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

## TL;DR

This study identifies distinct cancer stem cell subpopulations in oral cancer, showing they impact different clinical outcomes and can change over time.

## Contribution

The study reveals dynamic, phenotypically distinct CSC subpopulations in oral cancer with unique clinical correlations.

## Key findings

- CSC subpopulations marked by p75NTR and ALDH1A1 show distinct clinical associations in oral cancer.
- CSC markers can switch over time, indicating dynamic and evolving stem cell heterogeneity.
- Higher p75NTR+ cells correlate with poor survival in patients with otherwise better prognosis.

## Abstract

Cancer stem cells (CSCs) have been suggested to be the subset of cancer cells that drive tumor growth and progression and fuel the tumor with heterogeneous populations of cells, including those resistant to therapy. Different proteins have been proposed as biomarkers of CSCs in oral squamous cell carcinoma (OSCC). In this study, we simultaneously investigated multiple putative biomarkers of CSCs that have been previously proposed in OSCC, with the aim of identifying a pure CSC subpopulation. Our results suggest that several stem cell subpopulations may co-exist in a tumor, each having an impact on different clinical parameters. The cell subpopulations identified by use of different CSC markers were found to be dynamic and able to switch their protein markers over time. The inter-tumoral diversity of cancer stem cells demonstrated here raises novel challenges in terms of their use as predictive biomarkers and as efficient therapeutic targets for OSCC.

Background: Several markers have been shown to define subpopulations enriched for cancer stem cells (CSCs) and correlate with poor clinical outcomes 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) and 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 and 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 subpopulation after FACS and in vitro growth, but with different kinetics. Conclusions: Here, we show that several stem cell subpopulations with distinct phenotypes co-exist in a tumor, each having impact on different clinical parameters. The cell subpopulations identified by the use of different CSC markers were found to be dynamic populations, able to switch between phenotypes. In addition, our data suggest that the stem cell heterogeneity is acquired and evolves parallel with carcinoma progression.