TRPC3 signalling contributes to the biogenesis of extracellular vesicles.

Extracellular vesicles (EVs) contribute to a wide range of pathological processes including cancer progression, yet the molecular mechanisms underlying their biogenesis remain incompletely characterized. The development of tetraspanin-based pHluorin reporters has enabled the real-time analysis of EV release at the plasma membrane. Here, we employed CD81-pHluorin to investigate mechanisms of EV release in ovarian cancer (OC) cells and report a novel role for the Ca2+-permeable transient receptor potential (TRP) channel TRPC3 in EV-mediated communication. We found that specific activation of TRPC3 increased Ca2+ signalling in SKOV3 cells and stimulated an immediate increase in EV release. Ca2+-stimulants histamine and ionomycin likewise induced EV release, and imaging analysis revealed distinct stimulation-dependent temporal and spatial release dynamics. Interestingly, inhibition of TRPC3 attenuated histamine-stimulated Ca2+-entry and EV release, indicating that TRPC3 is likely to act downstream of histamine signalling in EV biogenesis. Furthermore, we found that direct activation of TRPC3 as well as the application of EVs derived from TRPC3-activated cells increased SKOV3 proliferation. Our data provides insights into the molecular mechanisms and dynamics underlying EV release in OC cells, proposing a key role for TRPC3 in EV biogenesis.

Adipocyte-Derived Extracellular Vesicles Promote Prostate Cancer Cell Aggressiveness by Enabling Multiple Phenotypic and Metabolic Changes.

BACKGROUND: In recent decades, obesity has widely emerged as an important risk factor for prostate cancer (PCa). Adipose tissue and PCa cells have been shown to orchestrate a complex interaction network to support tumor growth and evolution; nonetheless, the study of this communication has only been focused on soluble factors, although increasing evidence highlights the key role of extracellular vesicles (EVs) in the modulation of tumor progression. METHODS AND RESULTS: In the present study, we found that EVs derived from 3T3-L1 adipocytes could affect PC3 and DU145 PCa cell traits, inducing increased proliferation, migration and invasion. Furthermore, conditioning of both PCa cell lines with adipocyte-released EVs resulted in lower sensitivity to docetaxel, with reduced phosphatidylserine externalization and decreased caspase 3 and PARP cleavage. In particular, these alterations were paralleled by an Akt/HIF-1α axis-related Warburg effect, characterized by enhanced glucose consumption, lactate release and ATP production. CONCLUSIONS: Collectively, these findings demonstrate that EV-mediated crosstalk exists between adipocytes and PCa, driving tumor aggressiveness.

Extracellular Vesicles: Emerging Modulators of Cancer Drug Resistance.

Extracellular vesicles (EVs) have recently emerged as crucial modulators of cancer drug resistance. Indeed, it has been shown that they can directly sequester anti-tumor drugs, decreasing their effective concentration at target sites. Moreover, they facilitate the horizontal transfer of specific bioactive cargoes able to regulate proliferative, apoptotic, and stemness programs in recipient cells, potentially conferring a resistant phenotype to drug-sensitive cancer cells. Finally, EVs can mediate the communication between the tumor and both stromal and immune cells within the microenvironment, promoting treatment escape. In this context, clarifying the EV-driven resistance mechanisms might improve not only tumor diagnosis and prognosis but also therapeutic outcomes. Detailed cellular and molecular events occurring during the development of EV-mediated cancer drug resistance are described in this review article.

A practical toolkit to study aspects of the metastatic cascade in vitro.

While metastasis - the spread of cancer from the primary location to distant sites in the body - remains the principle cause of cancer death, it is incompletely understood. It is a complex process, requiring the metastatically successful cancer cell to negotiate a formidable series of interconnected steps, which are described in this paper. For each step, we review the range of in vitro assays that may be used to study them. We also provide a range of detailed, step-by-step protocols that can be undertaken in most modestly-equipped laboratories, including methods for converting qualitative observations into quantitative data for analysis. Assays include: (1) a gelatin degradation assay to study the ability of endothelial cells to degrade extracellular matrix during tumour angiogenesis; (2) the morphological characterisation of cells undergoing epithelial-mesenchymal transition (EMT) as they acquire motility; (3) a 'scratch' or 'wound-healing' assay to study cancer cell migration; (4) a transwell assay to study cancer cell invasion through extracellular matrix; and (5) a static adhesion assay to examine cancer cell interactions with, and adhesion to, endothelial monolayers. This toolkit of protocols will enable researchers who are interested in metastasis to begin to focus on defined aspects of the process. It is only by further understanding this complex, fascinating and clinically relevant series of events that we may ultimately devise ways of better treating, or even preventing, cancer metastasis. The assays may also be of more broad interest to researchers interested in studying aspects of cellular behaviour in relation to other developmental and disease processes.

The Challenges and Possibilities of Extracellular Vesicles as Therapeutic Vehicles.

Extracellular vesicles (EVs) are small lipid-enclosed particles that can carry various types of cargo, including proteins, nucleic acids and metabolites. They are known to be released by all cell types and can be taken up by other cells, leading to the transfer of the cargo they carry. As such, they represent an important type of intercellular signalling and a natural mechanism for transferring macromolecules between cells. This ability to transfer cargo could be harnessed to deliver therapeutic molecules. Indeed, a growing body of work has described the attempt by the field to utilise EVs to deliver a range of therapeutics including RNAi, CRISPR/Cas9 and chemotherapeutics, to a specific target tissue. However, there are numerous barriers associated with the use of EVs as therapeutic vehicles, including the challenge of efficiently loading therapeutics into EVs, avoiding clearance of the EVs from circulation, targeting the correct tissue type and the inefficiency of internalisation and functional delivery of the cargo. Despite these difficulties, EVs represent a tremendous therapeutic opportunity, both for the delivery of exogenous cargo, as well as the therapeutic benefit of targeting aberrant EV signalling or treating patients with natural EVs, such as those released by mesenchymal stem cells. This review describes current knowledge on the therapeutic potential of EVs and the challenges faced by the field. Many of these challenges are due to a lack of complete understanding of EV function, but further research in this area should continue to yield new solutions that will lead to the use of EVs in the clinic.

Detecting ovarian cancer using extracellular vesicles: progress and possibilities.

Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumor growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so, they represent an excellent potential source of novel biomarkers for the disease. In this review, we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but many studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insights into the stage of the disease and predict the likely therapeutic outcome. There remain many challenges to the use of EVs as biomarkers, but, through ongoing research and innovation in this exciting field, there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome.