Coopman P J, Do M T, Thompson E W, Mueller S C
Department of Cell Biology, Georgetown University Medical School, Washington, DC 20007, USA.
Clin Cancer Res. 1998 Feb;4(2):507-15.
During invasion and metastasis, cancer cells interact closely with the extracellular matrix molecules by attachment, degradation, and migration. We demonstrated previously the local degradation of fluorescently labeled gelatin matrix by cancer cells at invasive membrane protrusions, called invadopodia. Using the newly developed quantitative fluorescence-activated cell sorting-phagocytosis assay and image analysis of localized degradation of fluorescently labeled matrix, we document here that degradation and site-specific removal of cross-linked gelatin matrix is correlated with the extent of phagocytosis in human breast cancer cells. A higher phagocytic capacity is generally associated with increasing invasiveness, documented in other invasion and motility assays as well. Gelatin phagocytosis is time and cell density dependent, and it is mediated by the actin cytoskeleton. Most of the intracellular gelatin is routed to actively acidified vesicles, as demonstrated by the fluorescent colocalization of gelatin with acidic vesicles, indicating the intracellular degradation of the phagocytosed matrix in lysosomes. We show here that normal intracellular routing is blocked after treatment with acidification inhibitors. In addition, the need for partial proteolytic degradation of the matrix prior to phagocytosis is demonstrated by the inhibition of gelatin phagocytosis with different serine and metalloproteinase inhibitors and its stimulation by conditioned medium containing the matrix metalloproteinases MMP-2 and MMP-9. Our results demonstrate that phagocytosis of extracellular matrix is an inherent feature of breast tumor cells that correlates with and may even directly contribute to their invasive capacity. This assay is useful for screening and evaluating potential anti-invasive agents because it is fast, reproducible, and versatile.
在侵袭和转移过程中，癌细胞通过附着、降解和迁移与细胞外基质分子密切相互作用。我们之前已证明，癌细胞在侵袭性膜突出物（即侵袭伪足）处对荧光标记的明胶基质进行局部降解。利用新开发的定量荧光激活细胞分选-吞噬分析以及对荧光标记基质局部降解的图像分析，我们在此记录到，交联明胶基质的降解和位点特异性清除与人乳腺癌细胞中的吞噬程度相关。较高的吞噬能力通常与侵袭性增加相关，这在其他侵袭和运动分析中也得到了证实。明胶吞噬作用具有时间和细胞密度依赖性，且由肌动蛋白细胞骨架介导。如明胶与酸性囊泡的荧光共定位所示，大多数细胞内的明胶被转运至活跃酸化的囊泡，这表明吞噬的基质在溶酶体中进行细胞内降解。我们在此表明，用酸化抑制剂处理后，正常的细胞内转运被阻断。此外，不同的丝氨酸和金属蛋白酶抑制剂抑制明胶吞噬作用，而含有基质金属蛋白酶MMP-2和MMP-9的条件培养基刺激明胶吞噬作用，这证明了在吞噬作用之前对基质进行部分蛋白水解降解的必要性。我们的结果表明，细胞外基质的吞噬作用是乳腺肿瘤细胞的一个固有特征，并与其侵袭能力相关，甚至可能直接促成其侵袭能力。该分析方法对于筛选和评估潜在的抗侵袭药物很有用，因为它快速、可重复且用途广泛。
