A novel system is developed for high-throughput multiplexed biomarker detection in single extracellular vesicles.

Pleural effusion (PE) is commonly observed in advanced lung cancer and was suggested to contain both cell-free tumor DNA and tumor cells. Molecular profiling of PE represents a minimally invasive approach of detecting tumor driver mutations for clinical decision making, especially when tumor tissues are not available. The objective of this study is to investigate the efficacy and precision of detecting gene alterations in PE samples to address the feasibility in clinical use. Methods: Sixty-three metastatic lung cancer patients with (n=30, cohort 1) or without (n=33, cohort 2) matched tumor tissues were enrolled in this study. PE and plasma samples of each patient were collected simultaneously. Supernatant and cell precipitate of PE were processed separately to extract cfDNA (PE-cfDNA) and sediment DNA (sDNA). All samples were subjected to targeted next-generation sequencing (NGS) of 416 cancer-related genes. Results: PE supernatants contain more abundant tumor DNA than PE sediments and plasma samples, suggested by higher mutant allele frequencies (MAF) and elevated mutation detection rate in PE-cfDNA (98.4% vs. 90.5% in PE sDNA vs. 87% in plasma cfDNA). In Cohort 1 with matched tumor tissue, tumor mutational burden (TMB) of PE-cfDNA was similar as tumor tissues (6.4 vs. 5.6), but significantly higher than PE sDNA (median TMB: 3.3) and plasma cfDNA (median TMB: 3.4). Ninety-three percent (27 out of 29) of tissue-determined driver mutations were detected in PE-cfDNA, including alterations in ALK, BRAF, EGFR, ERBB2, KRAS, NF1, PIK3CA, and RET, while only 62% were captured in plasma cfDNA. PE-cfDNA also has the highest detection rate of EGFR driver mutations in the full cohort (71% vs. 68% in PE sDNA vs. 59% in plasma cfDNA). Mutation detection from cytological negative and hemorrhagic PE is challenging. Comparatively, PE-cfDNA demonstrated absolute superiority than PE sDNA in such a scenario, suggesting that it is an independent source of tumor DNA and therefore less influenced by the abundance of tumor cells. Conclusion: Genomic profiling of PE-cfDNA offers an alternative, and potentially more meticulous approach in assessing tumor genomics in advanced lung cancer when tumor tissue is not available. Our data further demonstrate that in hemorrhagic or cytologically negative PE samples, PE-cfDNA has higher mutation detection sensitivity than sDNA and plasma cfDNA, and therefore is a more reliable source for genetic testing.

Epidemiological studies have shown that urban particulate matter (PM) increases the risk of respiratory infection. However, the underlying mechanisms are poorly understood. PM has been postulated to suppress the activation of airway epithelial innate defence in response to infection. The effects of PM on antibacterial defence were studied using an in vitro infection model. The levels of antimicrobial peptides were measured using RT-PCR and ELISA. In addition to performing colony-forming unit counts and flow cytometry, confocal microscopy was performed to directly observe bacterial invasion upon PM exposure. We found that PM PM increased bacterial invasion by impairing the induction of β-defensin-2 (hBD-2), but not the other antimicrobial peptides (APMs) secreted by airway epithelium. PM further increases bacteria-induced ROS production, which is accompanied by an accelerated cell senescence and a decrease in bacteria-induced hBD-2 production, and the antioxidant NAC treatment attenuates these effects. The PM exposure further upregulated the expression of IL-8 but downregulated the expression of IL-13 upon infection. PM promotes bacterial invasion of airway epithelial cells by attenuating the induction of hBD-2 via an oxidative burst. These findings associate PM with an increased susceptibility to infection. These findings provide insight into the underlying mechanisms regarding the pathogenesis of particulate matter.

Background: Chromodomain helicase DNA-binding protein 4 (CHD4) has been shown to contribute to DNA repair and cell cycle promotion; however, its roles in cancer initiation and progression remain largely unknown. This study aimed to demonstrate the role of CHD4 in the development of non-small cell lung cancer (NSCLC) and determine the potential mechanisms of action. Methods: By using immunohistochemistry, the expression levels were evaluated in both cancer and non-cancerous tissues. Subsequently, CHD4 knockdown and overexpression strategies were employed to investigate the effects of CHD4 on cell proliferation, migration, along with the growth and formation of tumors in a xenografts mouse model. The protein expression levels of CHD4, PHF5A and ROCK/RhoA markers were determined by Western blot analysis. Results: Compared with non-cancerous tissues, CHD4 was overexpressed in cancer tissues and CHD4 expression levels were closely related to clinical parameters of NSCLC patients. In H292 and PC-9 cell lines, CHD4 overexpression could promote the proliferative and migratory potential of NSCLC cells. Furthermore, down-regulation of CHD4 could reduce the proliferative and migratory ability in A549 and H1299 cell lines. Meanwhile, knockdown of CHD4 could decrease the tumorigenicity in nude mice. Finally, we demonstrated that one of the mechanisms underlying the promotive effect of CHD4 on NSCLC proliferation and migration may be through its interaction with PHD finger protein 5A (PHF5A) and subsequent activation of the RhoA/ROCK signaling pathway. Conclusions: CHD4, which is highly expressed in cancer tissue, could be an independent prognostic factor for NSCLC patients. CHD4 plays an important role in regulating the proliferative and migratory abilities of NSCLC via likely the RhoA/ROCK pathway by regulating PHF5A.

Background: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are syndromes of acute respiratory failure with extremely high mortality and few effective treatments. Mesenchymal stem cells (MSCs) may reportedly contribute to tissue repair in ALI and ARDS. However, applications of MSCs have been restricted due to safety considerations and limitations in terms of large-scale production and industrial delivery. Alternatively, the MSC secretome has been considered promising for use in therapeutic approaches and has been advanced in pre-clinical and clinical trials. Furthermore, the MSC secretome can be freeze-dried into a stable and ready-to-use supernatant lyophilized powder (SLP) form. Currently, there are no studies on the role of MSC SLP in ALI. Methods: Intratracheal bleomycin was used to induce ALI in mice, and intratracheal MSC SLP was administered as a treatment. Histopathological assessment was performed by hematoxylin and eosin, immunohistochemistry, and immunofluorescence staining. Apoptosis, inflammatory infiltration, immunological cell counts, cytokine levels, and mRNA- and protein-expression levels of relevant targets were measured by performing terminal deoxynucleotidyl transferase dUTP nick-end labeling assays, determining total cell and protein levels in bronchoalveolar lavage fluids, flow cytometry, multiple cytokine-detection techniques, and reverse transcriptase-quantitative polymerase chain reaction and western blot analysis, respectively. Results: We found that intratracheal MSC SLP considerably promoted cell survival, inhibited epithelial cell apoptosis, attenuated inflammatory cell recruitment, and reversed immunological imbalances induced by bleomycin. MSC SLP inhibited the interleukin 6–phosphorylated signal transducer and activator of transcription signaling pathway to activate tumor protein 63–jagged 2 signaling in basal cells, suppress T helper 17 cell differentiation, promote p63⁺ cell proliferation and lung damage repair, and attenuate inflammatory responses. Conclusions: MSC SLP ameliorated ALI by activating p63 and promoting p63⁺ cell proliferation and the repair of damaged epithelial cells. The findings of this study also shed insight into ALI pathogenesis and imply that MSC SLP shows considerable therapeutic promise for treating ALI and ARDS.