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Abstract Lung diseases, including lung cancer, are rising causes of global mortality. Despite novel imaging technologies and the development of biomarker assays, the detection of lung cancer remains a significant challenge. However, the lung communicates directly with the external environment and releases aerosolized droplets during normal tidal respiration, which can be collected, stored and analzsed as exhaled breath condensate (EBC). A few studies have suggested that EBC contains extracellular vesicles (EVs) whose microRNA (miRNA) cargos may be useful for evaluating different lung conditions, but the cellular origin of these EVs remains unknown. In this study, we used nanoparticle tracking, transmission electron microscopy, Western blot analyses and super resolution nanoimaging (ONi) to detect and validate the identity of exhaled EVs (exh‐EVs). Using our customizable antibody‐purification assay, EV‐CATCHER, we initially determined that exh‐EVs can be selectively enriched from EBC using antibodies against three tetraspanins (CD9, CD63 and CD81). Using ONi we also revealed that some exh‐EVs harbour lung‐specific proteins expressed in bronchiolar Clara cells (Clara Cell Secretory Protein [CCSP]) and Alveolar Type II cells (Surfactant protein C [SFTPC]). When conducting miRNA next generation sequencing (NGS) of airway samples collected at five different anatomic levels (i.e., mouth rinse, mouth wash, bronchial brush, bronchoalveolar lavage [BAL] and EBC) from 18 subjects, we determined that miRNA profiles of exh‐EVs clustered closely to those of BAL EVs but not to those of other airway samples. When comparing the miRNA profiles of EVs purified from matched BAL and EBC samples with our three tetraspanins EV‐CATCHER assay, we captured significant miRNA expression differences associated with smoking, asthma and lung tumor status of our subjects, which were also reproducibly detected in EVs selectively purified with our anti‐CCSP/SFTPC EV‐CATCHER assay from the same samples, but that confirmed their lung tissue origin. Our findings underscore that enriching exh‐EV subpopulations from EBC allows non‐invasive sampling of EVs produced by lung tissues.

Abstract Background Current clinical criteria do not discriminate well between women who will or those who will not develop ipsilateral invasive breast cancer (IBC), or a DCIS recurrence after a ductal carcinoma in situ (DCIS) diagnosis. The 12-gene Oncotype DX® DCIS assay (RT qPCR gene-based scoring system) was established and shown to predict the risk of subsequent ipsilateral IBC or DCIS recurrence. Recent studies have shown that microRNA (miRNA) expression deregulation can contribute to the development of IBC, but very few have evaluated miRNA deregulation in DCIS lesions. In this study, we sought to determine whether specific miRNA expression changes may correlate with Oncotype DX® DCIS scores. Methods For this study, we used archived formalin-fixed, paraffin-embedded (FFPE) specimens from 41 women diagnosed with DCIS between 2012 and 2018. The DCIS lesions were stratified into low (n = 26), intermediate (n = 10), and high (n = 5) risk score groups using the Oncotype DX® DCIS assay. Total RNA was extracted from DCIS lesions by macro-dissection of unstained FFPE sections, and next-generation small-RNA sequencing was performed. We evaluated the correlation between miRNA expression data and Oncotype score, as well as patient age. RT-qPCR validations were performed to validate the topmost differentially expressed miRNAs identified between the different risk score groups. Results MiRNA sequencing of 32 FFPE DCIS specimens from the three different risk group scores identified a correlation between expression deregulation of 17 miRNAs and Oncotype scores. Our analyses also revealed a correlation between the expression deregulation of 9 miRNAs and the patient’s age. Based on these results, a total of 15 miRNAs were selected for RT-qPCR validation. Of these, miR-190b (p = 0.043), miR-135a (p = 0.05), miR-205 (p = 0.00056), miR-30c (p = 0.011), and miR-744 (p = 0.038) showed a decreased expression in the intermediate/high Oncotype group when compared to the low-risk score group. A composite risk score was established using these 5 miRNAs and indicated a significant association between miRNA expression deregulation and the Oncotype DX® DCIS Score (p

Human tumors are increasingly being described as a complex “ecosystem”, that includes many different cell types, secreted growth factors, extracellular matrix (ECM) components, and microvessels, that altogether create the tumor microenvironment (TME). Within the TME, epithelial cancer cells control the function of surrounding stromal cells and the non-cellular ECM components in an intricate orchestra of signaling networks specifically designed for cancer cells to exploit surrounding cells for their own benefit. Tumor-derived extracellular vesicles (EVs) released into the tumor microenvironment are essential mediators in the reprogramming of surrounding stromal cells, which include cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), tumor-infiltrating lymphocytes (TILs), and tumor endothelial cells (TECs), which are responsible for the promotion of neo-angiogenesis, immune cell evasion, and invasion which are essential for cancer progression. Perhaps most importantly, tumor-derived EVs play critical roles in the metastatic dissemination of tumor cells through their two-fold role in initiating cancer cell invasion and the establishment of the pre-metastatic niche, both of which are vital for tumor cell migration, homing, and colonization at secondary tumor sites. This review discusses extracellular vesicle trafficking within the tumor microenvironment and pre-metastatic niche formation, focusing on the complex role that EVs play in orchestrating cancer-to-stromal cell communication in order to promote the metastatic dissemination of cancer cells.

Abstract Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory‐based purification techniques rely on the physical properties of small‐EVs rather than their inherited cellular fingerprints. We established a highly‐selective purification assay, termed EV‐CATCHER, initially designed for high‐throughput analysis of low‐abundance small‐RNA cargos by next‐generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small‐RNAs from mouse small‐EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small‐EVs. As proof‐of‐principle for sensitive detection of circulating miRNAs, we compared small‐RNA sequencing data from a subset of small‐EVs serum‐purified with EV‐CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid‐19 patients. We identified and validated, only in small‐EVs, hsa‐miR‐146a and hsa‐miR‐126‐3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid‐19 patients with high anti‐spike IgG titers, we confirmed the neutralizing properties, against SARS‐CoV‐2 in vitro, of a subset of small‐EVs serum‐purified by EV‐CATCHER, as initially observed with ultracentrifuged small‐EVs. Altogether our data highlight the sensitivity and versatility of EV‐CATCHER.

Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of various cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects on both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. This study was therefore aimed at characterizing the role of Per2 in normal breast epithelia (MCF-12A) and in ER− breast cancer cells (MDA-MB-231) and also at determining the role of Per2 in doxorubicin-induced cell death. In both cell lines Per2 protein expression displayed a 24-hour circadian rhythm in both cell lines. Per2 was located predominantly in the cytoplasm, with nuclear localization observed with lower cytoplasmic fluorescent intensities. Our results show that Per2 silencing effectively sensitizes the chemoresistant MDA-MB-231 breast cancer cells to the cytotoxic effects of doxorubicin.