Extracellular Vesicles (EVs) have received considerable attention in recent years, both as mediators of intercellular communication pathways that lead to tumor progression, and as potential sources for discovery of novel cancer biomarkers. For many years, research on EVs has mainly investigated either the mechanism of biogenesis and cargo selection and incorporation, or the methods of EV isolation from available body fluids for biomarker discovery. Recent studies have highlighted the existence of different populations of cancer-derived EVs, with distinct molecular cargo, thus pointing to the possibility that the various EV populations might play diverse roles in cancer and that this does not happen randomly. However, data attributing cancer specific intercellular functions to given populations of EVs are still limited. A deeper functional, biochemical and molecular characterization of the various EV classes might identify more selective clinical markers, and significantly advance our knowledge of the pathogenesis and disease progression of many cancer types.

Cell deformability is a label-free biomarker of cell state in physiological and disease contexts ranging from stem cell differentiation to cancer progression. Harnessing deformability as a phenotype for screening applications requires a method that can simultaneously measure the deformability of hundreds of cell samples and can interface with existing high throughput facilities. Here we present a scalable cell filtration device, which relies on the pressure-driven deformation of cells through a series of pillars that are separated by micron-scale gaps on the timescale of seconds: less deformable cells occlude the gaps more readily than more deformable cells, resulting in decreased filtrate volume which is measured using a plate reader. The key innovation in this method is that we design customized arrays of individual filtration devices in a standard 96-well format using soft lithography, which enables multiwell input samples and filtrate outputs to be processed with higher throughput using automated pipette arrays and plate readers. To validate high throughput filtration to detect changes in cell deformability, we show the differential filtration of human ovarian cancer cells that have acquired cisplatin-resistance, which is corroborated with cell stiffness measurements using quantitative deformability cytometry. We also demonstrate differences in the filtration of human cancer cell lines, including ovarian cancer cells that overexpress transcription factors (Snail, Slug), which are implicated in epithelial-to-mesenchymal transition; breast cancer cells (malignant versus benign); and prostate cancer cells (highly versus weekly metastatic). We additionally show how the filtration of ovarian cancer cells is affected by treatment with drugs known to perturb the cytoskeleton and the nucleus. Our results across multiple cancer cell types with both genetic and pharmacologic manipulations demonstrate the potential of this scalable filtration device to screen cells based on their deformability.

Treatment of prostate cancer (PC) by androgen suppression promotes the emergence of aggressive variants that are androgen receptor (AR) independent. Here we identify the transcription factor ONECUT2 (OC2) as a master regulator of AR networks in metastatic castration-resistant prostate cancer (mCRPC). OC2 acts as a survival factor in mCRPC models, suppresses the AR transcriptional program by direct regulation of AR target genes and the AR licensing factor FOXA1, and activates genes associated with neural differentiation and progression to lethal disease. OC2 appears active in a substantial subset of human prostate adenocarcinoma and neuroendocrine tumors. Inhibition of OC2 by a newly identified small molecule suppresses metastasis in mice. These findings suggest that OC2 displaces AR-dependent growth and survival mechanisms in many cases where AR remains expressed, but where its activity is bypassed. OC2 is also a potential drug target in the metastatic phase of aggressive PC.