Tumor-associated Mesenchymal Stem Cells Research Articles

Non-small cell lung cancer cells and concomitant cancer therapy induce a resistance-promoting phenotype of tumor-associated mesenchymal stem cells.

The tumor microenvironment gained attraction over the last decades as stromal cells significantly impact on tumor development, progression and metastasis, and immune evasion as well as on cancer therapy resistance. We previously reported that lung-resident mesenchymal stem cells (MSCs) were mobilized and activated in non-small cell lung cancer (NSCLC) progression and could even mediate radiation resistance in co-cultured NSCLC cells. We investigated how MSCs were affected by NSCLC cells in combination with cancer (radiation) therapy in indirect co-cultures using tumor-conditioned medium and Transwells or direct three-dimensional NSCLC-MSC spheroid co-cultures in order to unravel the resistance-mediating action of tumor-associated MSCs. Although no obvious phenotypic and functional alterations in MSCs following NSCLC co-culture could be observed, MSC senescence was induced following co-applied radiotherapy (RT). Global gene expression profiling, in combination with gene set enrichment analysis upon treatment, was used to confirm the senescent phenotype of irradiated MSC and to reveal relevant senescence-associated secretory phenotype (SASP) factors that could meditate NSCLC RT resistance. We identified senescent tumor-associated MSC-derived serine proteinase inhibitor (serpin) E1/PAI1 as potential SASP factor mediating NSCLC progression and RT resistance. Specified intra-tumor-stroma interactions and cell type-specific pro-tumorigenic functions could not only improve lung cancer classification but could even be used for a more precise profiling of individual patients, finally paving an additional way for the discovery of potential drug targets for NSCLC patients.

Micromeria fruticosa and Foeniculum vulgare essential oils inhibit melanoma cell growth and migration by targeting MMP9 and NFkB signaling

BackgroundFennel (Fe), also known scientifically as Foeniculum vulgare, and Micromeria fruticose are herbaceous plants endemic to the Mediterranean Region. The use of their essential oils for their health-promoting effects has been seen in Middle-Eastern societies, where they have been used as a type of traditional medicine. These oils have been used to treat a variety of diseases, including headaches, abdominal pains, skin and eye infections, colds, and wounds. This study looks at the chemical makeup of essential oils extracted from Palestine-grown fennel seeds and Micromeria fruticose leaves. The anticancer properties of each essential oil, as well as the combined mixture of both oils, were evaluated against the melanoma cell line.ResultsGC–MS was used to study the essential oils (EOs) from Micromeria fruticose leaves and fennel seeds that were extracted by hydrodistillation. Analysis of M. fruticose EO allowed the identification of 20 compounds, accounting for 97.73% of the EO's overall composition, with pulegone (81.77%), β-caryophyllene (2.95%), isomenthone (2.17%), piperitenone oxide (1.78%), and p-mentha-3-en-8-ol (1.38%) being the primary components. 24 phytochemicals were identified in the essential oil of fennel seeds, accounting for 100% of its composition, of which trans-anethole (93.69%), fenchone (3.93%), and sylvestrene (0.83%) were the major constituents. Although the EOs derived from M. fruticosa leaves and fennel seeds have shown potential for inhibiting the growth of several types of cancer, their impact on the proliferation and migration of melanoma cells has not been investigated. The results of our study demonstrate that the application of both oils, either separately or in combination (referred to as Mix-EO therapy), effectively suppressed the growth of melanoma cells in a manner that was dependent on the dosage. Furthermore, both treatments resulted in the upregulation of pro-apoptotic Bax and the downregulation of apoptosis-inhibiting Bcl2 expression in an in vitro setting. The anti-proliferative effects were confirmed using a murine melanoma model in vivo. Furthermore, both the individual EOs that were assessed and their combination (Mix-EO) exhibited inhibitory properties against the migration of melanoma cells. Both EOs and Mix-EO were found to decrease the intracellular levels of the transcription factor nuclear factor kappa B (NFkB) and one of its downstream targets, matrix metalloproteinase9 (MMP9), in melanoma and tumor-associated mesenchymal stem cells. Finally, we demonstrate for the first time that EOs from M. fruticose leaves, fennel seeds, and their combination exert anti-cancer characteristics by inhibiting NFkB and MMP9 in melanoma (autocrine) and tumor-associated mesenchymal stem (paracrine) cells, thereby inducing melanoma cell apoptosis and preventing cell migration.ConclusionsThe findings of our investigation indicate that the EOs derived from the fennel seeds and the M. fruticose leaves encompass a diverse array of biologically active chemicals, which have robust anticancer effects. The results of this study hold promise for application in the advancement of innovative natural medications. Additional investigation is required to delve into the medicinal capacity of these essential oils in biological entities.Graphical

Cigarette smoke-promoted increases in osteopontin expression attract mesenchymal stem cell recruitment and facilitate lung cancer metastasis

IntroductionCigarette smoking is the main risk factor for lung cancer. MSCs in the TME promoting tumor angiogenesis, growth, and metastasis. SIBLING proteins enable cancer cells to extend, invade and metastasize. ObjectivesCigarette smoke promotes the progression and metastasis of lung cancer, although how this occurs is poorly understood. We evaluated the impact of whether cigarette smoking motivates SIBLING protein expression and is involved in MSC-mediated lung tumor metastasis. MethodsWe investigated the expression of OPN in the Gene Expression Omnibus (GEO) databases and confirmed the results by immunohistochemistry (IHC), qPCR and Western blotting (WB) of lung cancer cells and tissues. The effect of OPN on the recruitment and adhesion of mesenchymal stem cells (MSCs) to lung cancer cells and lung cancers metastasis was investigated by Transwell, adhesion assays. A series of in vitro and in vivo experiments were conducted to demonstrate the mechanisms by which OPN modulates recruitment and adhesion of MSCs to lung cancer cells and lung cancer metastasis. ResultsCigarette smoke extract (CSE) and benzo[α]pyrene (B[α]P) increased levels of OPN expression and facilitated the recruitment and adhesion of MSCs to lung cancer cells via JAK2/STAT3 signaling. We also observed that OPN promotes tumor-associated MSC (TA-MSC) formation through the OPN receptor (integrins αvβ1, αvβ3, αvβ5 or CD44), inducing lung cancer cell migration and invasion. In an orthotopic mouse model of lung cancer, increases in OPN expression promoted by cigarette smoke upregulated MSC recruitment and facilitated lung cancer metastasis. Knockdown of OPN expression inhibited cigarette smoke-induced lung cancer metastasis in vivo. ConclusionCigarette smoke increases OPN expression through the JAK2/STAT3 signaling pathway to attract MSC cell recruitment and promote lung cancer metastasis. Our findings offer important insights into how lung cancer metastasis develops in smokers.

Retinoic Acid Inhibits Tumor-Associated Mesenchymal Stromal Cell Transformation in Melanoma.

Bone marrow mesenchymal stem/stromal cells (BMSCs) can be transformed into tumor-associated MSCs (TA-MSCs) within the tumor microenvironment to facilitate tumor progression. However, the underline mechanism and potential therapeutic strategy remain unclear. Here, we explored that interleukin 17 (IL-17) cooperating with IFNγ transforms BMSCs into TA-MSCs, which promotes tumor progression by recruiting macrophages/monocytes and myeloid-derived suppressor cells (MDSCs) in murine melanoma. IL-17 and IFNγ transformed TA-MSCs have high expression levels of myelocyte-recruiting chemokines (CCL2, CCL5, CCL7, and CCL20) mediated by activated NF-κB signaling pathway. Furthermore, retinoic acid inhibits NF-κB signaling, decreases chemokine expression, and suppresses the tumor-promoting function of transformed TA-MSCs by prohibiting the recruitment of macrophages/monocytes and MDSCs in the tumor microenvironment. Overall, our findings demonstrate that IL-17 collaborating with IFNγ to induce TA-MSC transformation, which can be targeted by RA for melanoma treatment.

Tumor-associated mesenchymal stem cells promote hepatocellular carcinoma metastasis via a DNM3OS/KDM6B/TIAM1 axis.

Tumor-associated mesenchymal stem cells (MSCs) play a critical role in the growth and metastasis of hepatocellular carcinoma (HCC). However, the mechanism underlying the crosstalk between MSCs and HCC cells is not completely understood. Here, HCC cells were treated with or without conditioned medium of MSCs (CM-MSC), and examined for differential expression of long non-coding RNAs (lncRNAs). Knockdown and overexpression experiments were conducted to explore the function of the lncRNA DNM3OS in MSC-induced HCC growth and metastasis. CM-MSC treatment led to a concentration-dependent induction of DNM3OS in HCC cells. DNM3OS was significantly upregulated in HCC compared to adjacent liver tissues. High DNM3OS expression was associated with TNM stage, vascular invasion, and poor prognosis of HCC patients. Silencing of DNM3OS inhibited HCC cell proliferation and invasion in vitro and tumorigenesis and metastasis in vivo. Overexpression of DNM3OS enhanced HCC cell proliferation, invasion, and metastasis. Biochemically, DNM3OS was mainly localized in the nucleus and physically interacted with KDM6B. The association of DNM3OS with KDM6B induced the expression of TIAM1 through reduction of H3K27me3 at the TIAM1 promoter. TIAM1 overexpression restored the proliferation and invasion of DNM3OS-depleted HCC cells. Our data delineate a mechanism by which MSCs accelerate HCC growth and metastasis through a DNM3OS/KDM6B/TIAM1 axis.

Consistent Inclusion of Mesenchymal Stem Cells into In Vitro Tumor Models.

Over recent years, the role of distinct mesenchymal stem cell populations in cancer progression has become increasingly evident. In this regard, developing in vitro preclinical tumor models capable of portraying tumor-associated mesenchymal stem cells (TA-MSCs) interactions with the tumor microenvironment (TME), cellular and extracellular components, would allow to improve the predictive potential of these platforms and expedite preclinical drug screening. Although recent studies successfully developed in vitro tumor models in which the biomolecular and cellular behaviors of TA-MSCs were recapitulated in the context of their interactions with specific TME components, no consensus has yet been reached regarding distinct TA-MSCs influence in the evolution of solid tumors. The paradoxical observations regarding the roles of MSCs on in vitro tumor models can in part be associated to a lack of standardization in how MSCs integration is performed. Herein, we summarize some of the main parameters linked to phenotypic variations established upon MSCs inclusion and interaction within in vitro tumor models. Acritical overview of recent studies and how standardization of key parameters could improve the reproducibility and predictability of current preclinical validation models containing MSCsis also provided.

Reversibility of hAT-MSCs phenotypic and metabolic changes after exposure to and withdrawal from HCC-conditioned medium through regulation of the ROS/MAPK/HIF-1\u03b1 signaling pathway

BackgroundMesenchymal stem cells (MSCs) play an important role in tumor progression; concomitantly, MSCs also undergo profound changes in the tumor microenvironment (TME). These changes can directly impact the application and efficacy of MSC-based anti-tumor therapy. However, few studies have focused on the regulation of MSC fate in TME, which will limit the progress of MSC-based anti-tumor therapy. Herein, we investigated the effects of conditioned medium from human hepatocellular carcinoma cells (HCC-CM) on the phenotype and glucose metabolism of human adipose tissue-derived MSCs (hAT-MSCs).MethodsThe passage 2 (P2) to passage 3 (P3) hAT-MSCs were exposed to conditioned medium from Hep3B, Huh7 and HCCLM3 cells for 4–8 weeks in vitro. Then, immunofluorescent, CCK-8 assay, EdU assay, Transwell assay, and flow cytometry were used to assess the alterations in cell phenotype in terms of cell morphology, secretory profiles, proliferation, migration, invasion, cell cycle, and apoptosis. In addition, glucose metabolism was evaluated by related kits. Next, the treated hAT-MSCs were subjected to withdrawal from HCC-CM for 2–4 weeks, and alterations in phenotype and glucose metabolism were reevaluated. Finally, the molecular mechanism was clarified by Western blotting.ResultsThe results revealed that after exposure to HCC-CM, hAT-MSCs developed a stellate-shaped morphology. In association with cytoskeleton remodeling, hAT-MSCs showed enhanced capacities for migration and invasion, while cell proliferation was inhibited by regulating the cell cycle by downregulating cyclins and cyclin-dependent kinases and activating the mitochondrial apoptosis pathway. In terms of glucose metabolism, our results showed mitochondrial dysfunction and elevated glycolysis of hAT-MSCs. However, interestingly, when the treated hAT-MSCs were subjected to withdrawal from HCC-CM, the alterations in phenotype and glucose metabolism could be reversed, but secretory phenotype and tumor-promoting properties appear to be permanent. Further studies showed that these changes in hAT-MSCs may be regulated by the ROS/MAPK/HIF-1α signaling pathway.ConclusionTaken together, the effects of long-term HCC-CM treatment on phenotype and glucose metabolism in hAT-MSCs are modest and largely reversible after withdrawal, but HCC-CM endow hAT-MSCs with permanent secretory phenotype and tumor-promoting properties. This is the first report on the reversal of phenotype and glucose metabolism in tumor-associated MSCs (TA-MSCs), it is anticipated that new insights into TA-MSCs will lead to the development of novel strategies for MSC-based anti-tumor therapy.

The Emerging Role of GC-MSCs in the Gastric Cancer Microenvironment: From Tumor to Tumor Immunity.

Mesenchymal stem cells (MSCs) have been declared to not only participate in wound repair but also affect tumor progression. Tumor-associated MSCs, directly existing in the tumor microenvironment, play a critical role in tumor initiation, progression, and development. And different tumor-derived MSCs have their own unique characteristics. In this review, we mainly describe and discuss recent advances in our understanding of the emerging role of gastric cancer-derived MSC-like cells (GC-MSCs) in regulating gastric cancer progression and development, as well as the bidirectional influence between GC-MSCs and immune cells of the tumor microenvironment. Moreover, we also discuss the potential biomarker and therapeutic role of GC-MSCs. It is anticipated that new and deep insights into the functionality of GC-MSCs and the underlying mechanisms will promote the novel and promising therapeutic strategies against gastric cancer.

NLGP counterbalances the immunosuppressive effect of tumor-associated mesenchymal stem cells to restore effector T cell functions

BackgroundA dynamic interaction between tumor cells and its surrounding stroma promotes the initiation, progression, metastasis, and chemoresistance of solid tumors. Emerging evidences suggest that targeting the stromal events could improve the efficacies of current therapeutics. Within tumor microenvironment (TME), stromal progenitor cells, i.e., MSCs, interact and eventually modulate the biology and functions of cancer and immune cells. Our recent finding disclosed a novel mechanism stating that tumor-associated MSCs inhibit the T cell proliferation and effector functions by blocking cysteine transport to T cells by dendritic cells (DCs), which makes MSCs as a compelling candidate as a therapeutic target. Immunomodulation by nontoxic neem leaf glycoprotein (NLGP) on dysfunctional cancer immunity offers significant therapeutic benefits to murine tumor host; however, its modulation on MSCs and its impact on T cell functions need to be elucidated.MethodsBone marrow-derived primary MSCs or murine 10 T1/2 MSCs were tumor-conditioned (TC-MSCs) and co-cultured with B16 melanoma antigen-specific DCs and MACS purified CD4+ and CD8+ T cells. T cell proliferation of T cells was checked by Ki67-based flow-cytometric and thymidine-incorporation assays. Cytokine secretion was measured by ELISA. The expression of cystathionase in DCs was assessed by RT-PCR. The STAT3/pSTAT3 levels in DCs were assessed by western blot, and STAT3 function was confirmed using specific SiRNA. Solid B16 melanoma tumor growth was monitored following adoptive transfer of conditioned CD8+ T cells.ResultsNLGP possesses an ability to restore anti-tumor T cell functions by modulating TC-MSCs. Supplementation of NLGP in DC-T cell co-culture significantly restored the inhibition in T cell proliferation and IFNγ secretion almost towards normal in the presence of TC-MSCs. Adoptive transfer of NLGP-treated TC-MSC supernatant educated CD8+ T cells in solid B16 melanoma bearing mice resulted in better tumor growth restriction than TC-MSC conditioned CD8+ T cells. NLGP downregulates IL-10 secretion by TC-MSCs, and concomitantly, pSTAT3 expression was downregulated in DCs in the presence of NLGP-treated TC-MSC supernatant. As pSTAT3 negatively regulates cystathionase expression in DCs, NLGP indirectly helps to maintain an almost normal level of cystathionase gene expression in DCs making them able to export sufficient amount of cysteine required for optimum T cell proliferation and effector functions within TME.ConclusionsNLGP could be a prospective immunotherapeutic agent to control the functions and behavior of highly immunosuppressive TC-MSCs providing optimum CD8+ T cell functions to showcase an important new approach that might be effective in overall cancer treatment.

Tumor Mesenchymal Stromal Cells Regulate Cell Migration of Atypical Teratoid Rhabdoid Tumor through Exosome-Mediated miR155/SMARCA4 Pathway

Atypical teratoid/rhabdoid tumor (ATRT) is a rare pediatric brain tumor with extremely high aggressiveness and poor prognosis. The tumor microenvironment is regulated by a complex interaction among distinct cell types, yet the crosstalk between tumor-associated mesenchymal stem cells (tMSCs) and naïve ATRT cells are unclear. In this study, we sought to identify the secretory factor(s) that is responsible for the tMSC-mediated regulation of ATRT migration. Comparing with ATRT cell alone, co-culture of tMSCs or addition of its conditioned medium (tMSC-CM) promoted the migration of ATRT, and this effect could be abrogated by exosome release inhibitor GW4869. The exosomes in tMSC-CM were detected by transmission electron microscope and flow cytometry. ATRT naïve cell-derived conditioned media (ATRT-CM) also enhanced the exosome secretion from tMSCs, indicating the interplay between ATRT cells and tMSCs. Microarray analysis revealed that, compared with that in bone marrow-derived MSCs, microRNA155 is the most upregulated microRNA in the tMSC-CM. Tracing the PK67-labeled exosomes secreted from tMSCs confirmed their incorporation into naïve ATRT cells. After entering ATRT cells, miR155 promoted ATRT cell migration by directly targeting SMARCA4. Knockdown of SMARCA4 mimicked the miR155-driven ATRT cell migration, whereas SMARCA4 overexpression or the delivery of exosomes with miR155 knockdown suppressed the migration. Furthermore, abrogation of exosome release with GW4869 reduced the tumorigenesis of the xenograft containing naïve ATRT cells and tMSCs in immunocompromised recipients. In conclusion, our data have demonstrated that tMSCs secreted miR155-enriched exosomes, and the exosome incorporation and miR155 delivery further promoted migration in ATRT cells via a SMARCA4-dependent mechanism.

Tumor-associated mesenchymal stem cells in chemically-induced breast cancer in Wistar rats

Objective: to compare the morphological and functional properties of mesenchymal stem cells from mammary tissues and chemically-induced mammary tumor tissues. material and methods. The study included 25 female Wistar rats. In 20 rats, mammary carcinoma was induced by intramammary injection of N-methyl-N-nitrosourea after estrus synchronization with chorionic gonadotropin. The control group consisted of 5 rats. Mammary carcinoma was verified histologically and immunohistochemically. To examine, whether the cells isolated from normal tissue and tumor tissue belonged to mesenchymal stem cells, FACS Canto II flow cytofluorometer was used. The functional properties of mesenchymal stem cells were evaluated in MTT assay by the level of nitric oxide production in normal and by hydrogen peroxide-induced hypoxia. The levels of prolactin, luteinizing hormone and estradiol E2 in urine were studied using solid-phase enzyme-linked immunosorbent assay. results. Chemically-induced mammary tumor according to histological and immunohistochemical studies corresponded to luminal B type breast cancer in humans. In rats that developed mammary tumors, the urine prolactin levels after synchronization of estrus were increased. In rats that did not develop tumors, the levels of prolactin and luteinizing hormone were decreased, but the levels of estradiol E2 were increased. More mesenchymal stem cells with CD45-/CD90+phenotype were obtained from the breast tumor tissue. Mesenchymal stem cells from tumor tissue showed increased proliferative potential and were more resistant to hypoxia. conclusion. Tumor- associated mesenchymal stem cells having high proliferative potential and resistance to hypoxia were obtained from chemically-induced mammary tumor tissue. Morphologic and functional differences in mesenchymal stem cells obtained from mammary breast tissue and tumor tissue require further studies.

Tumor-associated mesenchymal stem cells inhibit naïve T cell expansion by blocking cysteine export from dendritic cells.

Mesenchymal stem cells (MSCs) represent an important cellular constituent of the tumor microenvironment, which along with tumor cells themselves, serve to regulate protective immune responses in support of progressive disease. We report that tumor MSCs prevent the ability of dendritic cells (DC) to promote naïve CD4(+) and CD8(+) T cell expansion, interferon gamma secretion and cytotoxicity against tumor cells, which are critical to immune-mediated tumor eradication. Notably, tumor MSCs fail to prevent DC-mediated early T cell activation events or the ability of responder T cells to produce IL-2. The immunoregulatory activity of tumor MSCs is IL-10- and STAT3-dependent, with STAT3 repressing DC expression of cystathionase, a critical enzyme that converts methionine-to-cysteine. Under cysteine-deficient priming conditions, naïve T cells exhibit defective cellular metabolism and proliferation. Bioinformatics analyses as well as in vitro observations suggest that STAT3 may directly bind to a GAS-like motif within the cystathionase promoter (-269 to -261) leading to IL-10-STAT3 mediated repression of cystathionase gene transcription. Our collective results provide evidence for a novel mechanism of tumor MSC-mediated T cell inhibition within tumor microenvironment.

Abstract 173: Tumor proximal mesenchymal stem cells initiate a pro-metastatic microRNA regulatory network which acts via convergent targeting of the speech-associated transcriptional repressor FOXP2

Abstract Tumor progression to metastasis is largely responsible for breast cancer mortalities, but the mechanisms underlying this process remain among the least understood in oncology. Cell-intrinsic mechanisms, such as accumulating genetic lesions, are well appreciated drivers of malignant progression; however, substantial evidence now supports a similarly critical role for cell-extrinsic stimuli, driving pro-malignant gene expression changes through epigenetic mechanisms. Such external stimuli derive from interactions with the tumor-associated stroma, a distinctive milieu of non-transformed stromal cells recruited to the tumor site by the growing neoplasm. Proximal interactions between cancer cells and components of this tumor-associated stroma have been demonstrated to drive angiogenesis, invasion and metastasis. Understanding these interactions will inform our knowledge of tumor progression and metastasis in general, and presents potential for developing therapeutic approaches based on interdicting the tumor-stroma crosstalk. Mesenchymal stem cells (MSCs) are ambulatory stromal progenitor cells that are normally dispatched to wound sites to aid in the repair of damaged tissues. MSCs similarly home with great affinity to tissues perturbed growing epithelial neoplasms, where they have been found to serve as stromal catalysts of carcinoma progression and metastasis in multiple experimental tumor models of a variety of human cancer contexts including lung, breast, melanoma, prostate, and colon. Despite expanding knowledge of how tumor-associated MSCs contribute to tumor malignancy, the programs and pathways deregulated in cancer cells as a consequence of their interactions with MSCs remain incompletely understood. We conducted genome-wide microRNA (miRNA) profiling analyses on MSC-exposed breast cancer cells (BCCs) to investigate how miRNA deregulation might contribute to malignant breast cancer progression. Our investigation revealed that MSCs cause aberrant expression of a distinct set of miRNAs that accelerate metastasis and are sufficient to provide cancer cells with enhanced tumor initiation capacities, including phenotypes and markers characteristic of putative cancer stem cells. We demonstrate that these miRNAs collectively constitute a regulatory network whose members converge upon a common target: the speech-associated transcriptional repressor FOXP2. FOXP2 knockdown phenocopied increases in metastasis and tumor-initiating ability of BCCs, including CSC-consistent markers, genes and phenotypes. Importantly, elevated MSC-induced miRNA levels and depressed FOXP2 levels were observed in human breast cancers as correlating with increased malignancy. Altogether, our results implicate FOXP2 and its MSC-induced miRNA regulators as novel determinants of malignant breast cancer progression. Citation Format: Benjamin Cuiffo, Antoine Campagne, George W. Bell, Evan Lien, Manoj K. Bhasin, Odette Mariani, Anne Vincent-Salomon, Antoine Karnoub. Tumor proximal mesenchymal stem cells initiate a pro-metastatic microRNA regulatory network which acts via convergent targeting of the speech-associated transcriptional repressor FOXP2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 173. doi:10.1158/1538-7445.AM2014-173

Tumor associated mesenchymal stem cells protect ovarian cancer cells from hyperthermia through CXCL12

Hyperthermic intraperitoneal chemotherapy (HIPEC) has shown promise in the treatment of ovarian carcinosis. Despite its efficiency for the treatment of peritoneal carcinosis from digestive tract neoplasia, it has failed to demonstrate significant benefit in ovarian cancers. It is therefore essential to understand the mechanism underlying the resistance to HIPEC in ovarian cancers. Mesenchymal Stem Cells (MSC) play an important role in the development of ovarian cancer metastasis and resistance to treatments. A recent study suggests that MSCs may be cytotoxic for cancer cells upon heat shock. In contrast, we describe the protective role of MSC against hyperthermia. Using cytokine arrays we determined that tumor associated MSC (TAMC) secrete pro-tumoral cytokines. We studied the effect of hyperthermia in co-culture setting of TAMC or bone marrow derived-MSC (BM-MSC) associated with ovarian cancer cell lines (SKOV3 and CaOV3) with polyvariate flow cytometry. We demonstrate that hyperthermia does not challenge survival of TAMC or BM-MSC. Both TAMC and BM-MSC displayed strong protective effect inducing thermotolerance in ovarian cancer cells (OCC). Transwell experiments demonstrated the role of secreted factors. We showed that CXCL12 was inducing thermotolerance and that inhibition of CXCL12/CXCR4 interaction restored cytotoxicity of hyperthermia in co-culture experiments. Targeting the interaction between stromal and cancer cells through CXCL12 inhibition might restore hyperthermia sensitivity in ovarian cancers, and thus improve HIPEC efficiency.

Tumor associated mesenchymal stem cells protects ovarian cancer cells from hyperthermia through CXCL12

Hyperthermic intraperitoneal chemotherapy (HIPEC) has shown promise in treatment of ovarian carcinosis. Despite its efficiency for the treatment of peritoneal carcinosis from digestive tract neoplasia, it has failed to demonstrate significant benefit in ovarian cancers. It is therefore essential to understand the mechanism underlying resistance to HIPEC in ovarian cancers. Mesenchymal stem cells (MSC) play an important role in the development of ovarian cancer metastasis and resistance to treatments. A recent study suggests that MSCs may be cytotoxic for cancer cells upon heat shock. In contrast, we describe the protective role of MSC against hyperthermia. Using cytokine arrays we determined that the tumor associated MSC (TAMC) secrete pro-tumoral cytokines. We studied the effect of hyperthermia in co-culture setting of TAMC or BM-MCS associated with ovarian cancer cell lines (SKOV3 and CaOV3) with polyvariate flow cytometry. We demonstrate that hyperthermia does not challenge survival of TAMC or bone marrow derived MSC (BM-MSC). Both TAMC and BM-MSC displayed strong protective effect inducing thermotolerance in ovarian cancer cells (OCC). Transwell experiments demonstrated the role of secreted factors. We showed that CXCL12 was inducing thermotolerance and that inhibition of CXCL12/CXCR4 interaction restored cytotoxicity of hyperthermia in co-culture experiments. Contrary to the previous published study we demonstrated that TAMC and BM-MSC co-cultured with OCC induced thermotolerance in a CXCL12 dependant manner. Targeting the interaction between stromal and cancer cells through CXCL12 inhibition might restore hyperthermia sensitivity in ovarian cancers, and thus improve HIPEC efficiency.

Abstract 1006: Tumor associated mesenchymal stem cells protect ovarian cancer cells from hyperthermia through stromal cell derived factor-1 (SDF-1) secretion

Abstract Background: Hyperthermia therapy is a medical procedure where local body tissues are exposed to high temperatures ranging from 40oC to 43oC with the aim of killing microscopic tumor residue, being more susceptible to heat than normal cells. When combined with hyperthermia during cytoreductive surgeries, intraperitoneal chemotherapy was clinically proved to be more effective. While hyperthermia's cytotoxicity was tested on cancer cells, its effect on interactions between cancer cells and tumor stroma cells was minimally scrutinized. In this study, we investigate the effect of tumor associated mesenchymal stem cells, on inducing resistance of ovarian cancer cells (OCC) to hyperthermia. Methods: OCC viability was challenges by heat shock at 42 degrees, in simple or co-culture systems. Cell viability was determined using Calcein Violet AM by Fluorescence Activated Cell Sorting, FACS. The same experiment was replicated using a transwell system. Using the trogocytosis assay we measured the interaction between the different cell types. Multiparameter flow cytometry was used to study the relation between intercellular interaction and thermoresistance. Finally using inhibitors and blocking antibodies we were able to analyse the role of SDF1 pathway in thermoresistance. Results: At 42oC, an increase in viability of OCC, co-cultured with ‘MSC, was detected in comparison to cells cultured alone. The same result was attained using the transwell experiment. Antibody and siRNA for CXCR4 reversed the heat resistant effect of MSC cells on OCC. More interestingly we were able to demonstrate an increased expression of CXCR4 in cancer cells co-cultured with the MSC, and increased thermoresistance in the subset of cancer cells with more interaction (as measured by oncologic trogocytosis). Conclusions: This study proved that contact and non contact-based cell to cell interactions between MSC cells and OCC help ovarian cancer cells resist hyperthermia. Within these interactions, MSC-secreted SDF-1 could be the major inducer of heat resistance. Combining monoclonal antibody against CXCR4 with hyperthermic intraperitoneal chemotherapy could eliminate the protective effect of tumor associated mesenchymal stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1006.

952. Human Mesenchymal Stem Cells Migrate to Colon Cancer Xenografts: Potential Carriers of Cancer Therapeutics

Mesenchymal stem cells (MSC) are found in the bone marrow and give rise to adipocytes, osteocytes, chondrocytes, and potentially other tissues. It has recently been shown that MSCs have affinity for lung and glioma tumors in xenograft models. We investigated MSC migration to colon cancer xenografts in conjunction with MSC transduction by a lentiviral vector as a way to deliver cancer therapeutics to the local tumor microenvironment. Subcutaneous colon or glioma tumor xenografts were generated in nude mice followed by intravenous infusion of DiOC18 fluorescently labeled MSCs. MSC pluripotentcy was confirmed by in vitro differentiation into osteocytes and adipocytes. Tumors and other organs were harvested 1 wk following MSC infusion and cryosections prepared in order to observe the presence of MSCs. We found that colon cancer tumors were selectively populated by labeled MSCs, compared to liver and spleen. MSCs were also observed to some extent in the lung. The distribution of tumor-associated MSCs were predominately at the tumor periphery and within loose clusters. In glioma xenografts, labeled MSCs were detected to a lesser extent and were mainly distributed as single cells. MSCs were then analyzed for their susceptibility to lentiviral transduction for eventual use to target cancer therapeutics to the tumor mass. We analyzed MSC transduction by a lentiviral vector harboring a luciferase-IRES-GFP cassette over a range of MOI. MSCs were easily transduced, with essentially complete transduction at an MOI of 18, and substantial transduction observed at MOIs as low as 2 when analyzed for GFP expression by flow cytometry. Luciferase expression was likewise high. These studies show that MSCs have the capacity for high lentiviral transduction and the ability to migrate to colon cancer xenografts, suggesting potential use to deliver transgene-expressed anti-cancer therapeutics to solid tumors in vivo.