Abstract 2810: Epithelial-mesenchymal hybrid population changes from monolayer, spheroid, and tumoroid ex vivo culture of syngeneic murine mammary tumors
Authors: Hum Nicholas R. , Martin Kelly A. , Wheeler Elizabeth , Coleman Matthew A. , Loots Gabriela G. 
Published: September 2020

Epithelial to mesenchymal transition (EMT) of cancer cells during tumor progression has been implicated in tumor initiation, growth, invasion, metastasis, colonization and resistance to therapy. In building a successful in vitro tumor model, it is critical to recapitulate in vivo cancer cell heterogeneity inclusive of both cell types and transition state present. This study investigates the EMT hybrid states of mouse triple negative breast cancer cells during ex vivo culturing across multiple methodologies.

Mouse mammary carcinoma (4T1) tumors were harvested from syngeneic, orthotopic mice and subsequently cultured using various methods over the course of 12 days. Ex vivo cultures were first assessed for retention of tumor cellular heterogeneity using endogenous Thy1.1 (CD90.1) expression via flow cytometry to distinguish 4T1 cancer cells from stromal derived cells. Cancer cell populations rapidly became the majority of cells in culture. After 3 days of ex vivo culture, we found the cancer cell population to have significantly expanded ~2-3 fold compared to the original tumor population, while other stromal cellular subtypes decreased. Additionally, monolayer culture of ex vivo cells contained significantly more cancer cells relative to the spheroid or tumoroid (tumor fragment) culturing techniques.

Cancer cells from each culturing technique were also evaluated for loss of Epcam expression and mesenchymal cell fate in reference to the initial EMT distribution at time of isolation. 4T1 cells cast into hydrogel retained high proportions of cells undergoing EMT, evidenced by fewer cells expressing Epcam. However, all other in vitro conditions favored the expansion of cancer cells in an epithelial state compared to in vivo tumors. Subsequent analysis of EMT populations for transitional hybrid states based on CD51, CD61, and CD106 expression was conducted using flow cytometric analysis. We found that in vitro culturing promoted mesenchymal character and this selection was time dependent. Additionally, we found that spheroids cultured in hydrogel for 7 days most closely resembled early hybrid EMT cell states ratios found in vivo

Future research aims to optimize ex vivo culturing methodologies to best retain cancer cell characteristics and behavior of tumors obtained from human biopsies. Alterations in growth conditions and identifying critical stromal populations of interest will be critical in development of optimized preclinical ex vivo tumor culture models for drug discovery or personalized treatment.

This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM: LLNL-ABS-798441

Citation Format: Nicholas R. Hum, Kelly A. Martin, Elizabeth Wheeler, Matthew A. Coleman, Gabriela G. Loots. Epithelial-mesenchymal hybrid population changes from monolayer, spheroid, and tumoroid ex vivo culture of syngeneic murine mammary tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2810.