Abstract
Cancer stem-like side population (SP) cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC) cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs) were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07%) was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC)-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP) LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.
Highlights
In this study we described the successful isolation and identification of cancer stem-like side population (SP) cells directly from a surgically resected Laryngeal squamous cell carcinoma (LSCC) specimen derived from a well-differentiated epiglottic neoplasm in a Chinese male patient
We purified the cultured LSCCs by repeated differential trypsinization to eliminate coexisting fibroblasts
The monolayer and cobblestone pattern of LSCCs identified by phase-contrast microscopy, positive staining of CK(pan) and cytokeratin 5 (CK5), negative staining of vimentin, and the epithelial ultrastructure revealed by transmission electron microscopy confirmed the epithelial lineage of the cultured LSCCs (Fig. 1)
Summary
Cancer stem-like side population (SP) cells have been successfully identified in a wide range of solid tumors, including breast cancer [1,2], hepatocellular carcinoma [3,4,5,6,7], lung cancer [8,9], gastrointestinal cancer [10,11,12], prostate cancer [13], gallbladder cancer [14], ovarian cancer [15], endometrial cancer [16], pancreatic cancer [17,18], urological cancer [19,20], glioblastoma [21], melanoma [22], osteosarcoma [23,24], mesenchymal neoplasms [25], nasopharyngeal cancer [26], oral cancer [27,28], and other head and neck cancers [29,30] Most of these investigations have been performed using established cancer cell lines. Ongoing research on SP cells to develop new agents that target cancer stem cells (CSCs) is urgently needed
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