Identification of Phenotypically Distinct Cancer Stem Cell Subpopulations in Oral Squamous Cell Carcinoma

Background: Several markers have been shown to define subpopulations enriched for cancer stem cells (CSCs) and correlate with poor clinical outcome in oral squamous cell carcinoma (OSCC). Objective: To investigate the pattern of expression and correlation with clinical parameters of two CSC markers, namely p75NTR and ALDH1A1, in both patient samples and cell lines. Methods: Archival formalin-fixed paraffin embedded samples from normal human oral mucosa (NHOM, n=31), oral dysplasia (OD, n=10) or OSCC (n=177) were subjected to multiple immunohistochemistry and some to qRT-PCR for expression of CSC and proliferation-related markers, BMI1 and Ki67. Correlations between CSC marker expression and clinical parameters were investigated. Primary cells and cell lines derived from NHOM, OD or OSCC were FACS- analyzed for the same markers. Results: A higher frequency of cells positive for CSC markers was detected in OD and OSCC compared to NHOM. Co-localization of the two markers was a rare finding in OSCC as compared to NHOM or OD and was more heterogeneous in OSCC cell lines than in OD and NHOM cells. Cells positive for p75NTR exhibited higher expression of proliferative and self-renewal markers in comparison to ALDH1A1+ or double ALDH1A1+/p75NTR+ cells. Cells positive for p75NTR were more frequent in small size tumors, poorly to moderately differentiated tumors, and correlated with poor survival of patients otherwise (clinically) deemed as of better prognosis. Higher frequency of ALDH1A1+ cells was found to be associated with lymph node metastasis. Both p75NTR+ cells and ALDH1A1+ cells could emerge de novo from the respective negative sub-population after FACS sorting and in vitro growth, but with different kinetics. Conclusion: Here we show that several stem cell sub-populations with distinct phenotypes co-exist in a tumor, each having impact on different clinical parameters. The cell subpopulations identified by use of different CSC markers were found to be dynamic populations, able to switch between phenotypes. In addition, our data suggest also that the stem cell heterogeneity is acquired and evolve parallel with carcinoma progression.