Abstract PD4-09: PD4-09 Single cell RNA-sequencing identifies intra-tumoral cellular heterogeneity and drug-induced subpopulation shifts in Triple Negative Breast Cancer mouse models
Authors: Glodowski Cherise R. , Mott Kevin R. , Okumu Denis , East Michael P. , Elston Timothy C. , Johnson Gary L. , Perou Charles M. 
Published: March 2023

Abstract
Introduction: Triple Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis that accounts for 10-20% of breast cancer cases worldwide. Intra-tumoral heterogeneity and tumor cell plasticity are thought to contribute to drug resistance in TNBCs. Our work aims to: 1) precisely identify the intra-tumoral heterogeneity in cellular states present within TNBC, and 2) test whether drugs can block or initiate plasticity between subpopulations to improve drug sensitivity. We hypothesize that treatment(s) induce cellular plasticity, thus causing cells to shift into resistant states that persist until treatment is removed. We further hypothesize that these resistant subpopulations give rise to new tumor outgrowths once the treatment stops. Methods: To test this, we are treating TP53-/- Genetically Engineered Mouse Model (GEMM) syngeneic transplant tumors of the basal-like TNBC phenotype with the chemotherapeutic doublet of carboplatin/paclitaxel, and targeted agents implicated in plasticity including the MEK inhibitor trametinib, a chromatin remodeling inhibitor I-BET151, and the dihydroorotate dehydrogenase inhibitor brequinar. To identify cellular subpopulations and examine their response to treatment, we performed both in vivo and in vitro drug sensitivity testing, as well as gene expression profiling using single cell RNA-sequencing (scRNAseq). Results: We have identified clear intra-tumoral heterogeneity with at least 6 distinct cell states present, including basal, mesenchymal/claudin-low, and proliferative subpopulations in vivo in most TNBC GEMM models. We performed 18 individual scRNAseq experiments on the TP53-/- 2225L GEMM transplant line with the aforementioned treatments and untreated controls in triplicate and compared subpopulation frequencies in treated versus untreated tumors. Notably, treatment with trametinib and brequinar caused the rise of two rare subpopulations (i.e. 1% to 3-4% of total tumor cells) that express genes consistent with previously described drug-tolerant persisters (DTP), which we have called “Epithelial-DTP” (Tacstd2, Krt6a, and Cryab enriched) and “Mesenchymal-DTP” (Snai2 and Sca-1 enriched). A gene signature generated from the Epithelial-DTP subpopulation predicted poor patient outcomes in neoadjuvant chemotherapy treated TNBC patients. Further, in TNBC patient-derived xenografts (PDX), these two DTP subpopulations are also present and induced by treatment to an even greater frequency. Ongoing experiments include the use of fluorescence-activated cell sorting to isolate and functionally test the tumor-initiating capabilities of these two rare cell subpopulations. In addition, many experiments are underway to identify means to therapeutically target these DTP cells, with these results to be presented. Ultimately, identifying these rare drug resistant subpopulations, and identifying means to eradicate them, could vastly improve therapeutic regimens and outcomes for patients with TNBCs.
Citation Format: Cherise R. Glodowski, Kevin R. Mott, Denis Okumu, Michael P. East, Timothy C. Elston, Gary L. Johnson, Charles M. Perou. PD4-09 Single cell RNA-sequencing identifies intra-tumoral cellular heterogeneity and drug-induced subpopulation shifts in Triple Negative Breast Cancer mouse models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-09.