Figure 2. CD276 is an enrichment surface marker for CSCs of HNSCC.

(A) Sorting strategy for CD276^high and CD276^low tumor cells.

(B) Representative image of tumorspheres derived from CD276^high and CD276^low tumor cells from HNSCC PDXs. PS2, patient sample 2; PS6, patient sample 6. Scale bar, 50 μM (upper panels), 100μm (lower panels).

(C) Quantification of number of tumorspheres from CD276^high and CD276^low tumor cells from HNSCC PDXs. **p<0.01, ***p<0.001 by Student’s t test.

(D) Quantification of diameter of tumorspheres from CD276^high and CD276^low tumor cells from HNSCC PDXs. **p<0.05 by Student’s t test.

(E) In vivo limiting dilution analysis of CD276^high and CD276^low tumor cells from HNSCC PDXs. The frequency of allograft formation at each cell dose injected is shown. The data were analyzed using ELDA software.

(F) Representative image of tumorspheres from CD276^high and CD276^low tumor cells isolated from murine HNSCC. Scale bar, 50μm

(G) Quantification of number and diameter of tumor spheres from mouse CD276^high and CD276^low tumor cells. ***p<0.001 and **p <0.01 by Student’s t test.

(H) In vivo limiting dilution analysis of murine CD276^high and CD276^low tumor cells. The frequency of allograft formation at each cell dose injected is shown. The data were analyzed using ELDA software.

(I) Heatmap showing the gene expression profiles of EpCAM⁺CD276^high and EpCAM⁺CD276^low tumor cells by RNA-seq.

(J) GSEA showed that the stemness gene signature was significantly enriched in EpCAM⁺CD276^high cells.

(K) GSEA showed that the EMT signature was significantly enriched in EpCAM⁺CD276^high cells.

(L) GSEA showed that the stemness gene signature was significantly enriched in human HNSCC with high expression of CD276 from TCGA datasets.

(M) GSEA showed that the EMT signature was significantly enriched in human HNSCC with high expression of CD276 from TCGA datasets.