Abstract
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.
Highlights
Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide, with ~15% of patients surviving 5 years after diagnosis[1]
There is strong evidence suggesting that cancer cells with stem properties selectively resist current cancer therapies, indicating the important role that cancer stem-like cells (CSCs) play in tumor evolution, relapse, and metastasis[8,9]
Starting material for these cultures can be commercial cell lines, but using cells directly isolated from surgical resections specimens reflect in vivo conditions better than they do, since immortalized cell lines do not behave as primary cultures and long-term manipulation alters phenotype, functions, and responsiveness to stimuli
Summary
Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide, with ~15% of patients surviving 5 years after diagnosis[1]. Curative surgery is the standard of care for early-stage patients with a good performance status, but the recurrence rate ranges from 35 to 50% and, after an apparently successful surgical treatment, appearance of secondary tumors often leads to the relapse of resected patients[7] This poor prognosis greatly supports the efforts to establish prognostic biomarkers and therapeutic targets for improving the management of NSCLC. Among these targets, cancer stem-like cells (CSCs) are proposed as a promising tumor population, since they are believed to survive after conventional cancer treatments and regenerate tumors even when are undetectable[8]. Specific strategies against CSCs are not approved in clinical practice and a better understanding of their impact on patients’ prognosis is required
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