Park D W, Ryu H S, Choi D S, Park Y H, Chang K H, Min C K
Department of Obstetrics and Gynecology, Ajou University School of Medicine, Suwon, Korea.
Gynecol Oncol. 2001 Sep;82(3):442-9. doi: 10.1006/gyno.2001.6294.
In this study, we have established an in vitro three-dimensional (3D) coculture, where normal endometrial stromal cells and endometrial cancer cells were cocultured under defined hormonal conditions, to investigate the potential paracrine effects on synthesis and secretion of matrix metalloproteinases (MMPs) and, thus, cancer invasion.
Endometrial stromal cells were obtained by biopsy, cultured in the presence of 100 nM estrogen for 3 days, and then mixed with extracellular matrix (ECM) composed of collagen I and matrigel in a 4:1 ratio. After 3 more days in culture, a 3D coculture was established with HEC-1A cells, an endometrial adenocarcinoma cell line, grown on top of the mixture under various ovarian steroids (no steroid, 100 nM beta-estradiol (E2), or 1.0 microM progesterone (P4)) for 10 days. The expression and localization of MMP-2, MMP-9, and integrin beta 3 subunit were visualized by immunocytochemistry and analyzed by reverse transcription polymerase chain reaction (RT-PCR). The extent of cancer invasion was quantified by Boyden's chamber assay.
Integrin beta 3 subunit was localized along the cell surface of HEC-1A cell under all three hormonal conditions. MMP-2 was located in the cytoplasm of stromal cells and on the surface of HEC-1A cells. Synthesis and secretion of stromal MMP-2 were increased in the presence of ovarian steroids. In contrast, expression of stromal MMP-9 was suppressed in the presence of ovarian steroids. No MMPs were synthesized in HEC-1A cells. Invasion assay revealed that HEC-1A cells achieved high tumoral invasiveness in the presence of beta-estradiol.
These findings suggest that stromal cell-derived MMP-2 is translocated to the surface of HEC-1A cells. Integrin beta 3 subunit might contribute, in part, to providing a binding site for MMP-2. Thus, HEC-1A cells invade by recruiting MMP-2 secreted by stromal cells, which is greatly enhanced in the presence of beta-estradiol. The 3D coculture provides an excellent experimental system in which single parameters can be isolated from a complex in vivo system in the process of endometrial cancer invasion.
在本研究中，我们建立了一种体外三维（3D）共培养体系，将正常子宫内膜基质细胞和子宫内膜癌细胞在特定激素条件下进行共培养，以研究其对基质金属蛋白酶（MMPs）合成和分泌的潜在旁分泌作用，进而探究对癌症侵袭的影响。
通过活检获取子宫内膜基质细胞，在100 nM雌激素存在下培养3天，然后与由I型胶原蛋白和基质胶按4:1比例组成的细胞外基质（ECM）混合。再培养3天后，建立3D共培养体系，将子宫内膜腺癌细胞系HEC-1A细胞接种在混合物上方，在不同卵巢类固醇（无类固醇、100 nMβ-雌二醇（E2）或1.0 μM孕酮（P4））条件下培养10天。通过免疫细胞化学观察MMP-2、MMP-9和整合素β3亚基的表达及定位，并通过逆转录聚合酶链反应（RT-PCR）进行分析。通过Boyden小室试验定量癌症侵袭程度。
在所有三种激素条件下，整合素β3亚基均定位于HEC-1A细胞的细胞表面。MMP-2定位于基质细胞的细胞质以及HEC-1A细胞的表面。在卵巢类固醇存在的情况下，基质MMP-2的合成和分泌增加。相反，在卵巢类固醇存在的情况下，基质MMP-9的表达受到抑制。HEC-1A细胞中未合成MMPs。侵袭试验显示，在β-雌二醇存在的情况下，HEC-1A细胞具有较高的肿瘤侵袭性。
这些发现表明，基质细胞来源的MMP-2转移至HEC-1A细胞表面。整合素β3亚基可能部分有助于为MMP-2提供结合位点。因此，HEC-1A细胞通过募集基质细胞分泌的MMP-2进行侵袭，在β-雌二醇存在的情况下这种侵袭作用大大增强。这种3D共培养提供了一个出色的实验系统，在子宫内膜癌侵袭过程中，可以从复杂的体内系统中分离出单一参数进行研究。
