Factors affecting the loss of MED12-mutated leiomyoma cells during in vitro growth

Abstract

Uterine leiomyomas (UL) are the most prevalent symptomatic human tumors at all and somatic mutations of the gene encoding mediator subcomplex 12 (MED12) constitute the most frequent driver mutations in UL. Recently, a rapid loss of mutated cells during in vitro growth of UL-derived cell cultures was reported, resulting in doubts about the benefits of UL-derived cell cultures. To evaluate if the rapid loss of MED12-mutated cells in UL cell cultures depends on in vitro passaging, we set up cell cultures from nine UL from 40–50 year old Caucasian patients with at least one UL. Cultured UL cells were investigated for loss of MED12-mutated cells. Genetic characterization of native tumor samples and adjacent myometrium was done by array analysis. “Aged” primary cultures without passaging were compared to cells of three subsequent passages. Comparative analyses of the mutated/non-mutated ratios between native tissue, primary cells, and cultured tumor cells revealed a clear decrease of MED12-mutated cells. None of the tumors showed gross alterations of the array profiles, excluding the presence of gross genomic imbalances besides the MED12 mutations as a reason for the intertumoral variation in the loss of MED12-mutated cells. Albeit at a lesser rate, loss of MED12-mutated cells from cell cultures of UL occurs even without passaging thus indicating the requirement of soluble factors or matrix components lacking in vitro. Identification of these factors can help to understand the mechanisms of the growth of the most frequent type of uterine leiomyomas and to decipher novel drug targets.

Subtype-Specific Tumor-Associated Fibroblasts Contribute to the Pathogenesis of Uterine Leiomyoma

“…Our current study questions the validity of previous studies that utilized LM cell culture as the primary research model: these studies likely analyzed the cell cultures that mostly consisted of TAFs (26). Indeed, LM and MM cells rapidly change their gene expression profiles in vitro (41).…”

“…The high concentration of TAFs explains why MED12 mutant cells are quickly lost from the primary culture of LMs (25,26); fibroblasts attach the culture dish better and grow faster than SMCs in standard culture conditions, conditions originally developed to optimize fibroblast growth (27). Overgrowth of fibroblasts is a classic problem for the primary culturing of SMCs including MM, and many techniques to repress the growth of fibroblasts in SMC culture have been proposed (20,28–32).…”

“…Given the high initial concentration of TAFs with superior in vitro growth potential, freshly prepared primary cultures of MED12-LMs would contain more fibroblasts than SMCs, and the concentration of SMCs should further decrease, as TAFs grow faster to reach confluence. Indeed, Bloch et al recently demonstrated that the concentration of MED12 mutant cells gradually decreases in the primary culture of MED12-LMs even without passaging (26). The study by Bloch et al also suggested that the loss of MED12 mutant cells is partially due to the detachment of mutant cells from the culture flask, which likely reflects the weaker adherence of SMCs to the plastic surface compared to fibroblasts (40).…”

Altered chromatin landscape and enhancer engagement underlie transcriptional dysregulation in MED12 mutant uterine leiomyomas

“…Previous studies suggest that culturing of primary cells from tissue samples may partially alter the gene expression profiles of cells 21 . Additionally, in primary cell culture of cells obtained from leiomyoma patient tissue samples, a rapid loss of MED12 mutant cells was observed, suggesting a very limited viability of this cell population in culture 22,23 .…”

Reasons to Reconsider Risk Associated With Power Morcellation of Uterine Fibroids

“…Accordingly, both types of mutations characterize independent tumor entities that can also be distinguished based on certain clinical and histological parameters e.g. their size and stromal content (18,19), their gene-expression patterns (20), and metabolome (21), and may even differ with respect to their cellular growth capacity in vitro (22). Moreover, they even very rarely coexist among the individual tumors of single patients (23).…”