Extracellular Vesicles-A Source of RNA Biomarkers for the Detection of Breast Cancer in Liquid Biopsies.

Over the past decade, extracellular vesicles (EVs) have emerged as a promising source of cancer-derived RNAs for liquid biopsies. However, blood contains a pool of heterogeneous EVs released by a variety of cell types, making the identification of cancer RNA biomarkers challenging. Here, we performed deep sequencing of plasma EV RNA cargo in 32 patients with locally advanced breast cancer (BC) at diagnosis and 7 days after breast surgery and in 30 cancer-free healthy controls (HCs). To identify BC-derived RNA biomarkers, we searched for RNAs that had higher levels in BC EVs at the time of diagnosis compared with HCs and decreased after surgery. Data analysis showed that the fractions of miRNAs, snRNAs, snoRNAs, and tRFs were increased, but the fraction of lncRNAs was decreased in BC EVs as compared to HCs. BC-derived biomarker candidates were identified across various RNA biotypes. Considered individually, they had very high specificity but moderate sensitivity for the detection of BC, whereas a biomarker model composed of eight RNAs: SNORD3H, SNORD1C, SNORA74D, miR-224-5p, piR-32949, lnc-IFT-122-2, lnc-C9orf50-4, and lnc-FAM122C-3 was able to distinguish BC from HC EVs with an AUC of 0.902 (95% CI = 0.872-0.931, p = 3.4 × 10-9) in leave-one-out cross-validation. Furthermore, a number of RNA biomarkers were correlated with the ER and HER2 expression and additional biomarker models were created to predict hormone receptor and HER2 status. Overall, this study demonstrated that the RNA composition of plasma EVs is altered in BC patients and that they contain cancer-derived RNA biomarkers that can be used for BC detection and monitoring using liquid biopsies.

Comprehensive characterization of RNA cargo of extracellular vesicles in breast cancer patients undergoing neoadjuvant chemotherapy.

Extracellular vesicles (EVs) are g7aining increased attention as carriers of cancer-derived molecules for liquid biopsies. Here, we studied the dynamics of EV levels in the plasma of breast cancer (BC) patients undergoing neoadjuvant chemotherapy (NAC) and explored the relevance of their RNA cargo for the prediction of patients' response to the therapy. EVs were isolated from serial blood samples collected at the time of diagnosis, at the end of NAC, and 7 days, 6, and 12 months after the surgery from 32 patients with locally advanced BC, and 30 cancer-free healthy controls (HCs) and quantified by nanoparticle tracking analysis. The pre-treatment levels of EVs in BC patients were higher than in HCs, significantly increased during the NAC and surgery, and decreased to the levels found in HCs 6 months after surgery, thus showing that a substantial fraction of plasma EVs in BC patients are produced due to the disease processes and treatment. RNA sequencing analysis revealed that the changes in the EV levels were associated with the alterations in the proportions of various RNA biotypes in EVs. To search for RNA biomarkers that predict response to the NAC, patients were dichotomized as responders and non-responders based on Miller-Payne grades and differential expression analyses were carried out between responders and non-responders, and HCs. This resulted in the identification of 6 miRNAs, 4 lncRNAs, and 1 snoRNA that had significantly higher levels in EVs from non-responders than responders at the time of diagnosis and throughout the NAC, and significantly lower levels in HCs, thus representing biomarkers for the prediction of response to NAC at the time of diagnosis. In addition, we found 14 RNAs representing piRNA, miRNA, lncRNA, snoRNA, and snRNA biotypes that were induced by NAC in non-responders and 2 snoRNAs and 1 piRNA that were induced by NAC in patients with early disease progression, thus warranting further functional studies on their role in chemoresistance and metastasis.

Effects of urinary extracellular vesicles from prostate cancer patients on the transcriptomes of cancer-associated and normal fibroblasts.

BACKGROUND: Increasing evidence suggests that cancer-derived extracellular vesicles (EVs) alter the phenotype and functions of fibroblasts and trigger the reprogramming of normal fibroblasts into cancer-associated fibroblasts (CAFs). Here, we for the first time studied the effects of urinary EVs from PC patients and healthy males on the transcriptional landscape of prostate CAFs and normal foreskin fibroblasts. METHODS: Patient-derived prostate fibroblast primary cultures PCF-54 and PCF-55 were established from two specimens of PC tissues. EVs were isolated from urine samples of 3 patients with PC and 2 healthy males and used for the treatment of prostate fibroblast primary cultures and normal foreskin fibroblasts. The EV-treated fibroblasts were subjected to RNA sequencing analysis. RESULTS: RNA sequencing analysis showed that the fibroblast cultures differed significantly in their response to urinary EVs. The transcriptional response of foreskin fibroblasts to the urinary EVs isolated from PC patients and healthy controls was very similar and mostly related to the normal functions of fibroblasts. On the contrary, PCF-54 cells responded very differently - EVs from PC patients elicited transcriptional changes related to the regulation of the cell division and chromosome segregation, whereas EVs from healthy males affected mitochondrial respiration. In PCF-55 cells, EVs from both, PC-patients and controls induced the expression of a number of chemokines such as CCL2, CCL13, CXCL1, CXCL8, whereas pathways related to regulation of apoptotic signaling and production of cell adhesion molecules were triggered specifically by EVs from PC patients. CONCLUSION: This study demonstrates that urinary EVs from PC patients and healthy controls elicit distinct transcriptional responses in prostate CAFs and supports the idea that EVs contribute to the generation of functional heterogeneity of CAFs. Moreover, this study suggests that the changes in the gene expression pattern in EV recipient cells might serve as a novel type of functional cancer biomarkers.

Exercise-Induced Extracellular Vesicles Delay the Progression of Prostate Cancer.

Increasing evidence suggests that regular physical exercise not only reduces the risk of cancer but also improves functional capacity, treatment efficacy and disease outcome in cancer patients. At least partially, these effects are mediated by the secretome of the tissues responding to exercise. The secreted molecules can be released in a carrier-free form or enclosed into extracellular vesicles (EVs). Several recent studies have shown that EVs are actively released into circulation during physical exercise. Here, we for the first time investigated the effects of exercise-induced EVs on the progression of cancer in an F344 rat model of metastatic prostate cancer. Although we did not observe a consistent increase in the circulating EV levels, RNA sequencing analysis demonstrated substantial changes in the RNA content of EVs collected before and immediately after forced wheel running exercise as well as differences between EVs from runners at resting state and sedentary rats. The major RNA biotype in EVs was mRNA, followed by miRNA and rRNA. Molecular functions of differentially expressed RNAs reflected various physiological processes including protein folding, metabolism and regulation of immune responses triggered by the exercise in the parental cells. Intravenous administration of exercise-induced EVs into F344 rats with orthotopically injected syngeneic prostate cancer cells PLS10, demonstrated reduction of the primary tumor volume by 35% and possibly-attenuation of lung metastases. Hence, our data provide the first evidence that exercise-induced EVs may modulate tumor physiology and delay the progression of cancer.

A novel 3D heterotypic spheroid model for studying extracellular vesicle-mediated tumour and immune cell communication.

Cancer-derived extracellular vesicles (EVs) have emerged as important mediators of tumour-host interactions, and they have been shown to exert various functional effects in immune cells. In most of the studies on human immune cells, EVs have been isolated from cancer cell culture medium or patients' body fluids and added to the immune cell cultures. In such a setting, the physiological relevance of the chosen EV concentration is unknown and the EV isolation method and the timing of EV administration may bias the results. In the current study we aimed to develop an experimental cell culture model to study EV-mediated effects in human T and B cells at conditions mimicking the tumour microenvironment. We constructed a human prostate cancer cell line PC3 producing GFP-tagged EVs (PC3-CD63-GFP cells) and developed a 3D heterotypic spheroid model composed of PC3-CD63-GFP cells and human peripheral blood mononuclear cells (PBMCs). The transfer of GFP-tagged EVs from PC3-CD63-GFP cells to the lymphocytes was analysed by flow cytometry and fluorescence imaging. The endocytic pathway was investigated using three endocytosis inhibitors. Our results showed that GFP-tagged EVs interacted with a large fraction of B cells, however, the majority of EVs were not internalised by B cells but rather remained bound at the cell surface. T cell subsets differed in their ability to interact with the EVs - 15.7-24.1% of the total CD3+ T cell population interacted with GFP-tagged EVs, while only 0.3-5.8% of CD8+ T were GFP positive. Furthermore, a fraction of EVs were internalised in CD3+ T cells via macropinocytosis. Taken together, the heterotypic PC3-CD63-GFP and PBMC spheroid model provides the opportunity to study the interactions and functional effects of cancer-derived EVs in human immune cells at conditions mimicking the tumour microenvironment.

Extracellular Vesicles as Biomarkers and Therapeutic Targets in Breast Cancer.

Cancer-derived extracellular vesicles (EVs) contain various cancer-associated molecules, such as mutated or overexpressed oncoproteins, glycoproteins, mRNAs, various non-coding RNAs and DNA fragments. They have been shown to propagate phenotypic traits, such as drug resistance, increased proliferation rate, invasiveness and stemness across cancer cells and to mediate cancer-induced immunosuppression. Therefore, cancer-derived EVs have gained increasing attention as cancer biomarkers and therapeutic targets. Unlike circulating tumor cells they are highly abundant in biofluids and, on the contrary to single-molecule circulating biomarkers, they protect their molecular cargo against degradation and may carry molecular signatures associated with specific phenotypes. Herein, we summarize studies investigating EVs as biomarkers in breast cancer and propose scenarios for various clinical applications of EV-based biomarkers in the management of breast cancer. Furthermore, we provide an overview of recent findings regarding the cancer-promoting effects of breast cancer-derived EVs and discuss opportunities for blocking EV-mediated signaling as a therapeutic strategy for breast cancer.

Biodistribution, Uptake and Effects Caused by Cancer-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) have recently emerged as important mediators of intercellular communication. They are released in the extracellular space by a variety of normal and cancerous cell types and have been found in all human body fluids. Cancer-derived EVs have been shown to carry lipids, proteins, mRNAs, non-coding and structural RNAs and even extra-chromosomal DNA, which can be taken up by recipient cells and trigger diverse physiological and pathological responses. An increasing body of evidence suggests that cancer-derived EVs mediate paracrine signalling between cancer cells. This leads to the increased invasiveness, proliferation rate and chemoresistance, as well as the acquisition of the cancer stem cell phenotype. This stimulates angiogenesis and the reprogramming of normal stromal cells into cancer-promoting cell types. Furthermore, cancer-derived EVs contribute to the formation of the pre-metastatic niche and modulation of anti-tumour immune response. However, as most of these data are obtained by in vitro studies, it is not entirely clear which of these effects are recapitulated in vivo. In the current review, we summarize studies that assess the tissue distribution, trafficking, clearance and uptake of cancer-derived EVs in vivo and discuss the impact they have, both locally and systemically.