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.