Abstract PO-116: High resolution single cell microscopy analyses identifies the cellular redox state that supports conversion to a neoplastic stem cell state

Abstract

Abstract High resolution single cell microscopy analyses identifies the cellular redox state that supports conversion to a neoplastic stem cell state Brian Spurlock and Kasturi Mitra University of Alabama at Birmingham Introduction: Single cell analyses lie at the core of teasing out tumor cell heterogeneity at the level of their self-renewal and differentiation ability, gene mutation and expression, energetics, metabolism, redox status etc. Cellular redox states are modulated by Reactive Oxygen Species (ROS) that are primarily generated by mitochondria. The neoplastic stem cells, which lie at the base of tumor cell hierarchy, are supported by optimally elevated mitochondrial ROS levels. However, induction of higher levels of mitochondrial ROS can eliminate the neoplastic stem cells. Therefore, there appears to be a sweet spot of mitochondrial ROS levels for maintenance of neoplastic stem cells. Given our longstanding interest in mitochondrial biology, we aimed at probing mitochondrial redox modulation at a single cell level towards understanding cellular susceptibility for neoplastic transformation. Experimental procedures: For this study we have used our high resolution confocal microscopy based single cell assay for mitochondrial properties, cell based Seahorse respirometry assay, tumorsphere assay, xenograft assays and shRNA mediated gene knockdown. Summary of data: We have used carcinogen induced cell transformation model to study neoplastic transformation. We found that carcinogen induced enrichment of neoplastic stem cells was associated with a certain level of mitochondrial ROS beyond which the number of neoplastic stem cell frequency decreases dramatically. The increase in the lineage specific stem cell marker, Sox2, happens only in cells with repression of a mitochondrial property called mitochondrial fission. Knockdown of the master regulator of mitochondrial fission, Drp1 (Dynamin Regulated Protein 1), alters mitochondrial ROS levels and causes increase in Sox2 levels and accelerates carcinogen induced cell transformation. Therefore, our data suggested that repression of mitochondrial fission sustains mitochondrial ROS at a certain level to allow enrichment of neoplastic stem cells. Most importantly, our single cell microscopy analyses of mitochondrial ROS and mitochondrial fission in the same cells identifies the intermediate mitochondrial ROS levels that associates with a minimum mito-fission state that supports neoplastic stem cells. Conclusion: We conclude that the cells with a certain quantifiable level of mitochondrial-ROS status are maximally susceptible to (carcinogen induced) conversion to neoplastic stem cells and can be identified by our novel high resolution microscopy based single cell analyses. Currently, we are performing single cell RNAseq experiments with the goal of integrated understanding of gene expression and mitochondrial redox regulation in neoplastic stem cells. Citation Format: Brian Spurlock, Kasturi Mitra. High resolution single cell microscopy analyses identifies the cellular redox state that supports conversion to a neoplastic stem cell state [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-116.