Stromal-epithelial crosstalk provides a suitable microenvironment for the progression of ovarian cancer cells in vitro.
ABSTRACT: The tumor microenvironment plays an important role in the progression of cancer. This study focused on carcinoma-associated fibroblasts (CAFs) and stromal-epithelial interaction between CAFs and epithelial ovarian carcinoma (EOC) cells. We isolated and established primary cultures of CAFs and co-cultured CAFs and EOC cells in vitro. The co-culture conditioned medium (CC-CM) was harvested and its influence on EOC cells was examined. Cytokine, chemokine, and growth factor levels were screened using a biotin label-based human antibody array system. We found that the stromal-epithelial crosstalk provided a suitable microenvironment for the progression of ovarian cancer cells in vitro.
Project description:The Cancer Genome Atlas network has revealed that the 'mesenchymal' epithelial ovarian cancer (EOC) subtype represents the poorest outcome, indicating a crucial role of stromal cancer-associated fibroblasts (CAFs) in disease progression. The cooperative role of CAFs in EOC metastasis has long been recognized, but the mechanisms of stromal CAFs activation are still obscure. Therefore, we carried out an integrative analysis to identify the regulator genes that are responsible for CAFs activation in microdissected tumor stroma profiles. Here, we determined that myristoylated alanine-rich C-kinase substrate (MARCKS) was highly expressed in ovarian stroma, and was required for the differentiation and tumor promoting function of CAFs. Suppression of MARCKS resulted in the loss of CAF features, and diminished role of CAFs in supporting tumor cell growth in 3D organotypic cultures and in murine xenograft model. Mechanistically, we found that MARCKS maintained CAF activation through suppression of cellular senescence and activation of the AKT/Twist1 signaling. Moreover, high MARCKS expression was associated with poor patient survival in EOC. Collectively, our findings identify the potential of MARCKS inhibition as a novel stroma-oriented therapy in EOC.
Project description:Cancer has long been considered a disease that mimics an "unhealed wound," with oncogene-induced secretory activation signals from epithelial cancer cells facilitating stromal fibroblast, endothelial, and inflammatory cell participation in tumor progression. However, the underlying mechanisms that orchestrate cooperative interaction between malignant epithelium and the stroma remain largely unknown. Here, we identified interleukin-1? (IL-1?) as a stromal-acting chemokine secreted by ovarian cancer cells, which suppresses p53 protein expression in cancer-associated fibroblasts (CAFs). Elevated expression of IL-1? and cognate receptor IL-1R1 in ovarian cancer epithelial cells and CAFs independently predicted reduced overall patient survival, as did repressed nuclear p53 in ovarian CAFs. Knockdown of p53 expression in ovarian fibroblasts significantly enhanced the expression and secretion of chemokines IL-8, growth regulated oncogene-alpha (GRO-?), IL-6, IL-1?, and vascular endothelial growth factor (VEGF), significantly increased in vivo mouse xenograft ovarian cancer tumor growth, and was entirely dependent on interaction with, and transcriptional up-regulation of, nuclear factor-kappaB (NF-?B) p65. Our results have uncovered a previously unrecognized circuit whereby epithelial cancer cells use IL-1? as a communication factor instructing stromal fibroblasts through p53 to generate a protumorigenic inflammatory microenvironment. Attenuation of p53 protein expression in stromal fibroblasts generates critical protumorigenic functionality, reminiscent of the role that oncogenic p53 mutations play in cancer cells. These findings implicate CAFs as an important target for blocking inflammation in the tumor microenvironment and reducing tumor growth.
Project description:Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-?2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.
Project description:BACKGROUND:The microenvironment plays a major role in the onset and progression of metastasis. Epithelial ovarian cancer (EOC) tends to metastasize to the peritoneal cavity where interactions within the microenvironment might lead to chemoresistance. Mesothelial cells are important actors of the peritoneal homeostasis; we determined their role in the acquisition of chemoresistance of ovarian tumours. METHODOLOGY/PRINCIPAL FINDINGS:We isolated an original type of stromal cells, referred to as "Hospicells" from ascitis of patients with ovarian carcinosis using limiting dilution. We studied their ability to confer chemoresistance through heterocellular interactions. These stromal cells displayed a new phenotype with positive immunostaining for CD9, CD10, CD29, CD146, CD166 and Multi drug resistance protein. They preferentially interacted with epithelial ovarian cancer cells. This interaction induced chemoresistance to platin and taxans with the implication of multi-drug resistance proteins. This contact enabled EOC cells to capture patches of the Hospicells membrane through oncologic trogocytosis, therefore acquiring their functional P-gp proteins and thus developing chemoresistance. Presence of Hospicells on ovarian cancer tissue micro-array from patients with neo-adjuvant chemotherapy was also significantly associated to chemoresistance. CONCLUSIONS/SIGNIFICANCE:This is the first report of trogocytosis occurring between a cancer cell and an original type of stromal cell. This interaction induced autonomous acquisition of chemoresistance. The presence of stromal cells within patient's tumour might be predictive of chemoresistance. The specific interaction between cancer cells and stromal cells might be targeted during chemotherapy.
Project description:Most solid tumors contain cancer-associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF-like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF-specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n?=?145) but not in the stroma of normal ovaries (n?=?4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n?=?8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC.
Project description:TGF-? has limited effects on ovarian cancer cells, but its contributions to ovarian tumor growth might be mediated through elements of the tumor microenvironment. In the present study, we tested the hypothesis that TGF modulates ovarian cancer progression by modulating the contribution of cancer-associated fibroblasts (CAF) that are present in the microenvironment. Transcriptome profiling of microdissected stromal and epithelial components of high-grade serous ovarian tumors and TGF-?-treated normal ovarian fibroblasts identified versican (VCAN) as a key upregulated target gene in CAFs. Functional evaluations in coculture experiments showed that TGF-? enhanced the aggressiveness of ovarian cancer cells by upregulating VCAN in CAFs. VCAN expression was regulated in CAFs through TGF-? receptor type II and SMAD signaling. Upregulated VCAN promoted the motility and invasion of ovarian cancer cells by activating the NF-?B signaling pathway and by upregulating expression of CD44, matrix metalloproteinase-9, and the hyaluronan-mediated motility receptor. Our work identified a TGF-?-inducible gene signature specific to CAFs in advanced high-grade serous ovarian tumors, and showed how TGF-? stimulates ovarian cancer cell motility and invasion by upregulating the CAF-specific gene VCAN. These findings suggest insights to develop or refine strategies for TGF-?-targeted therapy of ovarian cancer.
Project description:There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinoma-promoting phenotypes of breast and ovarian CAFs.
Project description:The interplay between tumor microenvironment and cancer that causes chemoresistance remains unclear. By analyzing public available microarray datasets, we identified that periostin (POSTN) was overexpressed in cancer stroma in epithelial ovarian cancer (EOC) patients. Immunohistochemistry analysis showed overexpression of stromal POSTN is a powerful independent poor prognostic predictor for EOC patients. Furthermore, patients with high levels of stromal POSTN tend to have higher percentage of cisplatin resistance compared to those with low levels of stromal POSTN. Moreover, we found POSTN treatment can induce cisplatin resistant and activate AKT pathway in A2780 cells in vitro. Inhibition of AKT activity by AKT inhibitor MK-2206 abolished POSTN-induced AKT activation and cisplatin resistance in vitro. Taken together, we found high POSTN expression in cancer microenvironment is correlated with poor prognosis in EOC patients and associated with platinum resistance. The effect of POSTN in cancer stroma cells may activate AKT pathway in tumor and AKT inhibitor can be beneficial to augment the efficacy of existing cancer therapeutics.
Project description:Tumorigenesis depends on intricate interactions between genetically altered tumor cells and their surrounding microenvironment. While oncogenic drivers in lung squamous carcinoma (LUSC) have been described, the role of stroma in modulating tissue architecture, particularly cell polarity, remains unclear. Here, we report the establishment of a 3D coculture system of LUSC epithelial cells with cancer-associated fibroblasts (CAFs) and extracellular matrix that together capture key components of the tumor microenvironment (TME). Single LUSC epithelial cells develop into acinar-like structures with 0.02% efficiency, and addition of CAFs provides proper tumor-stromal interactions within an appropriate 3D architectural context. Using this model, we recapitulate key pathological changes during tumorigenesis, from hyperplasia to dysplasia and eventually invasion, in malignant LUSC spheroids that undergo phenotypic switching in response to cell intrinsic and extrinsic changes. Overexpression of SOX2 is sufficient to mediate the transition from hyperplasia to dysplasia in LUSC spheroids, while the presence of CAFs makes them invasive. Unexpectedly, CAFs suppress the activity of high SOX2 levels, restore hyperplasia, and enhance the formation of acinar-like structures. Taken together, these observations suggest that stromal factors can override cell intrinsic oncogenic changes in determining the disease phenotype, thus providing fundamental evidence for the existence of dynamic reciprocity between the nucleus and the TME of LUSC.
Project description:Fibroblasts are a major component of cancer tissue and known to contribute to cancer progression. However, it remains unknown whether they are derived from local fibroblasts or of other origin. This study was designed to identify the contribution of local stromal cells to cancer stroma in human epithelial ovarian cancer. Seventy-six cases of surgically resected primary ovarian carcinoma (48 cases confined to the ovaries and 28 cases with distant metastases) and 17 cases of secondary ovarian tumor (e.g. colon cancer metastasized to the ovary) were enrolled in this study. The tissues were immunostained for forkhead box protein L2 (FOXL2), a transcription factor crucial for ovarian development and function, and markers for cancer-associated fibroblasts (CAFs) and inflammatory cells. Under normal condition, FOXL2 expression was restricted to ovarian stromal cells and some other types of cells in female genital tracts and never found in other sites of the body. FOXL2-positive cells were found in all primary and secondary tumors in the ovary, and were the dominant stromal cells in most cases. In contrast, only a few FOXL2-positive cells were found in peritoneal seeding sites of four serous carcinoma cases, and all the other tumors at extraovarian sites had no FOXL2-positive cells. FOXL2-positive cells in the ovarian lesion variably expressed CAFs markers, such as alpha-smooth muscle actin and fibroblast activating protein, as determined by double immunostaining. Background inflammation, but not histological subtype or origin of the neoplasm seemed to correlate with the proportion of FOXL2-positive cells. These results suggest that ovarian stromal cells are the main source of cancer stroma in the ovary but do not seem to move to distant sites via circulation together with tumor cells. Our results also support the hypothesis that cancer-associated fibroblasts may originate locally, which was previously demonstrated using animal models.