Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Microsc Res Tech. 2010 Feb;73(2):99-108. doi: 10.1002/jemt.20761.
Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type 1 matrix metalloproteinase (MT1-MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent-substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel-gelatin-FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1-MMP. Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1-MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After 1 h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1-MMP. Furthermore, MMP9 would be required in the initial steps of invadopodia formation.
迁移、入侵和蛋白酶活性对于肿瘤的进展和转移至关重要。转移性细胞依赖侵袭伪足来降解和侵袭细胞外基质 (ECM)。侵袭伪足是细胞膜突起，包含降解 ECM 所需的酶。这些突起包含重叠在消化基质区域的桩蛋白和膜型 1 基质金属蛋白酶 (MT1-MMP)。在这里，我们在源自人类腺样囊性癌的细胞系 (CAC2) 中对侵袭伪足进行了表征。我们进行了荧光底物降解测定以评估 CAC2 细胞的原位蛋白酶活性。荧光底物中的消化斑点在绿色背景下呈现为黑色区域。细胞在 Matrigel-明胶-FITC 上培养，1 小时和 3 小时后固定。CAC2 细胞被双重标记为肌动蛋白和桩蛋白。细胞也被双重染色为肌动蛋白和 MT1-MMP。通过激光共聚焦扫描显微镜研究样本。在所有时间点，CAC2 细胞均显示肌动蛋白、桩蛋白和 MT1-MMP 与荧光底物中的消化斑点共定位。我们寻找其他参与侵袭伪足活性的蛋白酶。我们之前已经证明 MMP9 影响腺样囊性癌的行为。这促使我们研究 MMP9 在侵袭伪足形成中的作用。CAC2 细胞通过 siRNA 沉默 MMP9。在荧光底物中孵育 1 小时后，与对照组相比，沉默 MMP9 的细胞的基质消化明显减少。在荧光底物上培养 3 小时的沉默 MMP9 的细胞没有发现差异。我们的结果表明，CAC2 细胞表现出含有桩蛋白和 MT1-MMP 的功能性侵袭伪足。此外，MMP9 在侵袭伪足形成的初始步骤中是必需的。
