Indirect Immunofluorescence and Tumorspheres Enrichment Technique for Identifying Cancer Stem Cell Markers in Cancer Cell Lines From Primary Oral Cancer Tissues: An In Vitro Study

Abstract

Aim: The cancer stem cells (CSCs) are known to be responsible for drug resistance and cancer relapse in the treatment of cancer. Identification and isolation of CSCs and study of their properties will play a crucial role in developing an effective drug against these targets. The aim of the study was to isolate CSCs from primary cancer by the tumorspheres enrichment method, to confirm by indirect immunofluorescence and gene expression of stem cell markers by using real-time polymerase chain reaction (RT-PCR) technique. Materials and Methods: In this in vitro study, we enriched oral CSCs through tumorsphere formation assay from seven primary cultures of OSCC patients with defined serum media. The expression and localization of the cell surface markers of CD133 and CD44 were tested by indirect immunofluorescence. Gene expression of stem cell markers such as CD44, CD133, Oct4, Sox2, and Nanog were quantified by RT-PCR technique. One-way analysis of variance was applied to analyze gene expression. Results: Tumorsphere formation has been used to isolate the CSCs from the OSCC tissue culture. Both CD133 and CD44 antibody confirmed the presence of CSCs through indirect immunofluorescence. In comparison to parental cell lines, the expression levels of CD133, CD44, Oct4, Sox2, and Nanog stem cell were significantly higher in CSC-enriched subpopulations. Conclusions: The cost-effective spheroid enrichment and the indirect immunofluorescence methods are useful for the isolation of CSCs from the primary tumor.