This study presents a novel in vitro bilayer 3D co‐culture platform designed to obtain cancer‐associated fibroblasts (CAFs)‐like cells. The platform consists of a bilayer hydrogel structure with a collagen/polyethylene glycol (PEG) hydrogel for fibroblasts as the upper layer and an alginate hydrogel for tumor cells as the lower layer. The platform enabled paracrine interactions between fibroblasts and cancer cells, which allowed for selective retrieval of activated fibroblasts through collagenase treatment for further study. Fibroblasts remained viable throughout the culture periods and showed enhanced proliferation when co‐cultured with cancer cells. Morphological changes in the co‐cultured fibroblasts resembling CAFs were observed, especially in the 3D microenvironment. The mRNA expression levels of CAF‐related markers were significantly upregulated in 3D, but not in 2D co‐culture. Proteomic analysis identified upregulated proteins associated with CAFs, further confirming the transformation of normal fibroblasts into CAF within the proposed 3D co‐culture platform. Moreover, co‐culture with CAF induced radio‐ and chemoresistance in pancreatic cancer cells (PANC‐1). Survival rate of cancer cells post‐irradiation and gemcitabine resistance increased significantly in the co‐culture setting, highlighting the role of CAFs in promoting cancer cell survival and therapeutic resistance. These findings would contribute to understanding molecular and phenotypic changes associated with CAF activation and provide insights into potential therapeutic strategies targeting the tumor microenvironment.

中文翻译：

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双层3D共培养平台诱导正常成纤维细胞分化为癌症相关成纤维细胞样细胞：获得癌症相关成纤维细胞的新体外来源


本研究提出了一种新型体外双层 3D 共培养平台，旨在获得癌症相关成纤维细胞 (CAF) 样细胞。该平台由双层水凝胶结构组成，上层为用于成纤维细胞的胶原/聚乙二醇（PEG）水凝胶，下层为用于肿瘤细胞的藻酸盐水凝胶。该平台实现了成纤维细胞和癌细胞之间的旁分泌相互作用，从而可以通过胶原酶处理选择性地回收活化的成纤维细胞以进行进一步研究。成纤维细胞在整个培养期间保持活力，并且在与癌细胞共培养时表现出增强的增殖。观察到共培养的成纤维细胞类似于 CAF 的形态变化，特别是在 3D 微环境中。 CAF 相关标记物的 mRNA 表达水平在 3D 共培养中显着上调，但在 2D 共培养中则没有。蛋白质组学分析确定了与 CAF 相关的上调蛋白质，进一步证实了在所提出的 3D 共培养平台内正常成纤维细胞转化为 CAF。此外，与 CAF 共培养可诱导胰腺癌细胞 (PANC-1) 产生放射和化疗耐药性。在共培养环境中，放射后癌细胞的存活率和吉西他滨耐药性显着增加，凸显了 CAF 在促进癌细胞存活和治疗耐药性中的作用。这些发现将有助于理解与 CAF 激活相关的分子和表型变化，并为针对肿瘤微环境的潜在治疗策略提供见解。