Introduction: Frequent locoregional recurrence and poor five-year survival of head and neck squamous cell cancer (HNSCC) persist as treatment challenges. Disease-free margins are critical in surgical management of HNSCC, but tumor behavior at the margin is ill-defined. Investigations into molecular markers at the surgical margin have drawn attention to the role of cancer stem cells (CSCs) in disease aggression and recurrence. Previously, our group characterized a CSC population (ALDH+/CD44high) with enhanced tumorigenicity, chemoresistance, and radioresistance in patient-derived xenograft (PDX) tumors. This study seeks to understand the biological and clinical impact of CSCs at the tumor invasive front.
CSC Distribution by FACS: Representative PDXs were sectioned into “leading edge (LE)” (outer 2-3mm) and “core” compartments. The CSC percentage (ALDH+/CD44high) by compartment was measured.
Determination of SOX2 Staining Pattern by IHC: Oral cavity (OC) and oropharynx (OP) HNSCC patients with three years of follow-up were selected from a prospective database (n=74). SOX2 staining architecture was classified as “leading-edge enriched (LEE)” or “diffuse staining (DS)” and compared with FACS CSC distribution.
Functional Analysis of Stemness by Tissue Compartment in a Leading-Edge Enriched PDX Case: PDX tumors were sectioned into “LE” and “core” compartments. CSCs (ALDH+/CD44high) from each compartment were identically sorted via FACS. Sorted CSCs were seeded (5,000 cells/well) and cultured in suspension in serum-free media, and the resulting spheres were counted.
Patient Outcomes Analysis: Kaplan-Meier curves were generated for disease-free and overall survival for “LEE” and “DS” SOX2 expression groups. Subset analysis was performed on HPV negative cases. Curves were compared via the log-rank method.
Results: To assess CSC distribution, three PDX cases underwent FACS analysis for CSC enrichment by tumor compartment. CUHN111 and CUHN109 demonstrated CSC enrichment of the LE (0.26% LE vs 0.04% core, p=0.004; 0.18% LE vs 0.07% Core, p = 0.034) while one PDX, CUHN013, demonstrated no difference (0.712 ± 0.3087 LE vs 3.56 ± 3.164 Core, p = 0.4). The CSC distribution in these PDXs mirrored the “LEE” or “DS” SOX2 staining patterns found in their respective originating patient resection specimens. All 74 OC/OP patient specimens were stained for SOX2 by IHC. 22 of 74 were SOX2+, 9/22 were “LEE”, and 13/22 were “DS.”
Functional differences in the CSCs isolated from the LE vs the Core were assessed using a sphere formation assay. In the LE-enriched case CUHN111, LE CSCs demonstrated greater sphere-forming efficiency, with a LE:Core efficiency ratio of 1.8:1 (n=5, p = 0.011).
Outcomes were assessed in SOX2 expressing cases. HPV-negative, “LEE” cases demonstrated improved survival vs “DS” cases (HR 3.2, p < 0.05). No other statistically significant differences were identified.
Conclusions: Here, we demonstrate that the distribution of SOX2+ cells by IHC mirrors that of ALDH+/CD44high CSCs by FACS. CSC behavior varies by tissue compartment, as LE CSCs demonstrate greater proliferative potential. Core CSCs may play a distinct functional role, as non-LE SOX cases demonstrated poorer overall survival. These data support further investigation into the impact of CSC localization and CSC-microenvironment interactions on CSC proliferation, CSC treatment resistance, and patient survival.