Abstract 4251: Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium
Authors: Yankaskas Chris , Balhouse Brittany , Paul Colin , Thakuri Pradip Shahi , Salen Shyanne , Kennedy Mark , Dallas Matt , Kuninger David 
Published: March 2024

Tumoroids, also known as cancer organoids, are self-assembled three-dimensional cultures of patient-derived tumor cells. Compared to traditional 2D cancer cell lines, tumoroids better maintain key characteristics of the original tumor including the genotype and transcriptome, which lead to better in vitro modeling of clinically relevant functional responses such as drug sensitivity. An under-studied aspect of tumoroid cultures has been their ability to recapitulate dependence on hormone signaling in vitro, with some reports indicating hormone dependence is lost over time in culture. To investigate this, we utilized Gibco™ OncoPro™ Tumoroid Culture Medium and defined tissue-specific supplements, FGF10 and beta-estradiol, that when added to the medium enabled the derivation of endometrial tumoroids from tumor resections. Derivation of tumoroid lines compatible with long-term growth depended on a variety of factors, including tissue quality and time from resection to culture initiation. Each of the 15 samples processed formed tumoroids when initially seeded into culture. 11/15 samples reformed tumoroids through multiple passages (typically 1-2 weeks per passage) over 1-3 months in culture. 7/15 samples met our high criteria for stable tumoroid line growth of &gt;5 passages and reaching &gt;5 cumulative population doublings (from initial number of cells seeded post-resection) in culture. Next-generation sequencing of the established tumoroids and the uncultured tumor material demonstrated a high degree of correlation (pearson’s r&gt;0.9) of genomic mutations in a targeted panel; few differentially expressed genes were detected beyond down-regulation of immune-related genes, presumably because the medium is optimized for outgrowth of cancer epithelial cells, and immune cells do not persist during long-term culture. Endometrial tumoroids were capable of growing embedded in basement membrane extract (BME) or in suspension with dilute BME added and could be cryopreserved and recovered. Endometrial tumoroids retained gene expression levels (both presence and absence) of estrogen receptor and progesterone receptor for up to the 20 passages tested in vitro. A subset of the models was tested and showed increased growth rates in response to increased concentrations of beta-estradiol. Of those tested, 2/3 tumoroid cultures stopped growing within 1-2 weeks after removal of beta-estradiol from the medium, demonstrating dependence on this hormone for growth. Maintenance of hormone receptor expression and dependency should enable in vitro studies to understand the signaling mechanisms and potential ways to modulate them in the context of cancer. Overall, this method provides a toolset for studying endometrial cancer, particularly hormone dependency, and may be more broadly applicable to other gynecological or hormone dependent cancers.

Citation Format: Chris Yankaskas, Brittany Balhouse, Colin Paul, Pradip Shahi Thakuri, Shyanne Salen, Mark Kennedy, Matt Dallas, David Kuninger. Establishment of hormone-dependent endometrial tumoroids in a conditioned-medium free, serum-free medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4251.