A New Method for Identifying Stem-Like Cells in Esophageal Cancer Cell Lines

Abstract

Cancer stem cells (CSCs) appear to resist chemo-radiotherapy and initiate tumor recurrence in patients. Isolation and further characterization of this subpopulation is important for targeting CSCs. Flow cytometry using Aldefluor, a fluorescent substrate of aldehyde dehydrogenase, has been used to isolate CSCs from various cancer cell lines. However, new techniques are needed to locate and identify CSCs in culture for live-cell analyses such as fluorescence microscopy without introducing artifacts during cell sorting and to observe CSC and non-CSC interactions. Previously, we characterized a distinct CSC subpopulation within human esophageal cancer cell lines (ESCC). In this study we introduce the attached-cell Aldefluor method (ACAM) to detect CSCs in ESCC cell lines (KY-5, KY-10, TE-1, TE-8, YES-1, YES-2). To validate this technique, we isolated CSCs from the YES-2 parental line using standard Aldefluor flow cytometry to create a cell line enriched in CSCs (YES-2CSC). This line showed significantly greater ACAM staining and higher CD44 levels than YES-2. ACAM also showed significantly higher ALDH activity in YES-2CSC than in YES-2S, a cell line that has a diminished CSC subpopulation after having survived treatment with curcumin. ACAM stained cells within tumorspheres made from the CSC-enriched line but not differentiating cells from the tumorspheres. This study also demonstrates a new method for generating and growing tumorspheres without the growth factor supplements normally used in medium to form tumorspheres. ACAM should be evaluated using other cancer cell lines to further substantiate its effectiveness and to characterize CSCs in culture through various imaging techniques.

The PI3K/AKT/c-MYC Axis Promotes the Acquisition of Cancer Stem-Like Features in Esophageal Squamous Cell Carcinoma

“…To our knowledge, intratumoral heterogeneity has not been extensively studied in ESCC. To our knowledge, 7 studies have identified CSC‐like cells based on the detection of specific markers, such as P75 NTR , CD44, CD90, and ALDH1 , and the activity of the ATP‐binding cassette transporters . In these studies, evidence in support of the CSC‐like features came from both in vitro studies and mouse xenograft models .…”

Cancer stem cell vaccine inhibits metastases of primary tumors and induces humoral immune responses against cancer stem cells

“…ALDH (aldehyde dehydrogenase) activity, often measured via ALDEFLUOR assay, has been successfully used as a marker to enrich CSC populations 11,[17][18][19][20][21][22] in a variety of cancers including human melanoma 23 and head and neck squamous cell cancer. 18 We characterized CSC-enriched populations in 2 histologically distinct murine tumors (melanoma D5 and squamous cell cancer SCC7) and evaluated their immunogenicity by administering CSC-based vaccines in 2 genetically different syngeneic immunocompetent hosts followed by tumor challenge.…”

Emerging therapeutic targets in esophageal adenocarcinoma

“…Similar research has been done in glioma, where CSCs (CD133-positive cells) were able to repair DNA damage more efficiently and rapidly than CD133 negative cells [38]. Most of this cancer stem cell research has been performed in other solid organ tumors and ESCC [29, 39], with limited studies on EAC [30, 40, 41]. Recognizing that EAC tumors can harbor a CSC population that manages to escape chemotherapy and/or radiation should open opportunities for scientists to explore and understand the pivotal role of CSCs and indeed exploit the underlying mechanisms in its treatment.…”