The high resistance against current therapies found in non-small-cell lung cancer (NSCLC) has been associated to cancer stem-like cells (CSCs), a population for which the identification of targets and biomarkers is still under development. In this study, primary cultures from early-stage NSCLC patients were established, using sphere-forming assays for CSC enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using quantitative PCR, gene expression profiles were analyzed and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in a NSCLC cohort (N = 661) from The Cancer Genome Atlas. Based on a Cox regression analysis, CDKN1A, SNAI1, and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSC score. Kaplan–Meier survival analysis showed that patients with high CSC score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months (mo), p = 0.001] and the adenocarcinoma subcohort [36.6 vs. 53.5 mo, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167–1.922; p = 0.001] and the adenocarcinoma subcohort [HR: 1.869; 95% CI, 1.275–2.738; p = 0.001]. This score was also analyzed in an independent cohort of 114 adenocarcinoma patients, confirming its prognostic value [42.90 vs. not reached (NR) mo, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung adenocarcinoma patients and the basis for developing novel therapies. Further studies are required to identify suitable markers and targets for lung squamous cell carcinoma patients.

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reached humane endpoints 10-14 days post injection. Immunophenotyping of dissociated tumours revealed changes in the proportions of myeloid and lymphocyte populations relative to tumour-naïve lungs. CD8+ T-cells were present and tumour cells expressed PDL1 suggesting LLC has the capacity to respond to ICI. Consistent with these observations, orthotopic LLC growth was delayed in mice treated with anti-PD1 therapeutic antibody compared to anti-IgG2a isotype control, demonstrating that LLC is an appropriate model for identifying mechanisms that confer sensitivity and/or resistance to ICI therapy. Based on these findings, we generated LLC-Luciferase cells stably expressing Cas9. Since genome-wide screens are not feasible with this in vivo tumour model, we are synthesizing a custom, focussed guide RNA (gRNA) library. Genomic analyses have identified w500 candidate immunomodulatory genes expressed in LLC and clinical lung tumours that will be targeted to determine the effects of inactivating these candidates on anti-PD1 response. Conclusion: This platform will enable high-throughput genetic screens to elucidate novel tumourintrinsic determinants of ICI response in vivo. Our discoveries will have potential to inform novel biomarkers predictive of response, and putative targets for new combination therapies to enhance the antitumour effects of ICIs. Collectively, this work will improve our understanding of the biological mechanisms governing ICI sensitivity, thereby stimulating the development of new strategies to maximize therapeutic benefit from ICIs in lung cancer patients.