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Background: Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines. Methods: We examined several lung adenocarcinoma cell lines for the ability of CD44v and/or ALDH expression to enrich for cells with CSC characteristics such as in vitro differential proliferation rate, chemotherapeutic-resistance, tumorsphere formation, and in vivo tumorigenicity. We also compared their in vivo secondary tumor formation, and histological characteristics of their xenograft tumors, and examined their expression of PD-L1, EGFR, xCT, and reactive oxygen species (ROS). Results: Both CD44v high /ALDH high and CD44v high /ALDH low cells were enriched in cells with CSC characteristics, with the CD44v high /ALDH low cells being more proliferative and more resistant to chemotherapeutics, whereas CD44v high /ALDH high cells were more efficient in forming tumorspheres in vitro, in making primary xenograft tumors, and in propagating secondary tumors in vivo . Applying stricter sorting gates to select for cells with the highest CD44v/ALDH expression caused the CD44v high /ALDH low cells to lose their high proliferation rates and chemotherapeutic resistance ability, but enriched for the tumorsphere-forming cells among the CD44v high /ALDH high and CD44v high /ALDH low cells. CD44v high expression was associated with PD-L1 and xCT expression in both H1650 and HCC827 cells. This association was not modified by ALDH expression in the H1650 cell line. However, in the HCC827 cell line, ALDH expression was negatively associated with PD-L1 and positively associated with xCT expression. Conclusion: Lung adenocarcinoma cells with high CD44v expression are enriched for CSCs. Addition of ALDH as an enrichment marker bestowed some CSCs characteristics to CD44v high /ALDH low cells and others to CD44v high /ALDH high cells. We propose that lung adenocarcinoma contains different CSCs, each of them endowed with different CSC characteristics.

Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma