Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, Blindern, 0316, Oslo, Norway.
Department of Hepato-Pancreato-Biliary Surgery, Institute of Clinical Medicine, University of Oslo, PO Box 1171, Blindern, 0318, Oslo, Norway.
BMC Cancer. 2019 Jun 17;19(1):596. doi: 10.1186/s12885-019-5803-1.
Gemcitabine remains a cornerstone in chemotherapy of pancreatic ductal adenocarcinoma (PDAC) despite suboptimal clinical effects that are partly due to the development of chemoresistance. Pancreatic stellate cells (PSCs) of the tumor stroma are known to interact with pancreatic cancer cells (PCCs) and influence the progression of PDAC through a complex network of signaling molecules that involve extracellular matrix (ECM) proteins. To understand tumor-stroma interactions regulating chemosensitivity, the role of PSC-secreted fibronectin (FN) in the development of gemcitabine resistance in PDAC was examined.
PSC cultures obtained from ten different human PDAC tumors were co-cultured with PCC lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, PANC-1 and SW-1990) either directly, or indirectly via incubation with PSC-conditioned medium (PSC-CM). Gemcitabine dose response cytotoxicity was determined using MTT based cell viability assays. Protein expression was assessed by western blotting and immunofluorescence. PSC-CM secretome analysis was performed by proteomics-based LC-MS/MS, and FN content in PSC-CM was determined with ELISA. Radiolabeled gemcitabine was used to determine the capacity of PCCs to uptake the drug.
In both direct and indirect co-culture, PSCs induced varying degrees of resistance to the cytotoxic effects of gemcitabine among all cancer cell lines examined. A variable degree of increased phosphorylation of ERK1/2 was observed across all PCC lines upon incubation with PSC-CM, while activation of AKT was not detected. Secretome analysis of PSC-CM identified 796 different proteins, including several ECM-related proteins such as FN and collagens. Soluble FN content in PSC-CM was detected in the range 175-350 ng/ml. Neither FN nor PSC-CM showed any effect on PCC uptake capacity of gemcitabine. PCCs grown on FN-coated surface displayed higher resistance to gemcitabine compared to cells grown on non-coated surface. Furthermore, a FN inhibitor, synthetic Arg-Gly-Asp-Ser (RGDS) peptide significantly inhibited PSC-CM-induced chemoresistance in PCCs via downregulation of ERK1/2 phosphorylation.
The findings of this study suggest that FN secreted by PSCs in the ECM plays a key role in the development of resistance to gemcitabine via activation of ERK1/2. FN-blocking agents added to gemcitabine-based chemotherapy might counteract chemoresistance in PDAC and provide better clinical outcomes.
吉西他滨仍然是胰腺导管腺癌（PDAC）化疗的基石，尽管临床效果不佳，部分原因是化疗耐药的发展。肿瘤基质中的胰腺星状细胞（PSC）已知与胰腺癌细胞（PCC）相互作用，并通过涉及细胞外基质（ECM）蛋白的复杂信号分子网络影响 PDAC 的进展。为了了解调节化疗敏感性的肿瘤-基质相互作用，研究了 PSC 分泌的纤维连接蛋白（FN）在 PDAC 吉西他滨耐药发展中的作用。
从 10 个人类 PDAC 肿瘤中获得的 PSC 培养物与 PCC 系（AsPC-1、BxPC-3、Capan-2、HPAF-II、MIA PaCa-2、PANC-1 和 SW-1990）直接或间接共培养，间接共培养是通过 PSC 条件培养基（PSC-CM）孵育。使用 MTT 基于细胞活力测定法确定吉西他滨剂量反应细胞毒性。通过蛋白质组学基于 LC-MS/MS 进行 PSC-CM 分泌组分析，并通过 ELISA 测定 PSC-CM 中的 FN 含量。放射性标记的吉西他滨用于确定 PCC 摄取药物的能力。
在直接和间接共培养中，PSC 在所有研究的癌细胞系中诱导了对吉西他滨细胞毒性作用的不同程度的耐药性。在用 PSC-CM 孵育时，所有 PCC 系均观察到 ERK1/2 的磷酸化程度不同程度增加，而 AKT 的激活未被检测到。PSC-CM 的分泌组分析鉴定出 796 种不同的蛋白质，包括几种 ECM 相关蛋白质，如 FN 和胶原蛋白。在 PSC-CM 中检测到可溶性 FN 含量在 175-350ng/ml 范围内。FN 或 PSC-CM 对 PCC 摄取吉西他滨的能力均没有影响。与未涂层表面相比，在 FN 涂层表面上生长的 PCC 对吉西他滨的耐药性更高。此外，FN 抑制剂，合成 Arg-Gly-Asp-Ser（RGDS）肽通过下调 ERK1/2 磷酸化显着抑制 PSC-CM 诱导的 PCC 化疗耐药性。
本研究的结果表明，ECM 中 PSC 分泌的 FN 通过激活 ERK1/2 在吉西他滨耐药的发展中起关键作用。在吉西他滨为基础的化疗中加入 FN 阻断剂可能会对抗 PDAC 中的化疗耐药性，并提供更好的临床结果。
