Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wrocław, Poland.
Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383, Wrocław, Poland.
Cell Commun Signal. 2022 May 10;20(1):63. doi: 10.1186/s12964-022-00871-x.
The tumor microenvironment consists of stromal cells, extracellular matrix, and physicochemical properties (e.g., oxygenation, acidification). An important element of the tumor niche are cancer-associated fibroblasts (CAFs). They may constitute up to 80% of the tumor mass and share some features with myofibroblasts involved in the process of wound healing. CAFs can facilitate cancer progression. However, their interaction with melanoma cells is still poorly understood.
We obtained CAFs using conditioned media derived from primary and metastatic melanoma cells, and via co-culture with melanoma cells on Transwell inserts. Using 2D and 3D wound healing assays and Transwell invasion method we evaluated CAFs' motile activities, while coverslips with FITC-labeled gelatin, gelatin zymography, and fluorescence-based activity assay were employed to determine the proteolytic activity of the examined cells. Western Blotting method was used for the identification of CAFs' markers as well as estimation of the mediators of MMPs' (matrix metalloproteinases) expression levels. Lastly, CAFs' secretome was evaluated with cytokine and angiogenesis proteomic arrays, and lactate chemiluminescence-based assay.
Acquired FAP-α/IL6-positive CAFs exhibited elevated motility expressed as increased migration and invasion ratio, as well as higher proteolytic activity (area of digestion, MMP2, MMP14). Furthermore, fibroblasts activated by melanoma cells showed upregulation of the MMPs' expression mediators' levels (pERK, p-p38, CD44, RUNX), enhanced secretion of lactate, several cytokines (IL8, IL6, CXCL1, CCL2, ICAM1), and proteins related to angiogenesis (GM-CSF, DPPIV, VEGFA, PIGF).
Observed changes in CAFs' biology were mainly driven by highly aggressive melanoma cells (A375, WM9, Hs294T) compared to the less aggressive WM1341D cells and could promote melanoma invasion, as well as impact inflammation, angiogenesis, and acidification of the tumor niche. Interestingly, different approaches to CAFs acquisition seem to complement each other showing interactions between studied cells. Video Abstract.
肿瘤微环境由基质细胞、细胞外基质和理化特性（如氧合、酸化）组成。肿瘤生态位的一个重要组成部分是癌相关成纤维细胞（CAFs）。它们可能构成肿瘤质量的 80%，并且与参与伤口愈合过程的肌成纤维细胞具有一些共同特征。CAFs 可以促进癌症的进展。然而，它们与黑色素瘤细胞的相互作用仍知之甚少。
我们使用从原发性和转移性黑色素瘤细胞衍生的条件培养基，以及通过与 Transwell 插入物上的黑色素瘤细胞共培养来获得 CAFs。我们使用 2D 和 3D 划痕愈合实验和 Transwell 侵袭方法评估 CAFs 的运动活性，同时使用带有 FITC 标记的明胶、明胶酶谱和荧光活性测定法来确定检查细胞的蛋白水解活性。Western Blotting 方法用于鉴定 CAFs 的标志物以及估计 MMPs（基质金属蛋白酶）表达水平的调节剂。最后，使用细胞因子和血管生成蛋白质组学阵列以及基于乳酸化学发光的测定法评估 CAFs 的分泌组。
获得的 FAP-α/IL6 阳性 CAFs 表现出较高的迁移和侵袭比值的迁移和侵袭比，以及较高的蛋白水解活性（消化面积、MMP2、MMP14）。此外，被黑色素瘤细胞激活的成纤维细胞表现出 MMPs 表达调节剂水平的上调（pERK、p-p38、CD44、RUNX），增强了乳酸、几种细胞因子（IL8、IL6、CXCL1、CCL2、ICAM1）和与血管生成相关的蛋白（GM-CSF、DPPIV、VEGFA、PIGF）的分泌。
观察到的 CAFs 生物学变化主要是由高度侵袭性黑色素瘤细胞（A375、WM9、Hs294T）驱动的，与侵袭性较低的 WM1341D 细胞相比，可以促进黑色素瘤的侵袭，以及影响炎症、血管生成和肿瘤生态位的酸化。有趣的是，获得 CAFs 的不同方法似乎相辅相成，显示出研究细胞之间的相互作用。视频摘要。
