Isolation and Identification of Cancer Stem-Like Cells in Adenocarcinoma and Squamous Cell Carcinoma of the Lung: A Pilot Study

Valentina Masciale; Giulia Grisendi; Federico Banchelli; Roberto D'Amico; Antonino Maiorana; Pamela Sighinolfi; Alessandro Stefani; Uliano Morandi; Massimo Dominici; Beatrice Aramini

doi:10.3389/fonc.2019.01394

Isolation and Identification of Cancer Stem-Like Cells in Adenocarcinoma and Squamous Cell Carcinoma of the Lung: A Pilot Study

Front Oncol. 2019 Dec 18:9:1394. doi: 10.3389/fonc.2019.01394. eCollection 2019.

Authors

Affiliations

¹ Division of Thoracic Surgery, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy.
² Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy.
³ Rigenerand SRL, Modena, Italy.
⁴ Department of Medical and Surgical Sciences for Children & Adults, Center of Medical Statistic, University of Modena and Reggio Emilia, Modena, Italy.
⁵ Department of Medical and Surgical Sciences for Children & Adults, Institute of Pathology, University of Modena and Reggio Emilia, Modena, Italy.

Abstract

Background: Lung cancer stem cells (CSCs) share many characteristics with normal stem cells, such as self-renewal and multipotentiality. High expression of aldehyde dehydrogenase (ALDH) has been detected in many tumors, particularly in the CSC compartment, and it plays an important role in tumor proliferation, metastasis, and drug resistance. CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumors. The aim of this study was to isolate and analyze cancer stem-like cells from surgically removed specimens to compare lung adenocarcinoma (ADENO) and squamous (SQUAMO) cell carcinoma. Methods: The ALDEFLUOR assay was used to identify and sort ALDH^high and ALDH^low human lung cancer cells following tissue digestion. Fluorescence-activated cell sorting analysis for CD44 was performed with tumor cells. Quantitative real-time PCR was performed to assess the expression of SOX2 and NANOG as stemness markers. ALDH1A1 expression was additionally determined by immunohistochemistry. Anchorage-independent ALDH^high cell growth was also evaluated. ALDH^high ADENO and SQUAMO cells were cultured to analyze spheroid formation. Results: All specimens contained 0.5-12.5% ALDH^high cells with 3.8-18.9% CD44-positive cells. SOX2 and NANOG relative expression in ALDH^high compared to ALDH^low cells in ADENO and SQUAMO was analyzed and compared between the histotypes. Immunohistochemistry confirmed the presence of ALDH1A1 in the sections. SOX2 and NANOG were expressed at higher levels in the ALDH^high subpopulation than in the ALDH^low subpopulation only in ADENO cells, and the opposite result was seen in SQUAMO cells. In vitro functional assays demonstrated that ALDH^high cells exhibited migration capacity with distinct behaviors between ALDH^high spheres in ADENO vs. SQUAMO samples. Conclusions: Our results highlight the importance of a better characterization of cancer stem-like cells in ADENO and SQUAMO histotypes. This may suggest new differential approaches for prognostic and therapeutic purposes in patients with non-small-cell lung cancer.

Keywords: CSC marker; aldehyde dehydrogenase; cancer stem-like cells; lung adenocarcinoma; lung squamous cell carcinoma; non-small-cell lung cancer.