Cancer associated fibroblasts (CAFs) are a major constituent of the tumor stroma, but little is known about how cancer cells transform normal fibroblasts into CAFs. miRNAs are small noncoding RNA molecules that negatively regulate gene expression at a posttranscriptional level. While it is clearly established that miRNAs are deregulated in human cancers, it is not known whether miRNA expression in resident fibroblasts is affected by their interaction with cancer cells. We found that in ovarian CAFs, miR-31 and miR-214 are downregulated while miR-155 is upregulated when compared to normal or tumor-adjacent fibroblasts. Mimicking this deregulation by transfecting miRNAs and miRNA inhibitors induced a functional conversion of normal fibroblasts into CAFs, and the reverse experiment resulted in the reversion of CAFs into normal fibroblasts. The miRNA-reprogrammed normal fibroblasts and patient-derived CAFs shared a large number of upregulated genes highly enriched in chemokines, which are known to be important for CAF function. The most highly upregulated chemokine, CCL5, was found to be a direct target of miR-214. These results indicate that ovarian cancer cells reprogram fibroblasts to become CAFs through the action of miRNAs. Targeting these miRNAs in stromal cells could have therapeutic benefit.

E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of A 5 -integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an A 5 B 1 -integrin-blocking antibody. When E-cadherin is silenced, A 5 -integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogenactivated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/B-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of A 5 -integrin, i.p. treatment with an A 5 B 1 -integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). A 5 -Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had A 5 -integrin overexpression, and 39% had some level of A 5 -integrin expression. The median survival for patients with high A 5 -integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified A 5 -integrin upregulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.

Ovarian cancer (OvCa) is characterized by widespread and rapid metastasis in the peritoneal cavity. Visceral adipocytes promote this process by providing fatty acids (FAs) for tumour growth. However, the exact mechanism of FA transfer from adipocytes to cancer cells remains unknown. This study shows that OvCa cells co-cultured with primary human omental adipocytes express high levels of the FA receptor, CD36, in the plasma membrane, thereby facilitating exogenous FA uptake. Depriving OvCa cells of adipocyte-derived FAs using CD36 inhibitors and short hairpin RNA knockdown prevented development of the adipocyte-induced malignant phenotype. Specifically, inhibition of CD36 attenuated adipocyte-induced cholesterol and lipid droplet accumulation and reduced intracellular reactive oxygen species (ROS) content. Metabolic analysis suggested that CD36 plays an essential role in the bioenergetic adaptation of OvCa cells in the adipocyte-rich microenvironment and governs their metabolic plasticity. Furthermore, the absence of CD36 affected cellular processes that play a causal role in peritoneal dissemination, including adhesion, invasion, migration and anchorage independent growth. Intraperitoneal injection of CD36-deficient cells or treatment with an anti-CD36 monoclonal antibody reduced tumour burden in mouse xenografts. Moreover, a matched cohort of primary and metastatic human ovarian tumours showed upregulation of CD36 in the metastatic tissues, a finding confirmed in three public gene expression datasets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in ovarian cancer cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

The role of the fibronectin receptor, α5β1-integrin, as an adhesion receptor and in angiogenesis, is well established. However, its role in cancer cell invasion and metastasis is less clear. We describe a novel mechanism by which fibronectin regulates ovarian cancer cell signaling and promotes metastasis. Fibronectin binding to α5β1-integrin led to a direct association of α5-integrin with the receptor tyrosine kinase, c-Met, activating it in a hepatocyte growth factor/scatter factor (HGF/SF) independent manner. Subsequently, c-Met associated with Src and activated Src and focal adhesion kinase (FAK). Inhibition of α5β1-integrin decreased the phosphorylation of c-Met, FAK and Src, both in vitro and in vivo. Independent activation of c-Met by its native ligand, HGF/SF, or overexpression of a constitutively active FAK in HeyA8 cells could overcome the effect of α5β1-integrin inhibition on tumor cell invasion, indicating that α5β1-integrin is upstream of c-Met, Src and FAK. Inhibition of α5β1-integrin on cancer cells in two xenograft models of ovarian cancer metastasis resulted in a significant decrease of tumor burden, which was independent of the effect of α5β1-integrin on angiogenesis. These data suggest that fibronectin promotes ovarian cancer invasion and metastasis through an α5β1-integrin/c-Met/FAK/Src dependent signaling pathway, transducing signals through c-Met in a HGF/SF independent manner.

Objective Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. Methods To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119, UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. Results Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. Conclusions Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community.

Cancer cells grow in an environment comprised of multiple components that support tumor growth and contribute to therapy resistance. Major cell types in the tumor micro-environment are fibroblasts, endothelial cells and infiltrating immune cells all of which communicate with cancer cells. One way that these cell types promote cancer progression is by altering expression of miRNAs, small noncoding RNAs that negatively regulate protein expression, either in the cancer cells or in associated normal cells. Changes in miRNA expression can be brought about by direct interaction between the stromal cells and cancer cells, by paracrine factors secreted by any of the cell types, or even through direct communication between cells through secreted miRNAs. Understanding the role of miRNAs in the complex interactions between the tumor and cells in its micro-environment is necessary if we are to understand tumor progression and devise new treatments.

The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.

The reliable detection, sizing, and sorting of viruses and nanoparticles is important for biosensing, environmental monitoring, and quality control. Here we introduce an optical detection scheme for the real-time and label-free detection and recognition of single viruses and larger proteins. The method makes use of nanofluidic channels in combination with optical interferometry. Elastically scattered light from single viruses traversing a stationary laser focus is detected with a differential heterodyne interferometer and the resulting signal allows single viruses to be characterized individually. Heterodyne detection eliminates phase variations due to different particle trajectories, thus improving the recognition accuracy as compared to standard optical interferometry. We demonstrate the practicality of our approach by resolving nanoparticles of various sizes, and detecting and recognizing different species of human viruses from a mixture. The detection system can be readily integrated into larger nanofluidic architectures for practical applications.