Tumour microenvironment in Non-Small-Cell Lung Cancer (NSCLC): a proteomic approach to gain insights on novel disease biomarkers and therapeutic approaches.

Lung cancer is the most frequently diagnosed malignancy and remains the leading cause of cancer-related mortality worldwide, accounting for approximately 1.8 million deaths annually. The majority of cases (approximately 85%) are classified as non-small cell lung cancer (NSCLC), which includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Despite therapeutic advances, including immunotherapy, five-year survival rates remain low, with only a subset of patients—identified through biomarkers such as PD-L1—achieving meaningful clinical benefit.A major challenge lies in the intrinsic heterogeneity of lung tumors, which is often underestimated by conventional biopsies. These provide only a limited snapshot of the disease and fail to capture its dynamic evolution or the emergence of therapy resistance. In this context, liquid biopsies have emerged as a minimally invasive and promising approach, enabling the detection of circulating tumor cells, nucleic acids, and extracellular vesicles (EVs). EVs, enriched with proteins, DNA, RNA, and lipids, represent a valuable source of biomarkers for early diagnosis, monitoring treatment response, and identifying novel driver mutations.Unlike genomics and transcriptomics, which are already widely applied in liquid biopsy, the proteomic profiling of blood remains relatively underexplored, despite the central role of proteins in intercellular communication and biological regulation. Proteomic analysis of the tumor microenvironment (TME), combined with three-dimensional (3D) in vitro models based on cancer stem cells and tumor spheroids, offers the opportunity to better recapitulate tumor heterogeneity and to study the secretome under physiologically relevant conditions.This project aims to identify novel biomarkers and potential therapeutic targets through mass spectrometry (MS)-based proteomic analysis of the TME focusing on EVs isolated from liquid biopsies of cancer patients and from 3D cell culture models that mimic the TME in vitro.