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| 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 proteinlevel expression of HIDDEN cells, specifically in HPV+ HNSCCs. Scale bars, 100 µm.
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Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infect...
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... and furthermore that C7 was composed of a greater proportion of cells derived from HPV+ vs HPV− specimens. C7 shares approximately one-quarter of genes (89 of 336) with the original C9 gene list (Supplementary Data 2). Altogether, this analysis confirms the increased presence of HIDDEN cells using a published dataset of HPV+ versus HPV− HNSCCs. (Fig. 9b). Tonsillar keratinocytes were derived from two separate donors, nucleofected with HPV16 episomes or maintained as negative controls, prior to generation of HPV+ and HPV− organotypic epithelial rafts ( Fig. 9c and Supplementary Fig. 6a). DNA-ISH for highrisk HPV confirmed the expected HPV status (Fig. 9d). RNA-ISH for ELF3 and GPR110 ( ...
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... Data 2). Altogether, this analysis confirms the increased presence of HIDDEN cells using a published dataset of HPV+ versus HPV− HNSCCs. (Fig. 9b). Tonsillar keratinocytes were derived from two separate donors, nucleofected with HPV16 episomes or maintained as negative controls, prior to generation of HPV+ and HPV− organotypic epithelial rafts ( Fig. 9c and Supplementary Fig. 6a). DNA-ISH for highrisk HPV confirmed the expected HPV status (Fig. 9d). RNA-ISH for ELF3 and GPR110 ( Fig. 9e and Supplementary Fig. 6b) and IF for ELF3 ( Fig. 9f and Supplementary Fig. 6c) validated significant upregulation of HIDDEN cells with HPV infection in tonsillar rafts derived from both donors. Next, ...
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... dataset of HPV+ versus HPV− HNSCCs. (Fig. 9b). Tonsillar keratinocytes were derived from two separate donors, nucleofected with HPV16 episomes or maintained as negative controls, prior to generation of HPV+ and HPV− organotypic epithelial rafts ( Fig. 9c and Supplementary Fig. 6a). DNA-ISH for highrisk HPV confirmed the expected HPV status (Fig. 9d). RNA-ISH for ELF3 and GPR110 ( Fig. 9e and Supplementary Fig. 6b) and IF for ELF3 ( Fig. 9f and Supplementary Fig. 6c) validated significant upregulation of HIDDEN cells with HPV infection in tonsillar rafts derived from both donors. Next, HNSCC patient biopsies were probed for gene and proteinlevel expression of HIDDEN cell ...
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... (Fig. 9b). Tonsillar keratinocytes were derived from two separate donors, nucleofected with HPV16 episomes or maintained as negative controls, prior to generation of HPV+ and HPV− organotypic epithelial rafts ( Fig. 9c and Supplementary Fig. 6a). DNA-ISH for highrisk HPV confirmed the expected HPV status (Fig. 9d). RNA-ISH for ELF3 and GPR110 ( Fig. 9e and Supplementary Fig. 6b) and IF for ELF3 ( Fig. 9f and Supplementary Fig. 6c) validated significant upregulation of HIDDEN cells with HPV infection in tonsillar rafts derived from both donors. Next, HNSCC patient biopsies were probed for gene and proteinlevel expression of HIDDEN cell biomarkers. Many cells with multiple puncta were ...
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... two separate donors, nucleofected with HPV16 episomes or maintained as negative controls, prior to generation of HPV+ and HPV− organotypic epithelial rafts ( Fig. 9c and Supplementary Fig. 6a). DNA-ISH for highrisk HPV confirmed the expected HPV status (Fig. 9d). RNA-ISH for ELF3 and GPR110 ( Fig. 9e and Supplementary Fig. 6b) and IF for ELF3 ( Fig. 9f and Supplementary Fig. 6c) validated significant upregulation of HIDDEN cells with HPV infection in tonsillar rafts derived from both donors. Next, HNSCC patient biopsies were probed for gene and proteinlevel expression of HIDDEN cell biomarkers. Many cells with multiple puncta were detected in two cases of p16+ HNSCCs by RNA-ISH but ...
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... upregulation of HIDDEN cells with HPV infection in tonsillar rafts derived from both donors. Next, HNSCC patient biopsies were probed for gene and proteinlevel expression of HIDDEN cell biomarkers. Many cells with multiple puncta were detected in two cases of p16+ HNSCCs by RNA-ISH but were largely absent in two cases of p16-negative HNSCCs (Fig. 9g). Similarly, ELF3 protein was robustly detected in HPV+, but minimally in HPV−, HNSCCs with HPV status confirmed by p16 staining (Fig. 9h). These Supplementary Data Suggest HIDDEN cells persist through stages of HPV-driven carcinogenesis into SCCs. Altogether, we show enrichment of HIDDEN cell biomarkers in patient-derived 3D in vitro ...
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... for gene and proteinlevel expression of HIDDEN cell biomarkers. Many cells with multiple puncta were detected in two cases of p16+ HNSCCs by RNA-ISH but were largely absent in two cases of p16-negative HNSCCs (Fig. 9g). Similarly, ELF3 protein was robustly detected in HPV+, but minimally in HPV−, HNSCCs with HPV status confirmed by p16 staining (Fig. 9h). These Supplementary Data Suggest HIDDEN cells persist through stages of HPV-driven carcinogenesis into SCCs. Altogether, we show enrichment of HIDDEN cell biomarkers in patient-derived 3D in vitro models of cutaneous, cervical, and tonsillar epithelium, as well as in HPV-driven premalignant and malignant patient biopsies that reflect ...
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... To visualize genomic regions of interest we used Bacterial Artificial Chromosome (BAC) clones RP11-990E14 (hg38 -chr3:169,692,858-169,870,368 mapping to Telomerase RNA Component TERC), and RP11-662P23 (hg38 -chr5:56,420,151-56,608,998 mapping to the specific region of HPV16 integration on chromosome 5 in Tumor 4). In addition to human BAC clones we also used a plasmid containing the entire genomic sequence of HPV16 as reported in 100,101 . BAC DNA and HPV16 plasmid DNA was isolated and labeled by nick translation as we previously described 102,103 using the following dUTPs from Dyomics (Jena, GE, USA): DY-495-dUTP (within the green spectrum to visualize RP11-662P23); DY-530-aadUTP (within the gold spectrum to visualize RP11-990E14); and DY-590-dUTP (within the red spectrum to visualize HPV16). ...
HPV infections are associated with a fraction of vulvar cancers. Through hybridization capture and DNA sequencing, HPV DNA was detected in five of thirteen vulvar cancers. HPV16 DNA was integrated into human DNA in three of the five. The insertions were in introns of human NCKAP1 , C5orf67 , and LRP1B . Integrations in NCKAP1 and C5orf67 were flanked by short direct repeats in the human DNA, consistent with HPV DNA insertions at sites of abortive, staggered, endonucleolytic incisions. The insertion in C5orf67 was present as a 36 kbp, human-HPV-hetero-catemeric DNA as either an extrachromosomal circle or a tandem repeat within the human genome. The human circularization/repeat junction was defined at single nucleotide resolution. The integrated viral DNA segments all retained an intact upstream regulatory region and the adjacent viral E6 and E7 oncogenes. RNA sequencing revealed that the only HPV genes consistently transcribed from the integrated viral DNAs were E7 and E6*I. The other two HPV DNA+ tumors had coinfections, but no evidence for integration. HPV-positive and HPV-negative vulvar cancers exhibited contrasting human, global gene expression patterns partially overlapping with previously observed differences between HPV-positive and HPV-negative cervical and oropharyngeal cancers. A substantial fraction of the differentially expressed genes involved immune system function. Thus, transcription and HPV DNA integration in vulvar cancers resemble those in other HPV-positive cancers. This study emphasizes the power of hybridization capture coupled with DNA and RNA sequencing to identify a broad spectrum of HPV types, determine human genome integration status of viral DNAs, and elucidate their structures.
... Using a 3D organotypic epithelial raft model, Bedard et al. discovered a keratinocyte subpopulation that was expanded and reprogrammed by HPV [106]. These cells (termed HPV-induced differentiation-dissonant epithelial nonconventional cells; HIDDEN) formed a distinct compartment in the infected epithelium and persisted from neoplastic lesions to outright cancer. ...
... These cells (termed HPV-induced differentiation-dissonant epithelial nonconventional cells; HIDDEN) formed a distinct compartment in the infected epithelium and persisted from neoplastic lesions to outright cancer. ELF3/ESE-1 was identified as a key regulator whose depletion in HPV + epithelium greatly reduced HIDDEN cell biomarker expression and compartment formation [106]. ...
Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent cancers worldwide. The main risk factors are consumption of tobacco products and alcohol, as well as infection with human papilloma virus. Approved therapeutic options comprise surgery, radiation, chemotherapy, targeted therapy through epidermal growth factor receptor inhibition, and immunotherapy, but outcome has remained unsatisfactory due to recurrence rates of ~50% and the frequent occurrence of second primaries. The availability of the human genome sequence at the beginning of the millennium heralded the omics era, in which rapid technological progress has advanced our knowledge of the molecular biology of malignant diseases, including HNSCC, at an unprecedented pace. Initially, microarray-based methods, followed by approaches based on next-generation sequencing, were applied to study the genetics, epigenetics, and gene expression patterns of bulk tumors. More recently, the advent of single-cell RNA sequencing (scRNAseq) and spatial transcriptomics methods has facilitated the investigation of the heterogeneity between and within different cell populations in the tumor microenvironment (e.g., cancer cells, fibroblasts, immune cells, endothelial cells), led to the discovery of novel cell types, and advanced the discovery of cell-cell communication within tumors. This review provides an overview of scRNAseq, spatial transcriptomics, and the associated bioinformatics methods, and summarizes how their application has promoted our understanding of the emergence, composition, progression, and therapy responsiveness of, and intercellular signaling within, HNSCC.
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