Single-cell Transcriptomic Analysis of Tumour Ecosystems in Oropharyngeal Cancer

Oropharyngeal squamous cell carcinoma (OPSCC) is a highly heterogenous cancer. Single-cell RNA sequencing (scRNASeq) enables mapping this heterogeneity through identifying small but biologically significant cell populations. Our aim was to characterise these subpopulations and their functions in OPSCC to improve biological understanding and aid patient stratification.

3 HPV- and 13 HPV16+ OPSCC patients were included in the study. Fresh tumour samples were dissociated into a single-cell suspension. Cells were barcoded using the Chromium 10x system, followed by Illumina sequencing. The generated sequences were aligned to the human transcriptome as well as the transcriptomes of HPV 16, 18, 31, 33 and 35. Cells were clustered and their gene expression patterns examined to define the major cell types. Cancer cells and genetic subclones were defined by the presence of inferred copy number aberrations (CNA) across chromosomal regions. Recurring genetic profiles were combined into metaprograms representing biological functions.

More than 60000 cells were identified and classified. In cancer cells, metaprograms representing senescence, proliferation, epithelial-mesenchymal transition and stemness were found. Clonal tumour evolution could be traced through identifying cells with differing CNA in the same tumour. Particularly interesting were the facts that one p16+ tumour could be reclassified as HPV- due to absence of HPV transcripts, while another tumour showed two highly distinct CNA patterns, suggesting two biologically unrelated tumours in the same location. We could also identify HPV transcripts and CNA in pathologically normal tissue. In conclusion, these findings suggest a role for scRNASeq in aiding pathological diagnosis.