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.

CD44+/EPCAM+ cells detect a subpopulation of ALDHhigh cells in human non-small cell lung cancer: A chance for targeting cancer stem cells?

“…As performed previously [12], we decided to assess the tumor sphere formation ability of CD44+/EPCAM+ cells. We observed a growth of tumor spheres for up to 3 weeks, with results that were similar to those previously recorded for ALDH high cells in terms of the number and size of spheres [7].…”

“…Samples were digested for 60 min at 37°C in a gentle MACS Octo dissociator, filtered through 70-μm sterile cell strainers, centrifuged at 300 × g for 5 min, and resuspended in a DMEM and HAM'S F12 media mixture (2:1) (Gibco) containing 50 IU/mL penicillin-streptomycin and 4 mM glutamine. Viable cells were counted using an optic phase-contrast microscope [7].…”

“…The frequency of CSCs varies from 27% to 100% in highly tumorigenic cancers, such as hematopoietic and melanoma primary tumors, as well as in some cancer cell lines [2]. Although CSCs account for less than 1% of the total cells in solid tumors [3], they have several roles in tumor generation and progression, such as in the capacity for self-renewal, asymmetric cell division, slow division kinetics, increased capacity of invasion, metastasis, tumor formation, proliferation, resistance to conventional chemotherapy, and radiotherapy and immunotherapy [4][5][6][7]. For the small amount of these cells inside the tumor, however, several studies have been conducted on the identification of CSC markers [8,9].…”

“…Nevertheless, although ALDH is considered an intracellular enzyme and is the most used marker to identify CSCs in lung cancer [7,11,13], the scientific community has never correlated this intracellular marker with an epithelial marker, which may be very useful for targeting lung cancer stem cells. The aim of the present research was to compare the ALDH high cells with the double-positive CD44/EPCAM cells, in order to better define the superficial antigens of ALDH high cells.…”

CircRNA circ_POLA2 promotes lung cancer cell stemness via regulating the miR‐326/GNB1 axis

“…The most efficient sh‐circ_POLA2‐2 was selected to evaluate the biological function of circ_POLA2 knockdown. Since CSCs have been considered to be the root of lung cancer progression, 22,23 here, we explored the effects of circ_POLA2 on the stemness of lung cancer cells. As shown in Figure 2C, D, knockdown of circ_POLA2 significantly dampened the sphere‐formation ability in A549 and H1650 cells.…”