Fig. 9: HIDDEN cells are detected in HPV+ tonsil-derived epithelium and in HNSCC patient biopsies.

a Representative H&E from n = 5 frames of the patient tonsil (Donor A) that was used to culture patient-derived tonsil keratinocytes to support experiments. Scale bar, 100 µm. b Representative IF showing few cells with light ELF3+ staining (arrows in inset) in tonsil tissue, indicating the relative absence of HIDDEN cells. Scale bar, 100 µm, n = 5 frames across tonsil tissue. c–f Representative images from n = 10 frames from a raft set generated from Donor A. c–f Scale bar, 50 µm. c Representative H&Es of epithelial rafts generated from tonsil-derived keratinocytes from Donor A that were either nucleofected with HPV16 or not nucleofected. Many more basal cells are seen in HPV+ tonsil epithelium. Scale bar, 50 µm d DNA-ISH for HPV genomes confirms HPV status of 3D rafts. e RNA-ISH for ELF3 and GPR110 shows few cells with puncta in HPV− rafts, and many puncta in HPV+ rafts. f IF staining reveals significant upregulation of ELF3+ HIDDEN cells in the suprabasal compartment. g, h Representative images from n = 10 frames from two p16+ and two p16− HNSCC biopsies. g RNA-ISH for HIDDEN cell biomarkers ELF3 and GPR110 shows many puncta in HPV+ vs HPV− HNSCC patient biopsies. Scale bar, 50 µm in the main image and 20 µm in an insert. h Representative images of HNSCC tumors stained by H&E stain and by IF. Stain for p16 confirms the HPV status of tumors and ELF3 staining demonstrates protein-level expression of HIDDEN cells, specifically in HPV+ HNSCCs. Scale bars, 100 µm.