Targeted spatial proteomic analysis of CD8+ T- and myeloid cells in tonsillar cancer.

Background: Tonsillar cancer is caused by high-risk human papillomavirus (HPV), tobacco smoking, and alcohol abuse. Aspects of the patient's immune response to this disease have arisen as prognostic factors and treatment targets, reflecting differences in the type and protein expression profile of immune cells. Because tonsillar cancers are heterogenous lesions such data need to be spatially resolved. Methods: In this study, we aim to explore inter-patient and intra-tumoral sources of variation in tonsillar cancer using immunofluorescence and targeted spatial proteomics to interrogate a cohort of 105 patients. Furthermore, we assess prognostic factors and elucidate molecular targets. We have used CD8, CD11c, and Pan-cytokeratin (PanCK) to quantify and locate immune cells driving antigen-specific cellular immunity. Guided by immunofluorescence information, we selected 355 CD8+, CD11c+, or PanCK+ areas inside and outside (i.e., stroma) cancer-cell islets, to quantify 43 immune-related proteins using digital spatial profiling. Results: Quantitative analysis of immunofluorescence in combination with clinical data revealed that the abundance of total CD8+ cells and CD8+ cells infiltrating cancer-cell islets, respectively, were associated with higher 5-year disease-free survival and overall survival, independently of HPV-status and clinical stage. Comparison of CD8+ cells inside and outside cancer-cell islets revealed an upregulation of effector CD8+ T-cell and immune checkpoint molecules in the former. Among these, the expression of PD-L1 by CD8+ T-cells was associated with lower all-cause mortality in a univariate proportional hazards model. Similarly, a comparison of tumor boundary and stroma CD11c+ cells showed upregulation of both co-stimulatory and immune checkpoint molecules with proximity to tumor cell islets. Conclusion: Our findings highlight the relevance of analyzing aspects of tumor micro-architecture in the search of prognostic markers and molecular targets for tonsillar cancer. The abundance of intra-tumoral CD8+ T-cells can be considered a positive predictive marker for tonsillar cancer, while the significance of PD-L1 expression by intra-tumoral CD8+ T-cells warrants further evaluation. Location-based differences in CD8+ and CD11c+ cells suggest an immune cell-altering effect on the tumor microenvironment, and grant new insight into which cells that can be targeted by novel therapeutic agents.

Human papillomavirus-associated head and neck squamous cell carcinoma cells rely on glycolysis and display reduced oxidative phosphorylation.

Introduction: Head and neck squamous cell carcinoma (HNSCC) constitutes a heterogeneous group of cancers. Human papilloma virus (HPV) is associated with a subtype of HNSCC with a better response to treatment and more favorable prognosis. Mitochondrial function and metabolism vary depending on cancer type and can be related to tumor aggressiveness. This study aims to characterize the metabolism of HPV-positive and HPV-negative HNSCC cell lines. Methods: Oxidative phosphorylation (OXPHOS) and glycolysis were assessed in intact cells, in four HNSCC cell lines using Seahorse XF Analyzer. OXPHOS was further studied in permeabilized cells using high-resolution respirometry in an Oroboros O2K. Metabolomic analysis was performed using mass spectroscopy. Results: The HPV-negative cell lines were found to display a higher OXPHOS capacity and were also able to upregulate glycolysis when needed. The HPV-positive cell line had a higher basal glycolytic rate but lower spare OXPHOS capacity. These cells were also unable to increase respiration in response to succinate, unlike the HPV-negative cells. In the metabolomic analysis, the HPV-positive cells showed a higher kynurenine/tryptophan ratio. Discussion: HPV-positive HNSCC preferred glycolysis to compensate for lower OXPHOS reserves, while the HPV-negative HNSCC displayed a more versatile metabolism, which might be related to increased tumor aggressiveness. The higher kynurenine/tryptophan ratio of HPV-positive HNSCC might be related to increased indoleamine 2,3-dioxygenase activity due to the carcinoma's viral origin. This study highlights important metabolic differences between HPV-positive and HPV-negative cancers and suggests that future metabolic targets for cancer treatment should be individualized based on specific tumor metabolism.

Single-cell analysis of myeloid cells in HPV+ tonsillar cancer.

The incidence of human papillomavirus-positive (HPV+) tonsillar cancer has been sharply rising during the last decades. Myeloid cells represent an appropriate therapeutic target due to their proximity to virus-infected tumor cells, and their ability to orchestrate antigen-specific immunity, within the tonsil. However, the interrelationship of steady-state and inflammatory myeloid cell subsets, and their impact on patient survival remains unexplored. Here, we used single-cell RNA-sequencing to map the myeloid compartment in HPV+ tonsillar cancer. We observed an expansion of the myeloid compartment in HPV+ tonsillar cancer, accompanied by interferon-induced cellular responses both in dendritic cells (DCs) and monocyte-macrophages. Our analysis unveiled the existence of four DC lineages, two macrophage polarization processes, and their sequential maturation profiles. Within the DC lineages, we described a balance shift in the frequency of progenitor and mature cDC favoring the cDC1 lineage in detriment of cDC2s. Furthermore, we observed that all DC lineages apart from DC5s matured into a common activated DC transcriptional program involving upregulation of interferon-inducible genes. In turn, the monocyte-macrophage lineage was subjected to early monocyte polarization events, which give rise to either interferon-activated or CXCL-producing macrophages, the latter enriched in advanced tumor stages. We validated the existence of most of the single-cell RNA-seq clusters using 26-plex flow cytometry, and described a positive impact of cDC1 and interferon-activated DCs and macrophages on patient survival using gene signature scoring. The current study contributes to the understanding of myeloid ontogeny and dynamics in HPV-driven tonsillar cancer, and highlights myeloid biomarkers that can be used to assess patient prognosis.

Tonsillar Cancer with High CD8+ T-Cell Infiltration Features Increased Levels of Dendritic Cells and Transcriptional Regulation Associated with an Inflamed Tumor Microenvironment.

Human papillomavirus (HPV) is the main causal agent of tonsillar cancer (TC) and HPV+ TC has a favorable prognosis compared to HPV- disease. In this study, we examined aspects of the tumor microenvironment of TC, focusing on T-cells, dendritic cells (DC), and macrophages. Fresh biopsies of TC and the contralateral healthy tonsil (HT) were obtained from 20 patients, analyzed by multiparameter flow cytometry, and assessed against a detailed HPV-status. Additionally, RNA-sequencing data from 38 TC samples available in the public database, The Cancer Genome Atlas (TCGA), were explored, focusing on the same leukocyte populations. HPV+ TC featured increased levels of CD8+ T-cells and antigen-presenting cells (cf. HPV- TC and HT, respectively). In HPV+ TC, CD8+ T-cell frequencies correlated to DC levels independently of tumor stage, HPV 16 copy number, and E7 oncogene expression as well as frequencies of other leukocytes. Similarly, RNA sequencing data were explored by dividing the HPV+ TCs according to predefined CD8+ T-cell scores in silico. Higher levels of genes expressed by antigen-presenting cells and effector T-cells, such as immune checkpoints and cytokines, were detected in the CD8HIGH HPV+ TC samples (cf. CD8LOW HPV+ TC). In conclusion, CD8HIGH HPV+ TC displays a unique inflammatory profile associated with increased effector T-cell functions and the presence of antigen-presenting cells in the tumor microenvironment. Further studies are warranted to assess if this information can be used on an individual basis to aid in prognosis and treatment decisions.

Immune Phenotypes of Nasopharyngeal Cancer.

Nasopharyngeal cancer (NPC) features intralesional immune cells, but data are lacking on presence/distribution of T-cells and dendritic cells (DCs). Based on intralesional distribution of lymphocytes, a series of NPC biopsies (n = 48) were classified into "inflamed", "excluded", and "deserted" phenotypes. In addition, CD8+ T-cells and CD207+ DCs were quantified. The data were analyzed in relation to Epstein-Barr virus-encoded small RNA (EBER), Epstein-Barr virus (EBV) DNA, and survival. Separately, data on gene expression from a public database were analyzed. 61.7% of NPC lesions were "inflamed", 29.8% were "excluded", and 8.5% were "deserted". While CD8+ cells were present in cancer cell areas and in surrounding stroma, CD207+ cells were observed largely in cancer cell areas. High CD8+ T-cell presence was associated with EBV+ disease, but no such pattern was observed for CD207+ DCs. There was a difference in disease-free survival in favor of "inflamed" over "excluded" NPC. Gene expression analysis revealed differences between NPC and control tissue (e.g., with regard to interferon activity) as well as between subgroups of NPC based on CD8 expression (high vs. low). In conclusion, NPC lesions are heterogeneous with regard to distribution of CD8+ T-cells and CD207+ DCs. NPC can be classified into immune phenotypes that carry prognostic information. CD207+ DCs may represent a target for immunotherapy with potential to facilitate the antigen cross-presentation necessary to execute cytotoxic T-lymphocyte responses.

A novel human in vitro papillomavirus type 16 positive tonsil cancer cell line with high sensitivity to radiation and cisplatin.

BACKGROUND: Human papillomavirus (HPV) is an established risk factor for oropharyngeal squamous cell carcinoma (OSCC). The aim was to establish cell lines from HPV-positive tonsil carcinomas to be used for treatment development. METHODS: Fresh samples from 23 HPV-positive tonsil carcinomas were cultivated in vitro. The established cell line was analyzed for viral characteristics, cell karyotype, TP53 status, and growth capabilities in nude mice. In vitro studies of sensitivities to radiation, cisplatin and cetuximab were performed. RESULTS: After 19 months (eight passages), one cell line, LU-HNSCC-26, was established in vitro and also grew as xenografts. The tumor was from a 48 year old non-smoking man with non-keratinizing, p16 positive tonsil OSCC, stage T2N0M0 with HPV16. It contained 19.5 (CV% 3.7) HPV16 copies/cell (passage 8). The complete HPV16 genome sequence was obtained. Episomal HPV16 was present with an E2/E7 ratio of 1.1 (CV% 2.6). In addition, HPV16 mRNA specific for the intact E2 gene was detected. The viral expression manifested 1.0 (CV% 0.1) E7 mRNA copies per HPV16 genome. The karyotype was determined and the cell line demonstrated wild type TP53. The ID50 for radiation was 0.90 Gy and the IC50 for cisplatin was 0.99 µmol/L. The cell line was inhibited to a maximum of 18% by cetuximab. CONCLUSIONS: We established an in vitro tonsil carcinoma cell line containing episomal HPV16. This is an important step towards efficient treatment development.