignatti, M.; Murgia, A.; Grisendi, G.; Candini, O.; Resca, E.; Bergamini, V.; Ganzerli, F.; Portone, A.; Mastrolia, I.; Chiavelli, C.; Castelli, I.; Bernabei, D.; Tagliazucchi, M.; Bonetti, E.; Lolli, F.; De Santis, G.; Dominici, M.; Veronesi, E.
abstract

The resorption rate of autologous fat transfer (AFT) is 40–60% of the implanted tissue, requiring new surgical strategies for tissue reconstruction. We previously demonstrated in a rabbit model that AFT may be empowered by adipose-derived mesenchymal stromal/stem cells (AD-MSCs), which improve graft persistence by exerting proangiogenic/anti-inflammatory effects. However, their fate after implantation requires more investigation. We report a xenograft model of adipose tissue engineering in which NOD/SCID mice underwent AFT with/without human autologous AD-MSCs and were monitored for 180 days (d). The effect of AD-MSCs on AFT grafting was also monitored by evaluating the expression of CD31 and F4/80 markers. Green fluorescent protein-positive AD-MSCs (AD-MSC-GFP) were detected in fibroblastoid cells 7 days after transplantation and in mature adipocytes at 60 days, indicating both persistence and differentiation of the implanted cells. This evidence also correlated with the persistence of a higher graft weight in AFT-AD-MSC compared to AFT alone treated mice. An observation up to 180 d revealed a lower resorption rate and reduced lipidic cyst formation in the AFT-AD-MSC group, suggesting a long-term action of AD-MSCs in support of AFT performance and an anti-inflammatory/proangiogenic activity. Together, these data indicate the protective role of adipose progenitors in autologous AFT tissue resorption.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; Reggiani Bonetti, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes.

2021 - Alternative splicing of NF-YA promotes prostate cancer aggressiveness and represents a new molecular marker for clinical stratification of patients [Poster]
Belluti, Silvia; Semeghini, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Benati, Daniela; Torricelli, Federica; REGGIANI BONETTI, Luca; Carnevale, Gianluca; Grisendi, Giulia; Ciarrocchi, Alessia; Dominici, Massimo; Recchia, Alessandra; Dolfini, Diletta; Imbriano, Carol
abstract

2021 - CRISPR-Mediated Genome Editing to Redirect T Cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Marchionni, Matteo; Ferrari, Tommaso; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2021 - CRISPR-mediated genome editing to redirect T cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Ferrari, Tommaso; Cattin, Eleonora; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2021 - CRISPR-mediated genome editing to redirect T cells against Non-Small Cell Lung Cancer [Abstract in Atti di Convegno]
Benati, Daniela; Masciale, Valentina; Grisendi, Giulia; Ferrari, Tommaso; Cattin, Eleonora; Marchionni, Matteo; Aramini, Beatrice; Dominici, Massimo; Recchia, Alessandra
abstract

2021 - Cancer stem cells and macrophages: molecular connections and future perspectives against cancer. [Articolo su rivista]
Aramini, Beatrice; Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Morandi, Uliano; Dominici, Massimo; Husnain Haider, Khawaja
abstract

Cancer stem cells (CSCs) have been considered the key drivers of cancer initiation and progression due to their unlimited self-renewal capacity and their ability to induce tumor formation. Macrophages, particularly tumor-associated macrophages (TAMs), establish a tumor microenvironment to protect and induce CSCs development and dissemination. Many studies in the past decade have been performed to understand the molecular mediators of CSCs and TAMs, and several studies have elucidated the complex crosstalk that occurs between these two cell types. The aim of this review is to define the complex crosstalk between these two cell types and to highlight potential future anti-cancer strategies.

2021 - Deepening the knowledge of ros1 rearrangements in non-small cell lung cancer: Diagnosis, treatment, resistance and concomitant alterations [Articolo su rivista]
Guaitoli, G.; Bertolini, F.; Bettelli, S.; Manfredini, S.; Maur, M.; Trudu, L.; Aramini, B.; Masciale, V.; Grisendi, G.; Dominici, M.; Barbieri, F.
abstract

ROS proto-oncogene 1 (ROS1) rearrangements are reported in about 1–2% of non-squamous non-small-cell lung cancer (NSCLC). After efficacy of crizotinib was demonstrated, identification of ROS1 translocations in advanced disease became fundamental to give patients the chance of specific and effective treatment. Different methods are available for detection of rearrangements, and probably the real prevalence of ROS1 rearrangements is higher than that reported in literature, as our capacity to detect gene rearrangements is improving. In particular, with next generation sequencing (NGS) techniques, we are currently able to assess multiple genes simultaneously with increasing sensitivity. This is leading to overcome the “single oncogenic driver” paradigm, and in the very near future, the co-existence of multiple drivers will probably emerge more frequently and represent a therapeutic issue. Since recently, crizotinib has been the only available therapy, but today, many other tyrosine kinase inhibitors (TKI) are emerging and seem promising both in first and subsequent lines of treatment. Indeed, novel inhibitors are also able to overcome resistance mutations to crizotinib, hypothesizing a possible sequential strategy also in ROS1-rearranged disease. In this review, we will focus on ROS1 rearrangements, dealing with diagnostic aspects, new therapeutic options, resistance issues and the coexistence of ROS1 translocations with other molecular alterations.

2021 - GD2 CAR T cells against human glioblastoma [Articolo su rivista]
Prapa, M.; Chiavelli, C.; Golinelli, G.; Grisendi, G.; Bestagno, M.; Di Tinco, R.; Dall'Ora, M.; Neri, G.; Candini, O.; Spano, C.; Petrachi, T.; Bertoni, L.; Carnevale, G.; Pugliese, G.; Depenni, R.; Feletti, A.; Iaccarino, C.; Pavesi, G.; Dominici, M.
abstract

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF β1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer.

2021 - New Perspectives in Different Gene Expression Profiles for Early and Locally Advanced Non-Small Cell Lung Cancer Stem Cells. [Articolo su rivista]
Masciale, Valentina; Banchelli, Federico; Grisendi, Giulia; D'Amico, Roberto; Maiorana, Antonino; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Introduction: Lung cancer is one of the most common cancers in the world, causing over 1.7 million deaths in 2018. Thus far, no effective treatments against lung cancer for advanced stages have been found. For early stages, although surgery is considered the gold standard treatment, 30–55% of patients develop recurrence within the first 5 years of surgery. Our aim is to assess whether cancer stem cells (CSC) display overexpression of a pool of genes that were previously identified for adenocarcinoma recurrence in patients with early and locally advanced stages of non-small cell lung cancer (NSCLC). Methods: This cross-sectional study was carried out by harvesting surgical tumor specimens obtained from patients harboring early (I-II) and locally advanced (IIIA) stages of NSCLC. For each patient, cell sorting was performed to identify and isolate the ALDHhigh (CSC) and ALDHlow (cancer cells) populations. The mRNA expressions of 31 recurrence-related genes (target genes) in both ALDHhigh and ALDHlow populations were then assessed and compared. Results: Surgical specimens were obtained from 22 patients harboring NSCLC. Sixteen (51.6%) out of 31 recurrence-related genes were significantly overexpressed in ALDHhigh cells in the early stages and 9 (29.0%) were overexpressed in the locally advanced stages of NSCLC. Overall, the relative mRNA expressions for these recurrence-related genes were higher in early-stage patients. The average fold change, considering all 31 recurrence-related genes together, was 4.5 (95% CI = 3.1-6.3) in early-stage patients and 1.6 (95% CI = 1.2-2.2) in locally advanced-stage patients. Conclusions: Our study represents the first attempt toward identifying genes associated with recurrence that are overexpressed in cancer stem cells in patients with early and ocally advanced stages of NSCLC. This finding may contribute to the identification of new target therapies tailored for NSCLC stages.

2021 - Persistency of Mesenchymal Stromal/Stem Cells in Lungs [Articolo su rivista]
Ferrini, E.; Stellari, F. F.; Franceschi, V.; Macchi, F.; Russo, L.; Murgia, A.; Grisendi, G.; Villetti, G.; Dominici, M.; Donofrio, G.
abstract

Mesenchymal stromal/stem cells (MSCs) are a fibroblast-like cell population with high regenerative potential that can be isolated from many different tissues. Several data suggest MSCs as a therapeutic tool capable of migrating to a site of injury and guide tissue regeneration mainly through their secretome. Pulmonary first-pass effect occurs during intravenous administration of MSCs, where 50 to 80% of the cells tend to localize in the lungs. This phenomenon has been exploited to study MSC potential therapeutic effects in several preclinical models of lung diseases. Data demonstrated that, regardless of the lung disease severity and the delivery route, MSCs were not able to survive longer than 24 h in the respiratory tract but still surprisingly determined a therapeutic effect. In this work, two different mouse bone marrow-derived mesenchymal stromal/stem cell (mBM-MSC) lines, stably transduced with a third-generation lentiviral vector expressing luciferase and green fluorescent protein reporter genes tracking MSCs in vivo biodistribution and persistency, have been generated. Cells within the engrafted lung were in vivo traced using the high-throughput bioluminescence imaging (BLI) technique, with no invasiveness on animal, minimizing biological variations and costs. In vivo BLI analysis allowed the detection and monitoring of the mBM-MSC clones up to 28 days after implantation independently from the delivery route. This longer persistency than previously observed (24 h) could have a strong impact in terms of pharmacokinetics and pharmacodynamics of MSCs as a therapeutic tool.

2021 - TRAIL receptors are expressed in both malignant and stromal cells in pancreatic ductal adenocarcinoma [Articolo su rivista]
Dall'Ora, Massimiliano; Rovesti, Giulia; Reggiani Bonetti, Luca; Casari, Giulia; Banchelli, Federico; Fabbiani, Luca; Veronesi, Elena; Petrachi, Tiziana; Magistri, Paolo; Di Benedetto, Fabrizio; Spallanzani, Andrea; Chiavelli, Chiara; Spano, Maria Carlotta; Maiorana, Antonino; Dominici, Massimo; Grisendi, Giulia
abstract

: This study assesses the expression of all TNF-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic ductal adenocarcinoma (PDAC) tumor tissue. We aimed to include TRAIL receptor expression as an inclusion parameter in a future clinical study using a TRAIL-based therapy approach for PDAC patients. Considering the emerging influence of PDAC desmoplastic stroma on the efficacy of anti-PDAC therapies, this analysis was extended to tumor stromal cells. Additionally, we performed PDAC stroma characterization. Our retrospective cohort study (N=50) included patients with histologically confirmed PDAC who underwent surgery. The expression of TRAIL receptors (DR4, DR5, DcR1, DcR2, and OPG) in tumor and stromal cells was evaluated by immunohistochemistry (IHC). The amount of tumor stroma was assessed by anti-vimentin IHC and Mallory's trichrome staining. The prognostic impact was determined by the univariate Cox proportional hazards regression model. An extensive expression of functional receptors DR4 and DR5 and a variable expression of decoy receptors were detected in PDAC tumor and stromal cells. Functional receptors were detected also in metastatic tumor and stromal cells. A poor prognosis was associated with low or absent expression of decoy receptors in tumor cells of primary PDAC. After assessing that almost 80% of tumor mass was composed of stroma, we correlated a cellular-dense stroma in primary PDAC with reduced relapse-free survival. We demonstrated that TRAIL functional receptors are widely expressed in PDAC, representing a promising target for TRAIL-based therapies. Further, we demonstrated that a low expression of DcR1 and the absence of OPG in tumor cells, as well as a cellular-dense tumor stroma, could negatively impact the prognosis of PDAC patients.

2021 - The release of inflammatory mediators from acid-stimulated mesenchymal stromal cells favours tumour invasiveness and metastasis in osteosarcoma [Articolo su rivista]
Avnet, S.; Lemma, S.; Cortini, M.; Di Pompo, G.; Perut, F.; Lipreri, M. V.; Roncuzzi, L.; Columbaro, M.; Errani, C.; Longhi, A.; Zini, N.; Heymann, D.; Dominici, M.; Grisendi, G.; Golinelli, G.; Consolino, L.; Longo, D. L.; Nanni, C.; Righi, A.; Baldini, N.
abstract

Osteosarcoma is the most frequent primary malignant bone tumour with an impressive tendency to metastasise. Highly proliferative tumour cells release a remarkable amount of protons into the extracellular space that activates the NF-kB inflammatory pathway in adjacent stromal cells. In this study, we further validated the correlation between tumour glycolysis/acidosis and its role in metastases. In patients, at diagnosis, we found high circulating levels of inflammatory mediators (IL6, IL8 and miR-136-5p-containing extracellular vesicles). IL6 serum levels significantly correlated with disease-free survival and18F-FDG PET/CT uptake, an indirect measurement of tumour glycolysis and, hence, of acidosis. In vivo subcutaneous and orthotopic models, co-injected with mesenchymal stromal (MSC) and osteosarcoma cells, formed an acidic tumour microenvironment (mean pH 6.86, as assessed by in vivo MRI-CEST pH imaging). In these xenografts, we enlightened the expression of both IL6 and the NF-kB complex subunit in stromal cells infiltrating the tumour acidic area. The co-injection with MSC also significantly increased lung metastases. Finally, by using 3D microfluidic models, we directly showed the promotion of osteosarcoma invasiveness by acidosis via IL6 and MSC. In conclusion, osteosarcoma-associated MSC react to intratumoural acidosis by triggering an inflammatory response that, in turn, promotes tumour invasiveness at the primary site toward metastasis development.

2020 - Arming Mesenchymal Stromal/Stem Cells Against Cancer: Has the Time Come? [Articolo su rivista]
Golinelli, Giulia; Mastrolia, Ilenia; Aramini, Beatrice; Masciale, Valentina; Pinelli, Massimo; Pacchioni, Lucrezia; Casari, Giulia; Dall’Ora, Massimiliano; Botelho Pereira Soares, Milena; Kauanna Fonseca Damasceno, Patrícia; Nascimento Silva, Daniela; Dominici, Massimo; Grisendi, Giulia
abstract

Since mesenchymal stromal/stem cells (MSCs) were discovered, researchers have been drawn to study their peculiar biological features, including their immune privileged status and their capacity to selectively migrate into inflammatory areas, including tumors. These properties make MSCs promising cellular vehicles for the delivery of therapeutic molecules in the clinical setting. In recent decades, the engineering of MSCs into biological vehicles carrying anticancer compounds has been achieved in different ways, including the loadingof MSCs with chemotherapeutics or drug functionalized nanoparticles (NPs), genetic modifications to force the production of anticancer proteins, and the use of oncolytic viruses. Recently, it has been demonstrated that wild-type and engineered MSCs can release extracellular vesicles (EVs) that contain therapeutic agents. Despite the enthusiasm for MSCs as cyto-pharmaceutical agents, many challenges, including controlling the fate of MSCs after administration, must still be considered. Preclinical results demonstrated that MSCs accumulate in lung, liver, and spleen, which could prevent their engraftment into tumor sites. For this reason, physical, physiological, and biological methods have been implemented to increase MSC concentration in the target tumors. Currently, there are more than 900 registered clinical trials using MSCs. Only a small fraction of these are investigating MSC-based therapies for cancer, but the number of these clinical trials is expected to increase as technology and our understanding of MSCs improve. This review will summarize MSC-based antitumor therapies to generate an increasing awareness of their potential and limits to accelerate their clinical translation.

2020 - Author Correction: A Novel 3D In Vitro Platform for Pre-Clinical Investigations in Drug Testing, Gene Therapy, and Immuno-oncology (Scientific Reports, (2019), 9, 1, (7154), 10.1038/s41598-019-43613-9) [Articolo su rivista]
Candini, O.; Grisendi, G.; Foppiani, E. M.; Brogli, M.; Aramini, B.; Masciale, V.; Spano, C.; Petrachi, T.; Veronesi, E.; Conte, P.; Mari, G.; Dominici, M.
abstract

The original version of this Article contained an error in Affiliation 5, which was incorrectly given as ‘Department of Surgery, Oncology and Gastroenterology, University of Padova, Istituto Oncologico Veneto IRCCS, Padova, Italy’. The correct affiliation is listed below: Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy In addition, the original version of this Article omitted an affiliation for Pierfranco Conte. The correct affiliations for Pierfranco Conte are listed below: Medical Oncology 2, Veneto Institute of Oncology IOV, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Padova, Italy Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy These errors have now been corrected in the HTML and PDF versions of this Article, and in the accompanying Supplementary Information.

2020 - CD44+/EPCAM+ cells detect a subpopulation of ALDHhigh cells in human non-small cell lung cancer: A chance for targeting cancer stem cells? [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Sighinolfi, Pamela; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Objectives: Several studies demonstrated that aldehyde dehydrogenase (ALDH) and CD44 are the most considered cancer stem cells (CSC) markers. However, a comparison between ALDH high cells and CD44+ cells have been previously described with no significant correlation. Indeed, the aim of the present research is to identify a superficial marker able to match with ALDH high cells population in freshly isolated human lung cancer cells. Materials and Methods: This cross-sectional study analyzed the expression of ALDHhigh/low cells and the positivity for CD44 and epithelium cell adhesion molecule (EPCAM) antigens in surgical lung cancer tissues. The main approach was a cytofluorimetric analysis of ALDH expression and positivity for CD44/EPCAM on primary cell population obtained from 23 patients harboring NSCLC. Results: There was a highly positive correlation between the expressions of ALDHhigh and CD44+/EPCAM+ cells, with a Pearson’s correlation coefficient equal to 0.69 (95% CI 0.39–0.86; P = 0.0002), and Spearman’s correlation coefficient equal to 0.52 (P = 0.0124). The average paired difference between the expression of ALDHhigh and CD44+/EPCAM+ cells was very close to 0, being 0.1% (SD 2.5%); there was no difference between these subpopulations in terms of means (95% CI = –1.0; 1.2%, P = 0.8464). These results highlight a strong similarity between ALDHhigh and CD44+/EPCAM+ cells. Conclusions: Our study is the first attempt which identifies a high correlation between the ALDHhigh and the CD44+/EPCAM+ cells, thus suggesting the possibility to use this superficial marker for future target treatments against lung cancer stem cells.

2020 - Cancer Stem-Like Cells in a Case of an Inflammatory Myofibroblastic Tumor of the Lung. [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Sighinolfi, Pamela; Brugioni, Lucio; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Background: Inflammatory myofibroblast tumor (IMT) is a rare tumor with obscure etiopathogenesis in which different inflammatory cells and myofibroblastic spindle cells are seen histologically. Although the majority of these neoplasms have a benign clinical course, the malignant form has also been reported. The gold standard is surgical treatment for complete removal. Our report describes a 50-year-old woman who underwent surgery for IMT of the lung. The aim is to determine whether cancer stem cells may be present in IMT of the lung. Methods: In April 2018, the patient underwent surgery for tumor mass asportation through lateral thoracotomy. The histology of the tumor was consistent with IMT of the lung. The ALDEFLUOR assay, after tissue digestion, was used to identify and sort human lung cancer cells expressing high and low aldehyde dehydrogenase (ALDH) activity. SOX2, NANOG, OCT-4, and c-MYC positivity were additionally determined by immunohistochemistry. Results: The specimen contained 1.10% ALDHhigh cells among all viable lung cancer cells, which indicates the population of cancer stem cells is not negligible. Immunohistochemically assessed cell positivity for ALDH1A1, SOX2, NANOG, OCT-4, and c-MYC, which are considered as lung cancer stem-like cells markers. Conclusion: For the first time, we demonstrated the presence of cancer stem cells in a case of IMT of the lung. This finding may provide a base for considering new pathological and molecular aspects of this tumor. This perspective suggests further studies to understand the possibility of developing recurrence depending on the presence of cancer stem cells.

2020 - Genetic Engineering as a Strategy to Improve the Therapeutic Efficacy of Mesenchymal Stem/Stromal Cells in Regenerative Medicine [Articolo su rivista]
Damasceno, P. K. F.; de Santana, T. A.; Santos, G. C.; Orge, I. D.; Silva, D. N.; Albuquerque, J. F.; Golinelli, G.; Grisendi, G.; Pinelli, M.; Ribeiro dos Santos, R.; Dominici, M.; Soares, M. B. P.
abstract

Mesenchymal stem/stromal cells (MSCs) have been widely studied in the field of regenerative medicine for applications in the treatment of several disease settings. The therapeutic potential of MSCs has been evaluated in studies in vitro and in vivo, especially based on their anti-inflammatory and pro-regenerative action, through the secretion of soluble mediators. In many cases, however, insufficient engraftment and limited beneficial effects of MSCs indicate the need of approaches to enhance their survival, migration and therapeutic potential. Genetic engineering emerges as a means to induce the expression of different proteins and soluble factors with a wide range of applications, such as growth factors, cytokines, chemokines, transcription factors, enzymes and microRNAs. Distinct strategies have been applied to induce genetic modifications with the goal to enhance the potential of MCSs. This review aims to contribute to the update of the different genetically engineered tools employed for MSCs modification, as well as the factors investigated in different fields in which genetically engineered MSCs have been tested.

2020 - Modulating endothelial adhesion and migration impacts stem cell therapies efficacy [Articolo su rivista]
Schafer, R.; Schwab, M.; Siegel, G.; von Ameln-Mayerhofer, A.; Buadze, M.; Lourhmati, A.; Wendel, H. -P.; Kluba, T.; Krueger, M. A.; Calaminus, C.; Scheer, E.; Dominici, M.; Grisendi, G.; Doeppner, T. R.; Schlechter, J.; Finzel, A. K.; Gross, D.; Klaffschenkel, R.; Gehring, F. K.; Spohn, G.; Kretschmer, A.; Bieback, K.; Kramer-Albers, E. -M.; Barth, K.; Eckert, A.; Elser, S.; Schmehl, J.; Claussen, C. D.; Seifried, E.; Hermann, D. M.; Northoff, H.; Danielyan, L.
abstract

Background: Limited knowledge of stem cell therapies‘ mechanisms of action hampers their sustainable implementation into the clinic. Specifically, the interactions of transplanted stem cells with the host vasculature and its implications for their therapeutic efficacy are not elucidated. We tested whether adhesion receptors and chemokine receptors on stem cells can be functionally modulated, and consequently if such modulation may substantially affect therapeutically relevant stem cell interactions with the host endothelium. Methods: We investigated the effects of cationic molecule polyethylenimine (PEI) treatment with or without nanoparticles on the functions of adhesion receptors and chemokine receptors of human bone marrow-derived Mesenchymal Stem Cells (MSC). Analyses included MSC functions in vitro, as well as homing and therapeutic efficacy in rodent models of central nervous system´s pathologies in vivo. Findings: PEI treatment did not affect viability, immunomodulation or differentiation potential of MSC, but increased the CCR4 expression and functionally blocked their adhesion receptors, thus decreasing their adhesion capacity in vitro. Intravenously applied in a rat model of brain injury, the homing rate of PEI-MSC in the brain was highly increased with decreased numbers of adherent PEI-MSC in the lung vasculature. Moreover, in comparison to untreated MSC, PEI-MSC featured increased tumour directed migration in a mouse glioblastoma model, and superior therapeutic efficacy in a murine model of stroke. Interpretation: Balanced stem cell adhesion and migration in different parts of the vasculature and tissues together with the local microenvironment impacts their therapeutic efficacy. Funding: Robert Bosch Stiftung, IZEPHA grant, EU grant 7 FP Health

2020 - Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors [Articolo su rivista]
Golinelli, Giulia; Grisendi, Giulia; Prapa, Malvina; Bestagno, Marco; Spano, Carlotta; Rossignoli, Filippo; Bambi, Franco; Sardi, Iacopo; Cellini, Monica; Horwitz, Edwin M.; Feletti, Alberto; Pavesi, Giacomo; Dominici, Massimo
abstract

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers.

2020 - WISP-2 expression induced by Teriparatide treatment affects in vitro osteoblast differentiation and improves in vivo osteogenesis [Articolo su rivista]
Smargiassi, A.; Bertacchini, J.; Checchi, M.; Poti, F.; Tenedini, E.; Montosi, G.; Magaro, M. S.; Amore, E.; Cavani, F.; Ferretti, M.; Grisendi, G.; Maurel, D. B.; Palumbo, C.
abstract

The Osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances occurring during bone remodeling, caused by hormone perturbations or by mechanical loading alterations, can induce bone pathologies such as osteoporosis. Recently, the active fraction of parathormone, PTH (1-34) or Teriparatide (TPTD), was chosen as election treatment for osteoporosis. The effect of such therapy is dependent on the temporal manner of administration. The molecular reasons why the type of administration regimen is so critical for the fate of bone remodeling are numerous and not yet well known. Our study attempts to analyze diverse signaling pathways directly activated in osteocytes upon TPTD treatment. By means of gene array analysis, we found many molecules upregulated or downregulated in osteocytes. Later, we paid attention to Wisp-2, a protein involved in the Wnt pathway, that is secreted by MLO-Y4 cells and increases upon TPTD treatment and that is able to positively influence the early phases of osteogenic differentiation. We also confirmed the pro osteogenic property of Wisp-2 during mesenchymal stem cell differentiation into the preliminary osteoblast phenotype. The same results were confirmed with an in vivo approach confirming a remarkable Wisp-2 expression in metaphyseal trabecular bone. These results highlighted the anabolic roles unrolled by osteocytes in controlling the action of neighboring cells, suggesting that the perturbation of certain signaling cascades, such as the Wnt pathway, is crucial for the positive regulation of bone formation.

2019 - A Novel 3D In Vitro Platform for Pre-Clinical Investigations in Drug Testing, Gene Therapy, and Immuno-oncology. [Articolo su rivista]
Candini, O; Grisendi, G; Foppiani, Em; Brogli, M; Aramini, B; Masciale, V; Spano, C; Petrachi, T; Veronesi, E; Conte, P; Mari, G; Dominici, M.
abstract

Tumors develop within complex cell-to-cell interactions, with accessory cells playing a relevant role starting in the early phases of cancer progression. This event occurs in a three-dimensional (3D) environment, which to date, has been difficult to reproduce in vitro due to its complexity. While bi-dimensional cultures have generated substantial data, there is a progressive awareness that 3D culture strategies may rapidly increase the understanding of tumor development and be used in anti-cancer compound screening and for predicting response to new drugs utilizing personalized approaches. However, simple systems capable of rapidly rebuilding cancer tissues ex-vivo in 3D are needed and could be used for a variety of applications. Therefore, we developed a flat, handheld and versatile 3D cell culture bioreactor that can be loaded with tumor and/or normal cells in combination which can be monitored using a variety of read-outs. This biocompatible device sustained 3D growth of tumor cell lines representative of various cancers, such as pancreatic and breast adenocarcinoma, sarcoma, and glioblastoma. The cells repopulated the thin matrix which was completely separated from the outer space by two gas-permeable membranes and was monitored in real-time using both microscopy and luminometry, even after transportation. The device was tested in 3D cytotoxicity assays to investigate the anti-cancer potential of chemotherapy, biologic agents, and cell-based therapy in co-cultures. The addition of luciferase in target cancer cells is suitable for comparative studies that may also involve parallel in vivo investigations. Notably, the system was challenged using primary tumor cells harvested from lung cancer patients as an innovative predictive functional assay for cancer responsiveness to checkpoint inhibitors, such as nivolumab. This bioreactor has several novel features in the 3D-culture field of research, representing a valid tool useful for cancer investigations, drug screenings, and other toxicology approaches.

2019 - Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway [Articolo su rivista]
Avnet, Sofia; Chano, Tokuhiro; Massa, Annamaria; Bonuccelli, Gloria; Lemma, Silvia; Falzetti, Luigi; Grisendi, Giulia; Dominici, Massimo; Baldini, Nicola
abstract

Extracellular acidification is a very common cause of stress in tumor microenvironment and of Darwinian pressure. In acid areas of the tumor, most cancer cells are-albeit slowly proliferating-more resistant to cell death than those in well-perfused regions. Tumor acidosis can directly regulate the expression of pro-survival proteins since a low extracellular pH activates the caspase-dependent cell death machinery. This mechanism has never been explored in bone sarcomas. We cultured osteosarcoma and Ewing sarcoma cells under low pH (pH 6.5), and we performed deep-sequencing and protein analysis. Both in in vitro and in vivo models, acidification activity enhanced tumor cells survival. However, we did not observe any change in ERK1 phosphorylation. On the contrary, both at the mRNA and protein level, we found a significant induction of TRAF adaptor proteins and of cIAP proteins (BIRC2 and/or BIRC3). As a consequence, the downstream nuclear transcription factor kappa B (NF-κB) survival pathway was increased. Furthermore, the treatment with the cIAP inhibitor LCL161 reverted the protection from apoptosis under low pH. In vitro results were confirmed both in Ewing sarcoma xenograft and in osteosarcoma patients, since the analysis of tumor tissues demonstrated that the levels of expression of TRAF1 or NF-κB1 significantly correlate with the level of expression of the vacuolar ATPase (V-ATPase), the most important proton pump in eukaryotes. Moreover, in the tissue sections of xenograft model, the nuclear translocation of RelB, a key subunit of the NF-κB transcriptional complex, localized in the tumor region that also corresponded to the acid microenvironment associated with the highest levels of expression of LAMP2 and V-ATPase, in the internal area of the tumor, as revealed by immunohistochemistry. Our data confirm that tumor acid microenvironment activates a stress-regulated switch to promote cell survival of bone sarcoma, and support the hypothesis that this mechanism is mediated by the recruitment of TRAF/cIAP complexes. Altogether, these results suggest that TRAF/cIAP can be considered as a target for anti-cancer therapies.

2019 - Cancer stem-neuroendocrine cells in an atypical carcinoid case report. [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Lung neuroendocrine cells tumor (NET) classification and diagnosis, particularly for typical and atypical carcinoids, are complicated by a variable natural history and nonspecific symptoms. Mechanisms for the development and progression of well-differentiated lung NETs are still unclear. An accurate and timely diagnosis can ensure the implementation of appropriate treatment and impact on prognosis. One of the main unclear point is the definition of these cells’ composition. In fact, it is known that carcinoids are mainly constituted by neuroendocrine cells. Aim of our report is to show for the first time the presence of a high percentage of cancer stem cells (CSCs) in an atypical carcinoid. The ALDEFLUOR assay was used to identify and sort ALDHhigh and ALDHlow human lung cancer cells following tissue digestion. SOX2 was additionally determined by immunohistochemistry. All specimens contained the 53.10% of ALDHhigh cells among all viable lung cancer cells, which indicates that more than half of the entire tumor cell population was composed by CSCs. As expected also in immunohistochemistry, about a half of the nuclei of the cells were positive for SOX2. We strongly support the hypothesis of the presence of cancer stem-neuroendocrine cells (CSCs-NETs) as subpopulation in these types of tumors.

2019 - Challenges in Clinical Development of Mesenchymal Stromal/Stem Cells: Concise Review [Articolo su rivista]
Mastrolia, I.; Foppiani, E. M.; Murgia, A.; Candini, O.; Samarelli, A. V.; Grisendi, G.; Veronesi, E.; Horwitz, E. M.; Dominici, M.
abstract

Identified 50 years ago, mesenchymal stromal/stem cells (MSCs) immediately generated a substantial interest among the scientific community because of their differentiation plasticity and hematopoietic supportive function. Early investigations provided evidence of a relatively low engraftment rate and a transient benefit for challenging congenital and acquired diseases. The reasons for these poor therapeutic benefits forced the entire field to reconsider MSC mechanisms of action together with their ex vivo manipulation procedures. This phase resulted in advances in MSCs processing and the hypothesis that MSC-tissue supportive functions may be prevailing their differentiation plasticity, broadening the spectrum of MSCs therapeutic potential far beyond their lineage-restricted commitments. Consequently, an increasing number of studies have been conducted for a variety of clinical indications, revealing additional challenges and suggesting that MSCs are still lagging behind for a solid clinical translation. For this reason, our aim was to dissect the current challenges in the development of still promising cell types that, after more than half a century, still need to reach their maturity. Stem Cells Translational Medicine 2019;8:1135–1148.

2019 - Correlating tumor-infiltrating lymphocytes and lung cancer stem cells: a cross-sectional study. [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Sighinolfi, Pamela; Pinelli, Massimo; Lovati, Eleonora; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Background: Lung cancer stem cells (LCSCs) are endowed with high aldehyde dehydrogenase (ALDH) expression and play roles in tumor proliferation, metastasis, and drug resistance. Their elusive nature may allow them to escape the immune response by tumor-infiltrating lymphocytes (TILs), which can positively affect the outcome in non-small cell lung cancer (NSCLC) patients. Despite independent investigations on both LCSCs and TILs, the relationship between the two has been very marginally considered. We analyzed whether these two cell types may be related as a prerequisite for novel diagnostic and therapeutic approaches. Methods: In this cross-sectional study, NSCLC human surgical specimens from 12 patients were tested by ALDEFLUOR assay to identify ALDHhigh cells. Fluorescence-activated cell sorting (FACS) analyses for CD3+, CD4+, and CD8+ TILs were performed in combination with immunohistochemistry evaluation. Results: Statistically positive correlations were found between ALDH+ and CD8+, and between ALDH+ and CD3+ cells populations; no correlation was found between ALDH+ and CD4+ cells. The expression of CD3+ and CD8+ by cells accounted for 40.1% and 58.7%, respectively, of the variability of ALDH+ cell expression by an R-squared index, which highlights the strong correlation between TILs and LCSCs. Immunohistochemistry revealed 6–25% positive cells. Conclusions: We report a correlation between cytotoxic TILs and LCSCs, which may contribute to the future development of targeted therapies focusing on the different roles of lymphocytes against lung cancer.

2019 - Impact of HOXB7 overexpression on human adipose-derived mesenchymal progenitors [Articolo su rivista]
Foppiani, E. M.; Candini, O.; Mastrolia, I.; Murgia, A.; Grisendi, G.; Samarelli, A. V.; Boscaini, G.; Pacchioni, L.; Pinelli, M.; De Santis, G.; Horwitz, E. M.; Veronesi, E.; Dominici, M.
abstract

Background: The ex vivo expansion potential of mesenchymal stromal/stem cells (MSC) together with their differentiation and secretion properties makes these cells an attractive tool for transplantation and tissue engineering. Although the use of MSC is currently being tested in a growing number of clinical trials, it is still desirable to identify molecular markers that may help improve their performance both in vitro and after transplantation. Methods: Recently, HOXB7 was identified as a master player driving the proliferation and differentiation of bone marrow mesenchymal progenitors. In this study, we investigated the effect of HOXB7 overexpression on the ex vivo features of adipose mesenchymal progenitors (AD-MSC). Results: HOXB7 increased AD-MSC proliferation potential, reduced senescence, and improved chondrogenesis together with a significant increase of basic fibroblast growth factor (bFGF) secretion. Conclusion: While further investigations and in vivo models shall be applied for better understanding, these data suggest that modulation of HOXB7 may be a strategy for innovative tissue regeneration applications.

2019 - Inducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy [Articolo su rivista]
Rossignoli, Filippo; Grisendi, Giulia; Spano, Carlotta; Golinelli, Giulia; Recchia, Alessandra; Rovesti, Giulia; Orsi, Giulia; Veronesi, Elena; Horwitz, Edwin M.; Dominici, Massimo
abstract

Cellular therapies based on mesenchymal stromal/stem cells (MSC) are promising strategies in regenerative medicine and oncology. Despite encouraging results, there is still some level of concerns on inoculating MSC in cancer patients. To face this issue, one possibility resides in engineering MSC by incorporating a suicide gene in order to control their fate once infused. Strategies based on Herpes Simplex Virus Thymidine Kinase (HSV-TK) and the Cytosine Deaminase genes have been developed and more recently a novel suicide gene, namely, iCasp9, has been proposed. This approach is based on a variant of human Caspase9 that binds with high affinity to a synthetic, bioinert small molecule (AP20187) leading to cell death. Based on this technology so far marginally applied to MSC, we tested the suitability of iCasp9 suicide strategy in MSC to further increase their safety. MSC have been transfected by a lentiviral vector carrying iCasp9 gene and then tested for viability after AP20187 treatment in comparison with mock-transfected cells. Moreover, accounting our anti-tumor approaches based on MSC expressing potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), we generated adipose MSC co-expressing iCasp9 and TRAIL successfully targeting an aggressive sarcoma type. These data show that anti-cancer and suicide mechanisms can coexist without affecting cells performance and hampering the tumoricidal activity mediated by TRAIL. In conclusion, this study originally indicates the suitability of combining a MSC-based anti-cancer gene approach with iCasp9 demonstrating efficiency and specificity.

2019 - Isolation and Identification of Cancer Stem-Like Cells in Adenocarcinoma and Squamous Cell Carcinoma of the Lung: A Pilot Study [Articolo su rivista]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Maiorana, Antonino; Sighinolfi, Pamela; Stefani, Alessandro; Morandi, Uliano; Dominici, Massimo; Aramini, Beatrice
abstract

Background: Lung cancer stem cells (CSCs) share many characteristics with normal stem cells, such as self-renewal and multipotentiality. High expression of aldehyde dehydrogenase (ALDH) has been detected in many tumors, particularly in the CSC compartment, and it plays an important role in tumor proliferation, metastasis, and drug resistance. CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumors. The aim of this study was to isolate and analyze cancer stem-like cells from surgically removed specimens to compare lung adenocarcinoma (ADENO) and squamous (SQUAMO) cell carcinoma. Methods: The ALDEFLUOR assay was used to identify and sort ALDHhigh and ALDHlow human lung cancer cells following tissue digestion. Fluorescence-activated cell sorting analysis for CD44 was performed with tumor cells. Quantitative real-time PCR was performed to assess the expression of SOX2 and NANOG as stemness markers. ALDH1A1 expression was additionally determined by immunohistochemistry. Anchorage-independent ALDHhigh cell growth was also evaluated. ALDHhigh ADENO and SQUAMO cells were cultured to analyze spheroid formation. Results: All specimens contained 0.5–12.5% ALDHhigh cells with 3.8–18.9% CD44-positive cells. SOX2 and NANOG relative expression in ALDHhigh compared to ALDHlow cells in ADENO and SQUAMO was analyzed and compared between the histotypes. Immunohistochemistry confirmed the presence of ALDH1A1 in the sections. SOX2 and NANOG were expressed at higher levels in the ALDHhigh subpopulation than in the ALDHlow subpopulation only in ADENO cells, and the opposite result was seen in SQUAMO cells. In vitro functional assays demonstrated that ALDHhigh cells exhibited migration capacity with distinct behaviors between ALDHhigh spheres in ADENO vs. SQUAMO samples. Conclusions: Our results highlight the importance of a better characterization of cancer stem-like cells in ADENO and SQUAMO histotypes. This may suggest new differential approaches for prognostic and therapeutic purposes in patients with non-small-cell lung cancer.

2019 - MSC-delivered soluble TRAIl and paclitaxel as novel combinatory treatment for pancreatic adenocarcinoma [Articolo su rivista]
Rossignoli, Filippo; Spano, Carlotta; Grisendi, Giulia; Foppiani, Elisabetta Manuela; Golinelli, Giulia; Mastrolia, Ilenia; Bestagno, Marco; Candini, Olivia; Petrachi, Tiziana; Recchia, Alessandra; Miselli, Francesca; Rovesti, Giulia; Orsi, Giulia; Veronesi, Elena; Medici, Gregorio; Petocchi, Benedetta; Pinelli, Massimo; Horwitz, Edwin M.; Conte, Pierfranco; Dominici, Massimo
abstract

Pancreatic cancer is the fourth leading cause of cancer death in western countries with more than 100,000 new cases per year in Europe and a mortality rate higher than 90%. In this scenario, advanced therapies based on gene therapies are emerging, thanks to a better understanding of tumour architecture and cancer cell alterations. We have demonstrated the efficacy of an innovative approach for pancreatic cancer based on mesenchymal stromal cells (MSC) genetically engineered to produce TNF-related Apoptosis Inducing Ligand (TRAIL). Here we investigated the combination of this MSC-based approach with the administration of a paclitaxel (PTX)-based chemotherapy to improve the potential of the treatment, also accounting for a possible resistance onset. Methods: Starting from the BXPC3 cell line, we generated and profiled a TRAIL-resistant model of pancreatic cancer, testing the impact of the combined treatment in vitro with specific cytotoxicity and metabolic assays. We then challenged the rationale in a subcutaneous mouse model of pancreatic cancer, assessing its effect on tumour size accounting stromal and parenchymal organization. Results: PTX was able to restore pancreatic cancer sensitivity to MSC-delivered TRAIL by reverting its pro-survival gene expression profile. The two compounds cooperate both in vitro and in vivo and the combined treatment resulted in an improved cytotoxicity on tumour cells. Conclusion: In summary, this study uncovers the potential of a combinatory approach between MSC-delivered TRAIL and PTX, supporting the combination of cell-based products and conventional chemotherapeutics as a tool to improve the efficacy of the treatments, also addressing possible mechanisms of resistance.

2019 - Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer [Articolo su rivista]
Spano, Carlotta; Grisendi, Giulia; Golinelli, Giulia; Rossignoli, Filippo; Prapa, Malvina; Bestagno, Marco; Candini, Olivia; Petrachi, Tiziana; Recchia, Alessandra; Miselli, Francesca; Rovesti, Giulia; Orsi, Giulia; Maiorana, Antonino; Manni, Paola; Veronesi, Elena; Piccinno, Maria Serena; Murgia, Alba; Pinelli, Massimo; Horwitz, Edwin M.; Cascinu, Stefano; Conte, Pierfranco; Dominici, Massimo
abstract

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.

2019 - Stem cells and lung cancer : between advanced diagnostics and new therapeutics. [Capitolo/Saggio]
Masciale, V; Grisendi, G; Morandi, U; Dominici, M; Aramini, B.
abstract

Lung cancers (LCs) remain a significant and devastating cause of morbidity and mortality worldwide. Despite the very recent success of immunotherapy, the diagnosis and treatment of LC remain one of the greatest challenges in chest surgery, clinical oncology, and molecular medicine. A growing number of investigations on normal/cancer stem cells and cellular therapies are offering exciting new avenues to advance knowledge on LC. Here, we will be focusing on the multiple relationships between LC and stem cells accounting for cancer stem cell (CSC) diagnostics and progenitor-based therapeutics for LC. Cancer cell repopulation after chemotherapy and/or radiotherapy still represents a major factor limiting the efficacy of treatment since CSCs play critical roles during this process by reciprocal connections between CSCs and tumor microenvironment. This calls for new opportunities to integrate advanced CSC diagnostics and targeted approaches also based on immunotherapy. In addition, recent discoveries on malignant pleural and other LC highlight that mesenchymal stromal/stem cells may be a novel platform for drug delivery within still unexplored gene therapy strategies. This chapter will dissect these two apparently distant technologies within a unified stem-cell-based vision aimed at providing better diagnostics and therapeutics for LC at the forefront of modern clinical oncology.

2018 - Cancer stem cells and their microenvironment. [Capitolo/Saggio]
Masciale, Valentina; Grisendi, Giulia; Banchelli, Federico; D'Amico, Roberto; Morandi, Uliano; Dominici, Massimo; Husnain Haider, Khawaja; Aramini, Beatrice
abstract

2018 - Challenging the efficacy of checkpoint inhibitors: a new 3D model for a precision medicine approach. [Abstract in Atti di Convegno]
Foppiani, ELISABETTA MANUELA; Candini, Olivia; Aramini, Beatrice; Masciale, Valentina; Grisendi, Giulia; Matteo, Brogli; Giorgio, Mari; Dominici, Massimo
abstract

Challenging the efficacy of checkpoint inhibitors: a new 3D model for a precision medicine approach.

2018 - Human Herpes simplex 1 virus infection of endometrial decidual tissue-derived MSC alters HLA-G expression and immunosuppressive functions [Articolo su rivista]
Bortolotti, Daria; Rossignoli, Filippo; Rotola, Antonella; Campioni, Diana; Cultrera, Rosario; Grisendi, Giulia; Dominici, Massimo; Rizzo, Roberta
abstract

Objectives: Mesenchymal stromal/stem cells have immunosuppressive functions. Our previous results demonstrated that one of the players of this immunomodulation can be ascribed to the Human Leukocyte Antigen-G. HLA-G, a non classical HLA class I antigen, is involved in immune tolerance during pregnancy, organ transplantation, autoimmune and inflammatory diseases. In this study we wanted to verify whether human endometrial decidual tissue derived (EDT)-MSC could express HLA-G. Additionally we assessed the permissivity to Human Herpesvirus infections, using HSV-1 as a model, and the possible effect on EDT-MSC immunosuppressive functions towards peripheral blood mononuclear cell (PBMC) proliferation. Methods: We analyzed immune-inhibitory functions and HLA-G expression in human EDT-MSC before and after HSV-1 infection. Results: We observed that EDT-MSC express HLA-G molecules, that partly are responsible for the immune-inhibitory functions of EDT-MSC towards PBMC proliferation. EDT-MSC are permissive for a productive infection by HSV-1, that decreases HLA-G expression and affects EDT-MSC immune-inhibitory functions. Conclusions: We demonstrate that EDT-MSC are susceptible to HSV-1 infection, that reduces HLA-G expression and their immune-inhibitory function. These data could have a clinical implication in the use of EDT-MSC as an immunosuppressant, in particular in steroid-refractory GvHD after allogeneic hematopoietic stem cell transplantation and in autoimmune diseases.

2018 - In vitro and in vivo discrepancy in inducing apoptosis by mesenchymal stromal cells delivering membrane-bound tumor necrosis factor–related apoptosis inducing ligand in osteosarcoma pre-clinical models [Articolo su rivista]
Guiho, Romain; Biteau, Kevin; Grisendi, Giulia; Chatelais, Mathias; Brion, Regis; Taurelle, Julien; Renault, Sarah; Heymann, Dominique; Dominici, Massimo; Redini, Françoise
abstract

Background: Osteosarcoma (OS) is the most frequent pediatric malignant bone tumor. OS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine Tumor necrosis factor (TNF)–Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, many OS cell lines appear resistant to recombinant human (rh)TRAIL-induced apoptosis. We, therefore, hypothesized that TRAIL presentation at the membrane level of carrier cells might overcome this resistance and trigger apoptosis. Methods: To address this, human adipose mesenchymal stromal cells (MSCs) transfected in a stable manner to express membrane-bound full-length human TRAIL (mbTRAIL) were co-cultured with several human OS cell lines. Results: This induced apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL. In contrast, mbTRAIL delivered by MSCs was not able to counteract tumor progression in this OS orthotopic model. Discussion: This was partly due to the fact that MSCs showed a potential to support tumor development. Moreover, the expression of mbTRAIL did not show caspase activation in adjacent tumor cells.

2018 - Proposal of a Novel Natural Biomaterial, the Scleral Ossicle, for the Development of Vascularized Bone Tissue In Vitro [Articolo su rivista]
Checchi, Marta; Bertacchini, Jessika; Grisendi, Giulia; Smargiassi, Alberto; Sola, Antonella; Messori, Massimo; Palumbo, Carla
abstract

Recovering of significant skeletal defects could be partially abortive due to the perturbations that affect the regenerative process when defects reach a critical size, thus resulting in a non-healed bone. The current standard treatments include allografting, autografting, and other bone implant techniques. However, although they are commonly used in orthopedic surgery, these treatments have some limitations concerning their costs and their side effects such as potential infections or malunions. On this account, the need for suitable constructs to fill the gap in wide fractures is still urgent. As an innovative solution, scleral ossicles (SOs) can be put forward as natural scaffolds for bone repair. SOs are peculiar bony plates forming a ring at the scleral-corneal border of the eyeball of lower vertebrates. In the preliminary phases of the study, these ossicles were structurally and functionally characterized. The morphological characterization was performed by SEM analysis, MicroCT analysis and optical profilometry. Then, UV sterilization was carried out to obtain a clean support, without neither contaminations nor modifications of the bone architecture. Subsequently, the SO biocompatibility was tested in culture with different cell lines, focusing the attention to the differentiation capability of endothelial and osteoblastic cells on the SO surface. The results obtained by the above mentioned analysis strongly suggest that SOs can be used as bio-scaffolds for functionalization processes, useful in regenerative medicine.

2017 - Blocking tumor-educated MSC paracrine activity halts osteosarcoma progression [Articolo su rivista]
Baglio, Sr; Lagerweij, T; Pérez Lanzón, M; Xuan Ho, D; Léveillé, N; Melo, Sa; Cleton-Jansen, Am; Jordanova, Es; Roncuzzi, L; Greco, M; van Eijndhoven, Ma; Grisendi, G; Dominici, M; Bonafede, R; Lougheed, S; de Gruijl, Td; Zini, N; Cervo, S; Steffan, A; Canzonieri, V; Martson, A; Maasalu, K; Koks, S; Wurdinger, T; Baldini, N; Pegtel, Dm.
abstract

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients.

2017 - Scleral ossicles as natural biomaterials on which vascular-like network is promoted from Mouse Aortic Endothelial cells (MAECs): preliminary results [Abstract in Rivista]
Checchi, Marta; Grisendi, Giulia; Bertacchini, Jessika; Magaro', MARIA SARA; Ferretti, Marzia; Benincasa, Marta; Sena, Paola; Cavani, Francesco; Palumbo, Carla
abstract

When a severe fracture is difficult to self-recovered, it is defined as “critical-size” bone defect. Till now, many efforts have been made by the tissue engineering (TE) to generate scaffolds suitable for recovering of this type of fracture, but the main obstacle remains the lack of an appropriate vascularization of the scaffolds. In the field of the regenerative medicine, the TE has developed many different biomaterials, with various features and peculiar functions, to be used in combination with cells and growth factors, in the generation of specialized constructs. Our proposal of natural scaffolds useful to obtain complex constructs concerns peculiar bony chips extracted from the eye bulb of adult chickens: the scleral ossicles (SOs). This proposed model is interesting because once SOs reach the definitive size in the adult animal, they are devoted only to mechanical stereotyped stress for their lifetime so that the activation of the bone remodelling should be avoided and, to do this, the osteocytes undergo massive apoptosis, making the ossicles like decellularized bones [1]. The novelty of our proposal is that the scaffolds do not require surface treatment (like further matrix deposition on the SO surface) since they are characterized, like all bones, by the well-known organic components such as type I-collagen fibres, proteoglycans and glycoproteins. The latter, for example, play the role of adhesion proteins and therefore can mediate the adhesion of the endothelial cells that should develop the vascular network. Our final goal is to obtain an in vitro 3D-vascularized natural constructs, from scaffolds easily available in nature to use in vivo for the healing of “critical-size” bone defeats. Previously [2] we identified the best preparation methods to obtain suitable SO surface for cell culture. Recently, we have performed a series of in vitro experiments to test the biocompatibility properties of the support; then, cell adhesion tests, viability and proliferation assay were carried out. Further, we tried to induce a vascular-like network organization of Mouse Aortic Endothelial Cells (MAECs) directly on the SOs surface, stimulating the cells with a known angiogenic factor, the Vascular Endothelial Growth Factor (VEGF), getting encouraging preliminary results.

2017 - Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma [Articolo su rivista]
Petrachi, Tiziana; Romagnani, Alessandra; Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia
abstract

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhighand ALDHlowderived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.

2016 - Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance [Articolo su rivista]
Avnet, Sofia; Lemma, Silvia; Cortini, Margherita; Pellegrini, Paola; Perut, Francesca; Zini, Nicoletta; Kusuzaki, Katsuyuki; Chano, Tokuhiro; Grisendi, Giulia; Dominici, Massimo; De Milito, Angelo; Baldini, Nicola
abstract

Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24-48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, shortterm acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance.

2016 - CD271 Down-Regulation Promotes Melanoma Progression and Invasion in Three-Dimensional Models and in Zebrafish [Articolo su rivista]
Saltari, Annalisa; Truzzi, Francesca; Quadri, Marika; Lotti, Roberta; Palazzo, Elisabetta; Grisendi, Giulia; Tiso, Natascia; Marconi, Alessandra; Pincelli, Carlo
abstract

CD271 is a neurotrophin receptor variably expressed in melanoma. Although contradictory data are reported on its role as a marker of tumor-initiating cells, little is known about its function in tumor progression. CD271 expression was higher in spheroids derived from freshly isolated cells of primary melanomas and in primary WM115 and WM793-B cell lines, and it decreased during progression to advanced stages in cells isolated from metastatic melanomas and in metastatic WM266-4 and 1205Lu cell lines. Moreover, CD271 was scarcely detected in the highly invasive spheroids (SKMEL28 and 1205Lu). CD271, originally expressed in the epidermis of skin reconstructs, disappeared when melanoma started to invade the dermis. SKMEL8 CD271(-) cells showed greater proliferation and invasiveness in vitro and were associated with a higher number of metastases in zebrafish compared with CD271(+) cells. CD271 silencing in WM115 induced a more aggressive phenotype in vitro and in vivo. On the contrary, CD271 overexpression in SKMEL28 cells reduced invasion in vitro, and CD271 overexpressing 1205Lu cells was associated with a lower percentage of metastases in zebrafish. A reduced cell-cell adhesion was also observed in the absence of CD271. Taken together, these results indicate that CD271 loss is critical for melanoma progression and metastasis.

2016 - Ibrutinib modifies the function of monocyte/macrophage population in chronic lymphocytic leukemia [Articolo su rivista]
Fiorcari, Stefania; Maffei, Rossana; Audrito, Valentina; Martinelli, Silvia; Hacken, Elisa Ten; Zucchini, Patrizia; Grisendi, Giulia; Potenza, Leonardo; Luppi, Mario; Burger, Jan A; Deaglio, Silvia; Marasca, Roberto
abstract

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.

2016 - In vivo editing of the human mutant Rhodopsin gene by electroporation of plasmid-based CRISPR/Cas9 in the mouse retina [Articolo su rivista]
Latella, Maria Carmela; Di Salvo, Maria Teresa; Cocchiarella, Fabienne; Benati, Daniela; Grisendi, Giulia; Comitato, Antonella; Marigo, Valeria; Recchia, Alessandra
abstract

The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/ Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells.

2016 - Resistance to neoplastic transformation of ex-vivo expanded human mesenchymal stromal cells after exposure to supramaximal physical and chemical stress [Articolo su rivista]
Conforti, Antonella; Starc, Nadia; Biagini, Simone; Tomao, Luigi; Pitisci, Angela; Algeri, Mattia; Sirleto, Pietro; Novelli, Antonio; Grisendi, Giulia; Candini, Olivia; Carella, Cintia; Dominici, Massimo; Locatelli, Franco; Bernardo, Maria Ester
abstract

The risk of malignant transformation of ex-vivo expanded human mesenchymal stromal cells (huMSCs) has been debated in the last years; however, the biosafety of these cells after exposure to supramaximal physical and chemical stress has never been systematically investigated.We established an experimental in vitro model to induce supramaximal physical (ionizing radiation, IR) and chemical (starvation) stress on ex-vivo expanded bone marrow (BM)-derived huMSCs and investigated their propensity to undergo malignant transformation. To this aim, we examined MSC morphology, proliferative capacity, immune-phenotype, differentiation potential, immunomodulatory properties and genetic profile before and after stressor exposure. Furthermore, we investigated the cellular mechanisms underlying MSC response to stress. MSCs were isolated from 20 healthy BM donors and expanded in culture medium supplemented with 5% platelet lysate (PL) up to passage 2 (P2). At this stage, MSCs were exposed first to escalating doses of IR (30, 100, 200 Gy) and then to starvation culture conditions (1% PL).With escalating doses of radiation, MSCs lost their typical spindle-shaped morphology, their growth rate markedly decreased and eventually stopped (at P4-P6) by reaching early senescence. Irradiated and starved MSCs maintained their typical immune-phenotype, ability to differentiate into adipocytes/osteoblasts and to inhibit mitogen-induced T-cell proliferation. The study of the genetic profile of irradiated/starved MSCs did not show any alteration. While the induction of supramaximal stress triggered production of ROS and activation of DNA damage response pathway via multiple mechanisms, our data indicate that irradiated/starved MSCs, although presenting altered morphology/growth rate, do not display increased propensity for malignant transformation.

2016 - TRAIL delivered by mesenchymal stromal/stem cells counteracts tumor development in orthotopic Ewing sarcoma models [Articolo su rivista]
Guiho, Romain; Biteau, Kevin; Grisendi, Giulia; Taurelle, Julien; Chatelais, Mathias; Gantier, Malika; Heymann, Dominique; Dominici, Massimo; Redini, Françoise
abstract

Ewing sarcoma (EWS) is the second most frequent pediatric malignant bone tumor. EWS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine TNF-Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, certain EWS cell lines appear resistant to recombinant human (rh) TRAIL-induced apoptosis. We therefore hypothesized that a TRAIL presentation at the surface of the carrier cells might overcome this resistance and trigger apoptosis. For this purpose, human adipose mesenchymal stromal/stem cells (MSC) transfected in a stable manner to express full-length human TRAIL were co-cultured with several human EWS cell lines, inducing apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL or AMG655, an antibody agonist to the death receptor, DR5. In vivo, TRAIL delivered by MSCs was able to counteract tumor progression in two orthotopic models of Ewing sarcoma, associated with caspase activation, indicating that a cell-based delivery of a potent apoptosis-inducing factor could be relevant in EWS.

2016 - Tumor Stroma Manipulation By MSC [Articolo su rivista]
Grisendi, Giulia; Spano, Carlotta; Rossignoli, Filippo; D. Souza, Naomi; Golinelli, Giulia; Fiori, Agnese; Horwitz, Edwin M; Guarneri, Valentina; Piacentini, Federico; Paolucci, Paolo; Dominici, Massimo
abstract

Tumor stroma (TS) plays relevant roles in all steps of cancer development. We here address several fundamental aspects related with the interaction between cancer cells and their stromal counterparts. Dissecting these players is of pivotal importance to understand oncogenesis, immunoescape and drug resistance. In addition, this better comprehension will allow the introduction of novel and more effective therapeutic approaches where manipulated stromal elements may become detrimental for tumor growth. Our group and others rely on the use of multipotent mesenchymal stromal/stem cells (MSC) as anti-cancer tools, since these putative TS cell precursors can deliver potent apoptosis-inducing agents. Multimodal-armed MSC can target a variety of cancers in vitro and, when injected in vivo, they localize into tumors mediating cell death without evident toxicities to normal tissues. While several aspects of these strategies shall require further investigations, these approaches collectively indicate how TS manipulation by MSC represents a tool to influence the fate of cancer cells, creating a new generation of anti-cancer strategies.

2015 - A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing [Articolo su rivista]
Prapa, Malvina; Caldrer, Sara; Spano, Maria Carlotta; Bestagno, Marco; Golinelli, Giulia; Grisendi, Giulia; Petrachi, Tiziana; Conte, Pierfranco; Horwitz, EDWIN MARK; Campana, Dario; Paolucci, Paolo; Dominici, Massimo
abstract

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.

2015 - CD271 Mediates Stem Cells to Early Progeny Transition in Human Epidermis [Articolo su rivista]
Truzzi, Francesca; Saltari, Annalisa; Palazzo, Elisabetta; Lotti, Roberta; Petrachi, Tiziana; Dallaglio, Katiuscia; Gemelli, Claudia; Grisendi, Giulia; Dominici, Massimo; Pincelli, Carlo; Marconi, Alessandra
abstract

CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the tumor necrosis factor receptor superfamily. Because in human epidermis, CD271 is predominantly expressed in transit-amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSCs) to progeny. Calcium induced an upregulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 small interfering RNA. Furthermore, CD271 overexpression provoked the switch of KSCs to TA cells, whereas silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of β1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and keratin 15 (K15) compared with CD271(-) cells and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in the skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSCs to TA cells.

2015 - Carbonic anhydrase IX inhibition is an effective strategy for osteosarcoma treatment [Articolo su rivista]
Perut, Francesca; Carta, Fabrizio; Bonuccelli, Gloria; Grisendi, Giulia; Di, Pompo Gemma; Avnet, Sofia; Sbrana, Francesca Vittoria; Hosogi, Shigekuni; Dominici, Massimo; Kusuzaki, Katsuyuki; Supuran, Claudiu T.; Baldini, Nicola
abstract

Objective: Hypoxia-inducible factor 1, a regulator of CA IX activity, is often overexpressed in human osteosarcoma (OS) but not in normal tissues, and its expression levels correlate with prognosis. In this study, we investigated the therapeutic potential of newly synthesized CA IX sulfonamide inhibitors in OS. Methods: CA IX expression was evaluated in OS cell lines and bone marrow stromal cells (BMSC). After treatment with CA IX inhibitors, cell proliferation, apoptosis, cell cycle, extracellular and cytosolic pH changes were evaluated both in vitro and in mouse OS xenografts. Results: CA IX expression levels were significantly higher in OS than in BMSC. Accordingly, CA IX inhibitor 3 induced remarkable cytotoxicity on OS cells without affecting BMSC proliferation. This activity was increased under hypoxia, and was mediated by cell cycle arrest and by the modulation of cytosolic and extracellular pH. In vivo, CA IX inhibitor 3 reduced tumor growth by inducing significant necrosis. Conclusions: Our results provide a strong rationale for the clinical use of the newly synthesized CA IX inhibitor 3 in human OS.

2015 - Detection of microparticles from human red blood cells by multiparametric flow cytometry [Articolo su rivista]
Grisendi, Giulia; Finetti, Elena; Manganaro, Daniele; Cordova, Nicoletta; Montagnani, Giuliano; Spano, Maria Carlotta; Prapa, Malvina; Guarneri, Valentina; Otsuru, Satoru; Horwitz, Edwin M.; Mari, Giorgio; Dominici, Massimo
abstract

Background: During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as "storage lesions". These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC. Material and methods: RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody. Results: We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected. Discussion: Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies.

2015 - Mesenchymal progenitors aging highlights a mir-196 switch targeting HOXB7 as master regulator of proliferation and osteogenesis [Articolo su rivista]
Candini, Olivia; Spano, Maria Carlotta; Murgia, Alba; Grisendi, Giulia; Veronesi, Elena; Piccinno, MARIA SERENA; Ferracin, Manuela; Negrini, Massimo; Giacobbi, Francesca; Bambi, Franco; Horwitz, Edwin Mark; Conte, Pierfranco; Paolucci, Paolo; Dominici, Massimo
abstract

Human aging is associated with a decrease in tissue functions combined with a decline in stem cells frequency and activity followed by a loss of regenerative capacity. The molecular mechanisms behind this senescence remain largely obscure, precluding targeted approaches to counteract aging. Focusing on mesenchymal stromal/stem cells (MSC) as known adult progenitors, we identified a specific switch in miRNA expression during aging, revealing a miR-196a up-regulation which was inversely correlated with MSC proliferation through HOXB7 targeting. A forced HOXB7 expression was associated with an improved cell growth, a reduction of senescence and an improved osteogenesis linked to a dramatic increase of autocrine bFGF secretion. These findings, along with the progressive decrease of HOXB7 levels observed during skeletal aging in mice, indicate HOXB7 as a master factor driving progenitors behavior lifetime, providing a better understanding of bone senescence and leading to an optimization of MSC performance.

2015 - Mesenchymal progenitors expressing TRAIL induce apoptosis in sarcomas [Articolo su rivista]
Grisendi, Giulia; Spano, Maria Carlotta; D'Souza, Naomi; Rasini, Valeria; Veronesi, Elena; Prapa, Malvina; Petrachi, Tiziana; Piccinno, MARIA SERENA; Rossignoli, Filippo; Burns, Jorge Phillip Joaquin Sans; Fiorcari, Stefania; Granchi, Donatella; Baldini, Nicola; Horwitz, EDWIN MARK; Guarneri, Valentina; Conte, Pierfranco; Paolucci, Paolo; Dominici, Massimo
abstract

Sarcomas are frequent tumors in children and young adults that, despite a relative chemo-sensitivity, show high relapse rates with up to 80% of metastatic patients dying in 5 years from diagnosis. The real ontogeny of sarcomas is still debated and evidences suggest they may derive from precursors identified within mesenchymal stromal/stem cells (MSC) fractions. Recent studies on sarcoma microenvironment additionally indicated that MSC could take active part in generation of a supportive stroma. Based on this knowledge, we conceived to use modified MSC to deliver tumor necrosis factor related apoptosis inducing ligand (TRAIL) targeting different sarcoma histotypes. Gene modified MSC expressing TRAIL were co-cultured with different osteosarcoma, rhabdomyosarcoma and Ewing's Sarcoma (ES) cell lines assessing viability and caspase-8 activation. An in vivo model focused on ES was then implemented considering the impact of MSC-TRAIL on tumor size, apoptosis and angiogenesis. MSC expressing TRAIL induced significantly high apoptosis in all tested lines. Sarcoma death was specifically associated with caspase-8 activation starting from 8 hours of co-culture with MSC-TRAIL. When injected into pre-established ES xenotransplants, MSC-TRAIL persisted within its stroma, causing significant tumor apoptosis versus control groups. Additional histological and in vitro studies reveal that MSC-TRAIL could also exert potent anti-angiogenic functions. Our results suggest that MSC as TRAIL vehicles could open novel therapeutic opportunities for sarcoma by multiple mechanisms.

2015 - Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies [Articolo su rivista]
D’Souza, Naomi; Rossignoli, Filippo; Golinelli, Giulia; Grisendi, Giulia; Spano, Maria Carlotta; Candini, Olivia; Osturu, Satoru; Catani, Fabio; Paolucci, Paolo; Horwitz, Edwin M.; Dominici, Massimo
abstract

Regenerative medicine relying on cell and gene therapies is one of the most promising approaches to repair tissues. Multipotent mesenchymal stem/stromal cells (MSC), a population of progenitors committing into mesoderm lineages, are progressively demonstrating therapeutic capabilities far beyond their differentiation capacities. The mechanisms by which MSC exert these actions include the release of biomolecules with anti-inflammatory, immunomodulating, anti-fibrogenic, and trophic functions. While we expect the spectra of these molecules with a therapeutic profile to progressively expand, several human pathological conditions have begun to benefit from these biomolecule-delivering properties. In addition, MSC have also been proposed to vehicle genes capable of further empowering these functions. This review deals with the therapeutic properties of MSC, focusing on their ability to secrete naturally produced or gene-induced factors that can be used in the treatment of kidney, lung, heart, liver, pancreas, nervous system, and skeletal diseases. We specifically focus on the different modalities by which MSC can exert these functions. We aim to provide an updated understanding of these paracrine mechanisms as a prerequisite to broadening the therapeutic potential and clinical impact of MSC.

2014 - Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells [Articolo su rivista]
Bonuccelli, Gloria; Avnet, Sofia; Grisendi, Giulia; Salerno, Manuela; Granchi, Donatella; Dominici, Massimo; Kusuzaki, Katsuyuki; Baldini, Nicola
abstract

The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.

2014 - Staminali per la cura del tumore del pancreas: tra evidenze scienti che e possibile trasferimento clinico [Articolo su rivista]
Golinelli, Giulia; Grisendi, Giulia; Spano, Carlotta; Rossignoli, Filippo; Dominici, Massimo
abstract

Una nuova promessa terapeutica arriva dall'utilizzo di cellule umane ca- paci di indurre la morte delle cellule tumorali tramite il rilascio di composti tossici.

2014 - Suppression of invasion and metastasis of triple-negative breast cancer lines by pharmacological or genetic inhibition of slug activity [Articolo su rivista]
Ferrari Amorotti, Giovanna; Chiodoni, Claudia; Shen, Fei; Cattelani, Sara; Soliera, Angela Rachele; Manzotti, Gloria; Grisendi, Giulia; Dominici, Massimo; Rivasi, Francesco; Colombo, Mario Paolo; Fatatis, Alessandro; Calabretta, Bruno
abstract

Most triple-negative breast cancers (TNBCs) exhibit gene expression patterns associated with epithelial-to-mesenchymal transition (EMT), a feature that correlates with a propensity for metastatic spread. Overexpression of the EMT regulator Slug is detected in basal and mesenchymal-type TNBCs and is associated with reduced E-cadherin expression and aggressive disease. The effects of Slug depend, in part, on the interaction of its N-terminal SNAG repressor domain with the chromatin-modifying protein lysine demethylase 1 (LSD1); thus, we investigated whether tranylcypromine [also known as trans-2-phenylcyclopropylamine hydrochloride (PCPA) or Parnate], an inhibitor of LSD1 that blocks its interaction with Slug, suppresses the migration, invasion, and metastatic spread of TNBC cell lines. We show here that PCPA treatment induces the expression of E-cadherin and other epithelial markers and markedly suppresses migration and invasion of TNBC cell lines MDA-MB-231 and BT-549. These effects were phenocopied by Slug or LSD1 silencing. In two models of orthotopic breast cancer, PCPA treatment reduced local tumor growth and the number of lung metastases. In mice injected directly in the blood circulation with MDA-MB-231 cells, PCPA treatment or Slug silencing markedly inhibited bone metastases but had no effect on lung infiltration. Thus, blocking Slug activity may suppress the metastatic spread of TNBC and, perhaps, specifically inhibit homing/colonization to the bone.

2014 - Surrounding pancreatic adenocarcinoma by killer mesenchymal stromal/stem cells [Articolo su rivista]
Golinelli, Giulia; Grisendi, Giulia; Spano, Maria Carlotta; Dominici, Massimo
abstract

N/A

2014 - Transportation conditions for prompt use of Ex Vivo expanded and freshly harvested clinical-grade bone marrow mesenchymal stromal/stem cells for bone regeneration [Articolo su rivista]
Veronesi, Elena; Murgia, Alba; Caselli, Anna; Grisendi, Giulia; Piccinno, MARIA SERENA; Rasini, Valeria; Giordano, Rosaria; Montemurro, Tiziana; Bourin, Philippe; Sensebé, Luc; Rojewski, Markus T.; Schrezenmeier, Hubert; Layrolle, Pierre; Ginebra, Maria Pau; Panaitescu, Carmen Bunu; Gómez Barrena, Enrique; Catani, Fabio; Paolucci, Paolo; Burns, Jorge Phillip Joaquin Sans; Dominici, Massimo
abstract

Successful preliminary studies have encouraged a more translational phase for stem cell research. Nevertheless, advances in the culture of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) and osteoconductive qualities of combined biomaterials can be undermined if necessary cell transportation procedures prove unviable. We aimed at evaluating the effect of transportation conditions on cell function, including the ability to form bone in vivo, using procedures suited to clinical application. hBM-MSC expanded in current Good Manufacturing Practice (cGMP) facilities (cGMP-hBM-MSC) to numbers suitable for therapy were transported overnight within syringes and subsequently tested for viability. Scaled-down experiments mimicking shipment for 18 h at 4°C tested the influence of three different clinical-grade transportation buffers (0.9\% saline alone or with 4\% human serum albumin [HSA] from two independent sources) compared with cell maintenance medium. Cell viability after shipment was >80\% in all cases, enabling evaluation of (1) adhesion to plastic flasks and hydroxyapatite tricalcium phosphate osteoconductive biomaterial (HA/β-TCP 3D scaffold); (2) proliferation rate; (3) ex vivo osteogenic differentiation in contexts of 2D monolayers on plastic and 3D HA/β-TCP scaffolds; and (4) in vivo ectopic bone formation after subcutaneous implantation of cells with HA/β-TCP scaffold into NOD/SCID mice. Von Kossa staining was used to assess ex vivo osteogenic differentiation in 3D cultures, providing a quantifiable test of 3D biomineralization ex vivo as a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all transportation buffers. Moreover, cGMP-hBM-MSC transported from a production facility under clinical-grade conditions of 4\% HSA in 0.9\% saline to a destination 18 h away showed prompt adhesion to HA/β-TCP 3D scaffold and subsequent in vivo bone formation. A successfully validated transportation protocol extends the applicability of fresh stem cells involving multicentric trials for regenerative medicine.

2013 - Adipose stromal/stem cells assist fat transplantation reducing necrosis and increasing graft performance. [Articolo su rivista]
PICCINNO, MARIA SERENA; VERONESI, Elena; LOSCHI, Pietro; M., Pignatti; MURGIA, ALBA; GRISENDI, Giulia; I., Castelli; D., Bernabei; CANDINI, Olivia; P., Conte; PAOLUCCI, Paolo; E. M., Horwitz; DE SANTIS, Giorgio; IUGHETTI, Lorenzo; DOMINICI, Massimo
abstract

Autologous fat transfer (AFT) is a procedure for adipose tissue (AT) repair after trauma, burns, post-tumor resections and lipodystrophies still negatively impacted by the lack of graft persistence. The reasons behind this poor outcome are unclear and seem to involve damages in either harvested/transplanted mature adipocytes or on their mesenchymal progenitors, namely adipose stromal/stem cells (ASC), and due to post-transplant AT apoptosis and involution. A rabbit subcutaneous AT regeneration model was here developed to first evaluate graft quality at different times after implant focusing on related parameters, such as necrosis and vasculogenesis. Standard AFT was compared with a strategy where purified autologous ASC, combined with hyaluronic acid (HA), assisted AFT. Five million of autologous ex vivo isolated CD29+, CD90+, CD49e+ ASC, loaded into HA, enriched 1 ml of AT generating an early significant protective effect in reducing AFT necrosis and increasing vasculogenesis with a preservation of transplanted AT architecture. This beneficial impact of ASC assisted AFT was then confirmed at three months with a robust lipopreservation and no signs of cellular transformation. By a novel ASC assisted AFT approach we ensure a reduction in early cell death favoring an enduring graft performance possibly for a more stable benefit in patients.

2013 - IGF-1-mediated osteoblastic niche expansion enhances long-term hematopoietic stem cell engraftment after murine bone marrow transplantation [Articolo su rivista]
Caselli, Anna; Olson, Timothy S.; Otsuru, Satoru; Chen, Xiaohua; Hofmann, Ted J.; Nah, Hyun Duck; Grisendi, Giulia; Paolucci, Paolo; Dominici, Massimo; Horwitz, Edwin Mark
abstract

The efficiency of hematopoietic stem cell (HSC) engraftment after bone marrow (BM) transplantation depends largely on the capacity of the marrow microenvironment to accept the transplanted cells. While radioablation of BM damages osteoblastic stem cell niches, little is known about their restoration and mechanisms governing their receptivity to engraft transplanted HSCs. We previously reported rapid restoration and profound expansion of the marrow endosteal microenvironment in response to marrow radioablation. Here, we show that this reorganization represents proliferation of mature endosteal osteoblasts which seem to arise from a small subset of high-proliferative, relatively radio-resistant endosteal cells. Multiple layers of osteoblasts form along the endosteal surface within 48 hours after total-body irradiation, concomitant with a peak in marrow cytokine expression. This niche reorganization fosters homing of the transplanted hematopoietic cells to the host marrow space and engraftment of long-term (LT)-HSC. Inhibition of insulin-like growth factor (IGF)-1-receptor tyrosine kinase signaling abrogates endosteal osteoblast proliferation and donor HSC engraftment, suggesting that the cytokine IGF-1 is a crucial mediator of endosteal niche reorganization and consequently donor HSC engraftment. Further understanding of this novel mechanism of IGF-1-dependent osteoblastic niche expansion and HSC engraftment may yield clinical applications for improving engraftment efficiency after clinical HSC transplantation.

2013 - Isolation, characterization, and transduction of endometrial decidual tissue multipotent mesenchymal stromal/stem cells from menstrual blood [Articolo su rivista]
Rossignoli, Filippo; Caselli, Anna; Grisendi, Giulia; Piccinno, MARIA SERENA; Burns, Jorge Phillip Joaquin Sans; Murgia, Alba; Veronesi, Elena; Loschi, Pietro; Masini, Cristina; Conte, Pierfranco; Paolucci, Paolo; Horwiz, Edwin M.; Dominici, Massimo
abstract

Mesenchymal stromal/stem cells (MSCs) reveal progenitor cells-like features including proliferation and differentiation capacities. One of the most historically recognized sources of MSC has been the bone marrow, while other sources recently include adipose tissue, teeth, bone, muscle, placenta, liver, pancreas, umbilical cord, and cord blood. Frequently, progenitor isolation requires traumatic procedures that are poorly feasible and associated with patient discomfort. In the attempt to identify a more approachable MSC source, we focused on endometrial decidual tissue (EDT) found within menstrual blood. Based also on recent literature findings, we hypothesized that EDT may contain heterogeneous populations including some having MSC-like features. Thus, we here sought to isolate EDT-MSC processing menstrual samples from multiple donors. Cytofluorimetric analyses revealed that resulting adherent cells were expressing mesenchymal surface markers, including CD56, CD73, CD90, CD105 and CD146, and pluripotency markers such as SSEA-4. Moreover, EDT-MSC showed a robust clonogenic potential and could be largely expanded in vitro as fibroblastoid elements. In addition, differentiation assays drove these cells towards osteogenic, adipogenic, and chondrogenic lineages. Finally, for the first time, we were able to gene modify these progenitors by a retroviral vector carrying the green fluorescent protein. From these data, we suggest that EDT-MSC could represent a new promising tool having potential within cell and gene therapy applications.

2013 - MSC and Tumors: Homing, Differentiation and Secretion Influence The Therapeutic Potentials [Capitolo/Saggio]
D'Souza, Naomi; Burns, Jorge Sans; Grisendi, Giulia; Candini, Olivia; Veronesi, Elena; Piccinno, Serena; Horwitz, EDWIN MARK; Paolucci, Paolo; Conte, Pierfranco; Dominici, Massimo
abstract

Mesenchymal stromal/stem cells (MSC) are adult multipotent progenitors with fibroblast-like morphology able to differentiate into adipocytic, osteogenic, chondrogenic, and myogenic lineages. Due to these properties, MSC have been studied and introduced as therapeutics in regenerative medicine. Preliminary studies have also shown a possible involvement of MSC as precursors of cellular elements within tumor microenvironments, in particular tumor-associated fibroblasts (TAF). Among a number of different possible origins, TAF may originate from a pool of circulating progenitors from bone marrow or adipose tissue-derived MSC. There is growing evidence to corroborate that cells immunophenotypically defined as MSC are able to reside as TAF influencing the tumor microenvironment in a potentially bi-phasic and obscure manner: either promoting or inhibiting growth depending on tumor context and MSC sources. Here we focus on relationships between the tumor microenvironment, cancer cells, and MSC, analyzing their diverse ability to influence neoplastic development. Associated activities include MSC homing driven by the secretion of various mediators, differentiation towards TAF phenotypes, and reciprocal interactions with the tumor cells. These are reviewed here with the aim of understanding the biological functions of MSC that can be exploited for innovative cancer therapy.

2012 - Combination of low doses of Enzastaurin and Lenalidomide has synergistic activity in B-non-Hodgkin lymphoma cell lines [Articolo su rivista]
Cosenza, Maria; Civallero, Monica; Grisendi, Giulia; Marcheselli, Luigi; Roat, Erika; Bari, Alessia; Sacchi, Stefano
abstract

Less toxic and more active treatments are needed for indolent lymphomas as there is no curative treatment, and patients eventually die due to complications related to their disease. The purpose of the present study was to assess the antitumour activity of the combination of low doses of Enzastaurin and Lenalidomide (Revlimid) on B-lymphoma cell lines. The combination of Enzastaurin and Lenalidomide, at doses as low as 1 μM, showed strong synergism against indolent lymphomas by reducing cell growth, producing an increase in G0-G1 phase followed by significant decrease in S phase, increasing apoptosis, and inhibiting PI3K/AKT, PKC and MAPK/ERK pathways. These preclinical findings, together with promising results obtained with Lenalidomide for the treatment of non-Hodgkin lymphoma, suggest that further evaluation of the combination of Enzastaurin and Lenalidomide for the treatment of indolent lymphomas is warranted. These compounds, with a favourable toxicity profile, are not classic chemotherapeutic agents, causing severe side effects, and could be considered an example of a new innovative attempt of an anti-cancer 'soft treatment'.

2012 - IFN-β expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling [Articolo su rivista]
Tamassia, N.; Bazzoni, F.; Le Moigne, V.; Calzetti, F.; Masala, C.; Grisendi, G.; Bussmeyer, U.; Scutera, S.; De Gironcoli, M.; Costantini, C.; Musso, T.; Cassatella, M. A.
abstract

Upon LPS binding, TLR4 activates a MyD88-dependent pathway leading to the transcriptional activation of proinflammatory genes, as well as a MyD88-independent/TRIF-dependent pathway, responsible for the transcriptional induction of IFN-β. Previous findings delineated that human neutrophils are unable to induce the transcription of IFN-β in response to TLR4 stimulation. Because neutrophils do not express protein kinase C ε, a molecule recently reported as essential for initiating the MyD88-independent/ TRIF-dependent pathway, we optimized an electroporation method to transfect PKCε into neutrophils with very high efficiency. By doing so, a significant IFN-β mRNA expression was induced, in the absence of LPS stimulation, not only in PKCε-overexpressing neutrophils but also in cells transfected with a series of empty DNA plasmids; however, LPS further upregulated the IFN-β transcript levels in plasmid-transfected neutrophils, regardless of PKCε overexpression. Phosphoimmunoblotting studies, as well as chromatin immunoprecipitation assays targeting the IFN-β promoter, revealed that IFN-β mRNA induction occurred through the cooperative action of IRF3, activated by transfected DNA, and NF-κB, activated by LPS. Additional immunoblotting and coimmunoprecipitation studies revealed that neutrophils constitutively express various cytosolic DNA sensors, including IFN-inducible protein 16, leucine-rich repeat (in Flightless I) interacting protein-1, and DDX41, as well as that IFN-inducible protein 16 is the intracellular receptor recognizing transfected DNA. Consistently, infection of neutrophils with intracellular pathogens, such as Bartonella henselae, Listeria monocytogenes, Legionella pneumophila, or adenovirus type 5, promoted a marked induction of IFN-β mRNA expression. Taken together, these data raise questions about the role of PKCεin driving the MyD88-independent/TRIF-dependent response and indicate that human neutrophils are able to recognize and respond to microbial cytosolic DNA. Copyright © 2012 by The American Association of Immunologists, Inc.

2012 - In vitro anti-myeloma activity of TRAIL-expressing adipose-derived mesenchymal stem cells. [Articolo su rivista]
S., Ciavarella; Grisendi, Giulia; Dominici, Massimo; M., Tucci; O., Brunetti; F., Dammacco; F., Silvesitris
abstract

Recently, genetically modified mesenchymal stem cells (MSCs) have beenexploited to deliver anti-cancer bio-drugs directly within the tumour mass.Here, we explored whether adipose-derived MSCs (AD-MSCs), engineeredto express the pro-apoptotic ligand TRAIL (also known as TNFSF10), killmultiple myeloma (MM) cells and migrate toward MM cells in vitro. DifferentMM cell lines were assessed for their sensitivity to recombinanthuman (rh) TRAIL alone and in combination with the proteasome inhibitorbortezomib, which was shown to enhance the effect of rhTRAIL.TRAIL+-AD-MSCs were co-cultured with bortezomib-pretreated MM cellsand their killing activity was evaluated in presence or absence of caspaseinhibition, whereas AD-MSC migration toward media conditioned by bothmyeloma cells and myeloma bone fragments was also investigated. Despitemoderate MM cell sensitivity to rhTRAIL, TRAIL+-AD-MSCs in combinationwith bortezomib significantly induced myeloma cell death. This effectwas associated with caspase-8 activation and abrogated by capsase inhibition.On the other hand, co-culture experiments were performed to evaluatewhether unmodified AD-MSCs affect myeloma cell growth in vitro.AD-MSCs appeared ineffective on myeloma cell growth and showed powerfulmigratory capacity toward MM cells in vitro. These data emphasize theanti-myeloma activity of TRAIL-engineered AD-MSCs and provide supportfor a future model of a cell-based approach against MM.

2012 - Inhibiting interactions of lysine demethylase LSD1 with Snail/Slug blocks cancer cell invasion. [Articolo su rivista]
Ferrari, Giovanna; Fragliasso, Valentina; Esteki, R; Prudente, Zelia; Soliera, Angela Rachele; Cattelani, Sara; Manzotti, Gloria; Grisendi, Giulia; Dominici, Massimo; Pieraccioli, M; Raschellà, G; Claudia, C; Colombo, Mp; Calabretta, Bruno
abstract

The process of epithelial-mesenchymal transition (EMT) which is required for cancer cell invasion is regulated by a family of E-box binding transcription repressors which include Snail (SNAI) and Slug (SNAI2). Snail appears to repress the expression of the EMT marker E-cadherin by epigenetic mechanisms dependent on the interaction of its N-terminal SNAG domain with chromatin modifying proteins including lysine specific demethylase 1 (LSD1/KDM1A). We assessed whether blocking Snail/Slug-LSD1 interaction by treatment with Parnate, an enzymatic inhibitor of LSD1, or TAT-SNAG, a cell-permeable peptide corresponding to the SNAG domain of Slug, suppresses the motility and invasiveness of cancer cells of different origin and genetic background. We show here that either treatment blocked Slug-dependent repression of the E-cadherin promoter and inhibited the motility and invasion of tumor cell lines without any effect on their proliferation. These effects correlated with induction of epithelial and repression of mesenchymal markers and were phenocopied by LSD1 or Slug down-regulation. Parnate treatment also inhibited bone marrow homing/engraftment of Slug-expressing K562 cells. Together, these studies support the concept that targeting Snail/Slug-dependent transcription repression complexes may lead to the development of novel drugs selectively inhibiting the invasive potential of cancer cells.

2012 - Phosphorylation of serine 21 modulates the proliferation inhibitory more than the differentiation inducing effects of C/EBPα in K562 cells. [Articolo su rivista]
Fragliasso, Valentina; Y., Chiodo; Ferrari, Giovanna; Soliera, Angela Rachele; Manzotti, Gloria; Cattelani, Sara; Candini, Olivia; Grisendi, Giulia; J., Vergalli; Sa, Mariani; Guerzoni, Clara; Calabretta, Bruno
abstract

The CCAAT/enhancer binding protein α (C/EBPα) is a transcription factor required for differentiation of myeloid progenitors. In acute myeloid leukemia (AML) cells expressing the constitutively active FLT3-ITD receptor tyrosine kinase, MAP kinase-dependent phosphorylation of serine 21 (S21) inhibits the ability of C/EBPα to induce granulocytic differentiation. To assess whether this post-translational modification also modulates the activity of C/EBPα in BCR/ABL-expressing cells, we tested the biological effects of wild-type and mutant C/EBPα mimicking phosphorylated or non-phosphorylatable serine 21 (S21D and S21A, respectively) in K562 cells ectopically expressing tamoxifen-regulated C/EBPα-ER chimeric proteins. We show here that S21D C/EBPα-ER induced terminal granulocytic differentiation of K562 cells almost as well as wild-type C/EBPα-ER, while S21A C/EBPα-ER was less efficient. Furthermore, wild-type C/EBPα suppressed the proliferation and colony formation of K562 cells vigorously, while S21D and S21A C/EBPα mutants had more modest anti-proliferative effects. Both mutants were less effective than wild-type C/EBPα in suppressing endogenous E2F-dependent transactivation and bound less E2F-2 and/or E2F-3 proteins in anti-C/EBPα immunoprecipitates. Together, these findings suggest that mutation of S21 more than its phosphorylation inhibits the anti-proliferative effects of C/EBPα due to reduced interaction with or impaired regulation of the activity of E2F proteins. By contrast, phosphorylation of serine 21 appears to have a modest role in modulating the differentiation-inducing effects of C/EBPα in K562 cells.

2012 - Sarcomas as a mise en abyme of Mesenchymal Stem Cells; exploiting interrelationships for cell mediated anticancer therapy [Articolo su rivista]
J. S., Burns; A., Safwat; Grisendi, Giulia; M., Kassem; Dominici, Massimo
abstract

Mise en abyme meaning "placed into abyss or infinite recurrence" is an apt paradigm for the relentless growth of sarcoma cells. Its alternative meaning, "self-reflexive embedding" fits the central role attributed to cancer stem cells (CSCs). Diversely sourced and defined, mesenchymal stem cells (MSCs) may be the cells of sarcoma origin, evolve a CSC phenotype and/or contribute to tumor growth through inherent qualities for homing, neovascularization, paracrine cross-feeding, microvesicle secretion, cell fusion, entosis and immune modulation. Exploiting these qualities, MSC expressing modified forms of the TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) are being developed to complement more conventional radiation and chemotherapy.

2011 - An adoptive immuno-gene therapy approach targeting neuroblastoma. [Abstract in Rivista]
Caldrer, Sara; Spano, Maria Carlotta; Grisendi, Giulia; Dominici, Massimo; Paolucci, Paolo
abstract

Although dose intensification of chemotherapy has increased initial response rates in neuroblastoma (NB), this effect has not translated into durable remissions in patients with disseminated disease. Immunotherapy may be an alternative approach following cytoreductive chemotherapy providing a long-term disease control (I). We have engineered human cytotoxic T lymphocytes (CTL) to express a chimeric receptor (CR) for the specific recognition of GD2 that is expressed in NB (II). Chimeric lymphocytes (CL) have been previously introduced towards several tumors (III). Thus, we have generated a novel CR consisting of a single-chain variable domain of an anti-GD2 antibody in conjunction with a signal domain of 4-1BB molecule (CD137). Retroviral particles encoding for the flag gene only (GFP), for the whole CR and for a truncated receptor were generated. Peripheral blood cells from buffy-coat were specifically stimulated and transuced obtaining a cytotoxic population of T cells having high level of transduction (50±25%). Co-culture experiments were then performed against NB cell lines. In short (4h) co-culture experiments (T:E=1:20) by Cr51 release assay, CLs exerted a powerful and specific cytotoxicity against GD2-positive NB cells, compared with truncated controls (35±5% and 10,8±4% ; p<0,05). Moreover, in a longer co-culture assay (5 days) in vitro at higher ratio (T:E =1:5), using as read-out FACS analyses targeting GD2 expressing cells, we observed a significant decrease (from 92±3% to 19±5% ; p<0.05) of the NB cells compared with controls (from 92±3% to 76,5±4% %; p<0.05). These preliminary data in vitro suggest that CL against GD2-positive NB cells may represent a powerful new tool for T-cell therapy in patients with GD2-positive NB or other GD2-positive malignancies.

2011 - Bone marrow derived mesenchymal stem/stromal cells transduced with full length human TRAIL repress the growth of rhabdomyosarcoma cells in vitro [Articolo su rivista]
Barti-Juhasz, H.; Mihalik, R.; Nagy, K.; Grisendi, G.; Dominici, M.; Petak, I.
abstract

Our results with BM-MSCs together with the results ofKuci et al. with CIK cells1 emphasize that cell mediateddelivery of TRAIL to metastatic RMS tumor sites can bea useful approach in RMS therapy. Targeted delivery ofTRAIL to tumors may allow systemic exposure ofpatients to drugs (e.g. the proteasome inhibitor bortezomib)that may overcome resistance for TRAIL-inducedapoptosis in RMS cells.

2011 - Mesenchymal Stromal/Stem Cells from Tissue Repair to Destruction of Tumor Cells [Capitolo/Saggio]
Bussolari, Rita; Grisendi, Giulia; L., Cafarelli; Loschi, Pietro; L., Scarabelli; A., Frassoldati; M., Maur; DE SANTIS, Giorgio; Paolucci, Paolo; P. F., Conte; Dominici, Massimo
abstract

A tumor is a complex framework composed of tumor cells (TC) and stroma where the extracellular matrix (ECM) and cellular components such as immune cells, vessels cells and fibroblasts interact closely together. Here these complex interactions are addressed in the light of the possible use of mesenchymal stomal cells to target tumors.

2011 - Understanding Tumor-Stroma Interplays for Targeted Therapies by Armed Mesenchymal Stromal Progenitors: The Mesenkillers. [Articolo su rivista]
Grisendi, Giulia; Bussolari, Rita; Veronesi, Elena; Piccinno, MARIA SERENA; Burns, J. S.; DE SANTIS, Giorgio; Loschi, Pietro; Pignatti, M.; DI BENEDETTO, Fabrizio; Ballarin, Roberto; C., Di Gregorio; V., Guarneri; Piccinini, Lino; Em, Horwitz; Paolucci, Paolo; P., Conte; Dominici, Massimo
abstract

Tumor represents a complex structure containing malignant cells strictly coupled with a large variety of surroundingcells constituting the tumor stroma (TS). In recent years, the importance of TS for cancer initiation, development,local invasion and metastases became increasingly clear allowing the identification of TS as one of the possibleways to indirectly target tumors. Inside the heterogeneous stromal cell population, tumor associated fibroblasts(TAF) play a crucial role providing both functional and supportive environments. During both tumor and stroma development,several findings suggest that TAF could be recruited from different sources such as locally derived host fibroblasts,via epithelial/endothelial mesenchymal transitions or from circulating pools of fibroblasts deriving form mesenchymalprogenitors, namely mesenchymal stem/stromal cells (MSC). These insights prompted scientists to identifymultimodal approaches to target TS by biomolecules, monoclonal antibodies and, more recently, via cell basedstrategies. These latter appear extremely promising, although associated with still debated and unclear findings. Thisreview discusses on crosstalk between cancers and their stroma, dissecting specific tumor types, such as sarcoma,pancreatic and breast carcinoma where stroma plays distinct paradigmatic roles. The recognition of these distinctstromal functions may help in planning effective and safer approaches aimed either to eradicate or to substitute TSby novel compounds and/or MSC having specific killing activities

2010 - Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy [Articolo su rivista]
Grisendi, Giulia; Bussolari, Rita; Cafarelli, Luigi; Petak, I.; Rasini, Valeria; Veronesi, Elena; DE SANTIS, Giorgio; Spano, Maria Carlotta; Tagliazzucchi, M.; Barti Juhasz, H.; Scarabelli, Laura; Bambi, F.; Frassoldati, A.; Rossi, G.; Casali, Christian; Morandi, Uliano; Horwitz, E. M.; Paolucci, Paolo; Conte, P.; Dominici, Massimo
abstract

Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based gene therapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules for cancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that induces apoptosis in a variety of human cancers but not normal tissues. Although several studies have documented the antitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted a variety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and, in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with activation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localized into tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, our results provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors.

2010 - Effects of enzastaurin, alone or in combination, on signaling pathway controlling growth and survival of B-cell lymphoma cell lines. [Articolo su rivista]
Civallero, Monica; Cosenza, Maria; Grisendi, Giulia; Marcheselli, L; Todoerti, K; Sacchi, Stefano
abstract

AbstractImmunochemotherapies have improved outcomes in indolent lymphoma. However, response durations progressively shorten following each treatment, and the majority of patients eventually die from the disease. Thus, new, less toxic, and more active treatments are needed. Protein kinase C (PKC), which has been repeatedly implicated in B-cell lymphoma progression, may be a new target for lymphoma cell growth inhibition. Enzastaurin, a PKC-beta inhibitor, has toxic effects on a variety of cancer cells. The purpose of the present study was to assess the antitumor activity of enzastaurin on B-cell lymphoma cell lines and to investigate the underlying antitumor mechanisms. Enzastaurin induced apoptosis and inhibited phosphorylation of PKC, RSK, AKT, and downstream proteins. Moreover, our results reveal a new mechanism for enzastaurin-induced apoptosis via BAD activation. Finally, enzastaurin synergizes in its effects with chlorambucil and fludarabine, respectively. Taken together, our results strongly support clinical evaluation of enzastaurin in patients with B-cell lymphoma.

2010 - GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion. [Articolo su rivista]
Grisendi, Giulia; Annerén, C; Sternieri, R; Veronesi, Elena; Cervo, Gl; Luminari, Stefano; Maur, M; Frassoldati, A; Palazzi, G; Otsuru, S; Bambi, F; Paolucci, Paolo; Conte, Pierfranco; Horwitz, E; Dominici, Massimo; Cafarelli, Luigi
abstract

BACKGROUND AIMS: Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance.METHODS: BM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential.RESULTS: No differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45(+) fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage.CONCLUSIONS: Both GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.

2009 - Refining GMP-manufactured density gradient media for optimised mesenchymal stromal/stem cell isolation and expansion. [Abstract in Rivista]
Grisendi, Giulia; C., Anneren; L., Cafarelli; Veronesi, Elena; R., Sternieri; G. L., Cervo; Luminari, Stefano; A., Frassoldati; M., Maur; G., Palazzi; Paolucci, Paolo; Conte, Pierfranco; Dominici, Massimo
abstract

not available

2008 - Effects of Enzastaurin, Alone or in Combination, on Signalling Pathway Controlling Growth and Survival of B-Cell Lymphoma Cell LinesBlood (ASH Annual Meeting Abstracts), Nov 2008; 112: 4978 [Abstract in Rivista]
Sacchi, Stefano; Civallero, Monica; Cosenza, Maria; Grisendi, Giulia; Luigi, Marcheselli; Antonino, Neri
abstract

Background. Although, the recent introduction of Rituximab in combination with chemotherapy has improved outcome of patients with indolent lymphomas, in particular FL, these diseases are still considered incurable and the majority of patients still have a fatal course. Enzastaurin, an acyclic bisindolylmaleimide, is a potent and selective competitive inhibitor of protein kinase C beta, which has been shown to inhibit cell proliferation and angiogenesis in human cancer cell lines. The purpose of the present study was to test enzastaurin for its effects on proliferation and survival of B-cell lines established from NHL patients. Methods. The WSU-NHL and Karpas 422 were kindly provided Dr M Introna ( Division of Haematology, Ospedali Riuniti, Bergamo, Italy); the RL was purchased by DSMZ. All three lines are carrying the t(14; 18) and Karpas 422 is EBV+. We decided to conduct all the experiments under the optimal culture conditions with 10% FCS to avoid subjecting the cells to further stress stimuli. IC50 values were calculated from curves based on enzastaurin concentration ranging from 1 to 10µM using MTT assay and cell viability assessment by Trypan Blue exclusion. Cell apoptosis was assessed by flow cytometer after staining with Annexin V-FITC and propidium iodide. The effects of enzastaurin on caspases activation as well as on AKT phosphorilation were evaluated by Western blotting. We also investigated the interaction of enzastaurin with chlorambucil and fludarabine. Results using MTT assay were expressed as fraction of cells killed by the individual drug or the combination in the drug-treated versus untreated cells. The interaction between drugs was analyzed by isobologram analysis using the StaCorp8.2 software program based upon the Chou-Talalay method to determine if the combinations were additive or synergistic. Results. We found that enzastaurin alone inhibits the proliferation of B-cell lymphomas cell lines at IC50 values ranging from 5 to 7.5 µM after 48 h and from 2.5 to 3.2 µM after 72 h. Induction of apoptosis by enzastaurin evaluated on WSU-NHL and RL, by flow cytometry analysis of membrane permeability, showed that enzastaurin induces an increase in the percentage of apoptotic cells compared with untreated controls in a time-dependent fashion. Furthermore, enzastaurin induces the appearance of the cleaved caspase-3 fragment in the same cell lines in a time dependent fashion. Activation of the apoptotic pathway was confirmed by cleavage of the PARP enzyme. The apoptosis is partially prevented by the ZVAD–fmk broad caspase inhibitor. Phosphorilation status analysis of AKT up to 72h of treatment showed a decrease of AKT phosphorilation starting from 48h after treatment. We tested the effect of enzastaurin combined with chlorambucil and fludarabine,drugs that are active against B-cell lymphoma and we demonstrated that these agents enhanced the cytotoxicity triggered by enzastaurin in a dose-dependent fashion. A clear synergistic interaction (CI=0.87) appeared using low concentrations of the drugs and increased (CI=0.1) at high concentrations. Conclusions. Our data suggest that in B lymphoma cell lines carrying t(14; 18), enzastaurin elicits its antitumor effect suppressing AKT phosphorilation, inducing apoptosis and inhibiting proliferation. Furthermore, enzastaurin synergizes with chlorambucil and fludarabine. These results support the potential use of enzastaurin in patients with NHL, and in particular provide a rationale for combination with chlorambucil and fludarabine

2004 - Dietary evaluation and metabolic alterations in HIV-related lipodystrophy [Abstract in Rivista]
Orlando, Gabriella; Guaraldi, Giovanni; Giuricin, F; Grisendi, G; Carubbi, Francesca; Zini, Isabella; Borghi, V; Esposito, Roberto; Galetti, S.
abstract

not available