P-356 establishment of a novel 3D spheroid culture system of uterine leiomyoma cells
Authors: Sakai T , Sato S , Tamehisa T , Tamura I , Maekawa R , Sugino N 
Published: June 2023

Abstract

Study question
To establish three-dimensional (3D) spheroid culture system of uterine leiomyoma (ULM) cells that responds to estrogen(E) and progesterone(P).

Summary answer
We established the 3D spheroid culture system of ULM cells, in which the ULM-spheroid grows in response to progesterone.

What is known already
ULMs proliferate in response to female hormones in the patients-derived xenograft model in vivo. However, it is controversial whether ULM cells proliferate in response to female hormones in in vitro monolayer culture system. We reported that ULM cells formed nodule-like aggregates of cells when the cells were grown in 3D culture using collagen gel. However, 3D spheroid culture system of ULM cells is not established yet. Recently, somatic mutations in the MED12 gene were detected in almost 70% of ULMs. It is also unclear whether responsiveness of ULM cells to female hormones differs between MED12-mutation positive and negative ULMs.

Study design, size, duration
Fourteen and 13 ULMs obtained from 18 premenopausal women were used for monolayer culture and 3D spheroid culture, respectively. The isolated ULM cells were confirmed to be smooth muscle cells with 80% purity and to have progesterone receptors by immunocytochemistry. The cells were preincubated for 2 days and then used for the following experiments below. MED12-mutation status was analyzed by Sanger sequencing. Monolayer and spheroid cultures were done on MED12-mutation positive and negative ULM cells.

Participants/materials, setting, methods
For the monolayer culture system, cells were incubated with E+P, E alone, P alone, and control (without E+P) for 7 days. For 3D culture system, cells were incubated in the low attachment well dish to induce floating cell culture, and after 24 h, we identified the floating cell aggregates as spheroid of ULM cells. Then, the ULM-spheroids were incubated with E+P, E alone, P alone, E+P+selective progesterone receptor modulator (SPRM), and control for 7 days.

Main results and the role of chance
Responsiveness to female hormones was assessed by counting cells for monolayer culture, and by measuring the cross-sectional area of the spheroid for 3D culture system. ULM cells did not proliferate by any female hormones in the monolayer culture system. In the 3D spheroid culture system, the ULM-spheroid cells showed expression of alpha-smooth muscle actin and vimentin, indicating that most of the spheroid cells are smooth muscle cells. In MED12-mutation negative ULMs, the mean of the cross-sectional area of the spheroid of E+P , P alone, E alone, E+P+SPRM and control were 0.375mm², 0.343mm², 0.237mm², 0.252mm² and 0.193mm², respectively, while in MED12-mutation positive ULMs, the area were 0.349mm², 0.338mm², 0.268mm², 0.264mm². and 0.215mm². The morphology of the spheroid of the E alone, E+P+SPRM and control groups showed the loss of smooth muscle cells. These results suggest that the growth of the ULM-spheroid is maintained by progesterone, and that the responsiveness of the spheroid to progesterone does not differ between MED-mutation negative and positive ULM cells. Our study showed that ULM cells of the 3D spheroid culture system acquire function to respond to progesterone.

Limitations, reasons for caution
Further studies are needed to investigate whether the 3D spheroid culture system established in this study accurately reflects the responsiveness to female hormones of the in vivo.

Wider implications of the findings
Our study showed that the 3D spheroid culture system of ULM cells has the responsiveness to progesterone whereas the monolayer culture system does not. The 3D spheroid culture system of ULM cells established in this study would be useful as a screening system for therapeutic agents.

Trial registration number
non-clinical trials