Articles de revues : « MSC, Tumor Immunology, Mesenchymal Stem Cell »

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Wirths, Stefan, Hans-Joerg Buehring, Lothar Kanz, Joerg T. Hartmann et Hans-Georg Kopp. « Mesenchymal Stem Cell Differentiation Markers Shared with Soft Tissue Sarcomas ». Blood 112, n^o 11 (16 novembre 2008) : 4755. http://dx.doi.org/10.1182/blood.v112.11.4755.4755.

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Abstract Malignant tumors are hypothesized to harbor small populations of self-renewing cancer stem cells. Targeting these cells may be the decisive step to overcome treatment resistance and achieve tumor eradication in cancer patients. Advanced soft tissue sarcomas (STS) are rare tumors with a dismal prognosis and a small number of systemic treatment options. STS may originate from mesenchymal stem cells (MSC); the latter have mainly been isolated from adult bone marrow (BM) as non-hematopoietic, self-renewing cells whose in vitro progeny comprises osteoblasts, chondroblasts, myocytes, and adipocytes. While in vitro expression profiles of MSC have been investigated extensively, the in vivo counterparts of MSC are still hypothetical. To target rare human cell BM populations including MSC, an exclusive antibody panel was developed. The target antigens include platelet-derived growth factor receptor-β (CD140b), HER-2/erbB2 (CD340), the recently described W8B2 antigen as well as several surface antigens identified by novel antibodies. To define the expression pattern of MSC-markers in STS, three STS cell lines were tested for expression of these antigens. In addition, snap-frozen primary STS sections were analyzed by immunohistochemistry using the same antibody panel. All cell lines revealed expression of selected markers including CD340, W8B2, and CD140b. Several MSC markers were restricted to a subpopulation of cells. In addition, leiomyosarcoma cells displayed a different expression pattern as compared to liposarcoma and Ewing’s sarcoma cells. Results of immunohistochemistry analysis of primary leiomyosarcoma tumor samples correlated strongly with expression patterns established by FACS analysis. However, important cytoarchitectural features regarding selected markers were revealed by immunohistochemistry: while primary leiomyosarcomas displayed uniform expression of W7C6, HEK3D6, CD10, and CD318, other markers such as CD34, W5C5, and 57D2 were expressed by tumor endothelia only. Moreover, a population of perivascular tumor cells was found to express the MSC-markers W4A5, W8B2, CD140b, W3D5, and W5C4. Novel MSC-markers are expressed by subpopulations in STS cell lines as well as in primary sarcoma tissue. Further studies on the functional significance of these phenotypical studies are underway and may help to identify novel specific targets recognizing the self-renewing STS-compartment.

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BISWAS, SUBIR, Gunjan Mandal, Sougata Roy Chowdhury, Suman Purohit, Kyle K. Payne, Carmen Maria Anadon Galindo, Arnab Gupta, Xiaoqing Yu, Jose R. Conejo-Garcia et Arindam Bhattacharyya. « Mesenchymal stem cells educate breast tumor associated macrophages to acquire increased immunosuppressive features ». Journal of Immunology 202, n^o 1_Supplement (1 mai 2019) : 135.25. http://dx.doi.org/10.4049/jimmunol.202.supp.135.25.

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Abstract Breast cancer metastasis is known to be extensively promoted by immunosuppression. Here we describe a novel mechanism by which mesenchymal stem cell (MSC)-derived exosomes drive an immunosuppressive program within the breast tumor microenvironment. Initial q-PCR and immunophenotyping of human breast tumor samples, and TCGA data analysis confirmed a positive correlation between infiltrations of MSC and M2 macrophage phenotype, therefore we hypothesized that MSC promote M2 macrophage differentiation in breast tumors. We then performed a series of experiments in vitro and in vivo, and found that MSC-derived exosomes significantly promote differentiation of macrophages into PD-L1 expressing ‘M2-like’ phenotype, while also enhancing malignant progression of breast tumors, in vivo. Additionally, we observed increased invasive potential of tumor cells with higher expression of mesenchymal markers when induced with MSC-derived exosomes. Importantly, our observation of a significantly higher TGF-β production by tumor associated macrophages in exosome-induced tumors, with stronger PD-1 expression by intra-tumoral T cells, suggests TGF-β driven PD-1 upregulation. Blocking of PD-L1 abrogates exosome-induced tumor growth signifying the PD-L1/PD-1 checkpoint pathway is vital to MSC-induced tumor progression. Together, infiltration of MSCs within breast tumors drives the increased invasive potential as well as immune-checkpoint-mediated immunosuppression through inducing PD-L1 expression by tumor associated macrophages and PD-1 expression by T cells. Targeting infiltration of MSCs into the breast tumor therefore is a potential approach to reduce breast tumor metastases and improve efficacy of immunotherapies.

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Ruvolo, Peter, Yihua Qiu, Vivian Ruvolo, Rui-Yu Wang, Zhihong Zeng, Jared Burks, Rongqing Pan et al. « Role of Mesenchymal Stem Cell Galectin 3 in the AML Tumor Microenvironment ». Blood 126, n^o 23 (3 décembre 2015) : 1198. http://dx.doi.org/10.1182/blood.v126.23.1198.1198.

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Abstract Background: Mesenchymal stem cells (MSC) protect acute myeloid leukemia (AML) cells from chemotherapy. Galectin 3 (LGALS3) is a beta-galactoside binding protein that regulates cell adhesion, apoptosis, cell cycle, and mRNA processing. When secreted, LGALS3 can modulate receptor kinase activity, influence cell adhesion, and can suppress immune cells. MSC are a rich source of LGALS3. In the present study we address the role of MSC derived LGALS3 in the leukemia microenvironment. Methods: The expression of LGALS3 and 150 other proteins was examined by reverse phase protein array analysis (RPPA) in AML (N = 106) and healthy donor (N = 71) MSC. To examine the putative role of MSC-derived proteins in therapy resistance, RPPA was used to determine levels of the 151 proteins in MSC obtained at diagnosis and from relapsed/refractory patients relapse. In vitro studies were performed to examine the effects of hypoxia or co-culture with AML cell lines on LGALS3 expression by immunofluorescence (IF) microscopy and immunoblot analysis. MSC containing lentiviral control shRNA or LGALS3 shRNA were used to assess effects of LGALS3 on cell viability, apoptosis, and cell adhesion by flow cytometry assays and effects on survival signaling by immunoblot analysis. Results: LGALS3 levels were found to be higher in AML MSC compared to normal MSC (p = 0.0001). The increase in protein expression was not correlated with gene expression as mRNA levels were similar in AML and normal MSC, suggesting a post-translational mechanism. To identify MSC proteins associated with relapse or refractory status, RPPA was utilized to compare protein expression in MSC from newly diagnosed with those from relapsed and refractory (salvage) patients. RPPA identified LGALS3 as one of only three proteins increased in MSC from relapse/refractory patients. Pearson Correlation of LGALS3 with the other 150 proteins in AML MSC revealed that LGALS3 was negatively correlated with expression of integrin beta 3 and LYN. These 2 proteins were also among the 6 proteins found to have lower expression in salvage as compared to MSC from newly diagnosed patients. LGALS3 positively correlated with 13 proteins including phosphorylated beta-catenin which was also higher in salvage as compared to newly diagnosed AML MSC. . The LGALS3 promoter contains HIF1 alpha response elements. MSC from healthy donors grown in 1% oxygen displayed > 4 fold increase in LGALS3 protein after 24 hours. Hypoxic MSC exhibited a re-localization of LGALS3 from the nucleus to the cytoplasm. LGALS3 activates RAS signaling so hypoxia induced expression and nuclear export of the galectin would be expected to activate diverse survival signaling pathways and would also allow the molecule to be secreted where it could then suppress immune cells. Using in vitro co-culture experiments of MSC with human OCI-AML3 cells to mimic the leukemia microenvironment, LGALS3 levels were induced in MSC suggesting that cross talk from leukemia cells could contribute to the increased LGALS3 levels in MSC cells. The consequences of suppressing LGALS3 in normal MSC were tested using lentivral shRNA. Suppression of LGALS3 resulted in a 2-fold reduction of MYC and S473 phosphorylated AKT, and a 2-fold increase in PPP2R2A (an AKT phosphatase) compared to control shRNA. LGALS3 suppression rendered MSC less adherent to OCI-AML3 cells (~ 50% reduction in MSC with LGALS3 shRNA compared to control shRNA). Conclusions: The data presented here demonstrate that LGALS3 is elevated in AML MSC especially in relapse/refractory samples. Possible mechanisms of up-regulation of LGALS3 in AML MSC may be due to the hypoxic nature of the leukemia microenvironment and/or contact with AML cell. Hypoxia induced nuclear export of the galectin to the cytoplasm could contribute to MSC survival and anti-tumor functions (e.g. suppression of immune cells by secreted galectin). Knock down of LGALS3 in MSC suppresses AKT activation and MYC expression suggesting it has a key role in MSC cell survival. Experiments are underway to determine if MSC derived LGALS3 might impact AML cell homing (as a regulator of cell adhesion) and/or immune surveillance (as a suppressor of T and NK cells). The identification of LGALS3 as an important component of AML MSC suggests that this molecule could be a target in a tumor microenvironment based therapy concept of AML. Disclosures No relevant conflicts of interest to declare.

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Andreeff, Michael, Jennifer Dembinski, Brett M. Hall, Matus Studeny, Xiaoyang Ling, Maria Cabreira-Hansen, A. Kate Sasser, Teresa McQueen, Frederick Lang et Frank C. Marini. « Mesenchymal Stem Cells Selectively Engraft into Tumor Stroma and Produce Potent Antitumor Proteins In Situ. » Blood 108, n^o 11 (16 novembre 2006) : 352. http://dx.doi.org/10.1182/blood.v108.11.352.352.

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Abstract The formation of stroma is essential for tumor growth and involves complex interactions between malignant tumor cells, and non-tumor stromal cells. We have previously demonstrated that mesenchymal stem cells (MSC) integrate into solid tumors as stromal elements (Cancer Res62:3603, 2002; JNCI96:1593, 2004,), suggesting the development of anti-cancer therapies based on the intratumoral production of agents by gene-modified MSC. However, no direct evidence has demonstrated this migration and selective engraftment into the tumor microenvironment. Therefore, we noninvasively visualized MSC using luciferase bioluminescence. MSC were labeled by a fiber modified Ad vector expressing firefly luciferase (AdLux-F/RGD) and these MSC-Lux were injected into normal (healthy) SCID mice or mice bearing established metastatic breast or ovarian tumors. Biodistributed MSC-Lux were imaged utilizing the Xenogen IVIS detection system. In normal mice, human MSC (hMSC) migrated to the lungs where they remained resident for 7–10 days. In animals bearing established metastatic lung tumors, IV injected hMSC again migrated to the lungs. However, in contrast to control mice, the Lux signal remained strong over a 15-day period with only a slight decrease over the first 10 days. After IP injection, hMSC-LUX were detected in the peritoneum, and after 7 days, no hMSC-LUX was detected in normal animals, while strong punctate regions of LUX-activity were observed in ovarian tumors. In contrast to SCID mice injected with hMSC, healthy Balb/C mice injected with Balb/C derived MSC-LUX initially migrated to the lungs and within 2.5 hrs had exited the lungs to remain liver and spleen resident for 5–7 days. When tumor cells were transduced with renilla luciferase constructs, the co-localization and dynamic interactions of firefly luciferase MSC and renilla luciferase tumors could be examined in detail. Mechanisms regulating the MSC-tumor interactions involve TGF-beta, HGF/c-Met, and EGFR and will be discussed. We then examined whether hMSC-producing interferon-beta (IFNb-MSC) could inhibit the growth of metastatic tumors in the lungs of SCID mice. When injected IV (4 doses of 106 MSC/week) into SCID mice bearing pulmonary metastases of carcinomas or melanomas, tumor growth was significantly inhibited as compared to untreated or vector-control MSC controls (p= 0.007), while recombinant IFNb protein (50,000 IU qod) was ineffective (p=0.14). IV injected IFNb-MSC prolonged the survival of mice bearing metastatic breast carcinomas (p=0.001) Intraperitoneal (IP) injections of IFN-MSC into mice carrying ovarian carcinomas resulted in doubling of survival in SKOV-3, and cures in 70% of mice carrying OVAR-3 tumors. MSC injected into the ipsilateral or contralateral carotid artery were found to localize to glioma xenografts in mice and IFNb-MSC significantly (p<0.05) prolonged survival of these mice. These data suggest that systemically administered gene-modified MSC selectively engraft into the tumor microenvironment and remain resident as part of the tumor architecture. MSC-expressing IFN-b inhibit the growth of melanomas, gliomas, metastatic breast and ovarian cancers in vivo and prolong the survival of mice bearing established tumors. Clinical trials are in preparation.

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Tolar, Jakub, Alma J. Nauta, Mark J. Osborn, Angela Panoskaltsis-Mortari, Ron T. McElmurry, Scott Bell, Lily Xia et al. « Osteosarcoma Derived from Cultured Mesenchymal Stem Cells. » Blood 108, n^o 11 (16 novembre 2006) : 2554. http://dx.doi.org/10.1182/blood.v108.11.2554.2554.

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Abstract The beneficial effects of Mesenchymal Stem Cells (MSC) are being tested clinically in attempts to improve hematopoietic engraftment, to treat osteogenesis imperfecta, graft-versus-host disease and autoimmune diseases, and to deliver therapy for malignancies. In early reports, phase I clinical studies have not been associated with toxicities. To study the biodistribution of MSC, we labeled adult murine C57BL/6 MSC with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and co-infused labeled MSC with bone marrow into irradiated allogeneic recipients. Using in vivo whole body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in lungs. Two mice also developed sarcomas in their extremities. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Mapping of the Sleeping Beauty transposition insertion sites did not identify an obvious transposon-related genetic abnormality. Importantly, the original MSC cultures not labeled with transposons, as well as cultured MSC independently isolated from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro. Even though not all MSC cultures formed tumors upon in vivo injection, these data indicate that MSC transformation was neither strain-specific nor a rare event following ex-vivo expansion. Karyotype analyses using fluorescence in situ hybridization with spectral karyotyping (SKY) as well as combined binary ratio labeling of nucleic acid probes (COBRA) showed clonal evolution of transformed MSC suggesting that the critical transformation event(s) occurred before MSC infusion. Collectively, we describe cytogenetic instability of murine MSC isolated in two independent laboratories, their cellular transformation, and potential for sarcoma formation. While the growth characteristics of human and murine MSC are not identical and murine cells are more prone to undergo immortalization and transformation in culture than human cells, our study highlights the importance of quality control measures needed for ongoing and future clinical trials using human MSC.

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Fakiruddin, Kamal Shaik, Moon Nian Lim, Norshariza Nordin, Rozita Rosli et Syahril Abdullah. « Chemo-Sensitization of CD133+ Cancer Stem Cell Enhances the Effect of Mesenchymal Stem Cell Expressing TRAIL in Non-Small Cell Lung Cancer Cell Lines ». Biology 10, n^o 11 (26 octobre 2021) : 1103. http://dx.doi.org/10.3390/biology10111103.

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Pre-clinical studies have demonstrated the efficacy of mesenchymal stem cells (MSCs) expressing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or MSC-TRAIL against several tumors. However, due to the existence of cancer stem cells (CSCs), some tumors, including non-small cell lung cancer (NSCLC), exhibit TRAIL resistance. This study was designed to evaluate the capacity of using first-line chemotherapies including cisplatin, 5-fluorouracil (5-FU) and vinorelbine to act as a chemo-sensitizer on CD133+ (prominin-1 positive) CSCs derived from NSCLC cell lines (A549, H460 and H2170) for the purpose of MSC-TRAIL-induced inhibition. We showed that MSC-TRAIL was resistant to all three chemotherapies compared to the NSCLC cell lines, suggesting that the chemotherapies had little effect on MSC-TRAIL viability. Pre-treatment using either cisplatin or 5-FU, but not with vinorelbine, was able to increase the efficacy of MSC-TRAIL to kill the TRAIL-resistant A549-derived CSCs. The study also demonstrated that both 5-FU and vinorelbine were an effective chemo-sensitizer, used to increase the anti-tumor effect of MSC-TRAIL against H460- and H2170-derived CSCs. Furthermore, pre-treatment using cisplatin was noted to enhance the effect of MSC-TRAIL in H460-derived CSCs; however, this effect was not detected in the H2170-derived CSCs. These findings suggest that a pre-treatment using certain chemotherapies in NSCLC could enhance the anti-tumor effect of MSC-TRAIL to target the CSCs, and therefore the combination of chemotherapies and MSC-TRAIL may serve as a novel approach for the treatment of NSCLC.

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Giallongo, Cesarina, Nunziatina L. Parrinello, Daniele Tibullo, Claudia Bellofiore, Piera La Cava, Alessandra Romano, Annalisa Chiarenza et al. « Mesenchymal STEM CELLS Favor Tumor Growth By Generating Granulocyte-like Myeloid Derived Suppressor CELLS in CML Patients ». Blood 126, n^o 23 (3 décembre 2015) : 4018. http://dx.doi.org/10.1182/blood.v126.23.4018.4018.

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Abstract INTRODUCTION. The complex interplay between cancer cells and immune system allows neoplastic cells to evade immune surveillance and expand. Recently, our and another group have demonstrated that a subpopulation of myeloid cells, defined as "granulocytic myeloid-derived suppressor cells" (G-MDSC), plays an important role for immune escape in chronic myeloid leukemia (CML) patients by reducing T cell activation. The aim of this study was to evaluate the influence of Mesenchymal stem cells (MSC) on generation of MDSCs by comparing CML MSCs (n=10) with healthy donors (HD) MSC (n=8). METHODS. G-MDSC (CD11b+CD33+CD14-HLADR- cells) were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of G-MDSC with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. For G-MDSC generation, human peripheral blood mononucleated cells (PBMC) from HD were cultured alone and with MSC of CML (n=10) or HD (n=8) (1:100 ratio). After one week, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype was confirmed by cytofluorimetric analysis. Expression of ARG1, NOS2, PTGS2, TNFα, TGFβ, IL6, IL10, IL1β was also evaluated using real time PCR. RESULTS. Percentage of cells with a G-MDSC phenotype was greater in PB obtained from CML patients than HD (82.5±9.6% vs 56,2±5.4%, p<0.0001). G-MDSC were able to inhibit T cell proliferation compared to positive control (25±5% vs 48±7.6%, p=0.0057). To investigate if CML MSC may be involved in G-MDSC generation, we incubated HD PBMC with CML or HD MSC for one week. After magnetic isolation, we found that only CML MSC-educated G-MDSC acquired immune-suppressive ability, inhibiting T cell proliferation compared to G-MDSCs control (isolated from PBMC cultured in medium alone) (32±12% vs 63±5.9%, p=0.003). On the contrary, HD MSC-educated G-MDSC did not show any suppressive effect. We also found that CML MSC-educated G-MDSC expressed higher level of the following immune modulatory factors: TNFα (20.8±19.3, p=0.006), IL1β (47.3±25.2, p=0.001), PTGS2 (20.7±10.9, p=0.002) and IL6 (33.8±13.9, p=0.004) compared to HD MSC-educated G-MDSCs (arbitrarily 2-ΔΔCt value: 1). MSC WE also observed ane an up-regulation of PTGS2 (19±4.4, p=0.04), TGFβ (6±3, p=0.01) and IL6 (5±2.8, p=0.04) in CML MSCs at time 0 with a great variability among the patients (calculated value of 2-ΔΔCt in HD MSC was 1). After 48 h of co-culture with PBMC, CML MSC showed statistically significant up-regulation of ARG1 (23.5±11.9, p=0.02), TGFβ (4.8±3, p=0.04), IL10 (5.6±2.8, p=0.03) and IL6 (54.3±23, p=0.02) expression, suggesting that multiple mechanisms are involved in MDSC induction by CML MSC. CONCLUSION. Our work demonstrates that CML MSCs are able to activate MDSCs favoring cancer immune evasion in CML patients. Disclosures Palumbo: Novartis: Honoraria, Other: Advisory Board.

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Martiniello-Wilks, Rosetta, Stephen R. Larsen, Stephane Flamant, Jessamy C. Tiffen, Charles G. Bailey et John E. J. Rasko. « Mesenchymal Stem Cells as Suicide Gene Therapy Vehicles for Organ-Confined and Metastatic Prostate Cancer (PCa). » Blood 110, n^o 11 (16 novembre 2007) : 5148. http://dx.doi.org/10.1182/blood.v110.11.5148.5148.

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Abstract The efficacy of mesenchymal stem cells (MSC) is currently being examined as a clinical regenerative medicine for multiple sclerosis, cirrhosis, liver disease, tibial fractures, heart failure and graft versus host disease. MSC display an inherent tumor-tropic property that has been exploited for the targeted delivery of therapeutic genes to metastatic melanoma, glioma, breast and colon carcinoma in animal models. Advantages of using MSC include their ability for: self-renewal, ease of propagation and gene modification ex vivo; secretion of high levels of therapeutic protein; evasion of immune rejection; differentiation into connective tissue and tumor stroma; and long-term engraftment in vivo. This study explores the utility of MSC to deliver reporter or suicide genes to advanced Pca which is currently incurable using the syngeneic RM1 mouse model. Sca-1bright CD45− cells sorted from adherent bone marrow cells were shown to be true MSC by their ability to undergo tri-lineage differentiation in adipogeneic, osteogenic and chondrogenic media and their characteristic CD44+, CD90+, CD73+ and CD106+ phenotype. Lentiviral vectors showed sustained green fluorescent protein (GFP) reporter gene expression in MSC (MSC-GFP) for 50 passages by flow cytometry. When MSC-GFP (10e6 cells) were implanted into the mouse prostate with or without RM1 tumor cells on day 0, examination by full body fluorescence imaging (IVIS 100; Xenogen/Caliper) showed MSC persisted only within the tumor-bearing prostate (p<0.05; day 18). No MSC were detected in any other organ examined. To test their systemic homing ability, MSC-GFP (10e6 cells) were infused every second day (2–14) via the tail vein of mice in the presence or absence of RM1 lung pseudometastases. MSC persisted within the lungs of RM1 tumor-bearing mice alone (p<0.01) with no detectable MSC in any other organ examined (day 18). These results suggest MSC engraft organ-confined Pca and home to metastatic Pca. Gene directed enzyme prodrug therapy (GDEPT) describes the transfer of a suicide gene, not expressed in mammalian cells, into tumors using viral vectors. This renders tumors sensitive to prodrugs that are non-toxic to normal tissues. In our pre-clinical study, prostate tumors established from RM1 tumor cells stably transfected with fusion suicide gene cytosine deaminase/uracil phosphoribosyltransferase (CDUPRT) followed by systemic treatment with prodrug 5-fluorocytosine, showed significant reductions in prostate tumor growth and pseudometastases in the lungs (1). More recently, we stably transfected MSC with CDUPRT prior to implantation into established RM1 prostate tumors. In the presence of prodrug MSC-CDUPRT showed similar levels of Pca killing observed in the published experiment. In both experiments CDUPRT in the presence of prodrug significantly reduced (∼75%; p<0.05) the weight of RM1 prostate tumors compared to the control gene or no prodrug control mice. These results demonstrate that MSC can deliver suicide genes to developing Pca and efficiently convert prodrug to a toxic diffusible metabolite to suppress tumor growth. MSC implantation was well tolerated without observed toxicity and therefore show promise as a novel form of cell-directed suicide gene therapy.

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Khakoo, Aarif Y., Shibani Pati, Stasia A. Anderson, William Reid, Mohamed F. Elshal, Ilsa I. Rovira, Ahn T. Nguyen et al. « Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma ». Journal of Experimental Medicine 203, n^o 5 (24 avril 2006) : 1235–47. http://dx.doi.org/10.1084/jem.20051921.

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Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell–cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSC administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies characterized by dysregulated Akt.

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Chen, Xiao Chen, De Pei Wu, Feng Chen et Wei Rong Chang. « Research on Biological Characteristics of Chronic Myelogenous Leukemia Patients’ Bone Marrow Mesenchymal Stem Cells. » Blood 110, n^o 11 (16 novembre 2007) : 4535. http://dx.doi.org/10.1182/blood.v110.11.4535.4535.

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Abstract Objective To isolate and culture bone marrow mesenchymal stem cells (MSC) from chronic myelogenous leukemia (CML) patients and examine their functional characteristics; To investigate their biological characteristics like proliferation, apoptosis and tumorigenicity;To observe their effects on proliferation and apoptosis of K562 cells. Methods Bone marrow was extracted from the anterior superior iliac spines of 20 patients with CML. MSC were isolated and cultured in vitro and subcultured for three to five generations. Cell morphology was observed under microscope. The ultrastructure was observed with electron microscope. The immunophenotype was detected by flow cytometry (FCM). Different agents were used to induce the MSC to differentiate into osteocyte and adipocyte, and Von Kossa staining, oil-red staining was used to examine the ability of differentiation. The growth curve, cell cycle and apoptosis were investigated. MSC were cultured in soft agar for 2 weeks to observe the clone growth. BALB/C nude mice were inoculated with MSC to observe the tumorigenicity. MSC and K562 cells were cocultured in vitro and coinjected into subcutaneous of BALB/C nude mice, to observe the effects of MSC on proliferation and apoptosis of K562 cells. Results Fibroblast-like, positive in CD44, CD73 and CD90,and negative in CD34, CD45 and HLA-DR, the CML derived MSC could differentiate into osteocyte and adipocyte. The MSC showed normal ultrastructure. After 2 weeks’ culture, no clone was formed from the MSC. Four weeks after, no tumor was seen in the mice inoculated with MSC. The population doubling time of P3 MSC is (32.61±1.54)h. The population doubling time of P4 MSC is (32.59±1.23)h, The population doubling time of P5 MSC is (32.41±0.75)h. To investigate the cell cycle of the third passage MSC by FCM, there were(93.67%±1.66%)cells in phase G0/G1,(6.33%±1.66%)in phase S+G2+M. There weren’t any apoptosis observed. K562 cells adhensively cultivated with MSC were accelerated and the population doubling time decreased([29.59±0.46]h vs [37.49±2.19]h, P<0.05),cells in G0/G1 decreased ([16.43%±1.67%] vs [32.23%±3.35%], P<0.01),cells in phase S increased ([69.63%±3.09%] vs [59.37%±4.40%], P<0.05),and those in G2/M increased([13.93%±1.45%] vs [8.40%±1.05%], P<0.01),compared with that cultivated in suspension. The apoptosis in both conditions were not observed. Coinjected with MSC, K562 cells developed tumors in 100% of the mice by (12.00±0.82)d compared to (15.50±0.58)d when implanted alone, indicating a statistically earlier onset of tumor growth in presence of MSC (P<0.01). The tumor volume and tumor weight were also increased when K562 cells coinjected with MSC compared with implanted alone (K562+MSC vs K562):tumor volume([75.70±7.30]mm2 vs [37.38±2.39]mm2, P<0.01),tumor weight([0.64±0.08]g vs [0.32±0.06]g, P<0.01). Conclusion MSC from the bone marrow of CML patients can be isolated and cultured;MSC derived from CML seem to have the abilities of powerful proliferation in vitro and multiple differentiation and have no tumorigenicity. The MSC can accelerate the proliferation of K562 cells both in vitro and vivo.

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Park, Sang In, Ho Yong Jung, Eui-Jin Lee et Lee-So Maeng. « THERAPEUTIC EFFECTS OF TRAIL-SECRETING MESENCHYMAL STEM CELLS WITH FOCUSED ULTRASOUND-INDUCED TEMPORARILY OPENING OF BLOOD-BRAIN BARRIERS IN THE BRAIN TUMOR ». Journal of Immunology 204, n^o 1_Supplement (1 mai 2020) : 239.39. http://dx.doi.org/10.4049/jimmunol.204.supp.239.39.

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Abstract Mesenchymal stem cell (MSC)-based gene therapy is a promising tool for the treatment of various neurological disease including brain tumor. The tumor necrosis factor-related apoptotic ligand (TRAIL) is believed to have promise as a cancer the therapy. For stem cell therapy in the brain tumor, therapy efficiency of stem cells by non-invasive method is extremely limited due to blood brain barrier (BBB). Noninvasive pulsed focused ultrasound (FUS) can temporarily opening the BBB of specific areas in the brain. Herein, we investigated the tumor-targeted BBB temporarily opening by FUS and therapeutic efficiency of NIR fluorescence-labeled MSCs-TRAIL by intravenous injection in brain tumor. In addition, we observed the inflammatory changes of microglia cells by FUS using PET-CT and histological analysis. As a results, In vivo survival experiments showed that FUS treated-MSC-TRAIL group has greater therapeutic efficacy than FUS untreated-groups. The number of MSCs accumulated specifically site in the tumor region. MSC migration toward FUS treated-tumor site greater compared to the FUS untreated-tumors. Furthermore, the changes of microglia activity by FUS was no difference between the other groups. Collectively, these results suggest that therapeutic efficacy of MSC-TRAIL increases by temporarily opening the BBB using the FUS, which may be a more useful strategy for cancer therapy.

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Feldman, Leonardo Julio, Valeria B. Fernández Vallone, Hosoon Choi, Vivian Labovsky, Leandro M. Martinez, raul H. Bordenave, Vera Milovic et al. « Bone Marrow Mesenchymal Stem Cells : Pre-Metastatic Niche For Breast Cancer ». Blood 122, n^o 21 (15 novembre 2013) : 4859. http://dx.doi.org/10.1182/blood.v122.21.4859.4859.

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It has been described that the hematopoietic niche has to be considered as a dynamic system that could easily behave as a tumor niche because they share many structural and functional components. Bone marrow (BM) mesenchymal stem cells (MSC) are one of the actors that regulate homing, migration, proliferation and survival, quiescence and latency of hematopoietic SC or tumor cells. Since one of the chosen sites for breast cancer (BC) metastasis is BM/bone, we have been trying for many years to address how BM´s MSC could play a role as permissive promoters of this type of metastasis. In this regard, we have previously described that BM-MSC from untreated advanced BC patients, without BM/bone metastasis (BCP), had lower cloning efficiency to give fibroblast colony forming units (CFU-F), lower plasticity capacity towards osteogenic and adipogenic lineages as well as lower capacity to regulate the hematopoietic process. CD146 expression over MSC/osteoprogenitors is diminished while they are enriched in CD49b. These last observations added to lower expression of VCAM-1 and lower levels of SDF-1 released by patient´s MSC plus the lower expression of phosphorylated β-catenin could be related to their poor transmigration capacity and so higher retention in BM. We tried, then, to analyze the migration capacity of BM-MSC of untreated advanced BCP (infiltrative ductal carcinoma, IIIB clinical stage) compared to those coming from healthy volunteers (HV) and the possible factors that influence it. Finally, since these MSC are the ones remaining in BM after the development of the primary tumor and could act as tumor niche we investigated how capable are those for tumor cells recruitment. Materials and Methods BCP (n=5) were in menopause with an age range between 50-65 years old. Patients were free of metabolic bone disease, such as vitamin D deficiency, thyroid disease, parathyroid disease or kidney damage. HV (n=6) were healthy females adult bone marrow donors for allogeneic BM transplantation. BM aspirates, peripheral blood plasma (PBP) and primary tumor tissue biopsies were used for this work. All individuals gave consent prior to participating in these studies. The investigations were approved by the IBYME Ethical Committee and performed in accordance with the principles of the Helsinki Declaration. Transwell co-culture assays were performed to observe BM-MSC migration capacity towards BC cells (human lines: MDA-MB231 and MCF-7) and vice versa. ELISA and immunohistochemistry assays were done to study the factors and receptors implicated in the process. IHC studies were carry out on paraffin blocks from 5 primary tumor tissue biopsies of these BCP and 5 non-malignant breast tissues. The controls “non- malignant” tissue were breast biopsies from women who had negative results of BC (non-neoplastic breast tissue). Also these women were in menopause and did not present compromise in their BM and bone metabolism. Results BM-MSC from BCP showed lower migration capacity towards both BC cell lines compared with MSC from HV. These observations could be related to the lower levels (mean±SE, pg/ml) of SDF-1 (54 ± 28 and 117 ± 25; unpaired t-test, p<0.05) and G-CSF (250 ± 113 and 1076 ± 325; unpaired t-test, p<0.05) as well as high levels of MIF (4,564±591 and 2,265±402; unpaired t-test, p<0.05) found in PBP from BCP compared with HV. Both type of BC cells migrated in higher proportion towards MSC from BCP than those from HV. The higher migration % was significant for the more invasive line MDA-MB231 (Mann Whitney test, p= 0.0159). These last observations could be related to the higher expression of membrane RANKL and IL-6 observed in BM-MSC of BCP respect to HV and the presence of its correspondent receptors in epithelial localization seen over primary breast tumor biopsies from advanced BCP compared with non-malignant breast tissues. Conclusion These findings suggest that early therapeutic intervention may be required to oppose the tumor-induced changes to the BM-MSC and haematopoietic microenvironment, and thus, future bone metastatic development Disclosures: No relevant conflicts of interest to declare.

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Pebrel-Richard, Céline, Richard Veyrat Masson, Frédérique Dubois-Galopin, Jean-Jacques Guérin, Laurent Guillouard, Jacques Chassagne, Jacques-Olivier Bay, Olivier Tournilhac, Karin Tarte et Marc G. Berger. « Bone Marrow Mesenchymal Stem Cells Are Altered in B-Cell Chronic Lymphocytic Leukemia. » Blood 112, n^o 11 (16 novembre 2008) : 2066. http://dx.doi.org/10.1182/blood.v112.11.2066.2066.

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Abstract In B-cell chronic lymphocytic leukemia (B-CLL), CD5+CD19+ malignant cells home into the bone marrow (BM) and circulate in the blood. While CLL tumor cells are not susceptible to apoptosis in vivo, they die rapidly in vitro in the absence of specialized non-hematopoietic feeder cells, such as mesenchymal stem cells (MSC). Recent observations have suggested that there is a functional relationship between B cell clone and the stroma. We have thus compared BM-MSC obtained from B-CLL patients and healthy subjects. We first evaluated the influence of in vitro culture conditions on the number of BM-derived CFU-F and the proliferation of MSC and, in parallel, we quantified in unmanipulated normal and malignant BM samples the CD45negCD14negCD73pos cell subset that was previously shown to contain CFU-F (Veyrat-Masson et al., BJH, 2007). Changes in the level of 42 cytokines/chemokines, were then evaluated in MSC-conditioned media (4 CLL vs 4 normal BM-MSCs) using protein-array (RayBio Human Cytokine Antibody Array IIITM, Tebu-bio SA,). In addition, total RNA was extracted (Rneasy MiniKit, Qiagen,) from 9 expanded MSC at passage 1 (P1) in the presence of bFGF (5 untreated B-CLL BM-MSC: 2 Binet stage A, 2 stage B and 1 stage C; 4 normal BM-MSC) and then reverse transcribed (High Capacity cDNA RT Kit, Applied BioSystems). Quantitative PCR reactions, using dedicated microfluid cards screening 384 selected genes, were then performed (TLDAs, Applied Biosystem Courtaboeuf, France). The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize gene expression level. Despite a 16-fold increase in total cell numbers tested, we found that most BM-MSC cultures from B-CLL patients failed under standard culture conditions (IMDM/10%FCS), in contrast with our experience with normal BM (69 % n = 13 vs <0.05 % n = 205 ; p <0.005). In agreement, CD45negCD14negCD73pos cells were under the threshold of detection in most of B-CLL BM samples (11/16). In productive cultures, we found more CFU-F from B-CLL BM formed by large, polygonal mesenchymal cells (58.1 ± 12.7 % vs 11.4 ± 3.6 % ; p = 0.008 ). These cells proliferated poorly and in most cases could not be further amplified. The use of normal human AB serum, CLL serum, or bFGF enabled us to detect CFU-F in most malignant samples and to amplify mesenchymal cells (19/21 (90 %)), but their frequency remained lower than in control BM. By using protein-array, we observed that MSC tended to release lower amounts of IL-6, IL-7, and MCP-1 and sometimes higher amounts of IL-8. The concentrations of these cytokines/chemokines in the MSC culture supernatant are under validation by ELISA. Finally, among the 384 genes tested by RT-qPCR, we identified 16 statistically up-regulated genes and 41 down-regulated genes (Mann Whitney U test, P< .05; and SAM permutation analysis, FDR<5%). Up-regulated genes included several growth and angiogenic factors as well as key players of the stroma - tumor cell crosstalk. Most down-regulated genes were involved in differentiation pathways. Conclusions: These results show that the BM-MSC from B-CLL patients were quantitatively and functionally altered and are dependent for their in vitro growth on circulating soluble factors or on growth factors like bFGF. Interestingly, from this small series, we observed 57 differentially expressed genes which could be involved in the B-CLL specific stromal cell alterations previously reported (dysregulation of cytokine secretion, angiogenesis, host-tumor relationships). These findings suggest the possible permissive role of MSC on B-cell clone progression and raise the question as to whether we are dealing with selection of a mesenchymal subset or with alteration of mesenchymal cells induced by malignant B-cells

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Zappia, Emanuela, Simona Casazza, Enrico Pedemonte, Federica Benvenuto, Ivan Bonanni, Ezio Gerdoni, Debora Giunti et al. « Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy ». Blood 106, n^o 5 (1 septembre 2005) : 1755–61. http://dx.doi.org/10.1182/blood-2005-04-1496.

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Abstract We studied the immunoregulatory features of murine mesenchymal stem cells (MSCs) in vitro and in vivo. MSCs inhibited T-cell receptor (TCR)-dependent and -independent proliferation but did not induce apoptosis on T cells. Such inhibition was paired with a decreased interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and was partially reversed by interleukin-2 (IL-2). Thus, we used MSCs to treat myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. We injected intravenously 1 × 106 MSCs before disease onset (preventive protocol) and at different time points after disease occurrence (therapeutic protocol). MSC administration before disease onset strikingly ameliorated EAE. The therapeutic scheme was effective when MSCs were administered at disease onset and at the peak of disease but not after disease stabilization. Central nervous system (CNS) pathology showed decreased inflammatory infiltrates and demyelination in mice that received transplants of MSCs. T-cell response to MOG and mitogens from MSC-treated mice was inhibited and restored by IL-2 administration. Upon MSC transfection with the enhanced green fluorescent protein (eGFP), eGFP+ cells were detected in the lymphoid organs of treated mice. These data suggest that the immunoregulatory properties of MSCs effectively interfere with the autoimmune attack in the course of EAE inducing an in vivo state of T-cell unresponsiveness occurring within secondary lymphoid organs. (Blood. 2005; 106:1755-1761)

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Yang, Chao, Deqiang Lei, Weixiang Ouyang, Jinghua Ren, Huiyu Li, Jingqiong Hu et Shiang Huang. « Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro ». BioMed Research International 2014 (2014) : 1–13. http://dx.doi.org/10.1155/2014/109389.

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Human mesenchymal stem cells (MSCs) have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy. But very limited studies investigated the antitumor properties of MSCs themselves. In this study we investigated the antiglioma properties of two easily accessible MSCs, namely, human adipose tissue-derived mesenchymal stem cells (ASCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs). We found (1) MSC conditioned media can significantly inhibit the growth of human U251 glioma cell line; (2) MSC conditioned media can significantly induce apoptosis in human U251 cell line; (3) real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 and significant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251 cells; (4) furthermore, MSCs conditioned media culture induced rapid and complete differentiation in U251 cells. These results indicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line. Whereas UC-MSCs are more efficient for apoptosis induction than ASCs, their capability of differentiation induction is not distinguishable from each other. Our findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future glioma therapy.

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Giallongo, Cesarina, Daniele Tibullo, Nunziatina Laura Parrinello, Piera La Cava, Giuseppina Camiolo, Nunzia Caporarello, Carmelina Daniela Anfuso et al. « Mesenchymal Stem Cells (MSC) from Patients with Multiple Myeloma Promote Myeloid Cells to Become Granulocytic-Myeloid-Derived Suppressor Cells (G-MDSC) with Immunosuppressive, Bone Resorption and Pro-Angiogenic Activity ». Blood 128, n^o 22 (2 décembre 2016) : 4458. http://dx.doi.org/10.1182/blood.v128.22.4458.4458.

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Abstract Purpose A well-recognized feature of MM is the intimate relationship between plasma cells and bone marrow microenvironment, which is mainly composed of MSC, endothelial cells, immune cells and extracellular matrix. G-MDSC accumulate in the tumor microenvironment during tumor development promoting tumor growth and immunosuppression. Aim Analyzing MSC from MGUS, Smoldering myeloma (SMM) and MM patients in promoting G-MDSC generation. Methods Human peripheral blood mononucleated cells (PBMC) isolated from healthy subjects (HS) were cultured alone and with HS- (n=10), MGUS- (n=10), SMM- (n=4) or MM-MSC (n=14)(1:100). After 6 days, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype(CD11b+CD33+CD14-HLADR-) was confirmed by cytofluorimetric analysis. Results Only G-MDSC educated by SMM- and MM-MSC co-cultures (MSCed-G-MDSC) were able to suppress T cell proliferation when cultured with normal lymphocytes (p<0.001) compared to G-MDSC control (isolated from PBMC cultured in medium alone). SMM- and MM-MSCed-G-MDSC significantly up-regulated Arg1, NOS2, TNFα and CEBPA, a transcription factor promoting suppressive phenotype. Since also the angiogenic factor BV8 was significantly up-regulated, we next investigated the pro-angiogenic effect in vitro co-culturing MSCed-G-MDSC with Human Brain Microvascular Endothelial Cells (HBMEC) (1:2). After 5 h, we observed that MM-MSCed-G-MDSC were able to increase both tube length and number of branch points compared to G-MDSC control (p<0.05). Moreover, MM-MSCed-G-MDSC were able to digest bone matrix in vitro (p<0.01). Adding Bortezomib (5 nM), Lenalidomide (10 μM) or Pomalidomide (1 nM) during co-culture with MM-MSC, isolated G-MDSC showed a significant reduction of pro-angiogenic and bone resorption activity (p<0.05) but did not lose immunosuppressive ability. Conclusion MSC play a key role promoting tumor microenvironment transformation in SMM and MM patients.Indeed, only SMM- and MM-MSC and not MGUS-MSC are able to activate myeloid cells in G-MDSC with immunosuppressive, pro-angiogenic, and bone resorptionactivity. Disclosures No relevant conflicts of interest to declare.

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Poggi, Alessandro, Anna-Maria Massaro, Simone Negrini, Ivana Pierri, Manuela Balocco, Gianluca Michelis, Sara Aquino, Alessandra Albarello, Marco Gobbi et Maria Raffaella Zocchi. « Evidence for Killing of Mesenchymal Stem Cells (MSC) by Autologous Natural Killer Lymphocytes. » Blood 104, n^o 11 (16 novembre 2004) : 1290. http://dx.doi.org/10.1182/blood.v104.11.1290.1290.

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Abstract In this study, Mesenchymal Stem Cells (MSC) were obtained from bone marrow of 10 patients suffering from acute myeloid leukemia (AML), six M0/1 two M2, and two M5 (according to the FAB classification), 8 out of 10 in post-chemotherapy complete remission. These cells differentiated into adipocytes or osteoblasts under appropriate culture conditions. MSC were CD44+, CD73a+ CD73b+ CD105+, beta1 integrin+, ICAM1+, HLA-I+, HLA-II+ (variable proportions), CD45−, CD31−, CD34− and they constitutively expressed the stress-inducible MHC-related molecules MIC-A and the UL16 (induced at the surface of cells infected by cytomegalovirus) binding protein ULBP3. These molecules are reported ligands for the NKG2D receptor expressed by natural killer (NK) and CD8+ T lymphocytes, effector cells that are thought to play a role in host defence against tumors. NK cells have also been shown to regulate normal differentiation of hemopoietic precursor into the myeloid or lymphoid cell lineage. Moreover, it has been stated that NK cells are not able to damage autologous cells, as they receive negative signals through inhibitory receptors, including killer Ig-like receptors (KIR) or C-type lectin inhibitory receptors (CLIR), which bind to HLA-I discrete alleles. Surprisingly, we found that autologous IL2-activated, but not freshly isolated, NK cells lysed MSC, while T lymphocytes did not kill self or non-self MSC. Binding of ICAM-1 expressed by MSC to its receptor, the integrin LFA-1, expressed by NK cells plays a key role in MSC/NK interaction. More importantly, NKG2D/MICA and/or NKG2D/ULBP3 engagement is responsible for the delivery of lethal hit. Conversely, it appears that HLA-I molecules do not protect MSC from NK cell-mediated injury. Taken together, these data suggest that NK cells, when activated as it may occur during the first response to viral infections, are able to eliminate MSC, thus altering the normal interactions with hemopoietic precursors and possibly affecting their differentiation. This mechanism might also contribute to the development of aberrant precursors as observed in acute leukaemias.

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Patel, Shyam Ajay, et Pranela Rameshwar. « Role of CD74 in Mesenchymal Stem Cell Dysfunction in Patients with Acute Myeloid Leukemia ». Blood 120, n^o 21 (16 novembre 2012) : 4344. http://dx.doi.org/10.1182/blood.v120.21.4344.4344.

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Abstract Abstract 4344 Mesenchymal stem cell (MSC) dysfunction has been implicated in the biology of the acute myeloid leukemia (AML). Preliminary studies from our laboratory have demonstrated that MSCs derived from AML patients have diminished T cell immunosuppressive capacity, and these MSCs demonstrate signs of transformation. In the current study, we seek to explore the mechanisms of MSC dysfunction in patients with AML. Recent reports have implicated migration inhibitory factor (MIF) and its receptor CD74 in cancer progression. CD74 is a cell surface glycoprotein that also serves as the invariant chain for the class II major histocompatibility complex (MHC-II). The significance of these studies is two-fold: (1) CD74 participates in intracellular signal transduction via NF-kappaB, ERK, and PI(3)K pathways, thereby promoting cell proliferation, and (2) MHC-II invariant chain expression in MSCs may prevent tumor antigen loading and presentation to T cells, allowing for immune subversion. Our results show increased expression of CD74 on MSCs derived from AML patients. The readily targetable phenotype and the availability of anti-CD74 antibody milatuzumab merits further investigation into the role of CD74 in AML biology. Disclosures: No relevant conflicts of interest to declare.

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Gruenloh, William, Amal Kambal, Claus Sondergaard, Jeannine McGee, Fernando Antonio Fierro, Scott Olson et Jan A. Nolta. « Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived From Human Embryonic Stem Cells. » Blood 114, n^o 22 (20 novembre 2009) : 1451. http://dx.doi.org/10.1182/blood.v114.22.1451.1451.

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Abstract Abstract 1451 Poster Board I-474 Mesenchymal stem cells (MSC, aka marrow stromal cells) present a promising tool for cell therapy, and have been shown to contribute to the recovery of tissues in myocardial infarction, stroke, meniscus injury, limb ischemia, and even neurodegenerative disorders. The percentage of engrafted MSC in these studies has been very low in comparison to the recipient tissue cells, suggesting that their efficacy relies upon actions other than differentiation. One theory of tissue repair and regeneration by adult MSC is that the injected stem cells home to the injured area, in particular to hypoxic, apoptotic, or inflamed areas, and release trophic factors that hasten endogenous repair. These secreted bioactive products can suppress the local immune system, enhance angiogenesis, inhibit fibrosis and apoptosis, and stimulate recruitment, retention, proliferation and differentiation of tissue-residing stem cells. Paracrine effects exerted by MSC are distinct from the classical model of direct differentiation of stem cells into the tissue to be regenerated. MSC can, however, directly contribute to the repair of bone, tendon and cartilage. In some cases, where the patient lacks expression of a critical gene product, genetic engineering of the MSC is desired. Using human embryonic stem cells (hESC), the integration site of a vector can be fully characterized and the clones with benign integration sites can be expanded. Homologous recombination is also now feasible for embryonic cells due to increased efficiencies, and clones with vectors targeted for gene correction can be expanded. For this reason we have generated MSC from the well-characterized human embryonic stem cell line H9. H9-derived MSC (H9-MSC) expressed CD105, CD90, CD73 and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR. H9-MSC also lacked expression of the hESC pluripotency markers SSEA-4 and Tra-1-81, which were expressed by the starting H9 line. Additionally, they lacked expression of SSEA-1, an early marker of hESC differentiation. Marrow-derived MSC showed a similar phenotype when analyzed concurrently with H9-MSC. Morphology was similar to adult MSC derived from marrow or adipose tissue. H9-MSC grew more robustly than MSC derived from marrow or adipose tissue, with a rapid doubling time and a failure to display contact inhibition. However, upon reaching maturity the cells did slow to the same rate as bone marrow-derived MSC, and then were finally subject to contact inhibition. Additionally, H9-MSC were injected with matrigel subcutaneously into the flank of immune deficient mice [NOD/SCID/IL2Rg-/- (NSG)] to assess their ability to form tumors due to possible growth abnormalities. No mice that received injections of H9-MSC formed teratoma or other tumors, whereas the parent H9 line robustly forms teratoma. H9-MSC could be robustly differentiated into bone, as shown by alizarin red staining of mineralized plaques after 21 days of culture in standard osteogenic differentiation medium containing 0.2 mM ascorbic acid, 0.1 m M dexamethasone, and 10 mM b-glycerophosphate with media changes every three days. H9-MSC morphology was noticeably different from that of H9-MSC undergoing differentiation as early as day 3 of the differentiation protocol. Finally, cells were tested for their capacity to respond to a hypoxic in vivo environment, using our standard hindlimb ischemia model in immune deficient [NOD/SCID/b2M-/-] mice. H9-MSC were found to have homed to the hypoxic muscle by 48 hours after injection into the bloodstream. In summary we have defined methods for differentiation of hESC into MSC, have defined their characteristics, and in vivo migratory properties. This system could be useful, following further safety studies, for production of large numbers of MSC from embryonic or induced pluripotent stem cells that have been corrected for gene defects by lentiviral vector integration with careful assessment of integration site, or by homologous integration, with subsequent expansion, characterization and banking of the line prior to differentiation into functional mesenchymal stem cells. Disclosures No relevant conflicts of interest to declare.

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Cen, Jian-nong, Zi-xing Chen, Jun Qian, Zhen-jiang Li et Qiao-cheng Qiu. « Human Mesenchymal Stem Cells Enhances the Growth of Acute Myeloid Leukemia Cells In Vivo. » Blood 106, n^o 11 (16 novembre 2005) : 2307. http://dx.doi.org/10.1182/blood.v106.11.2307.2307.

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Abstract Human mesenchymal stem cells (MSC) are adhering cells capable of self-renew, proliferation and multilineage differentiation. MSC have the capacity to differentiate into osteoblasts, chondroblasts, myocytes and adipocytes when exposed to appropriate in vivo or in vitro stimuli. MSC descendants are involved in the formation of stroma maintaining the hemopoiesis. In this study, we investigated whether in vivo coinjection of hMSCs could enhance acute myeloid leukemia cells growth. Acute myeloid leukemia line (SHI-1) cells were grown in IMDM with 10%FCS. The hMSCs in adult BM were isolated and cultured in 60%DMEM (low glucose) and 40% MCDB-201 medium, 5%FCS, ITS+1. The hMSCs were immunotyped by using FACS. The differentiation ability in vitro towards osteoblasts and adipocytes was examined. BABL/c nude mice, aged 5 to 6 weeks, were used to explore the hMSCs’ function in vivo. SHI-1cells and hMSCs were prepared either as single-cell type suspensions (3×106 cells in 100ul PBS) or a mixture of cells (3×106 SHI-1 cells plus 4×105 hMSCs in 100ul PBS ). Subcutaneous injection was performed in alar area. Injection of hMSCs alone was used as control. Mice were examined 3 times a week and tumor growth was evaluated by measuring the length and width of tumor mass. After 30 days, animals were sacrificed and tumor masses were weighed. Our experiments showed that the hMSCs have the potential of proliferation and the capacity to differentiate into osteoblasts and adipocytes in vitro. The immunophenotype of MSCs is CD34−,CD45−,CD105+,CD19−,CD13+,CD14−.10 days after injection with a mix of cells small tumor mass occurred in mice while no tumor mass could be observed in mice injected SHI-1 cells alone. Upon day 20, small tumor mass started to emerge in mice injected with SHI-1 cells alone. By the end of the experiment, mice were sacrificed and tumors were recovered. The weight of tumor masses from the mouse injected with mixed cells of SHI-1 and hMSCs was 0.42±0.18 g and from the mouse injected SHI-1 cells alone was 0.11±0.089 g, (P<0.01). No tumor masses could be found in mice injected with hMSCs alone. From this animal model, we proposed that human mesenchymal stem cells favors the growth of acute myeloid leukemia cells in vivo.

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McNiece, Ian K., et Santhosh Sivajothi. « Cardiac Derived Stromal Cells Inhibit Tumor Cell Proliferation : A Potential Role Of miR206 ». Blood 122, n^o 21 (15 novembre 2013) : 4861. http://dx.doi.org/10.1182/blood.v122.21.4861.4861.

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Abstract Stromal cells play an important role in control of proliferation and differentiation of stem cells and are a key component of the stem cell niche. Bone marrow (BM) stromal cells (also termed mesenchymal stem cells; MSC) have been extensively studied and shown to control differentiation of hematopoietic stem cells (HSCs) in part through secreted growth factors. Recent studies have demonstrated the presence of stromal cells in cardiac tissue, however the role of cardiac stromal cells (CStrC) is unclear. In this study we have compared human CStrCs to human BM-MSCs and demonstrate that CStrCs have a similar morphology and surface marker expression as BM-MSCs. To further characterize the CStrCs we performed micro array analysis of human CStrCs compared to human BM-MSCs. The CStrCs expressed a distinct cytokine and cytokine receptor profile compared to BM-MSC. In addition, a number of micro RNAs were expressed at very high levels in CStrCs compared to BM-MSC. Cardiac- associated microRNAs, including miR-1, miR-133a, and miR-206 were expressed at higher levels in CStrCs compared to BM-MSC. Given the lack of tumor development in cardiac tissue we hypothesized that CStrCs would fail to support tumor cell growth which has been described for BM-MSCs. Therefore, we cultured human tumor cell lines on CStrCs and compared the tumor cell proliferation to BM-MSCs. CStrCs inhibited the proliferation of a range of tumor cell lines including myeloid cell lines HL60, K562, myeloma cell lines U-266, RPMI 8266, ARP-1 and the B cell line Raji, while BM-MSCs supported the proliferation of tumor cells. Further we tested media conditioned by CStrCs and demonstrated inhibition of proliferation of both cell lines. Previous studies have implicated miR-206 in inhibition of proliferation of tumor cells and given the high levels of expression of miR-206 in CStrCs we hypothesize that miR-206 is a key player in the inhibitory effects of CStrCs and this effect is mediated via secreted molecules. Disclosures: McNiece: Proteonomix Inc: Consultancy.

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Zhao, Ke, Zhiping Fan, Fen Huang, Peng Xiang et Qifa Liu. « Mesenchymal Stem Cell As a Salvage Treatment for Patients with Refractory Bronchiolitis Obliterans Syndrome after Allogenetic Hematopoietic Stem Cell Transplantation ». Blood 126, n^o 23 (3 décembre 2015) : 855. http://dx.doi.org/10.1182/blood.v126.23.855.855.

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Abstract Background Bronchiolitis obliterans syndrome (BOS) is the most detrimental late pulmonary complications after allogeneic hematopoietic stem cell transplantation, as it is characterized by non-responsiveness to treatment, leading to high morbidity and mortality. Mesenchymal stem cell (MSC) has been considered as an effective treatment for refractory aGVHD, but the response to treat cGVHD, especially refractory BOS, is rarely reported. This study evaluated the efficacy of MSC from bone marrow (BM) of a third-party donor to BOS patients. Methods Fifty-three patients with refractory BOS were enrolled in our prospective study, including 29 patients in MSC group and 24 patients in non-MSC group. All patients had previously failed to at least 2 lines of immunosuppressive therapy. MSCs were given at a median dose of 1×106 cells/kg once weekly for 4 weeks as one cycle treatment. The responsiveness of MSC was evaluated after one cycle, non-responsive patients discontinued MSC treatments, others continued to accept another cycle of MSC treatments. Results Fifty-three patients developed BOS at a median time of 172 days (range, 94 to 398). After a total of 169 doses of MSC were administered, with a median of 6 (range:3-11) doses per patient, the overall response (OR) rate in MSC group was 75.9%, including CR in 13.8% and partial response (PR) in 62.1%. Compared with MSC group, OR rate in non-MSC group was 16.7%, including CR in 4.2% and PR in 12.5% (P=0.000; P=0.233; P=0.000, respectively). Furthermore, the efficacy of MSC to refractory BOS was significantly related with the severity of pulmonary function (mild VS severity, P=0.039). During the median follow-up time of 814 (range:228-2499) days post-transplantation, 7 patients were dead in MSC group, while 18 patients died in non-MSC group. The 5-year OS post-transplantation in MSC group were 68.6%±5.7%, compared with 21.2%±7.1% in non-MSC group (P=0.033). No patients experienced any toxic side effects and other secondary tumors after MSC treatment. Conclusion MSC derived from BM of third-party donors is a promising treatment for patients with refractory BOS. Disclosures No relevant conflicts of interest to declare.

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Gentilini, Chiara, Kathrin H. Al-Ali, Annette Reinhardt, Kristina Bartsch, Toralf Lange et Dietger W. Niederwieser. « Mesenchymal Stem Cells of Patients with Chronic Myeloid Leukemia Are Philadelphia Negative. » Blood 104, n^o 11 (16 novembre 2004) : 4683. http://dx.doi.org/10.1182/blood.v104.11.4683.4683.

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Abstract In the last years, focus of regenerational studies has pointed on mesenchymal stem cells (MSC) and their ability to differentiate into several mesenchymal tissues. MSC have been shown to play a pivotal role in the microenvironment of bone marrow cells and in the modulation of immune response as they can suppress lymphocytic proliferation in vitro. Moreover, some animal studies have suggested they could favor the proliferation of malignant cell clones in solid tumor models. Their role in hematological malignancies, however, remains to be further elucidated and little is known about the influence of MSC in the development and maintenance of the malignant clone in chronic myeloid leukemia (CML). This disease is characterized by the presence of the Philadelphia (Ph) chromosome, a fusion product generated by the reciprocal translocation between chromosomes 9 and 22. Previous reports showed that hepatocytes precursors, found in the liver of CML patients carry the Ph translocation. Our intent was to elucidate whether MSC isolated from patients with CML in different stages of the disease originate from the malignant clone. To this purpose bone marrow aspirates of 11 patients with CML were obtained after informed consent. Five patients were analyzed at diagnosis, two after allogenic stem cell transplantation, three on treatment with the tyrosine kinase inhibitor imatinib and one on treatment with interferon alpha in combination with hydroxyurea. MSC were then generated as previously described. Briefly, cells were isolated by density gradient methods, resuspended in RPMI1640 medium containing 10% fetal bovine serum and plated in culture flasks to adhere. After 4–5 weeks of culture cells were collected and characterized by the expression of several surface markers in a fluorescence activated cell sorter (FACS). The presence of the Ph chromosome was assessed by both fluorescence in situ hybridization (FISH) and polymerase chain reaction (nested PCR). Moreover whole bone marrow was analyzed and results compared with those obtained in the MSC population. MSC showed a typical morphological pattern, growing to confluence after a few weeks of culture and appearing as an adherent, spindle shaped cell layer. In FACS they stained positive for SH2 and SH3 and did not express CD34, CD45 and CD14. MSC were then analyzed by FISH using probes for BCR-ABL. We could not detect the Ph translocation in any of the analyzed patients, though it was present at variuos levels in the remnant bone marrow cells. Results did not change, if expression of BCR-ABL was measured by high sensitivity RT-PCR. Our results showh that MSC of patients with CML are Philadelphia negative irrespective of the stage of disease and the treatment given, suggesting that these cells are not involved in the development of the malignancy. However, their interactions with leukemic cells as well as their role in the immune response against the tumor remains to be further characterized.

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Tarte, Karin, Patricia Ame-Thomas, Hélène Maby-El Hajjami, Céline Monvoisin, Rachel Jean, Thierry Lamy et Thierry Fest. « Human Mesenchymal Stem Cells Isolated from Bone Marrow and Lymphoid Organs Support Tumor B-Cell Growth : Implications in Follicular Lymphoma Pathogenesis. » Blood 108, n^o 11 (1 novembre 2006) : 2409. http://dx.doi.org/10.1182/blood.v108.11.2409.2409.

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Abstract There is accumulating evidence that cellular microenvironment plays a key role in follicular lymphoma (FL) pathogenesis, both within tumor lymph nodes (LN) and in infiltrated bone marrow (BM) where ectopic LN-like reticular cells are integrated within malignant B-cell nodular aggregates. In normal secondary lymphoid organs, specific stromal cell subsets provide a highly specialized microenvironment that supports immune response. In particular, fibroblastic reticular cells (FRC) mediate immune cell migration, adhesion, and reciprocal interactions. The role of FRC and their postulated progenitors, i.e. bone marrow mesenchymal stem cells (MSC), in FL remains unexplored. In this study, we have investigated the relationships between FRC and MSC and their capacity to sustain malignant B-cell growth. Our findings strongly suggest that secondary lymphoid organs contain bona-fide MSC able to give rise at single-cell level to adipocytes, chondrocytes, and osteoblasts. These LN-derived MSC could also differentiate, in response to a combination of tumor necrosis factor-α (TNF) and lymphotoxin-α1β2 (LT), into fully functional FRC, able to construct a dense extracellular reticular meshwork positive for transglutaminase and fibronectin staining, to produce inflammatory (CXCL9, CXCL10, CCL5, CCL2) and LN-specific (CCL19) chemokines, and to favour lymphoma B-cell growth. Bone marrow-derived MSC (BM-MSC) acquire in vitro a complete FRC phenotype in the same culture conditions. As an exemple, BM-MSC had a strong, although not complete, protective effect on serum deprivation-induced apoptosis of BL2 cell line (mean percentage of CD20posCaspase-3pos cells: 24.8 +/− 17.5% in coculture with BM-MSC versus 80.7 +/- 10.4% in medium alone; P < .05; n =5) and pretreatment with TNF/LT fully restored BL2 viability (mean percentage of CD20posCaspase-3pos cells: 7.4 +/− 4.7%; P < .05; n = 5). Moreover, stimulation of stromal cells by TNF/LT before coculture enhanced the number of viable CD19pos primary FL B cells by 2.4-fold for BM-MSC and 2.3 fold for LN-MSC compared with the culture without stromal cells (P < .05; n = 6). Interestingly, cell contact with lymphoma B-cell lines or purified FL B cells trigger the differentiation of BM-MSC into FRC that, in turn, support malignant B-cell migration, adhesion and survival. Altogether, these new insights into the interactions between lymphoma cells and their microenvironment could offer original therapeutic strategies.

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Kanehira, Masahiko, Tohru Fujiwara, Shinji Nakajima, Yoko Okitsu, Yasushi Ohnishi, Noriko Fukuhara, Ryo Ichinohasama et Hideo Harigae. « Impaired Lysophosphatidic Acid Receptor 3 Signaling in Mesenchymal Stromal Cells Promotes Multiple Myeloma Progression through Cellular Senescence and Transdifferentiation into Tumor-Associated Fibroblasts ». Blood 126, n^o 23 (3 décembre 2015) : 1764. http://dx.doi.org/10.1182/blood.v126.23.1764.1764.

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Abstract Introduction Multiple myeloma (MM) is one of the hematologic malignancies characterized as accumulation of monoclonal tumor of plasma cells. Although novel therapeutic agents have significantly improved the survival of MM patients, MM is still a mostly incurable disease. The most common reason of relapsed and refractory MM is that myeloma cells intimately communicate with bone marrow stroma and acquire drug resistance. Stroma is known to secrete soluble factors and express some adhesion molecules advantageous for progression of MM. Recently, there are accumulating evidences that bone marrow-derived mesenchymal stem cells (MSC) serve as a component of stroma and support growth and drug resistance of myeloma cells. Although the exact etiology has not been clearly defined, it has been suggested that the incidence of MM increases with aging. In this study, we tried to explore the relationship between MM progression and cellular senescence in MSC. Methods Human myeloma cell lines used in this study were IM-9, OPM-2, and RPMI-8226. Human MSC used in this study were obtained from Texas A&M Health Science Center for the Preparation and Distribution of Adult Stem Cells. To establish a mouse xenograft model of human MM, 1.0 x 106 of IM-9 cells were co-injected with 4.0 x 105 of human MSC subcutaneously into the flank of BALB/c nude mice. Visible and palpable tumors were formed in all animals after 20 days. Tumor specimens were resected surgically and diagnosed pathologically as MM based on marker expression. For in vivo bioimaging, luciferase-expressing IM-9 (IM-9-luc+) cells were co-injected with human MSC subcutaneously into BALB/c-nude mice, and photon emission was detected with a sensitive CCD camera 10, 15 and 20 days later. Results In our earlier research, we have demonstrated that the signaling of lysophosphatidic acid (LPA), a bioactive lipid mediator, modulates cellular senescence in MSC (Kanehira et al. PLoS One. 2012). MSC produced autotaxin (ATX), a key enzyme in LPA synthesis, in response to myeloma cells via Toll-like receptor 4 (TLR4)/NF-kappaB-dependent pathway. To determine the LPA receptor responsible for cellular senescence in MSC, six LPA receptors (LPAR1-6) were individually knocked down using siRNA for each receptor. In BrdU incorpotaion assay, LPAR3 gene-silenced MSC (siLPAR3-MSC) were less proliferative than control MSC, and in cell cycle analysis with 7-AAD and Pyronin Y, siLPAR3-MSC was arrested in G1 phase. Interestingly, siLPAR3-MSC exhibited some characteristics of cellular senescence, such as up-regulation of senescence-associated beta-galactosidase activity, increased cell size, and flattened morphology. In a mouse xenograft model of MM, siLPAR3-MSC promoted progression of MM and also tumor-associated angiogenesis. In in vitro study, we confirmed that siLPAR3-MSC easily transdifferentiated into alpha-SMA+ tumor-associated fibroblast and secreted FGF2 in response to myeloma cells. The MM promoting effect and elevated tumor-associated angiogenesis observed in siLPAR3-MSC co-injection were both completely cancelled by FGF2 gene-silencing in siLPAR3-MSC (Figure 1 and 2). Conclusion and discussion In this study, we verified the impairment of LPAR3 signaling may accelerate cellular senescence in MSC. And senesced MSC can provide an advantageous microenvironment for MM progression by FGF2-dependent formation of tumor-stroma milieu. Here, we provide the possibility that LPAR3 signaling could be promising as a therapeutic target in MM. Disclosures Fujiwara: Chugai Pharmaceutical CO., LTD: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding.

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Aggarwal, Sudeepta, et Mark F. Pittenger. « Human mesenchymal stem cells modulate allogeneic immune cell responses ». Blood 105, n^o 4 (15 février 2005) : 1815–22. http://dx.doi.org/10.1182/blood-2004-04-1559.

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AbstractMesenchymal stem cells (MSCs) are multipotent cells found in several adult tissues. Transplanted allogeneic MSCs can be detected in recipients at extended time points, indicating a lack of immune recognition and clearance. As well, a role for bone marrow-derived MSCs in reducing the incidence and severity of graft-versus-host disease (GVHD) during allogeneic transplantation has recently been reported; however, the mechanisms remain to be investigated. We examined the immunomodulatory functions of human MSCs (hMSCs) by coculturing them with purified subpopulations of immune cells and report here that hMSCs altered the cytokine secretion profile of dendritic cells (DCs), naive and effector T cells (T helper 1 [TH1] and TH2), and natural killer (NK) cells to induce a more anti-inflammatory or tolerant phenotype. Specifically, the hMSCs caused mature DCs type 1 (DC1) to decrease tumor necrosis factor α (TNF-α) secretion and mature DC2 to increase interleukin-10 (IL-10) secretion; hMSCs caused TH1 cells to decrease interferon γ (IFN-γ) and caused the TH2 cells to increase secretion of IL-4; hMSCs caused an increase in the proportion of regulatory T cells (TRegs) present; and hMSCs decreased secretion of IFN-γ from the NK cells. Mechanistically, the hMSCs produced elevated prostaglandin E2 (PGE2) in co-cultures, and inhibitors of PGE2 production mitigated hMSC-mediated immune modulation. These data offer insight into the interactions between allogeneic MSCs and immune cells and provide mechanisms likely involved with the in vivo MSC-mediated induction of tolerance that could be therapeutic for reduction of GVHD, rejection, and modulation of inflammation. (Blood. 2005;105:1815-1822)

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Guo, Shuyuan, Yusen Zhang, Yanmin Zhang, Fanhua Meng, Minghua Li, Zhendong Yu, Yun Chen et Guanghui Cui. « Multiple Intravenous Injections of Valproic Acid-Induced Mesenchymal Stem Cell from Human-Induced Pluripotent Stem Cells Improved Cardiac Function in an Acute Myocardial Infarction Rat Model ». BioMed Research International 2020 (17 décembre 2020) : 1–13. http://dx.doi.org/10.1155/2020/2863501.

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Mounting evidence indicates that the mesenchymal stem cell (MSC) injection is safe and efficacious for treating cardiomyopathy; however, there is limited information relating to multiple intravenous injections of human-induced pluripotent stem cell-derived mesenchymal stem cell (hiPSC-MSC) and long-term evaluation of the cardiac function. In the current study, MSC-like cells were derived from human-induced pluripotent stem cells through valproic acid (VPA) induction and continuous cell passages. The derived spindle-like cells expressed MSC-related markers, secreted angiogenic and immune-regulatory factors, and could be induced to experience chondrogenic and adipogenic differentiation. During the induction process, expression of epithelial-to-mesenchymal transition- (EMT-) related gene N-cadherin and vimentin was upregulated to a very high level, and the expression of pluripotency-related genes Sox2 and Oct4 was downregulated or remained unchanged, indicating that VPA initiated EMT by upregulating the expression of EMT promoting genes and downregulating that of pluripotency-related genes. Two and four intravenous hiPSC-MSC injections (106 cells/per injections) were provided, respectively, to model rats one week after acute myocardial infarction (AMI). Cardiac function parameters were dynamically monitored during a 12-week period. Two and four cell injections significantly the improved left ventricular ejection fraction and left ventricular fractional shortening; four-injection markedly stimulated angiogenesis reduced the scar size and cell apoptosis number in the scar area in comparison with that of the untreated control model rats. Although the difference was insignificant, the hiPSC-MSC administration delayed the increase of left ventricular end-diastolic dimension to different extents compared with that of the PBS-injection control. No perceptible immune reaction symptom or hiPSC-MSC-induced tumour formation was found over 12 weeks. Compared with the PBS-injection control, four injections produced better outcome than two injections; as a result, at least four rounds of MSC injections were suggested for AMI treatment.

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Lee, Dean A., et William C. Choi. « Human Bone Marrow Mesenchymal Stem Cells Enhance Extramedullary Engraftment and Proliferation of Human AML Blasts in NOD-SCID Mice. » Blood 106, n^o 11 (16 novembre 2005) : 2312. http://dx.doi.org/10.1182/blood.v106.11.2312.2312.

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Abstract BACKGROUND: Inoculation of human normal or leukemic myeloblasts into sublethaly irradiated NOD/SCID mice often results in persistent low-level engraftment (< 5%), but significant proliferation (≥ 5-fold expansion) rarely occurs. Most malignant samples that engraft and proliferate are of FAB M4 subtype and exhibit rapid extramedullary growth at the site of injection without significant marrow or spleen involvement. We hypothesized that low engraftment and proliferation of less mature FAB subtypes results from an increased requirement of these cells for a marrow environment of cytokine and contact-dependent growth and survival factors not adequately provided across species by the mouse bone marrow stroma. Here we show that the subcutaneous injection of minimally-differentiated human mesenchymal stem cells (MSC) in a Matrigel matrix creates an artificial human marrow environment resulting in improved survival and proliferation of human myeloblasts. METHODS: Human leukemic myeloblasts were obtained from the marrow or peripheral blood of 14 newly diagnosed pediatric patients under an IRB-approved collection and banking protocol. MSC were obtained from sterile filters following processing of human marrow from healthy donors or from the NIH-funded MSC bank at Tulane University. 6-to-12 week old NOD-SCID mice were injected IV with 5x106 AML blasts via the retro-orbital sinus (N=38), subcutaneously in 0.5mL Matrigel (N=18), or subcutaneously with 5x105 MSC in 0.5mL of Matrigel (N=14). Mice were euthanized when evidence of tumor burden was present. Peripheral blood, bone marrow, spleen, and subcutaneous nodules were obtained for flow immunophenotyping, FISH, and histopathology. Percent engraftment was determined by flow cytometry for human CD33-APC and mouse H2Kd-PE. RESULTS: Median time from injection to necropsy was 12.5 weeks. 18% died of spontaneous murine thymomas. No animals died of progressive human AML if myeloblasts were injected IV or subcutaneously with Matrigel, and all had < 5% involvement of bone marrow, spleen, and blood. Six animals injected with AML and MSC (43%) developed visible tumors at a median of 8.5 weeks. These tumors were easily reduced to single cell suspensions of > 98% CD33+ by flow cytometry, with mean estimated recovery of 1.3x108 human myeloblasts per mouse tumor (mean 36-fold expansion, range 4 to 52-fold). For cases in which the AML and MSC were derived from subjects of disparate gender, the origin of the cells (leukemic donor vs. MSC donor) was validated by FISH for human X/Y chromosomes. Histopathology of the resulting mass revealed the central development of a stromal chondroid matrix similar to trabecular bone. Marrow, spleen, and blood for all these animals contained < 5% human myeloblasts. CONCLUSIONS: Here we describe an effective method for expanding immature human leukemic myeloblasts in the NOD/SCID mouse. These findings suggest that less mature myeloblasts require human MSC for survival and proliferation and appear to lack significant homing to or expansion in mouse marrow even in the presence of a significant ectopic tumor burden. This is a useful technique for expanding human AML cells for research, may be a model for more broad-based patient-oriented testing of chemotherapeutic and biologic therapies for AML, and represents a novel animal model for studying the stromal interactions and growth requirements of malignant and non-malignant myeloid precursors.

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Zhao, Ke, Zhiping Fan, Fen Huang, Shunqing Wang, Peng Xiang, Qi Zhang et Qifa Liu. « Bone Marrow Derived Mesenchymal Stromal Cells Reduce the Incidence of Chronic Graft-Versus-Host Disease after Allogenetic Hematopoietic Stem Cell Transplantation ». Blood 128, n^o 22 (2 décembre 2016) : 4578. http://dx.doi.org/10.1182/blood.v128.22.4578.4578.

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Abstract Background Chronic graft-versus-host disease (cGVHD) is the common long-term complication and the leading cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT), which has a significant impact on patient quality of life. Currently, mesenchymal stromal cells (MSCs) have been considered as a promising therapy for treating refractory acute/chronic GVHD and engraftment failure (EF) or poor graft function (PGF), but the efficacy of MSCs in the prophylaxis of cGVHD is rarely reported. Methods Eighty-two patients who received MSCs treatment after allo-HSCT were enrolled in our study, including 45 patients with refractory aGVHD, 33 patients with EF or PGF and 4 patients with virus infection. In order to evaluate the influence of bone marrow (BM) derived MSCs for the incidence and severity of cGVHD, 308 consecutive patients who underwent allo-HSCT in the same period but without MSCs treatment were considered as the control group. And no statistical significance was found between the two groups for the demographic and transplant characteristics. MSCs were given at a median dose of 1×106 cells/kg once weekly. Results All eighty-two patients in MSC group received a median of 5 (range:3-12) doses of MSCs per patient. In MSC group, seventeen patients (20.7%) had cGVHD, including 14 patients with limited cGVHD and 3 patients with extensive cGVHD. In the control group, 140 patients (45.5%) had cGVHD, including 95 patients with limited and 45 extensive cGVHD. The 2-year cumulative incidence of cGVHD was 26.7% (95%CI: 16.1%-37.5%) in MSC group and 50.4% (95%CI: 37%-63.8%) in the control group (P=0.037). Furthermore, the extensive cGVHD (3/82) in the MSC group were also significantly lower than those (45/308) in the control group (P=0.021). In addition, at a median follow-up time of 189 (range: 112-1035) days post-transplantation, 4 patients relapsed in MSC group and 13 patients relapsed in the control group. The incidence of tumor relapse was not different between the two groups (P=0.629). No short-term toxic side effects were observed and other secondary tumor occurred after MSCs treatment. Conclusion MSCs derived from BM reduce the incidence and severity of cGVHD after allo-HSCT, but not increase the risk of tumour relapse. Disclosures No relevant conflicts of interest to declare.

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Tan, Yuxin, Lu Ding, Can Can et Fuling Zhou. « Hematopoietic Reconstitution of Human Umbilical Cord Mesenchymal Stem Cells after Leukemia Chemotherapy : Effectiveness and Safety ». Blood 138, Supplement 1 (5 novembre 2021) : 2171. http://dx.doi.org/10.1182/blood-2021-149152.

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Abstract Objective: As the incidence of tumors increases, more patients need chemotherapy. Patients receiving chemotherapy also inevitably suffer side effects from treatment. Damage to the immune and hematopoietic system causes the failure of therapies. Mesenchymal stem cells have excellent capabilities in immune regulation and hematopoietic support. This study preliminarily explored the safety and effectiveness and of human umbilical cord mesenchymal stem cells (UCMSC) in myelosuppressive patient-derived tumor xenograft (PDX) models of acute myeloid leukemia (AML). Methods: B-NDG mice were used to establish the PDX models. The mice were randomly divided into three groups: AML group, AML+Ara-C group and AML+Ara-C+MSC group. Mice in the AML+Ara-C group and AML+Ara-C+MSC group were injected intraperitoneally with cytarabine (Ara-C) 60mg/kg on day1-day3 to induce myelosuppression. Mice in the AML+Ara-C+MSC group were injected 3×10 6 UCMSC through the tail vein on day4. We observed the changes in peripheral blood, bone marrow signaling pathways, and AML progression in mice. Results: The experiment found that UCMSC rescued the body weight and peripheral blood. We also found that UCMSC could increase the number of bone marrow CD117 + hematopoietic stem/progenitor cells and CD41 + megakaryocytes through flow cytometry. We verified at protein level that the hematopoiesis-related signaling pathway JAK2/STAT3 was up-regulated in AML+Ara-C+MSC group compared with AML+Ara-C group through flow cytometry and immunohistochemistry. At the same time, the infusion of UCMSC after Ara-C had no influence on disease progression. On day15, the proportion of human CD45 + cells in the bone marrow of between AML+Ara-C+MSC group and AML+Ara-C group was similar, and there was no statistical difference (21.96±3.10 vs. 23.04±1.51; P=0.6792). Conclusion: Infusion of exogenous UCMSC after chemotherapy in PDX models could promote the recovery of hematopoiesis without affecting the efficacy of Ara-C. A reasonable UCMSC infusion scheme is potential to be used in the treatment of AML patients and it requires a lot of preclinical exploration in the future. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Cytarabine (Ara-C) was administrated to mice models to induce myelosuppression.

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Ling, Xiaoyang, Ye Chen, Peter P. Ruvolo, Vivian Ruvolo, Zhiqiang Wang, Min Zhang, Yuexi Shi, Marina Konopleva, Richard E. Davis et Michael Andreeff. « Unique Effects of p53−/− Leukemic Cells On Mesenchymal Stromal Cell Gene Expression Profile in Vitro ». Blood 120, n^o 21 (16 novembre 2012) : 3468. http://dx.doi.org/10.1182/blood.v120.21.3468.3468.

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Abstract Abstract 3468 Mesenchymal stromal cells (MSC) participate in the generation of the microenvironmental bone marrow niche which protects normal and leukemic stem cells from injuries, including chemotherapy. MSC produce numerous factors that aid in this function; however, little is known about how leukemic cells affect MSC. In this study, paired murine AML cells, MLL/ENL/FIT3-ITD/p53−/− and MLL/ENL/FIT3-ITD/p53wt, originally derived from C57BL/6 mice (Zuber et al. Genes & Dev. 2009), were co-cultured with MSC from the same strain. After 48 hrs, MSC were isolated by FACS sorting using CD45−/PDGFr+ as markers. Total RNA was profiled on Illumina WG6 mouse whole-genome bead arrays by standard procedures. The significance analysis of microarrays (SAM) method identified 429 differentially-expressed genes (DEG) whose expression in MSC differed significantly (false discovery rate, 10%) in co-cultures with p53−/− (C78) vs. p53wt (C147) leukemic cells. Differences in these DEG were highly consistent in replicates (Figure 1). The results demonstrate that: 1) p53 status (p53−/− vs. p53wt) of AML cells affects GEP patterns in co-cultured MSC. Comparison of the GEP in MSC co-cultured with p53−/− (78) or p53wt (147) (Fig 1) identified the following 5 genes that showed the most significant differences (up- or down-regulated): up-regulated: WNT16, WNT5, IGFBp5, GCNT1, ATP1B1; down-regulated: NOS2, DCN, CCL7, CCL2, CAR9, CCL4. These were selected for qPCR validation, and the results confirmed the array data. In addition, immunohistochemical staining showed that WNT16 was up-regulated in MSC co-cultured with p53wt leukemic cells. In addition, CXCL5 was found up-regulated in MSC co-cultured with p53−/− leukemic cells. These results were consistent with the GEP data. 2) Leukemic cells alter MSC Signaling proteins in vitro: Western blotting showed that Stat3, Akt, PTEN, CXCL5 and HIF-1α were up- regulated in MSC co-cultured with p53−/− leukemic cells as compared to p53wt leukemic cells (48 hrs). Additional analyses showed that the downstream targets of HIF-1α, VEGFa and VEGFc, but not VEGFb, were up-regulated. Taken together, these results suggest that 1) leukemic cells with different p53 genetic background co-cultured with normal MSC have profoundly differential effects on GEP of normal MSC; 2) MSC co-cultured with p53−/− leukemic cells resulted in increased levels of onco-proteins such as Akt and HIF-1α when compared to MSC co-cultured with p53wt leukemic cells. Results suggest, for the first time, that the genetics of leukemic cells determines gene expression in co-cultured MSC. In vivo experiments are in progress to provide in vivo evidence for the existence of a novel model of leukemia-stroma interactions where the genetics of the tumor cell impacts stromal cell biology. Disclosures: No relevant conflicts of interest to declare.

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Brune, Jan Claas, Ariane Tormin, Ulrike Bauer, Ulf Neumann, Pehr Rissler, Otte Brosjö, Richard Löfvenberg et al. « Mesenchymal Stromal Cells (MSC) Isolated from Human Osteosarcomas Show a High Progenitor Cell Frequency, Typical MSC Morphology, Surface Marker Profile, and Differentiation Capacity, and They Are Considerably Affected by Tyrosine Kinase Inhibitors in Vitro. » Blood 112, n^o 11 (16 novembre 2008) : 1360. http://dx.doi.org/10.1182/blood.v112.11.1360.1360.

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Abstract Bone-marrow derived mesenchymal stromal cells (MSC, also referred to as mesenchymal stem cells) are multipotent cells with intriguing properties (proliferation and differentiation capacity, stroma function, immune modulation) making them promising candidates for clinical use. Recently, it has been reported that culture-derived MSC can acquire chromosomal abnormalities, and abnormal culture-derived murine MSC have been found to give rise to osteosarcomas in transplantation experiments. Furthermore, MSC have been implicated in osteogenic sarcoma, and we therefore aimed to isolate and characterize MSC from primary human osteosarcomas (OS). Single cell suspensions were prepared from OS specimens and cells could be clearly identified morphologically as tumor cells. Stromal progenitor content was assessed using the CFU-F assay. CFU-F frequencies in the OS specimens were 980 ± 450 colonies per 1 × 105 cells (n=6), which is considerably higher than in normal bone marrow (1.3 ± 0.2 colonies per 1 × 105 cells, n=8, bone marrow from healthy volunteer donors). Culture-derived MSC could be generated from every OS sample tested using standard tissue culture flasks and serum-containing MSC medium. OS-derived MSC were similar to bone marrow (BM) derived MSC showing typical MSC morphology and typical MSC surface marker profile, i.e. cells were positive for CD105, CD73, CD90, CD44, HLA-class I, CD166, and negative for CD45, CD34, CD14, CD19, HLA-DR, and CD31. Furthermore, three of three tested OS-derived MSC samples could be differentiated into the osteoblastic and chondroblastic lineages, and all but one showed adipocytic differentiation capacity. Karyotyping of OS-derived MSC showed that the majority of MSC samples were normal, as were the results of FISH analyses for chromosomes 7, 10, 13, and 17. MSC derived from one OS specimen contained cells that showed a complex abnormal karyotype, which, however, was different from the karyotype of the primary tumor. BM- and OS-derived MSC cultures exposed to the first and second generation tyrosine-kinase inhibitors (TKI) imatinib (IM) and nilotinib (NI) (0 – 10 μM) showed a significant inhibition of MSC growth at every time point studied (0 – 4 weeks). After 4 weeks, BM-derived MSC were reduced by IM to 31.4 ± 12.0% (0.625 μM), 24.6 ± 9.1% (1.25 μM), 23.8 ± 12.3% (2.5 μM), 10.6 ± 0.9% (5.0 μM), and 0.9 ± 0.1% (10.0 μM) compared to controls. The lowest numbers of OS-MSC were observed after 2 weeks exposure to IM (0.625 μM, 6.9 ± 4.6%; 1.25 μM, 8.5 ± 4.5%;2.5 μM, 10.3 ± 6.4%; 5.0 μM, 9.2 ± 4.9%; 10 μM, 0.6 ± 0.1%). BM-MSC and OS-MSC growth was also considerably inhibited by NI, but the effects were less pronounced for most doses and time points studied; minimum numbers of BM-MSC (0.8 ± 0.2% of control) and OS-MSC (0.6 ± 0.1% of control) were observed after 3 weeks culture with 10 μM NI. Taken together, our results show that osteosarcoma samples contain high numbers of mesenchymal progenitor cells and that MSC can be successfully generated from OS. OS-MSC are likely to represent stromal cells, i.e. cancer-associated fibroblasts, rather than the tumor cell population. Interestingly, strong similarities were observed between OSMSC and BM-MSC, implicating that these two cell types are closely related.

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Menendez, Pablo, Purificación Catalina, René Rodríguez, Gustavo J. Melen, Clara Bueno, Mar Arriero, Félix García-Sánchez, Alvaro Lassaletta, Ramón García-Sanz et Javier García-Castro. « Bone marrow mesenchymal stem cells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4 fusion gene ». Journal of Experimental Medicine 206, n^o 13 (7 décembre 2009) : 3131–41. http://dx.doi.org/10.1084/jem.20091050.

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MLL-AF4 fusion is a hallmark genetic abnormality in infant B-acute lymphoblastic leukemia (B-ALL) known to arise in utero. The cellular origin of leukemic fusion genes during human development is difficult to ascertain. The bone marrow (BM) microenvironment plays an important role in the pathogenesis of several hematological malignances. BM mesenchymal stem cells (BM-MSC) from 38 children diagnosed with cytogenetically different acute leukemias were screened for leukemic fusion genes. Fusion genes were absent in BM-MSCs of childhood leukemias carrying TEL-AML1, BCR-ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. However, MLL-AF4 was detected and expressed in BM-MSCs from all cases of MLL-AF4+ B-ALL. Unlike leukemic blasts, MLL-AF4+ BM-MSCs did not display monoclonal Ig gene rearrangements. Endogenous or ectopic expression of MLL-AF4 exerted no effect on MSC culture homeostasis. These findings suggest that MSCs may be in part tumor-related, highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4+ B-ALL. MLL-AF4 itself is not sufficient for MSC transformation and the expression of MLL-AF4 in MSCs is compatible with a mesenchymal phenotype, suggesting a differential impact in the hematopoietic system and mesenchyme. The absence of monoclonal rearrangements in MLL-AF4+ BM-MSCs precludes the possibility of cellular plasticity or de-differentiation of B-ALL blasts and suggests that MLL-AF4 might arise in a population of prehematopoietic precursors.

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Greiner, Jochen, Jan Torzewski, Peter Ponsaerts, Markus T. Rojewski, Desiree Kronawitter, Hubert Schrezenmeier, Vinzenz Hombach et al. « Highly Efficient mRNA- and cDNA-Based Transient Gene Delivery into Human Progenitor Cells. » Blood 108, n^o 11 (16 novembre 2006) : 5471. http://dx.doi.org/10.1182/blood.v108.11.5471.5471.

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Abstract The method of gene transfer into progenitor cells is critical as viral vector transduction involves the risk of tumor induction by non-specific genomic integration. Non-viral transfection systems often fail due to low transfection efficiency. However, gene transfer into human CD34+ hematopoietic progenitor (HPC) and mesenchymal stem cells (MSC) is an essential tool for in vitro- and in vivo-applications and therapeutic strategies such as tissue engineering and gene therapy. We recently reported an transient genetic labelling of human CD34+ HPC with deltaLNGFR-plasmid-DNA for in vivo application: Transient transfection was efficient for both, CD34+ HSC (41% ± 2%) and leukemia cell lines (55% ± 4.9%) using the method of nucleofection. Moreover, mature myeloid cells (CD66b+) derived from transfected human CD34+ HPC and leukemia cells maintained deltaLNGFR expression at a high percentage (70% ± 1.6% and 58% ± 2% respectively). In this work, we investigated labelling of CD34+ HPC with mRNA. Human CD34+ HPC and human MSC were transfected with in vitro-transcribed mRNA for deltaLNGFR, a marker gene approved for human in vivo-application, using nucleofection. EGFP was used as a control. 24h after nucleofection, FACS-analysis showed a higher transfection efficiency compared to plasmid transfected CD34+ HPC and MSC: A high transfection frequency was found for mRNA-transfected HPCs using deltaLNGFR (82.4±9.7%) and EGFP (88.7±2.6%). We found also a high transfection rate for MSC using the marker genes deltaLNGFR (92.4±3.6%) and EGFP (83.3±4.1%). Cell viability was not affected by mRNA-transfection. Moreover, differentiation assays of deltaLNGFR-selected MSC after transfection, showed that differentiation of MSC into mesenchymal cells like chondrocytes, adipocytes and osteoblasts was not affected by mRNA nucleofection. Taken together, mRNA based nucleofection is a powerful, highly efficient and non-toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may thus be useful to transiently manipulate stem cell characteristics and combine principles of gene therapy and tissue engineering.

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Djouad, Farida, Pascale Plence, Claire Bony, Philippe Tropel, Florence Apparailly, Jacques Sany, Danièle Noël et Christian Jorgensen. « Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals ». Blood 102, n^o 10 (15 novembre 2003) : 3837–44. http://dx.doi.org/10.1182/blood-2003-04-1193.

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Abstract Mesenchymal stem cells (MSCs) are largely studied for their potential clinical use. Recently, they have gained further interest after demonstration of an immunosuppressive role. In this study, we investigated whether in vivo injection of MSCs could display side effects related to systemic immunosuppression favoring tumor growth. We first showed in vitro that the murine C3H10T1/2 (C3) MSC line and primary MSCs exhibit immunosuppressive properties in mixed lymphocyte reaction. We demonstrated that this effect is mediated by soluble factors, secreted only on “activation” of MSCs in the presence of splenocytes. Moreover, the immunosuppression is mediated by CD8+ regulatory cells responsible for the inhibition of allogeneic lymphocyte proliferation. We then demonstrated that the C3 MSCs expressing the human bone morphogenetic protein 2 (hBMP-2) differentiation factor were not rejected when implanted in various allogeneic immunocompetent mice and were still able to differentiate into bone. Importantly, using a murine melanoma tumor model, we showed that the subcutaneous injection of B16 melanoma cells led to tumor growth in allogeneic recipients only when MSCs were coinjected. Although the potential side effects of immunosuppression induced by MSCs have to be considered in further clinical studies, the usefulness of MSCs for various therapeutic applications still remains of great interest. (Blood. 2003;102:3837-3844)

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Tolar, Jakub, Mark J. Osborn, Angela Panoskaltsis-Mortari, Ron T. McElmurry, Scott Bell, Lily Xia, Megan Riddle et al. « Mesenchymal Cancer Cells Can Arise from Ex Vivo Modified Mesenchymal Stem Cells. » Blood 106, n^o 11 (16 novembre 2005) : 4326. http://dx.doi.org/10.1182/blood.v106.11.4326.4326.

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Abstract Mesenchymal stem cells (MSCs) can differentiate into non-hematopoietic cell types, including adipocytes, chondrocytes and osteocytes. MSCs have been isolated from multiple species, including humans, and multiple organs, including bone marrow, adipose tissue and umbilical cord blood. The beneficial effects of MSCs are being tested clinically in attempts to: improve hematopoietic engraftment, to treat osteogenesis imperfecta, graft-versus-host disease and autoimmune diseases, and as antitumor agents to deliver therapy for malignancies. Phase I clinical studies have not been associated with toxicities. We aimed to investigate the capacity of MSCs to aid in tissue healing after radiation induced injury in irradiated bone marrow transplant (BMT) recipients. To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and co-infused them with allogeneic bone marrow into irradiated reipients. Using in vivo whole body bioluminenscent imaging luciferase signals were shown to be increased between weeks 3 and 12 indicating expansion of MSCs. Unexpectedly, some mice (N=8/17) with the highest luciferase signals died and all surviving mice (N=9/17) developed foci of ectopic ossification in lungs. Two of mice also developed osteosarcomas in their extremities. This prompted us to characterize the transformed MSCs that originated from the donor MSCs. The transformed cells were aneuploid, lost their capacity to differentiate into mesenchyme-derived adipocytes and chondrocytes, and histologically identified as osteosarcomas. In addition, infusion of tumor cells resulted in malignant lesions in secondary recipients. Mapping of transposition sites in the genome and karyotype analysis indicated that the critical transformation event(s) occurred before infusion of the MSCs. Even though we have not encountered a transformation event in >100 mice infused with MSC manipulated with transposons, we speculated that mutation by transposition was the inciting event. None of the identifiable transposition events occurred in a known proto-oncogene or tumor suppressor gene. This does not discount the possibility of insertional mutagenesis as the genomic lesion may have occurred on the chromosome which was subsequently disrupted or lost. Alternatively, genomic instability could have been a result of spontaneous unrepaired chromosomal lesion(s) that preceded the transposon insertion and resulted in osteosarcoma. These findings provide evidence of evolution of MSCs with osteogenic capacity into osteosarcoma in vivo and are clinically relevant as they document the potential of ex vivo manipulated MSCs for transformation into malignant disease.

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Iida, Yuichi, Rintaro Yoshikawa, Akihiko Murata, Hitoshi Kotani, Yasuhiro Kazuki, Mitsuo Oshimura, Yumi Matsuzaki et Mamoru Harada. « Local injection of CCL19-expressing mesenchymal stem cells augments the therapeutic efficacy of anti-PD-L1 antibody by promoting infiltration of immune cells ». Journal for ImmunoTherapy of Cancer 8, n^o 2 (juillet 2020) : e000582. http://dx.doi.org/10.1136/jitc-2020-000582.

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BackgroundMesenchymal stem/stromal cells (MSC) accumulate and reside in tumor sites.MethodsTaking advantage of this feature in anticancer therapy, immortalized murine MSC (iMSC) were genetically altered to produce chemokine (C-C motif) ligand 19 (iMSC/CCL19), which attracts dendritic cells (DC) and T lymphocytes. Thereafter, iMSC/CCL19 were examined for their therapeutic efficacy using a syngeneic CT26 colon carcinoma cell line.ResultsCo-injection of iMSC/CCL19 into mice significantly suppressed the in vivo growth of CT26 cells compared with that of CCL19-expressing immortalized fibroblasts (iFib/CCL19). This anticancer effect was not observed when injected in CT26-bearing nude mice. Co-injected iMSC/CCL19 survived longer than iFib/CCL19 in the tumor sites. In a therapeutic model, local injection of iMSC/CCL19 suppressed the tumor growth, and increased IFN (interferon)-γ+ CD8+ T cells and CCR7+ DC infiltration in tumor site was observed when treated with iMSC/CCL19, but not with iMSC. This antitumor effect was completely negated by depletion of CD4+ cells and partially negated by depletion of CD8+ cells. Furthermore, the antitumor effects induced by local injection of iMSC/CCL19 were augmented by additional therapy with anti-programmed death (PD)-ligand 1 (PD-L1) antibody, but not with anti-PD-1 antibody. This combination therapy cured most of the tumors in CT26-bearing mice.ConclusionThese results suggest that local therapy with iMSC/CCL19 can suppress tumor growth via effective recruitment of CCR7+ DC into tumor sites and increase IFN-γ+ CD8+ T cells, and that combination with anti-PD-L1 antibody therapy can be a powerful anticancer therapy.

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Laura, Desbourdes M., Suheyla Hasgur, Adam J. Guess, Minjun Yu, Satoru Otsuru et Edwin M. Horwitz. « Combination of Interferon α, Delivered By Engineered Mesenchymal Stromal Cells, and Cytarabine Limits the Development of Acute Myeloid Leukemia, Potentially Targeting Leukemic Stem Cells ». Blood 134, Supplement_1 (13 novembre 2019) : 5143. http://dx.doi.org/10.1182/blood-2019-127456.

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Background: While most acute myeloid leukemia (AML) patients achieve remission, approximately 50% will relapse which is generally attributed to the persistence of leukemic stem cells (LSCs). Indeed, those cells, regarded as the origin of leukemia, are chemoresistant. The Side Population (SP) cells of AML are known to be enriched in LSCs and to be more resistant to chemotherapy compared to non-SP AML cells. So, they are good candidate to target in the establishment of new protocol of treatment. Interestingly, it has been demonstrated that bone marrow mesenchymal stem/stromal cells (MSC) nesting promotes the proportion of AML SP cells (Malfuson et al., 2013). Aims: In this proof-of-concept study, we developed a two-step treatment based on the use of MSCs to activate the SP AML cells and of cytarabine, commonly used to treat AML, to eliminate them. However, as MSCs are suspected to stimulate leukemic growth and to protect against chemotherapies, MSCs have been engineered to deliver locally IFNα directly to an AML chloroma to enhance the anti-proliferative effect of cytarabine and to overcome the potential MSC pro-tumoral effect. Methods: Human MSCs were isolated and transduced with a lentiviral vector expressing human IFNα (IFNα MSCs) or a control vector (Ctrl MSCs). Flow cytometric analysis and ELISA confirmed the secretion of bio-active IFNα by transduced MSCs. The anti-AML activity of these MSCs was assessed by co-culture with the KG1a AML cell line. The in vivo validation was performed by subcutaneous injection of KG1a cells (chloroma model) with or without Ctrl MSCs or IFNα MSCs in NSG mice and monitoring of tumor volume. Results: MSC presence doubled the SP phenotype among AML cells in the co-culture system as expected. The IFNa MSCs significantly decreased the leukemic population by 30% by inducing more early and late apoptosis. The in vivo experiment confirmed this anti-leukemic activity with a lower tumor growth characterized by a tumor volume decreased by 40% compared to no MSC and Ctrl MSC conditions after 40 days post-injection. The effect of cytarabine on the IFNα MSC action was explored by the addition of the drug to the co-culture system or in the chloroma model. Thus, we showed that cytarabine treatment reduced by 7-fold leukemic SP cell proportion in regular culture of AML cells and by 26-fold in the IFNα MSC co-cultures demonstrating that MSC induced SP cells are more sensitive to the drug than basal SP cells. Combination of cytarabine with IFNa secreted by the modified MSCs enhances the anti-leukemic activity of those MSCs in vitro with clear additive effects on AML cell number reduction and early and late apoptosis induction, and with a synergistic effect on cell cycle arrest. The beneficial effect of using IFNa MSCs in addition to cytarabine was demonstrated in vivo with a 40% tumor volume reduction after 40 days post-injection and a longer survival compared to cytarabine or IFNa MSCs alone. Summary/conclusions: This study showed that IFNα MSCs in combination with cytarabine present a strong anti-leukemic effect, decreasing the leukemic burden and potentially leukemic stem cell pool. This new two-step therapeutic protocol represents a promising alternative for AML treatment. Disclosures No relevant conflicts of interest to declare.

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Platas, Julia, Maria Isabel Guillén, María Dolores Pérez del Caz, Francisco Gomar, Vicente Mirabet et Maria José Alcaraz. « Conditioned Media from Adipose-Tissue-Derived Mesenchymal Stem Cells Downregulate Degradative Mediators Induced by Interleukin-1βin Osteoarthritic Chondrocytes ». Mediators of Inflammation 2013 (2013) : 1–10. http://dx.doi.org/10.1155/2013/357014.

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Osteoarthritis (OA) is the most frequent joint disorder and an important cause of disability. Recent studies have shown the potential of adipose-tissue-derived mesenchymal stem cells (AD-MSC) for cartilage repair. We have investigated whether conditioned medium from AD-MSC (CM) may regulate in OA chondrocytes a number of key mediators involved in cartilage degeneration. CM enhanced type II collagen expression in OA chondrocytes while decreasing matrix metalloproteinase (MMP) activity in cell supernatants as well as the levels of MMP-3 and MMP-13 proteins and mRNA in OA chondrocytes stimulated with interleukin- (IL-) 1β. In addition, CM increased IL-10 levels and counteracted the stimulating effects of IL-1βon the production of tumor necrosis factor-α, IL-6, prostaglandin E2, and NO measured as nitrite and the mRNA expression of these cytokines, CCL-2, CCL-3, CCL-4, CCL-5, CCL-8, CCL-19, CCL-20, CXCL-1, CXCL-2, CXCL-3, CXCL-5, CXCL-8, cyclooxygenase-2, microsomal prostaglandin E synthase-1, and inducible NO synthase. These effects may be dependent on the inhibition of nuclear factor-κB activation by CM. Our data demonstrate the chondroprotective actions of CM and provide support for further studies of this approach in joint disease.

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Luo, Xiaohe, Shan Huang, Ningning He, Chen Liu, Yanan Chen, Yanhua Liu, Xue Mi et al. « Inflammatory Human Umbilical Cord-Derived Mesenchymal Stem Cells Promote Stem Cell-Like Characteristics of Cancer Cells in an IL-1β-Dependent Manner ». BioMed Research International 2018 (2018) : 1–12. http://dx.doi.org/10.1155/2018/7096707.

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To ensure the safety of clinical applications of MSCs, thorough understanding of their impacts on tumor initiation and progression is essential. Here, to further explore the complex dialog between MSCs and tumor cells, umbilical cord-derived mesenchymal stem cells (UC-MSCs) were employed to be cocultured with either breast or ovarian cancer cells. Though having no obvious influence on proliferation or apoptosis, UC-MSCs exerted intense stem cell-like properties promoting effects on both cancer models. Cocultured cancer cells showed enriched side population, enhanced sphere formation ability, and upregulated pluripotency-associated stem cell markers. Human cytokine array and real-time PCR revealed a panel of MSC-derived prostemness cytokines CCL2, CXCL1, IL-8, and IL-6 which were induced upon coculturing. We further revealed IL-1β, a well-characterized proinflammatory cytokine, to be the inducer of these prostemness cytokines, which was generated from inflammatory UC-MSCs in an autocrine manner. Additionally, with introduction of IL-1RA (an IL-1 receptor antagonist) into the coculturing system, the stem cell-like characteristics promoting effects of inflammatory UC-MSCs were partially blocked. Taken together, these findings suggest that transduced inflammatory MSCs work as a major source of IL-1β in tumor microenvironment and initiate the formation of prostemness niche via regulating their secretome in an IL-1β-dependent manner.

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Ding, Wei, Traci R. Knox, Justin C. Boysen et Neil E. Kay. « Platelet-Derived Growth Factor (PDGF) Secreted by Chronic Lymphocytic Leukemic B-Cells Is Capable of Regulating the Activation and Function of Mesenchymal Stem Cells : Implications for Leukemic Cell/Stromal Cell Crosstalk ». Blood 112, n^o 11 (16 novembre 2008) : 355. http://dx.doi.org/10.1182/blood.v112.11.355.355.

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Abstract Background: It is believed that malignant cells can “condition” microenvironment to facilitate tumor cell survival. Our previous finding demonstrated that the culture of chronic lymphocytic leukemic (CLL) B-cells and marrow-derived mesenchymal stem cells (MSC) impacts both cell types bi-directionally (ASH 2007, Blood, 110: 337). Thus, MSC are capable of promoting CLL B-cell activation and proliferation while soluble factors secreted from CLL B cells were found to induce MSC activation in terms of both Erk and Akt activation. However, the exact mechanism and extent of this leukemic cell-MSC crosstalk has not been explored yet in CLL. Methods: To investigate if factors secreted from the CLL B-cells can modulate the migration and activation of marrow derived MSC, the conditioned medium (CM) of CLL B-cells was generated by collecting the supernatant of freshly isolated PBMC from CLL patients cultured for approximately 4 days at a concentration of 5 × 106 per ml in AIM-V medium. The migration and proliferation capacities of MSC were measured when they were cultured with or without the CM of CLL cells. Subsequently, the membrane receptors activated in MSC after 30 minutes of exposure to the CM of CLL cells were tested using a receptor tyrosine kinase array assay. In addition, we studied the downstream signal pathways of MSC by immunoblot approaches pre and post exposure to CLL CM and in some experiments we added specific, commercially available inhibitors for individual signaling pathways of interest. ELISA was used to measure angiogenic cytokines including PDGF, VEGF, BFGF and TSP-1 levels from CLL plasma as well as from CM of CLL B cells. Results: To test if CLL B-cells were capable of secreting soluble factors that activate or signal MSC, we examined the migration and proliferation capacities of CLL-MSC when they were exposed to CM generated from cultured CLL B-cells compared to AIM-V medium. Bone marrow MSC of CLL patients exhibited an increased migration as measured by a modified Boyden chamber assay (n = 4, mean increase of migration: 15%, p = 0.01) and proliferation as measured by direct cell counting with trypan blue staining (n =4, mean increase of total cell levels: 3.2 fold, p = 0.02) when cultured with CM of CLL B-cells compared to AIM-V medium. Using a receptor tyrosine kinase array assay (R&D system), we found that the sole growth factor receptor activated on CLL –MSC was PDGFRα when MSC were exposed to CM of cultured CLL B-cells for 30 minutes. This finding was further confirmed by demonstrating that PDGFRα was phosphorylated by immunoprecipitating MSC lysates with anti-PDGFRα followed by immunoblot with anti-phosphotyrosine antibody. We subsequently found that both PDGFRα and Akt were activated within 10 minutes of exposure by CM of CLL B-cells. When CLL-MSC were pretreated with a PDGFR inhibitor (PDGFR tyrosine kinase inhibitor III, Calbiochem), an inhibitor known to block the ATP binding site of PDGFR, neither PDGFRα nor Akt activation was detectable when MSC were exposed to CM from CLL B-cells. These results imply that Akt is likely activated downstream of the PDGFR signal pathway in CLL-MSC. We next found that PDGF was secreted by CLL B-cells by detecting its presence using ELISA in both the CM (n = 12,113.7 ± 23.6 pg/ml, mean ± sem) and plasma of CLL patients (n = 21, 3296.7 ± 800.1 pg/ml). Since we have previously found that a switch in pro- vs. anti-angiogenic cytokines can occur when CLL B cells are added to MSC, we tested if PDGF can upregulate MSC angiogenic cytokine levels. When PDGF (5ng/ml, R&D system) was introduced to the CLL-MSC, we found that VEGF production, but not TSP-1 or BFGF significantly increased over control levels (n = 3, mean increase: 3.3 fold, p = 0.05) in the CM of the MSC harvested after a 72 h culture period. Conclusions: These results indicate that CLL B-cells are capable of activating MSC function by increasing their migration and proliferation capacity likely via signaling through the PDGF receptor. The PDGF activated MSC results in downstream Akt activation and the increased secretion of VEGF, a cytokine known to enhance CLL B-cellsurvival and drug resistance. Further interrogation of the mechanism(s) that regulate the interaction between leukemic CLL B cells and stromal cells should yield important information that can be used for therapeutic strategies in CLL.

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Krampera, Mauro, Annalisa Pasini, Antonella Rigo, Maria Teresa Scupoli, Cristina Tecchio, Giorgio Malpeli, Aldo Scarpa, Francesco Dazzi, Giovanni Pizzolo et Fabrizio Vinante. « HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells : inducing cell expansion and reversibly preventing multilineage differentiation ». Blood 106, n^o 1 (1 juillet 2005) : 59–66. http://dx.doi.org/10.1182/blood-2004-09-3645.

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Epidermal growth factor receptor-1 (EGFR-1/HER-1/ErbB-1) regulates proliferation and cell fate during epidermal development. HER-1 is activated by several EGF-family ligands including heparin-binding epidermal growth factor–like growth factor (HB-EGF), a mitogenic and chemotactic molecule that participates in tissue repair, tumor growth, and other tissue-modeling phenomena, such as angiogenesis and fibrogenesis. We found that mesenchymal stem cells (MSCs), the precursors of different mesenchymal tissues with a role in processes in which HB-EGF is often involved, normally express HER-1, but not HB-EGF itself. Under the effect of HB-EGF, MSCs proliferate more rapidly and persistently, without undergoing spontaneous differentiation. This effect occurs in a dose-dependent fashion, and is specific, direct, and HER-1 mediated, as it is inhibited by anti–HER-1 and anti–HB-EGF blocking antibodies. Moreover, HB-EGF reversibly prevents adipogenic, osteogenic, and chondrogenic differentiation induced with specific media. These data show that HB-EGF/HER-1 signaling is relevant to MSC biology, by regulating both proliferation and differentiation.

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Nityanand, Soniya, Naresh Kumar Tripathy, Chandra Prakash Chaturvedi, Ekta Minocha, Akhilesh Sharma et Khaliqur Rahman. « Altered Expression of Hematopoiesis Regulatory Genes in the Bone Marrow Mesenchymal Stem Cells of Patients of Aplastic Anemia ». Blood 128, n^o 22 (2 décembre 2016) : 3904. http://dx.doi.org/10.1182/blood.v128.22.3904.3904.

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Abstract Mesenchymal stem cells (MSC) are an important component of the hematopoietic niche in the bone marrow (BM) and regulate hematopoiesis by producing a variety of cytokines and growth factors. In aplastic anemia (AA), most of the studies have attributed the reduced hematopoiesis to a defect in hematopoietic stem cells (HSC) and limited data is available on the role of BM-MSC in AA. Therefore, the objective of the present study was to evaluate the expression of hematopoiesis regulatory genes, viz. granulocyte colony stimulating factor (G-CSF), stromal cell derived factor (SDF-1), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-α) macrophage inflammatory protein-1 alpha (MIP-1α) and transforming growth factor-beta (TGF-β) in BM-MSC of patients with AA and compare it with BM-MSC of control group. Twenty patients of idiopathic acquired AA with a median age of 25.5 years (range: 12-64 years) were included in the study. The control group consisted of 10 healthy volunteers and 10 patients with iron deficiency anemia or immune thrombocytopenic purpura. The median age of the control group was 20 years (range: 11-62 years). The BM-MSC were isolated and cultured as per protocol standardized and previously published by us. Third passage cells were used in the study. The MSC were characterized both by their phenotypic markers and by their ability to differentiate into adipogenic and osteogenic lineages. The expression of hematopoiesis regulatory genes was studied by real-time quantitative polymerase chain reaction (qRT-PCR). The GAPDH was used as the housekeeping gene to normalize the transcript levels and the fold change in the gene expression was calculated by 2-ΔΔCtmethod. The BM-MSC of AA patients and controls had similar morphology and expression of mesenchymal markers CD73, CD105, CD90 and CD166, absence of expression of hematopoietic markers CD13, CD34 and CD45 and of HLA-DR. However, the BM-MSC of AA patients exhibited a higher adipogenic and a lower osteogenic differentiation in comparison to those of controls. Further, the BM-MSC of AA patients in comparison to those of control group, had a higher expression of G-CSF (fold increase: 1.99; p<0.0001), SDF-1 (fold increase: 1.37; p<0.01) and TNF-α (fold increase: 10.68; p<0.0001) and a very low expression of MIP-1α (fold decease: 50.0; p<0.0001) transcripts. The expression of SCF and TGF-β transcripts were comparable in the BM-MSC of both the groups (p>0.05). Though AA patients have been shown to have elevated levels of G-CSF in the peripheral blood and BM but there is only one previous report on G-CSF gene expression in BM-MSC of AA, in which a higher expression was observed and thus corroborates with our data. There is no data available on SDF-1 levels in the peripheral blood and bone marrow of AA patients. We have observed higher gene expression of SDF-1 in BM-MSC of AA patients. The higher expression of G-CSF and SDF-1, pro-hematopoietic factors, in AA may be due to a compensatory response of the BM stroma to boost the hematopoiesis. Our observation of higher TNF-α gene expression in BM-MSC corroborates with previous reports on higher levels of this anti-hematopoietic cytokine in the BM plasma of patients with AA and indicates that MSC could contribute to the increase in the TNF-α level in the BM of AA patients. A conspicuous observation of our study was a markedly decreased expression of MIP-1α gene in BM-MSC of AA and to the best of our knowledge this is the first report on MIP-1α in AA. MIP-1α is a chemokine which has been shown to inhibit proliferation of HSC in vitro and thus may help to maintain HSC in an undifferentiated state. Furthermore, MIP-1α has also been reported to mediate interaction of HSC with stromal cells in BM and may have a role in supporting hematopoiesis. Its precise role in AA needs to be studied further. We are currently studying the levels of these cytokines/growth factors in the BM plasma of the same cohort of AA patients and controls and the data will be presented. Our study thus shows that BM-MSC of AA patients have altered expression of hematopoiesis regulatory genes which may contribute to the pathobiology of the disease. Disclosures Nityanand: Sanjay Gandhi Post Graduate Institute of Medical Sciences: Employment, Research Funding. Tripathy:Sanjay Gandhi Post Graduate Institute of Medical Sciences: Employment. Chaturvedi:Dept of Biotechnology, Govt of India: Employment. Minocha:Dept of Science and Technology, Govt of India: Other: PhD scholarship. Sharma:Sanjay Gandhi Post Graduate Institute of Medical Sciences: Employment. Rahman:SGPGI, Lucknow , India: Employment, Research Funding.

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Uchibori, Ryosuke, Takashi Okada, Takayuki Ito, Masashi Urabe, Hiroaki Mizukami, Akihiro Kume et Keiya Ozawa. « Retroviral Vector-Producing Mesenchymal Stem Cells for Tumor Tracking and Therapeutic Gene Amplification in Suicide Cancer Gene Therapy. » Blood 110, n^o 11 (16 novembre 2007) : 1917. http://dx.doi.org/10.1182/blood.v110.11.1917.1917.

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Abstract Mesenchymal stem cells (MSCs) are known to have a tendency to accumulate at the site of tumors, and therefore can be utilized as a platform for targeted delivery of anti-cancer agents. The MSC-based targeted cancer gene therapy can enhance the therapeutic efficacy, because MSCs are considered to reach tumors including metastatic lesions and to deliver therapeutic molecules in a concentrated fashion. This targeted therapy can also reduce systemic adverse side effects, because the anti-cancer agents act locally at the site of tumors without elevating their systemic concentrations. In the present study, we developed genetically-modified MSCs that produce retroviral vectors encoding HSVtk, aiming at augmenting therapeutic efficacy of systemic suicide cancer gene therapy. The tumor tropism and anti-tumor effects of vector-producing MSCs (VP-MSCs) were examined by intravascular injection in tumor-bearing nude mice. MSCs isolated from the bone marrow of SD rats were transfected with plasmid DNA expressing luciferase alone (=non-VP-MSCs) or whole retroviral vector components (LTR-Luc or LTR-HSVtk with Gag-pol and VSV-G) (=VP-MSCs) by nucleofection. To assess tumor tropism of MSCs, nude mice were subcutaneously inoculated with 9L rat glioma cells or Rat-1 fibroblasts, and were subsequently injected with luciferase-expressing MSCs through the left ventricular cavity. The transgene expression was periodically traced by using an in vivo imaging system. As a result, the transgene expression accumulated at the site of subcutaneous 9L tumors, but undetectable at the site of Rat-1 fibroblasts. In addition, the injection of luciferase-expressing VP-MSCs caused much stronger signal of bioluminescence at the site of 9L tumors compared with luciferease-expressing non-VP-MSCs. Immunostaining study showed that luciferase-positive cells (injected MSCs and transduced glioma cells) were detected at the periphery of tumors. To evaluate the therapeutic efficacy, tumor-bearing nude mice were treated with non-VP-MSCs or VP-MSCs combined with HSVtk/GCV system and then the size of subcutaneous tumors was periodically measured. In this model experiments, tumor growth was more efficiently suppressed by injecting VP-MSCs compared with non-VP-MSCs. The present study suggests the effectiveness of VP-MSCs in suicide cancer gene therapy. The therapeutic benefit of this strategy should be further examined in orthotopic and metastatic tumor models.

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Chulpanova, Daria, Valeriya Soloveva, Leisan G. Tazetdinova, Marina O. Gomzikova, Svetlana Khaiboullina et Albert Rizvanov. « Analysis of the Effect of Mesenchymal Stem Cells Culture with Interleukin 2 Overexpression on Human Mononuclear Cells Activation in Vitro ». Blood 132, Supplement 1 (29 novembre 2018) : 3712. http://dx.doi.org/10.1182/blood-2018-99-111228.

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Abstract Introduction. Cell and gene therapy are emerging novel approaches for cancer treatment. Mesenchymal stem cells (MSCs) are non-hematopoietic progenitor cells, which can be isolated from different types of adult tissues. Due to their tropism to the tumor site, MSCs could be used to deliver various antitumor agents, including cytokines, which could prevent and delay metastases. Interleukin-2 (IL-2) is an immunomodulating cytokine, which regulates the activities of white blood cells. This cytokine is often used as immunomodulatory in cancer treatment. In this study we investigated whether expression of IL-2 in MSCs will affect the expression of key cancer-related genes. Methods. Human MSCs were isolated from adipose tissue and transduced with recombinant lentiviruses encoding human IL-2 gene. The IL-2 expressing cells were selected using blasticidin S (5 μg/ml) for 10 days. The IL-2 gene expression was confirmed by quantitative PCR. The effect of the conditioned medium (CM) from native MSCs or MSC-IL2 on peripheral blood mononuclear cells (PBMCs) activation was investigated. CM was harvested 72 hours after culture PBMCs in MSC-IL2 CM. CM from native MSCs or fresh DMEM/F12 were used as control. PBMC were collected and used for flow cytometry analysis. Results. We found that 6.3±0.4% of PBMCs cultured in the presence of MSC-IL2 CM were positive for CD3/HLA-DR, typical for late T-cell activation. The percentage of CD3/HLA-DR-positive PBMCs cultured in the presence of CM from native MSCs and DMEM/F12 medium was half as much, 2.4±0.3% and 2.8±0.6%, respectively (Fig. 1A). We have shown that 3.0±0.3% of PBMCs cultured in the presence of CM from MSC-IL2 were positive for CD3/CD56 markers, typical for a population of TNK cells with both T-cell and NK-cell functions. The percentage of CD3/CD56-positive PBMCs cultured in the presence of CM from native MSCs and DMEM/F12 medium was 1.3±0.5% and 1.6±0.4%, respectively (Fig. 1B). We observed an increased number of NK cells after culturing in the presence of MSC-IL2 CM with 8.8±0.5% of PBMCs were CD56-positive and CD3-negative. The percentage of CD56+/CD3- PBMCs cultured in the presence of CM from native MSCs and DMEM/F12 was about two fold less, 4.3±0.7% and 3.1±0.3% respectively (Fig. 1C). Number of CD3+/CD25+ cells, characteristic of early activation of T-cell, was 7.1±0.3% when PBMCs were cultured in the presence of MSC-IL2 CM. The percentage of CD3/CD25-positive PBMCs cultured in the presence of CM from native MSCs and DMEM/F12 medium was higher, 6.9±0.4% and 8.0±0.5%, respectively (Fig. 1D). This could be explained by prolonged cultivation of the samples (72 hours). Analysis of activated T-killer population, CD8+ and CD38+, revealed that 33.4±1.4% of PBMCs cultured in the presence of MSC-IL2 CM were CD8+/CD38+. The percentage of CD8+/CD38+ PBMCs cultured in the presence of CM from native MSCs and DMEM/F12 was about two fold less, 13.7±1.8% and 12.4±1.2%, respectively (Fig. 1E). Conclusion. Conditioned medium from MSC expressing IL-2 increased number of key leukocyte populations, such as late-activated T-cells, NK cells, and activated T killers. The ability of IL-2 to activate cells of the immune system, as well as the natural tropism of MSCs to tumor sites, could make IL-2 expressing MSCs useful gene delivery tool for cancer immune therapy. This study was supported by Russian Science Foundation grant 18-74-10044 and Program of Competitive Growth of KFU. RAA was supported by state assignment 20.5175.2017/6.7. Disclosures No relevant conflicts of interest to declare.

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Tarte, Karin, Patricia Ame-Thomas, Hélène Maby, Sylvie Caulet-Maugendre, Rachel Jean, Céline Monvoisin, Thierry Lamy et Thierry Fest. « Human Mesenchymal Stem Cells as Precursors of Functional Fibroblastic Reticular Cells : Implications for the Physiopathology of Secondary Lymphoid Organs. » Blood 106, n^o 11 (16 novembre 2005) : 2310. http://dx.doi.org/10.1182/blood.v106.11.2310.2310.

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Abstract Several subsets of stromal cells are found among secondary lymphoid organs where they play a key role in the initiation and maintenance of immune response. In particular, fibroblastic reticular cells (FRC) of the paracortex secrete extracellular matrix (ECM) components that constitute a dense network of conduits allowing antigens carried within the subcapsular afferent lymph to reach the lumen of the medullary high endothelial venules. FRC produce also several chemokines that recruit T, B, and dendritic cells from blood and favour their reciprocal interactions. In addition, follicular dendritic cells (FDC) are located exclusively into germinal centers and allow normal B-cell selection through a complex set of survival signals, including BCR-mediated signal, chemokines and adhesion molecules. FRC and FDC networks are phenotypically and probably functionally altered during development of follicular lymphomas and diffuse large B cell lymphomas, the two most frequent Non-Hodgkin Lymphomas. FRC and FDC are supposed to be of mesenchymal origin even if no conclusive work has been conducted to date in human. We have obtained 15 tonsil-derived stromal cell lines, that displayed all the morphologic, phenotypic, and functional characteristics of FRC, including synthesis of inflammatory (CXCL10, CXCL9, CCL5) and lymph-node specific (CCL19, CCL21) chemokines, and secretion of ECM organized in a reticular meshwork after long-term culture in the presence of TNF-α and lymphotoxin-α1β2 (LT). These cells induced tonsil leukocyte migration and adhesion in vitro. Tonsil-derived stromal cells expressed LTβR, TNFR, and CD40 but were negative for FDC specific markers, such as CD21 or CXCL13, even following in vitro stimulation by TNF-α, LT, and trimeric CD40L. Interestingly, such TNF and LT-dependent FRC differentiation could also be induced in adult bone marrow-derived mesenchymal stem cells (MSC). In addition, MSC-like cells able to differentiate along osteogenic, adipogenic, and chondrogenic lineages at the clonal level were found in normal tonsils. These data shed new lights on our current understanding of lymph node stromal cell origin and strongly suggest that MSC are the precursors of FRC in secondary lymphoid organs, and perhaps in bone marrow in case of FL involvement where ectopic lymph node-like stromal cells are detected in close association with tumor cells. In conclusion, MSC and their progeny trigger differential immune effects, depending on cytokine context, localization and cell contact with immune cells. These properties are probably modified during lymphomas where the contact between malignant B cells and stromal cells is crucial for tumor development.

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Dhawan, Abhishek, Jens Friedrichs, Laura Bray, Lorenz C. Hofbauer, Manja Wobus et Martin Bornhäuser. « Interaction of Tumor Cells with the Hematopoietic Stem and Progenitor Cell Niche ». Blood 124, n^o 21 (6 décembre 2014) : 5139. http://dx.doi.org/10.1182/blood.v124.21.5139.5139.

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Abstract Introduction The bone marrow microenvironment regulates the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), through a network dependent on cell-cell interaction. This interaction is mediated by morphogens, the extracellular matrix and cell adhesion molecules expressed and secreted by various cell types in the HSPC niche. Mesenchymal stromal cells (MSCs), as the major cellular component, maintain the stemness properties of the niche. The microenvironment thus becomes conducive for HSPCs to remain quiescent, thereby enabling long term self-renewal. Therefore, the safe haven in the bone marrow microenvironment and its constituent cell types can be targeted during tumorigenesis, thus making the niche neoplastic. Dissemination of breast cancer cells into the bone marrow has been described even in the early stages of the disease. The present study focuses on the influence of breast carcinomas on the genetic and functional profile of mesenchymal and hematopoietic progenitor cells of the bone marrow niche. Methods In vitro coculture models of breast cancer cell lines- MDA-MB231, MCF-7 and primary MSCs derived from the bone marrow of healthy donors were used in the study. Atomic- force microscopy based single-cell force spectroscopy (AFM-SCFS) and fluorescence based assays were used for cell adhesion experiments. Hydrogel based culture systems were used for 3-dimensional cocultures of breast cancer cells and MSCs. Hypoxic and normoxic culture conditions (0.5% and 20% oxygen respectively) were used for the experiments. Results The breast cancer cell lines caused a significant reduction in HSPC adhesion to MSCs (88% by MDA-MB 231 cells; p<0.005 and 73% by MCF-7 cells; p<0.005). AFM-SCFS studies also indicated a higher binding force between breast cancer cells and MSCs, as compared to HSPCs (MDA-MB231 cells-0.13nN, MCF-7 cells-0.074nN and HSPCs-0.05nN). MDA-MB231and MCF-7 cells express Intercellular adhesion molecule-1(ICAM-1), which has been shown to promote breast cancer metastasis (Hanlon et al, 2002; Rosette et al, 2005; Schröder C. et al, 2011). There was a significant difference in reduction of HSPC adhesion towards MSCs by ICAM-1 knockdown (ICAM-1 KD) tumor cells as compared to MDA-MB231 cells (84.83% by MDA-MB231 cells versus 28.11% by ICAM-1KD tumor cells, p<0.001). AFM-SCFS studies also showed a reduced binding force between ICAM-1 KD tumor cells and MSCs as compared to MDA-MB231cells (MDA-MB231 cells-0.14nN versus ICAM-1-KD tumor cells-0.05nN, p value<0.001). ICAM-1 KD studies thus showed that reduction in HSPC adhesion to MSCs by breast cancer cells was mediated through ICAM-1 signaling. A cytokine array was performed to investigate if breast cancer cell lines affect the cytokine profile of MSCs. The array showed altered expression of growth factors- Basic fibroblast growth factor (bFGF) and Platelet derived growth factor–beta (PDGF-BB) (2.2 fold upregulation and 0.5 fold downregulation in breast cancer cells- MSC cocultures respectively). Based on the array, a bFGF-mediated increase in the proliferation of MSCs and breast cancer cells in coculture was observed. The bFGF upregulation also caused an increased migration of MDA-MB231 cells towards MSCs in a transwell migration assay. An upregulation in the phosphorylation status of Akt was observed in breast cancer cells – MSC cocultures, as a downstream effect of upregulated bFGF levels. The bFGF-mediated increase in the proliferation of breast cancer cells and MSCs in coculture was shown to be dependent on the activation of PI3K-Akt pathway. The bFGF- mediated increase in the migration of MDA-MB231 cells towards MSCs was also inhibited upon addition of the PI3K blocker. Interestingly, the breast cancer cells caused a reduction in osteoblastic differentiation of MSCs by downregulation of PDGF-BB. Studies with 3-dimensional cocultures of breast cancer cells and MSCs also showed a reduction in osteoblastic differentiation of MSCs. Furthermore, long-term cocultures of breast cancer cells, HSPCs and MSCs showed reduced support for primitive HSPCs in the neoplastic niche. Conclusions These findings indicate a perturbed HSPC niche upon tumor invasion. The possible role of altered cytokine expression, consecutive downstream signaling in niche activation and bone turnover will be further studied using in vitro and in vivo approaches to recapitulate tumor micrometastases to the HSPC niche. Disclosures No relevant conflicts of interest to declare.

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Li, Shenjie, Wei Xiang, Junjie Tian, Haorun Wang, Shuiwang Hu, Ke Wang, Ligang Chen, Changren Huang et Jie Zhou. « Bone Marrow-Derived Mesenchymal Stem Cells Differentially Affect Glioblastoma Cell Proliferation, Migration, and Invasion : A 2D-DIGE Proteomic Analysis ». BioMed Research International 2021 (11 février 2021) : 1–13. http://dx.doi.org/10.1155/2021/4952876.

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Bone marrow-derived mesenchymal stem cells (BM-MSCs) display high tumor tropism and cause indirect effects through the cytokines they secrete. However, the effects of BM-MSCs on the biological behaviors of glioblastoma multiforme remain unclear. In this study, the conditioned medium from BM-MSCs significantly inhibited the proliferation of C6 cells ( P < 0.05 ) but promoted their migration and invasion ( P < 0.05 ). Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) proteomic analysis revealed 17 proteins differentially expressed in C6 cells exposed to the BM-MSC-conditioned medium including five upregulated proteins and 12 downregulated proteins. Among these, six differentially expressed proteins (Calr, Set, Oat, Npm1, Ddah1, and Tardbp) were closely related to cell proliferation and differentiation, and nine proteins (Pdia6, Sphk1, Anxa4, Vim, Tuba1c, Actr1b, Actn4, Rap2c, and Tpm2) were associated with motility and the cytoskeleton, which may modulate the invasion and migration of tumor cells. Above all, by identifying the differentially expressed proteins using proteomics and bioinformatics analysis, BM-MSCs could be genetically modified to specifically express tumor-suppressive factors when BM-MSCs are to be used as tumor-selective targeting carriers in the future.

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Nguyen, Duong, Alberto Gomez, Forrest Neuharth, Ashley Alamillo, Thomas Herrmann, Barbara Härtl, Laura Schneider et al. « 716 Cell-based virotherapy for targeting cancers ». Journal for ImmunoTherapy of Cancer 8, Suppl 3 (novembre 2020) : A758. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0716.

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BackgroundOncolytic virotherapy has been recognized as a promising new therapy for cancer for decades but only few viruses have been approved worldwide. The therapeutic potential of oncolytic viruses can be severely restricted by innate and adaptive immune barriers making oncolytic virus clinically inefficient. To overcome this obstacle, we utilized adipose-derived stem cells (AD-MSC) loaded with tumor selective CAL1 oncolytic vaccinia virus to generate a new therapeutic agent called SNV1 (SuperNova-1).MethodsCAL1 vaccinia virus was tested for its ability to replicate and selectively kill various human cancer cell lines in vitro and in vivo. Additionally, CAL1 was loaded into adipose-derived mesenchymal stem cells to generate SuperNova1 (SNV1). Both CAL1 and SNV1 were tested for their ability to kill cancer cells in the presence of active complement and neutralizing antibodies in cell culture as well as in mice. Immune cell infiltration of the treated and untreated tumors was analyzed by flow cytometry.ResultsCAL1 showed preferential amplification and killed various tested human (PC3, FaDu, MDA-MB-231, RPMI) and mouse cancer cells (CT26, EMT6, TRAMP-C2, RM1). In animals, CAL1 caused tumor regression in PC3 and CT26 mouse models without signs of toxicity. SNV1 significantly enhanced protection of CAL1 virus from clearance by the immune system as compared to naked CAL1 virus, leading to higher therapeutic efficacy in animals. Five days after SNV1 administration, tumor infiltrating lymphocytes (TILs) from both treated and untreated tumors showed increased CD4 and CD8 T-cell infiltrations. Importantly, we documented a decreased frequency of Tregs, and improved effector to Treg ratios, which was associated with inhibition of tumor growth at the treated tumor site and also at distant untreated sites.ConclusionsCAL1 is potentially used as an oncolytic agent. In addition, SNV1 cell-based platform protects and potentiates oncolytic vaccinia virus by circumventing humoral innate and adaptive immune barriers, resulting in enhanced oncolytic virotherapy. Particularly, SNV1 provided instantly active viral particles for immediate infection and simultaneous release of therapeutic proteins in the injected tumors.

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de Haart, Sanne J., Monique C. Minnema, Julie H. Huang, Tineke Aarts-Riemens, Henk Lokhorst, Anton Martens, Constantine S. Mitsiades et Tuna Mutis. « The Survivin Suppressant YM155 Overcomes Microenvironment-Mediated Immune Resistance In a Humanized Multiple Myeloma Mouse Model ». Blood 122, n^o 21 (15 novembre 2013) : 3250. http://dx.doi.org/10.1182/blood.v122.21.3250.3250.

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Abstract Background Durable tumor regression can be achieved in Multiple Myeloma (MM) patients by immunotherapeutic approaches, such as allogeneic stem cell transplantation and donor lymphocyte infusion. However, the low rate of patients, who reach a sustained remission, impedes the success of this therapeutic approach. Currently, much emphasis is placed on the role of the tumor microenvironment in tumor cell immune escape. We previously described that different accessory cells of the microenvironment significantly inhibited the lysis of MM cell lines by T cells in a cell-cell contact dependent manner. Further analysis revealed that this immune escape could be attributed to the induction of a cell adhesion-mediated immune resistance (CAM-IR) of MM against T cell lysis in vitro. Further in vitro studies identified up-regulation of survivin in the MM cells as one of the potential mechanisms of immune resistance. Moreover, co-culture with YM155, a small molecule survivin suppressant, could abrogate accessory cell induced resistance in vitro. Methods and Results We investigated the influence of a human bone marrow microenvironment on the anti tumor activity of T cell therapy in a human-mouse hybrid model (Groen et al. Blood 2012). Immune-deficient RAG2-/- γc-/- -mice were implanted subcutaneously either with uncoated scaffolds or scaffolds coated with human mesenchymal stromal cells (MSC), which generates a human bone marrow environment. Luciferase transduced MM cell line UM9 injected directly in both types of scaffolds grew into MM tumors and were treated with Myeloma reactive minor Histocompatibility antigen specific T cell clones. The T cells induced effective anti-myeloma responses against tumors developed in the uncoated scaffolds. In contrast, and consistent with the in vitro results, no anti-tumor effect was observed in the MSC coated scaffolds. Thus confirming in vivo a microenvironment induced resistance of MM cells to T cell kill. We next investigated if the resistance could be abrogated by combination therapy with T cells and YM155.To this end, mice with tumors in human MSC coated scaffolds were treated with T cells alone, YM155 alone, or the combination of both. YM155- nor T cell-monotherapy induced tumor regression on evaluation of tumor growth based on bioluminescent signal. Moreover, the combination of YM155 with T cells demonstrated a substantial anti-tumor effect. Conclusion YM155 treatment can sensitize myeloma cells to T cell mediated anti-tumor effects and more importantly can overcome microenvironment-mediated resistance of MM cells to T cell treatment. These findings support further development of CAM-IR modulating agents in combination with immune therapy. Disclosures: Minnema: Janssen Cilag: Consultancy, Honoraria. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

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