Ovarian-Cancer-Associated Extracellular Vesicles: Microenvironmental Regulation and Potential Clinical Applications.

Ovarian cancer (OC) is one of the most diagnosed gynecological cancers in women. Due to the lack of effective early stage screening, women are more often diagnosed at an advanced stage; therefore, it is associated with poor patient outcomes. There are a lack of tools to identify patients at the highest risk of developing this cancer. Moreover, early detection strategies, therapeutic approaches, and real-time monitoring of responses to treatment to improve survival and quality of life are also inadequate. Tumor development and progression are dependent upon cell-to-cell communication, allowing cancer cells to re-program cells not only within the surrounding tumor microenvironment, but also at distant sites. Recent studies established that extracellular vesicles (EVs) mediate bi-directional communication between normal and cancerous cells. EVs are highly stable membrane vesicles that are released from a wide range of cells, including healthy and cancer cells. They contain tissue-specific signaling molecules (e.g., proteins and miRNA) and, once released, regulate target cell phenotypes, inducing a pro-tumorigenic and immunosuppressive phenotype to contribute to tumor growth and metastasis as well as proximal and distal cell function. Thus, EVs are a "fingerprint" of their cell of origin and reflect the metabolic status. Additionally, via the capacity to evade the immune system and remain stable over long periods in circulation, EVs can be potent therapeutic agents. This review examines the potential role of EVs in the different aspects of the tumor microenvironment in OC, as well as their application in diagnosis, delivery of therapeutic agents, and disease monitoring.

Altered Calcium Influx Pathways in Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) represent an important component of the tumour microenvironment and are implicated in disease progression. Two outstanding questions in cancer biology are how CAFs arise and how they might be targeted therapeutically. The calcium signal also has an important role in tumorigenesis. To date, the role of calcium signalling pathways in the induction of the CAF phenotype remains unexplored. A CAF model was generated through exogenous transforming growth factor beta 1 (TGFß1) stimulation of the normal human mammary fibroblast cell line, HMF3S (HMF3S-CAF), and changes in calcium signalling were investigated. Functional changes in HMF3S-CAF calcium signalling pathways were assessed using a fluorescent indicator, gene expression, gene-silencing and pharmacological approaches. HMF3S-CAF cells demonstrated functionally altered calcium influx pathways with reduced store-operated calcium entry. In support of a calcium signalling switch, two voltage-gated calcium channel (VGCC) family members, CaV1.2 and CaV3.2, were upregulated in HMF3S-CAFs and a subset of patient-derived breast CAFs. Both siRNA-mediated silencing and pharmacological inhibition of CaV1.2 or CaV3.2 significantly impaired CAF activation in HMF3S cells. Our findings show that VGCCs contribute to TGFß1-mediated induction of HMF3S-CAF cells and both transcriptional interference and pharmacological antagonism of CaV1.2 and CaV3.2 inhibit CAF induction. This suggests a potential therapeutic role for targeting calcium signalling in breast CAFs.

Characterization of Immune Cell Subsets of Tumor Infiltrating Lymphocytes in Brain Metastases.

The heterogeneity of tumor infiltrating lymphocytes (TILs) is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune-cell subsets in brain metastasis tissues to test immunosuppressive routes involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune-cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and IBA-1, and analyzed an average of 15,000 cells per sample. Classifying tumors as either high (>30%) or low (<30%) TILs, we found that increased TILs density correlated with survival. Phenotyping these TILs we found tumors with low TILs had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells (p < 0.01) as well as in their microenvironment (p < 0.001). Contrastingly, the tumors with high TILs displayed higher levels of microglia, as measured by IBA-1 expression. Low TILs-tumors displayed CD8+ T-cells that co-express VISTA (p < 0.01) significantly more compared to high TILs group, where CD8+cells significantly co-express IBA-11 (p < 0.05). These results were supported by RNA analysis of a publicly available, independent cohort. Our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.