Abstract 3520: Single cell-derived spheroids capture functional and genetic heterogeneity of head and neck cancer
Authors: Pandey Jyoti , Malik Md Jubbair , Shyanti Ritis K. , Lee Jee Min , Tailor Dhanir , Malhotra Sanjay V. , Chaturvedi Rupesh , Singh Rana P. 
Published: June 2023

Abstract
Introduction: Single cell transcriptome is used for mapping the genetic heterogeneity of a tumour; however, it does not reflect the functional heterogeneity. Currently, no model is available to provide a genotype to phenotype correlations for the heterogeneity. We developed the HNSCC spheroids from single cell that showed hyperproliferation/hypomigration or hypoproliferation/hypermigration properties, and performed transcriptome and proteomics approach to identify the underlaying mechanisms.
Materials and methods: Two cells/well from HNSCC cell line UMSCC-22B were cultured in ultra-low attachment plate to develop spheroids, which were identified for their functionality by monitoring growth and cell migration. Hyperproliferative/hypomigratory or hypoproliferative/hypermigratory spheroids were pooled to perform single cell transcriptome. sSpheroid were used for proteomic analysis.
Results: Single cell analysis showed four distinct clusters of cells in hyperproliferative/hypomigratory spheroids and six clusters in hypoproliferative/hypermigratory spheroids. Each cluster showed specific gene expression signature enriching a specific pathways. The hyperproliferative/hypomigratory spheroids clusters showed enrichment of cell cycle, anti-apoptosis, and pro-survival genes associated with EGFR, Wnt and Notch signalling pathways. Whereas hypoproliferative/hypermigratory spheroids cluster showed enrichment of TGF-beta, VEGF, and wound healing related pathways. Cell ontology tracing with gene signatures revealed the dominance of neoplastic cells with enrichment of EGFR, MAP Kinase, Wnt signalling pathways in the hyperproliferative/hypomigratory spheroids. The fibroblast cell cluster showed expression of stem cell markers and enrichment of ERK and EGFR related pathways. Bayesians and non-bayesians network analyses identified the surface proteins, ITGA6, Met, CD63, ICAM, and Kit as unique driver genes for hyperproliferative/hypomigratory spheroids whereas ITGB3, CD44, and EGFR were the driver genes for hypoproliferative/hypermigratory spheroids. Proteomic analysis also conformed the enrichment of antiapoptosis and cell survival related pathways in hyperproliferative/hypomigratory spheroids. While epithelial cell differentiation to keratinized cells and wound healing pathways were enriched in hypoproliferative/hypermigratory spheroids. TCGA data shows correlation with our analysis for survival/stage/progression of the disease.
Conclusions: Our results suggest that single cell-derived clonal spheroid represents the genetic heterogeneity of cell population and correlates with functionality of the spheroids. These spheroids can be used as tools and the identification of cell clusters in head and neck cancer patients can be used for deciding the optimal therapeutics as well as to predict the progression of the cancer.
Citation Format: Jyoti Pandey, Md Jubbair Malik, Ritis K. Shyanti, Jee Min Lee, Dhanir Tailor, Sanjay V. Malhotra, Rupesh Chaturvedi, Rana P. Singh. Single cell-derived spheroids capture functional and genetic heterogeneity of head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3520.