Abstract 10: Development of single cell spheroid as a tool to capture functional heterogeneity in head and neck cancer

Abstract

Abstract Introduction: Head and neck cancer squamous cell carcinomas (HNSCC) are diverse and complex with inter-tumor and intra-tumor heterogeneity and discrepancies in therapy response regardless of the clinical stage. Tumors are thought to be heterogeneous at the single-cell level involving cancer stem cells. Therefore, cancer diagnosis and treatment are difficult because single cells might have intrinsic functional and genetic heterogeneity. Single cell transcriptome is used for mapping the genetic heterogeneity of a tumor. Currently, no model is available to provide a genotype to phenotype correlations for the heterogeneity, a crucial step for successful treat of a cancer. Here, our focus is to develop a single-cell clonal spheroid model system as a valuable tool for studying functional heterogeneity, including growth, proliferation, angiogenesis, metastasis, and genetic heterogeneity. Material and methods: HNSCC cell lines namely, UMSCC-22B, FaDu and A-253, and patients derived primary cells were cultured in 96-well ultra-low attachment to develop spheroids. Spheroids were cultured from 2, 4 and 128 cells per well with media, growth factors and supplements. Confocal microscopy was done using cocktail dyes including Hoechst, Calcein-AM and Ethidium bromide to check the functionality of spheroids. Results: We have established a method for the development of single-cell derived clonal spheroids. The frequency of spheroids formation from 2, 4 and 128 UMSCC-22B cells per well were higher than FaDu and A-253 cell lines. The growth measurement showed clones of single cell derived spheroids with hyperproliferation as well as with low proliferative activity. UMSCC-22B and A-253 hypoproliferative spheroids had significant higher migration capabilities compared to spheroids with hyperproliferative activity. Confocal microscopy of single-cell derived clonally propagated spheroids revealed a different zone viz proliferating, quiescent and necrotic zone in intact spheroids. This condition mimics in vivo conditions and suitable tool for drug penetration studies. Conclusion: Together, our results suggest that single cell-derived clonal spheroid from different wells would represent the genetic and functional heterogeneity of the cell population. Such a spheroid model can reveal heterogeneity inside a tumor formed by the proliferation of a single-cell and heterogeneity between tumors resulting from a different single cell and thus has great potential for drug discovery and tumor biology research. Citation Format: Jyoti Pandey, Ritis Shyanti, Sanjay V. Malhotra, Rupesh Chaturvedi, Rana P. Singh. Development of single cell spheroid as a tool to capture functional heterogeneity in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 10.