Co-cultivation of colorectal cancer cells and human skin fibroblasts in 3D collagen gel and comparison of co-cultivated fibroblasts with CAFs. Co-cultivation of colorectal cancer cells and human skin fibroblasts in 3D collagen gel and comparison of co-cultivated fibroblasts with CAFs

Despite the growing recognition of the role of the stroma in cancer growth, invasive behavior and metastasis, the exact mechanisms of its participation remain unclear. We have explored the relationships between the epithelial/mesenchymal (E/M) state of colorectal cancer cells, their ability to activate fibroblasts, and the expression of collagen related genes. To this end, we studied (i) co-cultures of colorectal cancer cells with different hybrid E/M states and normal fibroblasts in a collagen matrix and (ii) patient-derived cancer-associated fibroblasts (CAFs). Using RNA-sequencing, we found that the different cancer cells can activate normal fibroblasts, which could form dense collagen networks. The functional enrichment analysis of differentially expressed genes indicates more mesenchymal phenotype and greater motility of SW480 cells compared to HT29 cells. The genes related to collagen biosynthesis and catabolism tend to be more active in SW480 cells rather than HT29 cells. Moreover, LOXL2 and LOXL3 genes, which are necessary for collagen fibril organization, are SW480 specific, which may indicate greater input of this cell line in collagen remodeling compared to HT29 cells. The expression of several CAF marker genes is activated in NFs upon co-cultivation with HT29 and SW480. Interestingly, a more-epithelial cell line HT29 activates the fibroblasts to a greater extent, than does SW480. The co-cultivation of colon cancer cell lines HT29 or SW480 with NFs leads to the activation of collagen biosynthesis and collagen fibril organization genes in NFs. Our findings suggest that the normal fibroblasts, activated by cancer cells, contribute to the organization of the extracellular matrix. Therefore, targeting the ability of cancer cells to activate normal fibroblasts can be considered as a new therapeutic strategy. Overall design: Examination of mRNA profiles of monocultures of colorectal cancer cell lines, HT29 and SW480, normal skin fibroblasts (NFs), two cultures of patient-derived colorectal CAFs (CAF1 and CAF2); mRNA profiles of HT29 or SW480 co-cultivated with NFs, and NFs co-cultivated with HT29 or SW480. The preparation of the libraries was performed in technical triplicate for mono- and co-cultivated HT29, SW480, and NFs, while for CAF1 and CAF2, the preparation of the libraries was performed in technical duplicate.

尽管基质（stroma）在癌症生长、侵袭行为与转移中的作用日益受到认可，但其参与调控的确切机制仍未阐明。我们探究了结直肠癌细胞的上皮/间质（epithelial/mesenchymal, E/M）状态、其激活成纤维细胞的能力，以及胶原相关基因表达之间的关联。为此，我们开展了两项研究：(i) 不同混合E/M状态的结直肠癌细胞与正常成纤维细胞在胶原基质中的共培养实验，以及(ii) 患者来源的癌症相关成纤维细胞（cancer-associated fibroblasts, CAFs）样本。通过RNA测序（RNA-sequencing），我们发现不同的癌细胞可激活正常成纤维细胞（normal fibroblasts, NFs），进而形成致密的胶原网络。对差异表达基因的功能富集分析结果显示，相较于HT29细胞，SW480细胞具有更强的间质表型与更高的迁移能力。胶原生物合成与分解代谢相关基因在SW480细胞中的活性普遍高于HT29细胞。此外，参与胶原纤维组织的LOXL2与LOXL3基因为SW480细胞所特有，这提示相较于HT29细胞，该细胞系在胶原重塑过程中发挥的作用更为显著。在与HT29或SW480共培养后，NFs中的多种癌症相关成纤维细胞标记基因表达被激活。值得注意的是，上皮表型更强的HT29细胞相比SW480细胞，更能有效激活成纤维细胞。结直肠癌细胞系HT29或SW480与NFs共培养，可促使NFs中胶原生物合成及胶原纤维组织相关基因的表达激活。本研究结果表明，被癌细胞激活的NFs可促进细胞外基质（extracellular matrix, ECM）的组织构建。因此，靶向癌细胞激活正常成纤维细胞的能力可作为一种全新的治疗策略。实验整体设计：本研究检测了以下样本的mRNA表达谱：结直肠癌细胞系HT29、SW480的单培养样本，正常皮肤成纤维细胞（normal fibroblasts, NFs），两株患者来源的结直肠癌相关成纤维细胞（CAF1与CAF2）；HT29或SW480与NFs共培养的样本，以及NFs与HT29或SW480共培养的样本。其中，单培养及共培养的HT29、SW480与NFs的文库构建均设置3次技术重复，而CAF1与CAF2的文库构建仅设置2次技术重复。