Is the Proteome of Bronchoalveolar Lavage Extracellular Vesicles a Marker of Advanced Lung Cancer?

Carvalho, Ana Sofia; Moraes, Maria Carolina Strano; Hyun Na, Chan; Fierro-Monti, Ivo; Henriques, Andreia; Zahedi, Sara; Bodo, Cristian; Tranfield, Erin M; Sousa, Ana Laura; Farinho, Ana; Rodrigues, Luís Vaz; Pinto, Paula; Bárbara, Cristina; Mota, Leonor; de Abreu, Tiago Tavares; Semedo, Júlio; Seixas, Susana; Kumar, Prashant; Costa-Silva, Bruno; Pandey, Akhilesh; Matthiesen, Rune

SIMPLE SUMMARY: Bronchoalveolar lavage is routinely collected during bronchoscopy for cytology analysis in the diagnostic of lung cancer. Due to low sensitivity of this method, early-stage cancers are undetected, lowering the treatment success. In this study, we analyzed extracellular vesicles isolated from bronchoalveolar lavage of lung cancer suspects by mass spectrometry-based proteomics. The protein composition of bronchoalveolar lavage extracellular vesicles of late-stage cancer showed a higher proteome complexity associated with mortality within the two year follow-up period. We identified a potential therapeutic target DNMT3B complex which was significantly expressed in bronchoalveolar lavage extracellular vesicles as well as in tumor tissue. Bronchoalveolar lavage extracellular vesicles proteome analysis of immune markers indicates the presence of markers of innate immune and fibroblast cells. ABSTRACT: Acellular bronchoalveolar lavage (BAL) proteomics can partially separate lung cancer from non-lung cancer patients based on principal component analysis and multivariate analysis. Furthermore, the variance in the proteomics data sets is correlated mainly with lung cancer status and, to a lesser extent, smoking status and gender. Despite these advances BAL small and large extracellular vehicles (EVs) proteomes reveal aberrant protein expression in paracrine signaling mechanisms in cancer initiation and progression. We consequently present a case-control study of 24 bronchoalveolar lavage extracellular vesicle samples which were analyzed by state-of-the-art liquid chromatography-mass spectrometry (LC-MS). We obtained evidence that BAL EVs proteome complexity correlated with lung cancer stage 4 and mortality within two years´ follow-up (p value = 0.006). The potential therapeutic target DNMT3B complex is significantly up-regulated in tumor tissue and BAL EVs. The computational analysis of the immune and fibroblast cell markers in EVs suggests that patients who deceased within the follow-up period display higher marker expression indicative of innate immune and fibroblast cells (four out of five cases). This study provides insights into the proteome content of BAL EVs and their correlation to clinical outcomes.