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.

Epithelial ovarian carcinoma (EOC) is the sixth most common malignancy in women and the leading cause of death from gynecological cancer worldwide. 1 EOC has a predisposi-tion to metastatic involvement of the peritoneal cavity. 2,3 Late stage EOC is characterized by widespread peritoneal dissemination, ascitis and a high rate of mortality with an overall survival ranging from 20 to 30% at 5 years. 4 Platinum associated to taxanes chemotherapy, is the standard treatment for ovarian cancers, and has achieved high response rate. The development of drug-resistant cancer cells exhibiting multidrug resistance phenotype is one of the major limitations of the efficacy illustrated in the literature for platinum or taxanes chemotherapy. 4,5 Therefore, new therapeutic modalities are critical to improve overall survival in ovarian cancer. Intra-peritoneal (IP) chemotherapy emerged as one therapeutic option from the natural history of ovarian cancers (e.g., local extension to the peritoneum, chemosensitivity). Indeed it has been demonstrated that IP delivery of certain chemotherapeutic agents leads to increased peritoneal cavity drug exposure. 6 Randomized control trials demonstrated superiority of IP chemotherapy over classical intravenous therapy in patients with optimally debulked Stage III ovarian cancer. 6,7 More recently, hyperthermic intraperitoneal chemotherapy (HIPEC) has emerged as a new option to increase the efficacy of chemotherapy. Its efficacy has already been demonstrated in carcinosis from colon cancer. 8 Hyperthermia is tumoricidal and increases the cytotoxicity of many chemotherapeutic agents in cell cultures as well as in animal models. 9,10 Synergistic effects of hyperthermia in conjunction with cisplatin treatment include increased DNA cross-linking, increased DNA adducts formation and deeper penetration into peritoneal tumor implants. 11,12 Furthermore, cancer cells operate under increased acidity due to their inability to expel waste created by the anaerobic metabolism and are thus sensitive to hyperthermia. Hyperthermia also disrupts the stability of cellular proteins, cell membranes and the cytoskeleton. Finally hyperthermia inhibits DNA replication forks, which leads to cancer cells displaying greater sensitivity to chemotherapy than normal cells. 13 Many clinical trials in peritoneal carcinosis studying different tumor types (gastric, mesothelioma, endometrial, colorectal) have been launched based on the described synergy between hyperthermia and chemotherapy, and possibility of concomitant IP delivery of these two modalities. 6,14,15 A Phase III randomized study of HIPEC following cyto-reductive surgery compared to traditional intravenous chemotherapy and palliative care, in patients with peritoneal spread of colorectal carcinoma showed a statistically significant prolongation of life in the experimental arm. 6 However despite different protocols, HIPEC could not demonstrate a real benefit compared to the standard treatment in ovarian cancer carcinosis. 16 In the case of ovarian cancers most of the studies are Phase I or II feasibility trials. The overall median survival following HIPEC ranged from 22 to 64 months with a median disease-free survival ranging from 10 to 57 months. In patients with optimal cytoreduction, a 5-year survival rate ranging from 12 to 66% could be achieved. The overall rate of severe perioperative morbidity and mortality is higher than standard treatment ranging from 0 to 40% and from 0 to 10%, respectively. 16,17 Hyperthermia by itself is not a treatment in ovarian cancers. However the growing amount of publication in the literature demonstrate that it might be of interest in the treatment of peritoneal carcinosis when associated to chemotherapy. Despite various modifications of the protocols, the optimal results have not been achieved and there are no demonstrated benefits for survival with associated high morbidity. 17 Several randomized trial are trying to address these issues. Some authors suggest that in patients with epithelial ovarian cancer with no residual disease, HIPEC should be considered as a consolidation treatment option. 18 Indeed surgical optimal cytoreduction immediately followed by HIPEC ensures intraperitoneal delivery of the drug to all peritoneal surfaces and the advantages of combined hyperthermia to be exploited to target microscopic residual disease. 19 A growing amount of evidence is underlying the role of microenvironment in EOC development. [20][21][22] Recently, our group was able to demonstrate that a subset of the mesothe-lial cells isolated from the peritoneal cavity of patients with ovarian cancer was able to induce chemoresistance through oncologic trogocytosis. 23 We went further in demonstrating that these cells were also able to sustain tumor growth in-vivo through the production of IL8 and their role in angiogenesis. 24 Finally we demonstrated that these cells could provide an immunotolerant microenvironment to cancer cells. 25 Evidences in the literature are suggesting a ''cross-talk'' between cancer cells and peritoneal stromal cells that might impact different therapeutic modalities. Thus far, there has been only one study on the role of hyperthermia on mesenchymal stem cells (MSCs) and their effect on ovarian cancer cells (OCC). Our study demonstrated that conditioned medium of hyperthermia treated MSCs exerted suppressive effects on the tumor progression and malignancy, suggesting that hyperthermia enables MSC to provide a sensitizing environment for tumor cells to undergo cell death. We choose a different approach and used a co-culture setting. We were able to display a significant protective effect of MSCs on OCC subjected to hyperthermia. We demonstrated that MSC-mediated thermotolerance upon cross-talk with the OCC was dependant on CXCL12.

Isolation of tumor associated mesenchymal cells (TAMC)
Mesothelial cells are known to exfoliate at the beginning of the metastatic process. This suggests that the ovarian cancerspecific mesothelial cells could interact with the epithelial ovarian cancer cell aggregates. TAMC were isolated from the ascitis of 3 untreated patients with Stage IIIc ovarian serous adenocarcinomas, who were undergoing ascitis evacuation for clinical discomfort, as previously described. [23][24][25] Lymphocytes and erythrocytes were separated from cancer cell-TAMC aggregates using a Ficoll procedure. These ascites-derived TAMC displayed a fibroblast-like morphology and could be cryopreserved and expanded in vitro. We determined the expression profile of surface marker proteins by fluorescenceactivated cell sorting. Cells expressed MSC related markers CD9, CD10, CD29, CD146, CD166 and HLA1. TAMC do not express any hematopoietic markers like CD45, CD31, CD34, CD3, CD4, CD8 and epithelial markers such as CD324 (E-Cadherin) and CD326 (EpCAM). 23 Polyvariate flow cytometry analysis was only able to define a unique population of TAMC, thus demonstrating that TAMC constitutes a pure population. [23][24][25] Ovarian cancer and mesenchymal stem cells

Heat treatments
Different cell populations were heat-shocked at 42 C for 1 to 2 hr (5% CO 2 , humid atmosphere). Cells were allowed to recover for a period of 2 hr before assessing cell viability.
Immunostaining and fluorescence activated cell sorting (FACS) analysis OCC were stained for the expression of CXCR4 using a rat anti-human CXCR4 antibody (BD Biosciences, clone 12G5) conjugated to APC and/or for EpCAM (CD326) using a mouse anti-human EpCAM antibody conjugated to PE (Miltenyi Biotec, clone HES125). Briefly, 2.10 5 cells were harvested and then blocked in PBS-5%FBS-1%BSA-10%FcR Blocking Reagent (Myltenyi Biotec) for 30 min on ice. Cell suspension was stained for the CXCR4-APC and/or the EpCAM-PE or with 0.01 mg of antibody per 1.10 6 cells for 45 min on ice. Filtered, single-cell suspension was analyzed by FACS on a SORP FACSAria2 (BD Biosciences). Data were processed with FACSDiva 6.3 software (BD Biosciences). Doublets were excluded by FSC-W Â FSC-H and SSC-W Â SSC-H analysis, single stained channels were used for compensation, and fluorophore minus one (FMO) controls were used for gating. Calcein-violet-AM fluorescence was acquired with 405 nm violet laser excitation and a 450/50 nm filter emission, eGFP fluorescence was acquired with 488 nm blue laser excitation and a 488/40 nm filter emission, EpCAM (CD326) conjugated to PhycoErythrin (EpCAM-PE) fluorescence was acquired with a 532 nm yellow-green laser and a 582/15 nm emission, CXCR4 (CD184) conjugated to AlloPhycoCyanin (CXCR4-APC) was acquired with 633 nm red laser excitation and a 670/14 nm emission. 45,000 events were acquired per sample. Charts display the mean of fluorescence intensity (MFI) relative to the control.

Cell viability, calcein staining
Calcein-AM indicates intracellular esterase activity. OCC were exposed to hyperthermia as described. The cells were then washed twice with Phosphate buffer saline (PBS). Cells were next stained with the 2 lM of calcein-violet-AM (Molecular Probes, Invitrogen, Leiden NL) for 45 min at 37 5% CO 2 according to manufacturers instructions. They were then immediately analyzed by FACS on a SORP FACSAria2 (BD Bioscience, San Jose, CA) as described.

Cytokines array
TAMC were cultivated in serum free media for 24 hr. TAMC were then heat-shocked as described earlier. Heat-shock conditioned media was collected and protein quantified based on sample absorbance at 280 nm using nanodrop device (Thermo-Scientific, Dubai, Emirates). 200 lg of protein was loaded on RayBio V R Human Cytokine Antibody Array G Se-ries 1000 (Raybiotec, Norcross, GA) according to manufacturer's instructions. Unconditioned media was used as a negative control sample. Arrays were revealed using Horse-Radish Peroxidase (HRP) and SuperSignal West Pico Chemiluminescent Substrate (Thermo-Scientific, Dubai, Emirates). Data were collected using Geliance CCD camera (Perkin Elmer, MA), and extracted using ImageJ software (NIH). Briefly, the pictures of the arrays were inverted and background subtracted. We then defined the area for signal capture for all spots as 110-120 micron diameter, using the same area for every spot. We defined our signal as the median pixel density value. For the comparison, the independent arrays values were normalized on their positive control intensity value.

Co-cultures
To demonstrate the protective effect of TAMC-eGFP or BM-MSC-eGFP on OCC during hyperthermia, we established cocultures of OCC with TAMC-eGFP or BM-MSC-eGFP at a ratio of 1:1. Co-cultures were established 24 hr prior to the beginning of the hyperthermia assay. OCC were differentiated from the TAMC or BM-MSC based on their eGFP and EpCAM expression (CD326) (restricted to OCC in our model). OCC were defined as the eGFP À EpCAMþ cells. Hyperthermia assays was driven as previously described and cell viability was assessed using calcein assay (see above). The same experiments were performed using a 0.2 lm trans-well system (BD Bioscience, San Jose, CA) to study the effect of intercellular contact on survival of OCC. To avoid donor-todonor variability, all experiments with BM-MSC involved two different lots of BM-MSC.

Statistical analysis
Student-t, Fisher exact or chi-square tests were performed as appropriate. All p-values are two-sided with statistical significance evaluated at the 0.05 alpha level. Ninety-five percent confidence intervals (95% CI) were calculated to assess the precision of the obtained estimates. All statistical analysis was done using the data analysis plug-in shipped into the Excel 2008 for the Mac (Microsoft). We first calculated the variance of two paired. Mean 6 SEM are shown on the graphs. All results are representative of the indicated number of independent experiments.

Characterization of heat-shocked TAMC and MSC
The TAMC or BM-MSC were either untreated or heatshocked as described in ''Material and Methods.'' Phase contrast microscopy analysis of TAMC submitted to heat shock for 2 hr did not display any morphological changes (Fig. 1a). We were able to demonstrate no cytotoxic effect of hyperthermia on BM-MSC or TAMC (Figs. 1b-1c). We went further and performed transcriptomic analysis of TAMC after heat-shock. As expected, genes involved in thermotolerance were upregulated (HSP-70, Id2). Accordingly to functional

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analysis, no genes involved in apoptosis or cell death were upregulated (Supporting Information Table 1). We then performed cytokine arrays of heat-shocked TAMC. Several cytokines involved in the inflammatory response (IL2, CCL1, CCL24 and CCL 26) as well as cytokines involved in negative regulation of cell death and cell proliferation (IFN-gamma, Angiogenin, BMP6, GM-CSF and GDNFm and CCl23) were upregulated. Cytokines involved in cell death signaling such as Fas, TNFSF18, TNFSF10C) were down-regulated (Supporting Information Figure 2). Comprehensive functional annotation chart was obtained by clustering more than 2-fold up or down regulated cytokines using DAVID Bioinformatic Resources (as described in Refs. 26,27). A number of functional groups or biological themes are identified as enriched in treated TAMC compared to the untreated control (Sup-porting Information Table 2). As shown in Figures 1d and 1e among all cytokines tested, cytokines involved in developmental process, regulation of cell death, anti-apoptosis and cell proliferation were specifically enriched. Having demonstrated a survival of TAMC and BM-MSC upon heat-shock treatment, we investigated the effect of co-culture of TAMC or BM-MSC with OCC.

TAMC and BM-MSC induced thermotolerance of OCC
The viability of OCC (SKOV3 and CaOV3) were challenged after 30 min of hyperthermia treatment and reached a plateau after 1 hr of treatment (Supporting Information Figure  3A and B). Timelapse study confirmed the kinetic observed in the cytotoxicity assay (Supporting Information Figure 3C). For all following experiments, heat shock duration of 2 hr with recovery period of 2 hr at 37 C was applied.
We used polyvariate flow cytometry to characterize the effect of hyperthermia in co-culture of mesenchymal cells with OCC. Figure 2a represents a typical scatter plot with selection of different sub-populations and the strategy used for analyzing OCC viability upon hyperthermia treatment. In the co-culture context, no significant increase in cell viability was observed prior to the heat-shock (Figs. 2a and 2b). When treated at 42 C for 2 hr OCC's viability was decreased by up to 40% (Figs. 2a and  2b). Twenty-four hours co-culture either with BM-MSC or TAMC was able to rescue OCC from hyperthermia-induced cell death (p < 0.05, n ¼ 5). To differentiate the role of intercellular contact from secreted factors, we reproduced the same set of experiments in the 0.2 lm transwell setting. The positive effect on cell viability persisted in the absence of a direct hetero-cellu-lar contact, highlighting the role of secreted factors in MSC mediated thermotolerance (Figs. 2c and 2d).

CXCR4 is a determinant of MSC induced thermotolerance
In human ovarian tumor microenvironment, chemokine CXCL12 (also known as Stromal Derived Factor 1, SDF1) stimulates proliferation and invasion of OCC by establishing a permissive network. 28 We first investigated the expression of one of CXCL12 receptors: CXCR4. As shown in Figure 3a, both CaOV3 and SKOV3 cell lines exhibit a high expression level of CXCR4. We were previously able to demonstrate that MSC rescued OCC form hyperthermia induced cell death (Fig. 2) here we checked whether rescued cells were expressing higher level of CXCR4. We first demonstrated that the OCC were expressing CXCR4 (Fig. 3a). Using polyvariate flow cytometry we discriminated BM-MSC-eGFP or TAMC-

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eGFP from OCC (CaOV3 or SKOV3) based on their eGFP fluorescence. Within the OCC population, we investigated the calcein violet fluorescence intensity and defined two populations: calcein low (nonviable) and calcein high (viable) (Fig. 3b). Then we determined the CXCR4 expression level in these two populations (Fig. 3b-3d). When co-cultivated with TAMC or BM-MSC, both CaOV3 and SKOV3 resistant to heat-shock exhibited a higher level of CXCR4 expression. According to these data, CXCR4 seems to play a role in the BM-MSC and TAMC induced thermotolerance.

CXCL12 mediates OCC thermotolerance
To further investigate the role of CXCL12 in thermotolerance induction, we stimulated the OCC (CaOV3 and SKOV3) with 100 ng/ml of CXCL12 24 hr prior to heat-shock. As shown in the Figure 4a, CXCL12 stimulation does not increase the percentage of viable cells when at 37 C. When heat shocked, untreated OCC underwent a major hyperthermia induced cell death (87% of calcein high cell against 29% after heat-shock). However, when treated with CXCL12, no significant hyperthermia induced cell death was observed (Figs. 4b and 4c). Hence, the CXCL12 stimulation is sufficient to induce thermotolerance of OCC.

CXCR4 inhibition reverses MSC induced thermotolerance
As CXCL12 is sufficient to protect OCC from hyperthermia induced cell death, we developed a strategy in which we inhibited CXCL12 signaling. OCC/TAMC or BM-MSC co- cultures were treated with blocking CXCR4 antibody 24 hr before heat-shock. Treatment with CXCR4 blocking antibody did not induce any cell death prior to heat-shock on the OCC, TAMC or BM-MSC (Figs. 5a and 5b and data not shown). When exposed to hyperthermia OCC/TAMC or BM-MSC co-cultures did not display major OCC death, as shown previously. However, treatment with a CXCR4 blocking antibody critically sensitized the OCC in the co-culture context (Figs. 5c and 5d). Here, we were able to demonstrate that the MSC-induced thermotolerance relies on CXCR4/ CXCL12 signaling axis.

Discussion
In our study, we were able to demonstrate that TAMC and BM-MSC are able to induce OCC thermotolerance.
Patients with peritoneal carcinosis have very poor survival. Indeed the rate of recurrence is quite high even in patients who underwent optimal debulking surgery and chemotherapy. HIPEC efficacy has been demonstrated in carcinosis from colon and gastric cancer. 8 However, most protocols used in the treatment of ovarian cancers failed to demonstrate significant benefit regarding the overall survival. Ovarian cancer is a different entity from digestive tract cancers. Some investigators even consider ovarian cancer to be a peritoneal disease. 29 Most of the patients (75%) display peritoneal metastasis at the time of diagnosis, compared to the digestive tract cancers (25%). Peritoneal metastasis seems to be an early event in the natural history of the disease. Identifying peritoneal components that play a role in therapeutic resistance is therefore of essence. The role of microenvironment in ovarian cancer has already been illustrated in the literature. [20][21][22] Several studies demonstrated that mesothelial cells from peritoneum of patients with ovarian cancer undergo phenotypic modification. Their transcriptomic profile demonstrated that they provide a pro-tumoral environment. 30,31 In these studies however, the tumor interacting cells were not specifically characterized.
MSC comprise a unique population of cells playing a central role in tissue maintenance and repair. 32 These cells have regenerative ability and multipotent capacity, and can differentiate into osteocytes, adipocytes, chondrocytes or myocytes. 33 Their tropism toward injury sites prompted investigators to study their ability to migrate toward neoplasic lesions considered as nonhealing wounds. Recently, it has been dem-onstrated that MSCs are able to relocate to ovarian neoplasic lesions, where they participate in the formation of the tumor stroma. Kidd et al. demonstrated that the MSCs injected in the peritoneal cavity co-localize to the ovarian tumor xenografts. 34 Furthermore Lee et al. 35 demonstrated that LPA is responsible for enhanced migration of human adipocyte derived stem cells in response to malignant ascites from ovarian cancer patients through activation of LPA-receptor. Finally Coffelt et al. 36 demonstrated that an inflammatory microenvironment promotes ovarian cancer progression through the recruitment of MSCs in an LL-27 dependant manner. We were recently able to demonstrate that TAMC induce increased proliferation and chemoresistance through IL8 and IL6 secretion and oncologic trogocytosis. 23 Therefore, we find the study of Cho et al. 37 demonstrating a cytotoxic effect of heated MSC on ovarian cancer cell lines quite intriguing.
In our study, we used TAMC isolated from cancer cell aggregates or BM-MSC in a co-culture context. It has been demonstrated that MSC undergo profound modification within peri-tumoral stroma. Indeed, co-culture of MSC with ovarian cancer cell lines induced the expression of tumor associated fibroblast markers such as TnC, TSP-a, FSP and increased the expression of alpha-SMA, FAP and desmin. 38,39 We therefore used the TAMC isolated from ovarian cancer aggregates. These cells have been modified by their contact with the OCC, and might therefore be more relevant. To avoid cell-type specific effects, we controlled their functional effect using BM-MSC from different donors in a co-culture setting.
We were able to demonstrate that neither TAMC nor BM-MSC underwent apoptosis when submitted to heat shock. Transcriptomic analysis of TAMC did not display upregulation of pro-apoptotic factors when exposed to heatshock. The cytokine arrays performed on TAMC after heatshock defined a pro-tumoral cytokine environment, rather than a cytotoxic one. For example, pro-survival cytokines such as FGF6, CSF2, IL2 were up-regulated. The tumor stroma is indeed a complex environment and it is hard to speculate on the role of each cytokine on their own. Therefore, we analyzed our cytokine arrays by clustering up-regulated and down-regulated cytokines using DAVID software. 26,27 We were able to demonstrate down regulation of cytokines involved in pro-apoptotic pathways as well as an up regulation of cytokines implicated in cell proliferation.
Our approach was very different from the approach of Cho et al. 37 To recapitulate a situation as close to the clinical setting as possible we used polyvariate flow cytometry, and were able to perform all hyperthermia experiments in a coculture context. While in vitro research is far from the clinical scenario, this was the best way to mimic the complex interaction between OCC and TAMC or MSC. We acknowledge that the complexity of the tumor stroma is significantly higher than our simple co-culture experiments may suggest. Other cell types such as endothelial cells and fibroblasts have important roles in tumor maintenance and resistance to therapy. The diverse components of the extra-cellular matrix also play an important role in tumor physiology. Finally, tri-dimensional aspect of the tumor should be considered when performing resistance assays. Nevertheless, a complex and practical tridimensional cultures have not yet been developed to allow for the reproducible assessment of therapeutic modalities.
In our co-culture context, we were able to demonstrate the induction of heat-shock resistance. This was concordant with our previously published results on the protective role of TAMC inducing chemoresistance and immunoevasion as well as with the data of other studies demonstrating supportive role of the MSC in ovarian tumor growth. 24,25 The discrepancy of our results with the results of the Cho et al. might be due to the methodological differences. Cho et al. 37 demonstrated that conditioned media from MSC submitted to heat shock presented toxicity. However it seems than when the thermo-resistance experiments were performed in the co-culture, the TAMC as well BM-MSC provided a protective environment.
Determining the role of TAMC and BM-MSC in inducing heat-shock resistance lead to us to identify the molecular basis of this effect. As the effect was not contact mediated, one or several cytokines qualified as strong candidates. Among these, CXCL12 has been widely implicated in cancer metastasis. 40 In particular, in the context of ovarian carcinoma it has been demonstrated that lysophosphatidic acid stimulates secretion of VEGF and CXCL12. 38,41 Kajiyama et al. 28 demonstrated the role of CXCL12 in the occurrence of peritoneal metastasis. Indeed CXCL12 increased cell adhesion to peritoneal mesothelial cells, and inhibiting CXCL12/CXCR4 axis would result in a reduction of peritoneal disease in mice. As in other tumors, CXCL12 contributed to the increased invasiveness of OCC through the activation of matrix metalloproteinase-2 and 9. 42 Human peritoneal mesothelial cells have been described to produce a significant quantity of CXCL12. 40 Next to the metastatic properties of the CXCL12, its role in the proliferation of OCC has also been described. Indeed, CXCL12 seems to work in collaboration with EGF receptor to enhance ovarian cancer cell proliferation. 43 We first demonstrated that CXCL12 was able to induce resistance to hyperthermia. In co-culture experiments, through the blocking of the CXCR4 axis, we were able to demonstrate the reversion of the thermotolerance. The effect was similar when using TAMC or BM-MSC ruling out a specific celltype effect. The role of CXCL12 does not rule out synergistic interaction or the role of other cytokines, and this should be addressed using a screening strategy. However as chemical inhibitors of CXCL12 have been tried in Phase I clinical trial, we suggest that CXCL12 blockade might be used as adjuvant therapy during HIPEC.
In this report, we demonstrate that TAMC and BM-MCS exert their thermo-protective effect by the elaboration of the CXCL12. Its role in resistance to therapeutic regimens in ovarian cancer has not yet been documented. We had previously demonstrated the role of TAMC in resistance to chemotherapy. Therefore in our study we specifically focused on the mechanism implicated in thermotolerance.
The results of the clinical trials are not yet convincing to consider HIPEC as standard treatment in ovarian cancers. HIPEC was used in ovarian cancer carinosis upon evidences accumulated in the treatment of colon cancer carcinosis or peritoneal mesotheliome. Indeed the high morbidity rate as well as the small benefits demonstrates the need for further pre-clinical studies. Moreover we also need to evaluate the role of hyperthermia associated to other modalities than chemotherapy, such as recent targeted therapies, or antiangiogenic therapies. Combining therapeutic modalities is mandatory to decrease the treatment failure rate in advanced

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ovarian carcinoma. 44 Several randomized trials have been launched to determine the role of HIPEC in advanced ovarian cancers. The differences in the trial outcomes between colon cancer and ovarian cancer prompt us to investigate treatment modalities in-vitro and in-vivo on animal models to set-up optimal clinical trials.
Whether CXCL12 induced resistance will play a role in the context of hyperthermia associated to chemotherapy should be further studied. However, from our data it seems that the inhibition of CXCL12 might provide sensitization of OCC to hyperthermia, which might therefore be of benefit in the context of HIPEC.