Development and Validation of a Three- Dimensional In Vitro Model, for Uterine Lelomyoma and Patient-Matched Myometrium

摘要

Leiomyomas are benign uterine tumors. They are distinguished from the surrounding myometrium by the presence of an excessive and disorganized extracellular matrix (ECM). Leiomyoma cells in vivo grow in a microenvironment high in ECM proteins such as collagen 1A1, which is a major contributor to the architecture of the tumor.This study describes the development of immortalized human patient-matched leiomyoma and myometrium 3-dimensional (3D) cultures and demonstrates the similarity of the morphologic and biochemical features of this model system to those of freshly excised surgical tissue. Features examined included quantitative reverse transcriptase—polymerase chain reaction, cytoimmunofhiorescence, and Western blot analysis. Expression of the following biomarker genes was measured in the surgical fibroid specimens and in the in vitro cell cultures grown in the 3D format: collagen 1A1, versican, fibronectin, dermatopontin, and transforming growth factor beta3 (TGF-beta3). Integrin-mediated 3D structural formation also was examined.The 3D cultures of both leiomyoma cells and the 3D myometrium leiomyoma cells maintained the characteristic spindle morphology of the progenitor smooth muscle. There was elevated expression of collagen 1A1 (6.66 ± 1.5), total versican (4.78 + 0.5), fibronectin (3.94 ± 0.3), and TGF-beta3 (2.21 + 0.1) similar to that seen in progenitor tissue. In addition, expression of the dermatopontin gene was down-regulated (0.29 ± 0.1); values were similar to those of the surgical tissue. Treatment of myometrial cells in 3D culture with TGF-beta3 for 3 days increased the expression of collagen 1 Al, fibronectin, and versican, and decreased expression of dermatopontin gene, indicating a fibrotic transformation of these cells to a leiomyoma phenotype. Treatment of the 3D leiomyoma cell cultures with integrin beta1-inhibiting antibody disrupted cell-ECM communication resulting in loss of adherence that led to induced apoptosis.These findings show that both myometrium and leiomyoma cells grown in 3D culture maintain the molecular characteristics that distinguish the 2 cell types in the progenitor tissue. This 3D model system can be used to assess the mechanism of aberrant ECM formation in fibroid tumors and to evaluate the effectiveness of potential therapies.