Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer

Cell.

2017 Nov 30

Puram SV, Tirosh I, Parikh AS, Patel AP, Yizhak K, Gillespie S, Rodman C, Luo CL, Mroz EA, Emerick KS, Deschler DG, Varvares MA, Mylvaganam R, Rozenblatt-Rosen O, Rocco JW, Faquin WC, Lin DT, Regev A, Bernstein BE.
PMID: 29198524 | DOI: 10.1016/j.cell.2017.10.044

The diverse malignant, stromal, and immune cells in tumors affect growth, metastasis, and response to therapy. We profiled transcriptomes of ∼6,000 single cells from 18 head and neck squamous cell carcinoma (HNSCC) patients, including five matched pairs of primary tumors and lymph node metastases. Stromal and immune cells had consistent expression programs across patients. Conversely, malignant cells varied within and between tumors in their expression of signatures related to cell cycle, stress, hypoxia, epithelial differentiation, and partial epithelial-to-mesenchymal transition (p-EMT). Cells expressing the p-EMT program spatially localized to the leading edge of primary tumors. By integrating single-cell transcriptomes with bulk expression profiles for hundreds of tumors, we refined HNSCC subtypes by their malignant and stromal composition and established p-EMT as an independent predictor of nodal metastasis, grade, and adverse pathologic features. Our results provide insight into the HNSCC ecosystem and define stromal interactions and a p-EMT program associated with metastasis.

MmuPV1-Induced Cutaneous Squamous Cell Carcinoma Arises Preferentially from Lgr5+ Epithelial Progenitor Cells

Viruses

2022 Aug 11

Moreno, R;Buehler, D;Lambert, PF;
PMID: 36016373 | DOI: 10.3390/v14081751

Murine papillomavirus, MmuPV1, causes natural infections in laboratory mice that can progress to squamous cell carcinoma (SCC) making it a useful preclinical model to study the role of papillomaviruses in cancer. Papillomavirus can infect cells within hair follicles, which contain multiple epithelial progenitor cell populations, including Lgr5+ progenitors, and transgenic mice expressing human papillomavirus oncogenes develop tumors derived from Lgr5 progenitors. We therefore tested the hypothesis that Lgr5+ progenitors contribute to neoplastic lesions arising in skins infected with MmuPV1 by performing lineage tracing experiments. Ears of 6-8-week-old Lgr5-eGFP-IRES-CreERT2/Rosa26LSLtdTomato mice were treated topically with 4-OH Tamoxifen to label Lgr5+ progenitor cells and their progeny with tdTomato and, 72 h later, infected with MmuPV1. Four months post-infection, tissue at the infection site was harvested for histopathological analysis and immunofluorescence to determine the percentage of tdTomato+ cells within the epithelial lesions caused by MmuPV1. Squamous cell dysplasia showed a low percentage of tdTomato+ cells (7%), indicating that it arises primarily from non-Lgr5 progenitor cells. In contrast, cutaneous SCC (cSCC) was substantially more positive for tdTomato+ cells (42%), indicating that cSCCs preferentially arise from Lgr5+ progenitors. Biomarker analyses of dysplasia vs. cSCC revealed further differences consistent with cSCC arising from LGR5+ progenitor cells.

Durable response in a patient with recurrent respiratory papillomatosis treated with immune checkpoint blockade

Head & neck

2022 Jul 11

Bai, K;Norberg, SM;Sievers, C;Meyer, T;Friedman, J;Hinrichs, C;Allen, CT;
PMID: 35815785 | DOI: 10.1002/hed.27144

Immune checkpoint blockade can provide clinical benefit for patients with advanced cancer. Here, we report durable disease control over many years following PD-L1 blockade through induction of a viral antigen-specific T cell response in an adult patient with recurrent respiratory papillomatosis.Antigen-specific T cell response assays, single cell RNA-sequencing, and RNA-scope was used to study clinical tissues.An HPV6 E2-specific T cell clone restricted to HLA-B*55, present at low frequency in the pre-treatment papilloma, significantly expanded after six doses of PD-L1 blockade and remained present and functional at the site of initial response in the larynx as a tissue resident memory T cell for 4 years. An associated reduction in E2 target gene was observed following treatment.Although demonstrated in a single exceptional responder, these results highlight that immune checkpoint blockade may induce durable, viral antigen-specific immunity of sufficient magnitude to control disease in patients with nonmalignant disorders.

Efficacy of Topically Administered Dihydroartemisinin in Treating Papillomavirus-Induced Anogenital Dysplasia in Preclinical Mouse Models

Viruses

2022 Jul 26

Gunder, LC;Blaine-Sauer, S;Johnson, HR;Shin, MK;Auyeung, AS;Zhang, W;Leverson, GE;Ward-Shaw, ET;King, RE;McGregor, SM;Matkowskyj, KA;Lambert, PF;Carchman, EH;
PMID: 35893697 | DOI: 10.3390/v14081632

The artemisinin family of compounds is cytopathic in certain cancer cell lines that are positive for human papillomaviruses (HPV) and can potentially drive the regression of dysplastic lesions. We evaluated the efficacy of topical dihydroartemisinin (DHA) on cervical dysplasia and anal dysplasia in two papillomavirus mouse models: K14E6/E7 transgenic mice, which express HPV16 oncogenes; and immunodeficient NOD/SCID gamma (NSG) mice infected with Mus musculus papillomavirus (MmuPV1). Mice started treatment with DHA at 25 weeks of age (K14E6/E7) or 20 weeks post infection (MmuPV1-infected), when the majority of mice are known to have papillomavirus-induced low- to high-grade dysplasia. Mice were treated with or without topical DHA at the cervix or anus and with or without topical treatment with the chemical carcinogen 7,12 dimethylbenz(a)anthracene (DMBA) at the anus of in transgenic mice to induce neoplastic progression. Mice were monitored for overt tumor growth, and tissue was harvested after 20 weeks of treatment and scored for severity of histological disease. For MmuPV1-infected mice, anogenital lavages were taken to monitor for viral clearance. Tissues were also evaluated for viral gene expression at the RNA and/or protein levels. Treatment with topical DHA did not reduce dysplasia in the anogenital tract in either papillomavirus-induced mouse model and did not prevent progression to anal cancer in the DMBA-treated K14E6/E7 mice.

Association of HPV42 with digital papillary adenocarcinoma and the use of in situ hybridization for its distinction from acral hidradenoma and diagnosis at non-acral sites

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

2022 May 10

Vanderbilt, C;Brenn, T;Moy, AP;Harloe, G;Ariyan, C;Athanasian, E;Busam, KJ;
PMID: 35538210 | DOI: 10.1038/s41379-022-01094-8

Digital papillary adenocarcinoma (DPAC) is a rare tumor of sweat gland origin that preferentially affects the digits and has the potential to metastasize. Its tumor diagnosis can be difficult. Well-differentiated variants of DPAC can be confused with a benign sweat gland tumor, in particular nodular hidradenoma. With the recent detection of HPV42 DNA in DPAC by next-generation sequence analysis, we reasoned that this association could be used for diagnostic purposes. To this end, we performed in situ hybridization for HPV42 on 10 tumors diagnosed as DPAC as well as 30 sweat gland tumors of various histology types, including 8 acral hidradenomas. All DPAC were positive for HPV42. Positive hybridization signals for HPV42 were seen in both primary and metastatic DPACs. All other tumors and normal tissues were negative. This study confirms the association of HPV42 with the tumor cells of DPAC through in situ hybridization. The positive test result in all lesions of DPAC and lack of detection of HPV42 in any of the acral hidradenomas or other sweat gland tumors examined in this series is encouraging for the potential diagnostic utility of the assay. As documented by two scrotal tumors of DPAC, the in situ hybridization test for HPV42 can also help support the rare occurrence of this tumor at a non-acral site.

A novel lineage-tracing mouse model for studying early MmuPV1 infections

eLife

2022 May 09

Yilmaz, V;Louca, P;Potamiti, L;Panayiotidis, M;Strati, K;
PMID: 35533001 | DOI: 10.7554/eLife.72638

Human papillomaviruses are DNA viruses that ubiquitously infect humans and have been associated with hyperproliferative lesions. The recently discovered mouse specific papillomavirus (MmuPV1) provides the opportunity to study papillomavirus infections in vivo in the context of a common laboratory mouse model (Mus musculus). To date, a major challenge in the field has been the lack of tools to identify, observe, and characterize individually the papillomavirus hosting cells and also trace the progeny of these cells over time. Here, we present the successful generation of an in vivo lineage-tracing model of MmuPV1-harboring cells and their progeny by means of genetic reporter activation. Following the validation of the system both in vitro and in vivo, we used it to provide a proof-of-concept of its utility. Using flow-cytometry analysis, we observed increased proliferation dynamics and decreased MHC-I cell surface expression in MmuPV1-treated tissues which could have implications in tissue regenerative capacity and ability to clear the virus. This model is a novel tool to study the biology of the MmuPV1 host-pathogen interactions.

Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining

PLoS pathogens

2021 Aug 01

Yu, L;Majerciak, V;Xue, XY;Uberoi, A;Lobanov, A;Chen, X;Cam, M;Hughes, SH;Lambert, PF;Zheng, ZM;
PMID: 34343212 | DOI: 10.1371/journal.ppat.1009812

MmuPV1 is a useful model for studying papillomavirus-induced tumorigenesis. We used RNA-seq to look for chimeric RNAs that map to both MmuPV1 and host genomes. In tumor tissues, a higher proportion of total viral reads were virus-host chimeric junction reads (CJRs) (1.9‰ - 7‰) than in tumor-free tissues (0.6‰ - 1.3‰): most CJRs mapped to the viral E2/E4 region. Although most of the MmuPV1 integration sites were mapped to intergenic regions and introns throughout the mouse genome, integrations were seen more than once in several genes: Malat1, Krt1, Krt10, Fabp5, Pard3, and Grip1; these data were confirmed by rapid amplification of cDNA ends (RACE)-Single Molecule Real-Time (SMRT)-seq or targeted DNA-seq. Microhomology sequences were frequently seen at host-virus DNA junctions. MmuPV1 infection and integration affected the expression of host genes. We found that factors for DNA double-stranded break repair and microhomology-mediated end-joining (MMEJ), such as H2ax, Fen1, DNA polymerase Polθ, Cdk1, and Plk1, exhibited a step-wise increase and Mdc1 a decrease in expression in MmuPV1-infected tissues and MmuPV1 tumors relative to normal tissues. Increased expression of mitotic kinases CDK1 and PLK1 appears to be correlated with CtIP phosphorylation in MmuPV1 tumors, suggesting a role for MMEJ-mediated DNA joining in the MmuPV1 integration events that are associated with MmuPV1-induced progression of tumors.

Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy

Cell

2021 Jun 22

Béziat, V;Rapaport, F;Hu, J;Titeux, M;Bonnet des Claustres, M;Bourgey, M;Griffin, H;Bandet, É;Ma, CS;Sherkat, R;Rokni-Zadeh, H;Louis, DM;Changi-Ashtiani, M;Delmonte, OM;Fukushima, T;Habib, T;Guennoun, A;Khan, T;Bender, N;Rahman, M;About, F;Yang, R;Rao, G;Rouzaud, C;Li, J;Shearer, D;Balogh, K;Al Ali, F;Ata, M;Dabiri, S;Momenilandi, M;Nammour, J;Alyanakian, MA;Leruez-Ville, M;Guenat, D;Materna, M;Marcot, L;Vladikine, N;Soret, C;Vahidnezhad, H;Youssefian, L;Saeidian, AH;Uitto, J;Catherinot, É;Navabi, SS;Zarhrate, M;Woodley, DT;Jeljeli, M;Abraham, T;Belkaya, S;Lorenzo, L;Rosain, J;Bayat, M;Lanternier, F;Lortholary, O;Zakavi, F;Gros, P;Orth, G;Abel, L;Prétet, JL;Fraitag, S;Jouanguy, E;Davis, MM;Tangye, SG;Notarangelo, LD;Marr, N;Waterboer, T;Langlais, D;Doorbar, J;Hovnanian, A;Christensen, N;Bossuyt, X;Shahrooei, M;Casanova, JL;
PMID: 34214472 | DOI: 10.1016/j.cell.2021.06.004

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

The Exon Junction Complex core factor eIF4A3 is a key regulator of HPV16 gene expression

Bioscience reports

2021 Mar 24

Meznad, K;Paget-Bailly, P;Jacquin, E;Peigney, A;Aubin, F;Guittaut, M;Mougin, C;Prétet, JL;Baguet, A;
PMID: 33760064 | DOI: 10.1042/BSR20203488

High-risk human papillomavirus (hrHPVs), particularly HPV16 and HPV18, are the etiologic factors of ano-genital cancers and some head and neck squamous cell carcinomas. Viral E6 and E7 oncoproteins, controlled at both transcriptional and post-transcriptional levels, drive hrHPVs-induced carcinogenesis. In this study, we investigated the implication of the DEAD-box helicase eIF4A3, an Exon Junction Complex factor, in the regulation of HPV16 gene expression. Our data revealed that the depletion of the factor eIF4A3 upregulated E7 oncoprotein levels. We also showed that the inhibition of the nonsense-mediated RNA decay (NMD) pathway, resulted in the upregulation of E7 at both RNA and protein levels. We therefore proposed that HPV16 transcripts might present different susceptibilities to NMD and that this pathway could play a key role in the levels of expression of these viral oncoproteins during the development of HPV-related cancers.

SOX10 Immunoexpression in Basaloid Squamous Cell Carcinomas: A Diagnostic Pitfall for Ruling out Salivary Differentiation.

Head Neck Pathol. 2018 Nov 29.

2018 Nov 29

Rooper LM, McCuiston AM, Westra WH, Bishop JA.
PMID: 30498968 | DOI: 10.1007/s12105-018-0990-7

SOX10 immunoexpression is increasingly recognized in salivary gland tumors, including but not limited to those with myoepithelial, serous acinar, and intercalated duct differentiation. However, SOX10 expression has not been extensively evaluated in other epithelial tumors that can mimic salivary origin. Basaloid squamous cell carcinoma (SCC) is a unique variant of SCC that shows morphologic overlap with several salivary tumors, including adenoid cystic carcinoma, basal cell adenocarcinoma, and myoepithelial carcinoma. We performed SOX10 immunohistochemistry on 22 basaloid SCCs and 280 non-basaloid SCCs. If tissue was available, we also performed immunohistochemistry for S100 and p16, and in-situ hybridization for high-risk HPV RNA. SOX10 was positive in 13/22 basaloid SCCs (59%), including 5/6 (83%) that were HPV-positive and 6/12 (50%) that were HPV-negative. Only 2/12 basaloid SCC (17%) demonstrated focal S100 expression. All non-basaloid SCCs were SOX10 negative. Frequent positivity for SOX10 in basaloid SCC presents a significant diagnostic pitfall for distinguishing these tumors from various basaloid salivary carcinomas. The preponderance of SOX10 expression in the basaloid variant of HPV-positive SCC also presents a diagnostic challenge in separating it from HPV-related multiphenotypic sinonasal carcinoma. SOX10 may be more broadly considered a marker of basal differentiation and should not be assumed to be specific for salivary origin in epithelial head and neck tumors.

Conditional Cell Reprogramming and Air-Liquid Interface Modeling Life Cycle of Oncogenic Viruses (HPV and EBV) in Epithelial Cells and Virus-Associated Human Carcinomas

Viruses

2023 Jun 17

Rani, AQ;Nurmemet, D;Liffick, J;Khan, A;Mitchell, D;Li, J;Zhao, B;Liu, X;
PMID: 37376685 | DOI: 10.3390/v15061388

Several oncogenic viruses are associated with approximately 20% of human cancers. Experimental models are crucial for studying the pathogenicity and biological aspects of oncogenic viruses and their potential mechanisms in tumorigenesis. Current cell models have considerable limitations such as: their low yield, genetic and epigenetic modification, and reduction in tumor heterogeneity during long propagation. Cancer cell lines are limited and not appropriate for studying the viral life cycle, for example, natural viral life cycles of HPV and EBV, and their persistence and latency in epithelial cells are poorly understood, since these processes are highly related to epithelial differentiation. Therefore, there is an urgent need of reliable human physiological cell models to study viral life cycle and cancer initiation. Conditional cell reprogramming (CCR) is a rapid and robust cell culture system, where the cells can be established from minimally invasive or noninvasive specimens and their lineage functions preserved during the long-term culture. These CR cells retain their ability to differentiate at air-liquid interface (ALI). Here, we recapitulated the applications of CR and ALI approaches in modeling host-virus interactions and viral-mediated tumorigenesis.

Single cell transcriptomic analysis of HPV16-infected epithelium identifies a keratinocyte subpopulation implicated in cancer

Nature communications

2023 Apr 08

Bedard, MC;Chihanga, T;Carlile, A;Jackson, R;Brusadelli, MG;Lee, D;VonHandorf, A;Rochman, M;Dexheimer, PJ;Chalmers, J;Nuovo, G;Lehn, M;Williams, DEJ;Kulkarni, A;Carey, M;Jackson, A;Billingsley, C;Tang, A;Zender, C;Patil, Y;Wise-Draper, TM;Herzog, TJ;Ferris, RL;Kendler, A;Aronow, BJ;Kofron, M;Rothenberg, ME;Weirauch, MT;Van Doorslaer, K;Wikenheiser-Brokamp, KA;Lambert, PF;Adam, M;Steven Potter, S;Wells, SI;
PMID: 37031202 | DOI: 10.1038/s41467-023-37377-0

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 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

Pages

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

Search form

Probes for HPV

Content for comparison

Gene

Product

Research area

Category

Pages

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China