Abstract B056: Chronic hypoxia in TNBC breast cancer induces development of a novel population of CD24+/CD49b-high cells characterized by high expression of CST1
Authors: Ke May Sin , Elshenawy Badran , Arora Anjali , Sheldon Helen , Harris Adrian , Buffa Francesca 
Published: February 2024

Abstract
Triple-negative breast cancer (TNBC) is the subtype of breast cancer that has the worst clinical outcome due to the absence of specific targeting agents. TNBC is highly heterogeneous and within the tumor microenvironment, hypoxia emerges as a pivotal factor contributing to its aggressive biology. Solid tumors experience a fluctuating oxygen supply, leading to the formation of zones with prolonged oxygen deprivation. There have been few studies describing the response to chronic hypoxia [7-14 days] and this project investigates how chronic hypoxia contributes to tumour heterogeneity in a temporal manner. We performed scRNA-seq analysis on MDA-MB-231 cells subjected to a 14-day hypoxia treatment at 1% oxygen. We collected samples from both normoxia and hypoxia conditions on Days 1, 2, 7, and 14. A total of 40,000 cells were sequenced. There were 6 hypoxic cell clusters which evolved over the time course. Interestingly, chronic hypoxia induces various transient subpopulations. Cells in hypoxia Day 7 grew more slowly, before recovering growth at Day 14, marking a crucial adaptation point at Day 7 in chronic hypoxia. Our study suggests that chronic hypoxia cells conserve energy through G1 cell cycle arrest and Myc suppression as an adaptive mechanism to cope with chronic hypoxia. We also observed a higher expression of cancer stem cell markers [CD44, CXCR4 and KLF4] in hypoxia subpopulations relative to normoxia subpopulations, especially in the chronic hypoxia subpopulations. Notably, we identified a distinct CD24+/CD49b-high population, comprising the largest proportion of the hypoxic Day 7 samples which disappears by Day 14. This population is characterised by the upregulation of cystatin family genes- CST 1, 4 and 7 and may be pro-metastatic [upregulation of EMP1, ST3GAL6 and KRT19 genes] compared to earlier and later hypoxia phases. Interestingly, KRT19 is more abundant in peripheral blood of breast cancer patients with node metastasis, although its role in stemness of breast cancer is controversial. The heterogeneity we found here may explain this, with the existence of a transient population. Chronic hypoxia could perhaps have a role in reprogramming cancer stem cell-like cells into a pro-metastatic state. In summary, our study provides insights on how hypoxia induces protective translational reprogramming and drives plasticity in cancer cells. Understanding these processes may facilitate the development of targeted treatments to combat therapy resistance in TNBC.
Citation Format: May Sin Ke, Badran Elshenawy, Anjali Arora, Helen Sheldon, Adrian Harris, Francesca Buffa. Chronic hypoxia in TNBC breast cancer induces development of a novel population of CD24+/CD49b-high cells characterized by high expression of CST1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B056.