Abstract PR03: The tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC): Investigating new aspects of known cell types using single-cell RNA sequencing
Authors: Kürten Cornelius , Kulkarni Aditi , Chen Xueer , Vujanovic Lazar , Cillo Anthony , Lu Xinghua , Ferris Robert L. 
Published: September 2020

Abstract
While immunotherapy has been a significant improvement to traditional head and neck squamous cell carcinoma (HNSCC) treatment, the response rate is only 15-20%. The resistance to immunotherapy is mediated by intrinsic and extrinsic factors produced in the tumor microenvironment (TME). Single-cell RNA sequencing can be employed to study heterogeneous populations (e.g., stroma, cancer, and immune cells) within the TME. We hypothesize that this transcriptomic mapping of single cells will improve our understanding of the complex interactions between the different cellular components of the TME. For this study, matched peripheral blood leukocytes (PBL) and tumors from treatment-naive patients were obtained from the operating room and were processed immediately. Following dissociation of tumors into single-cell suspensions, samples were sorted into CD45+ (tumor-infiltrating leukocytes: TIL) and CD45- (tumor and associated stromal) cells. 10x Genomics 3’ single-cell libraries were generated and sequenced on a NextSeq500 (Illumina). Cells from all samples were aggregated and normalized using the CellRanger pipeline. Downstream bioinformatic analyses were performed using the Scanpy package. 103,006 single cells that passed filtering criteria with a median of 1,105 genes per cell were analyzed. From these populations we identified 30 different clusters, of which 22 clusters were formed by PBL and TIL while CD45- nonimmune cells formed 8 clusters. By extracting and subclustering each cell type individually, subpopulations were identified and characterized. In the immune cell subsets, therapeutically important cell activation substates (e.g., activated regulatory T cells and exhausted CD8 T cells) were observed. Gene set enrichment analysis identified novel pathways between activated and exhausted lymphocytes. Among epithelial cells, transcriptomic profiles of HPV+ and HPV- specimens clustered separately and displayed a surprising degree of heterogeneity that was also seen using pseudotime analyses. The impact of patient-specific differences in epithelial cell profiles on the quality of immune infiltrate was evaluated by modeling interactions between epithelial and immune cell populations. Differences were observed in lymphocyte phenotypes based on HPV status. In conclusion, the TME of patients with HNSCC consists of heterogenous cell populations of immune, stroma, and cancer cells. These can be characterized transcriptomically using single-cell RNA sequencing, which allows to identify diverse cell states. In particular, we uncovered differences in tumor cell populations between HPV+ and HPV- patients, while the nontumor stromal compartment was similar.
Citation Format: Cornelius Kürten, Aditi Kulkarni, Xueer Chen, Lazar Vujanovic, Anthony Cillo, Xinghua Lu, Robert L. Ferris. The tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC): Investigating new aspects of known cell types using single-cell RNA sequencing [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR03.