LAUSR

Delayed relapse is attributable to residual tumor cells that remain minimally proliferative/quiescent until favorable conditions for growth are met, but what determines the overall duration of remission is unresolved. Recent evidence suggests that epithelial-mesenchymal plasticity (EMP) facilitates phenotypic switching between dormant and proliferative cell states, in addition to enabling chemoresistance, metastasis, and immune evasion. However, which features of EMP are responsible for each or multiple of these phenotypes, and to what extent EMP is restricted to certain tumor populations remains unclear. To address these questions, we initially enriched for features of EMP (CD44hi, CD104low) in the mouse mammary tumor cell line TSAE1. Interestingly, no difference in tumor growth was observed between parental or EMP-enriched mammary fat pad (MFP) tumors in either syngeneic Balb/c or immune-deficient SCID-beige mice. Individual tumors were then cultured ex vivo, revealing a significant EMP-enrichment in parental tumors after one round of selection in vivo. Similar results were observed in the mouse mammary cell line D2.OR, suggesting that implantation alone gives rise to EMP which may mask important tumor cell-intrinsic differences in a heterogeneous tumor. Thus, EMP-enriched TSAE1 cells were further divided by isolating nine clonal lines. Although each expressed less epithelial markers than parental cells (Epcam, E-cadherin), individual TSAE1 clones displayed remarkably divergent growth rates and patterns in 3D culture. In vivo all clones were capable of forming tumors, but some were significantly delayed regardless of being implanted into Balb/c or SCID-beige animals. Furthermore, individual clones recruited distinct immune and stromal populations. To assess if intrinsic phenotypes change over time, 3D culture assays and bulk RNA sequencing was performed on three representative clones before implantation (“Pre”) and on three Balb/c MFP-derived tumors expanded ex-vivo from each clone (“Post”). Surprisingly, for each clone the Post tumors closely resembled the Pre clone and much of the difference in up/downregulated genes between individual clones and parental TSAE1 cells was already found in Pre samples. Finally, MFP tumors from EMP-enriched TSAE1 clones were differently sensitive to Doxorubicin therapy, suggesting that specific aspects of EMP may be important during different stages of BC survival and progression. Together, these data reveal that proliferation itself is intrinsically determined at the clonal level, independent of adaptive immunity, and highlight the potential for individual tumor clones in various EMP states to shape the tumor microenvironment for long-term survival. Timothy N. Trotter, Jason McBane, Xing Gao, Carina Dagotto, Tao Wang, H. Kim Lyerly, Zachary C. Hartman. Tumor clones display intrinsic and stable rates of progression and coordination of the tumor microenvironment in association with epithelial mesenchymal plasticity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2675.