Intratumor transforming growth factor-β signaling with extracellular matrix-related gene regulation marks chemotherapy-resistant gastric cancer

Drug-tolerant persister (DTP) cells remain following chemotherapy and can cause cancer relapse. However, it is unclear when acquired resistance to chemotherapy emerges. Here, we compared the gene expression profiles of gastric cancer patient-derived cells (GC PDCs) and their respective xenograft tumors with different sensitivities to 5-fluorouracil (5-FU) by using immunodeficient female BALB/c-nu mice. RNA sequencing analysis of 5-FU-treated PDCs demonstrated that DNA replication/cell cycle-related genes were transiently induced in the earlier phase of DTP cell emergence, while extracellular matrix (ECM)-related genes were sustainably upregulated during long-term cell survival in 5-FU-resistant residual tumors. NicheNet analysis, which uncovers cell-cell signal interactions, indicated the transforming growth factor-β (TGF-β) pathway as the upstream regulator in response to 5-FU treatment. This induced ECM-related gene expression in the 5-FU-resistant tumor model. In the 5-FU-resistant residual tumors, there was a marked upregulation of cancer cell-derived TGF-β1 expression and increased phosphorylation of SMAD3, a downstream regulator of the TGF-β receptor. By contrast, these responses were not observed in a 5-FU-sensitive tumor model. We further found that TGF-β-related upregulation of ECM genes was preferentially observed in non-responders to chemotherapy with 5-FU and/or oxaliplatin among 22 patient-derived xenograft tumors. These observations suggest that chemotherapy-induced activation of the TGF-β1/SMAD3/ECM-related gene axis is a potential biomarker for the emergence of drug resistance in GCs. Examination of RNA-sequencing in (1) JSC18-1 cells were treated with or without 5-FU at IC50 values, 1 μM for 5 days, and (2) JSC15-3 and JSC18-1 cells were treated with or without 5-FU at IC50 values, 1 μM and 0.5 μM for 14 days, and (3) their xenograft tumors in BALB/c-nu mice were treated with PBS or 5-FU (100 mg/kg).

药物耐受持久细胞（drug-tolerant persister cells, DTP）在化疗后仍可残留，并可引发癌症复发。然而，目前尚不明确化疗获得性耐药究竟于何时产生。本研究采用免疫缺陷雌性BALB/c-nu小鼠，对比了对5-氟尿嘧啶（5-fluorouracil, 5-FU）敏感性各异的胃癌患者来源细胞（gastric cancer patient-derived cells, GC PDCs）及其对应异种移植瘤的基因表达谱。对经5-FU处理的GC PDCs进行RNA测序分析后发现，在DTP细胞形成的早期阶段，DNA复制/细胞周期相关基因会被瞬时诱导表达；而在5-FU耐药残留瘤的长期细胞存活过程中，细胞外基质（extracellular matrix, ECM）相关基因则持续上调。可揭示细胞间信号互作的NicheNet分析显示，转化生长因子-β（transforming growth factor-β, TGF-β）通路是响应5-FU处理的上游调控通路，该通路可诱导5-FU耐药瘤模型中ECM相关基因的表达。在5-FU耐药残留瘤中，癌细胞来源的TGF-β1表达显著上调，且TGF-β受体下游调控因子SMAD3的磷酸化水平升高；与之相反，在5-FU敏感瘤模型中未观察到上述变化。我们进一步在22例患者来源异种移植瘤中发现，TGF-β相关的ECM基因上调现象，优先出现在对5-FU和/或奥沙利铂（oxaliplatin）化疗无应答的样本中。上述结果表明，化疗诱导的TGF-β1/SMAD3/ECM相关基因轴激活，可作为胃癌化疗耐药产生的潜在生物标志物。本研究对以下样本开展了RNA测序分析：(1) 以IC50浓度（1μM）的5-FU处理JSC18-1细胞5天，设置未处理对照组；(2) 分别以IC50浓度的5-FU（1μM和0.5μM）处理JSC15-3和JSC18-1细胞14天，设置未处理对照组；(3) 对BALB/c-nu小鼠体内的对应异种移植瘤分别注射PBS或100mg/kg的5-FU进行处理。