Resumen de Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications
Lung cancer is the most commonly diagnosed type of cancer and the leading cause of cancer-related death worldwide, with approximately 15% of patients surviving 5 years after diagnosis. Curative surgery is the standard of care for early-stage patients with a good performance status, but 75% are diagnosed at advances stages, when surgery is not possible, and 35-50% of the resected patients relapse after an apparently successful surgical treatment. Significant advances in the development of therapies against driver mutations and immune-based treatments for these patients have been achieved in recent years, but many patients still develop treatment resistance, progress, and die. The high resistance against these therapies has been associated to cancer stem-like cells (CSCs), a population with stem properties which is able to survive after conventional treatments and regenerate tumor even when are undetectable.
In this thesis, primary cultures from early-stage non-small cell lung cancer (NSCLC) patients were established, using sphere-forming assays for CSCs 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 RTqPCR, gene expression profiles were analyzed, and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected as the best contributors 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 silico in a cohort of 661 NSCLC patients from TCGA. 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 CSCs score. Kaplan-Meier survival analysis showed that patients with high CSCs score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months, p = 0.001] and in the adenocarcinoma (ADC) subcohort [36.6 vs. 53.5 months, 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 ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. This score was also analyzed in an independent group of 245 patients from Consorci Hospital General Universitari de València, confirming its prognostic value in the ADC subtype [42.90 vs. not reached (NR) months, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung ADC patients and the basis for developing novel therapies.
