Organ preference of cancer metastasis, the role of cancer cell adhesion to the endothelial cell

许多癌症表现出独特的器官定植模式，不符合简单的肿瘤细胞扩散的解剖-机械陷阱理论。转移扩散的器官偏好似乎部分是通过肿瘤细胞选择性地附着到器官特异性的微血管内皮细胞来调节的。为了以有效和可重复性的方式研究这些肿瘤细胞与内皮细胞的相互作用，我们制作了器官特异性内皮细胞，由器官特异性细胞外基质调节。A549肺腺癌细胞更喜欢与培养于肺细胞外基质上的内皮细胞黏附。我们还研究了肿瘤细胞的趋化作用。趋化是肿瘤细胞侵袭转移过程中的关键步骤。我们利用A549细胞研究了非肌球蛋白轻链激酶(nm-MLCK)在肿瘤细胞对肝细胞生长因子(HGF)趋化中的作用。我们的分析包括：用Western印迹鉴定nm-MLCK的表达；在装有8μm孔聚碳酸酯膜的Boyden小室中检测对肝细胞生长因子的趋化作用；用抗肌球蛋白II抗血清免疫标记细胞以检测肌球蛋白II细丝的形成；通过肌球蛋白II免疫沉淀检测肌球蛋白轻链的磷酸化，然后用抗磷酸丝氨酸和抗磷苏氨酸抗体进行免疫印迹。为了评估MLCK活性的依赖性，在存在和不存在特定MLCK拮抗剂ML-7的情况下进行了实验。A549细胞表达214 kDa nm的MLCK，在HGF存在的情况下，使丝氨酸和苏氨酸残基上的MLC磷酸化。HGF还可诱导肌球蛋白II微丝形成和趋化作用，在30 ng/mlHGF作用24 h时达到最大。上述反应均被ML-7抑制。因此，似乎nm-MLCK活性调节HGF诱导的A549细胞的趋化作用。

Many cancers display characteristic organ colonization patterns that do not fit simple, anatomical-mechanical trapping theories of tumor cell dissemination. Organ preferences of metastatic spread appear to be mediated partly by the selective attachment of tumor cells to organ-specific, microvascular endothelium. To study these tumor cell-endothelial cell interactions in an efficient and reproducible manner, we made organ-specific endothelial cell modulated by the organ specific extracellular matrix. A549 lung adenocaricinoma cells prefer to adhere to the endothelial cell cultured on the lung extracellular matrix. We also investigated the tumor cell chemotaxis. Chemotaxis is a key step in the process of tumor cell invasion and metastasis. We studied the role of nonmuscle myosin light chain kinase (nm-MLCK) in tumor cell chemotaxis toward hepatocyte growth factor (HGF) using A549 cells. Our analysis entailed characterizing expression of nm-MLCK using Western blot; examining chemotaxis toward HGF in Boyden chambers equipped with 8 μm pore polycarbonate membranes; examining myosin II filament formation by immunolabeling cells with anti-myosin II antiserum; and examining myosin light chain (MLC) phosphorylation by myosin II immunoprecipitation, followed by immunoblotting with anti-phosphoserine and anti-phosphothreonine antibodies. To assess the dependency MLCK activity, experiments were performed in the presence and absence of the specific MLCK antagonist, ML-7. A549 cells were found to express a 214 kDa nm-MLCK, which in the presence of HGF, phosphorylated MLC on both serine and threonine residues. HGF also elicited myosin II filament formation and chemotaxis which was maximal at 24 h in the presence of 30 ng/mlHGF. All of the aforementioned reactions were inhibited by ML-7. Thus, it appears that nm-MLCK activity regulates HGF-induced A549 cell chemotaxis.