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

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.

Figure 1

Cells with high aldehyde dehydrogenase (ALDH) activity in one patient sample (1.2%). (A–C) The gating strategy of a representative fluorescence-activated cell sorting (FACS) analysis of a primary tumor cell suspension in one patient. 7-Amino-actinomycin D (7-AAD) was used to assess ALDH^highorlow on the live population of cells.

Figure 2

Cell positivity for CD44. (A–D) The gating strategy of a representative cytofluorimetric analysis of a primary tumor cell suspension. 7-Amino-actinomycin D (7-AAD) was used to exclude the CD45-positive cells in the live population to further analyze tumor cell positivity for CD44.

Figure 3

Relative messeger RNA (mRNA) expression of SOX2 and NANOG in ALDH^high compared to ALDH^low cell populations, using GAPDH as the reference gene. Expression was measured for the ALDH^low and ALDH^high cell populations in adenocarcinoma and squamous cell carcinoma histotypes using real-time PCR (RT-PCR), and relative expression comparing ALDH^high and ALDH^low was calculated by means of a linear mixed model. The two light gray bars represent ADENO, and the two dark gray bars represent SQUAMO. The error bars represent the 95% confidence intervals. ADENO, adenocarcinoma; SQUAMO, squamous cell carcinoma.

Figure 4

Immunohistochemical staining intensity of aldehyde dehydrogenase. Representative images of two patient samples in squamous cell carcinoma (A) and adenocarcinoma (B). Images were taken at 10× (upper panels) and 20× (lower panels) magnification; black arrows indicate positive cells.

Figure 5

Cell transformation assay. The MCF-7 cell line and ALDH^high cells were compared for growth ability in a semisolid agar substrate. Two different time points were evaluated: 0 and 8 days. In the vertical axis, DNA content of each sample was measured using a fluorescent signal released by Cyquant GR Dye. Time points are represented in the horizontal axis.

Figure 6

Cell proliferation assay. A colorimetric assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) based, was performed on ALDH^high cells after seeding. Six different time points were evaluated (0, 1, 2, 7, 14, and 21 days). Absorbance was measured at 490 nm.

Figure 7

General characteristics over time of tumor spheres in adenocarcinoma and squamous cell carcinoma. (1A–C) ALDH^high spheres from ADENO at 1, 2, and 3 weeks. (1D–F) ALDH^high spheres from SQUAMO at 1, 2, and 3 weeks. (2A–F) The number of spheres and their areas were calculated for two adenocarcinoma patients and two squamous cell carcinoma patients 1, 2, and 3 weeks after seeding. Points represent values for individual patients, and bars represent average values. ADENO, adenocarcinoma; SQUAMO, squamous cell carcinoma.

Figure 8

ALDH^low cells phase-contrast microscopy. ALDH^low cells in low-attachment serum-free culture showed difficult and slow growth that completely stopped at 3 days.