The crucial role of cancer-associated fibroblasts (CAFs) in promoting T-cell exclusion has a significant impact on tumor immune evasion and resistance to immunotherapy. Therefore, enhancing T-cell infiltration into solid tumors has emerged as a pivotal area of research. We achieved a conventional knockout of Shcbp1 (Shcbp1^−/−) through CRISPR/Cas9 gene editing and crossed these mice with spontaneous breast cancer MMTV-PyMT mice, resulting in PyMT Shcbp1^−/− mice. The different CAF subtypes were detected by flow cytometry analysis (FCA). We evaluated collagen and CAFs levels using Sirius red staining, immunohistochemistry (IHC), and immunofluorescence (IF). Primary tumor cells and CAFs were isolated from both PyMT Shcbp1^+/+ and PyMT Shcbp1^−/− mice. We analyzed CAFs’ proliferation, invasion, migration, apoptosis, and cell cycle. Transwell coculture experiments were performed with primary tumor cells and CAFs to evaluate the role of CAFs in increasing the sensitivity of tumor cells to Erdafitinib. Tumors from PyMT Shcbp1^+/+ and PyMT Shcbp1^−/− mice were orthotopically transplanted to assess the therapeutic effect of the Erdafitinib and PD-1 combination. CAFs and T-cell infiltration in these tumors were assessed using FCA and IF. Knockout of Shcbp1 leads to a significant reduction in tumor burden, promotes longer survival, and decreases CAFs in MMTV-PyMT. Moreover, knockout of Shcbp1 enhances the sensitivity of Erdafitinib, leading to effective inhibition of CAFs' proliferation and invasion, as well as the induction of apoptosis. Additionally, it results in cell cycle arrest at the G2/M phase in vitro. Meanwhile, Shcbp1^−/− CAFs change the sensitivity of Shcbp1^−/− tumor cells to Erdafitinib compared to Shcbp1^+/+ CAFs. Importantly, knockout of Shcbp1 boosts the effectiveness of Erdafitinib in combination with immune checkpoint blockade therapy by augmenting T-cell infiltration through CAFs regulation in vivo. Our findings demonstrate that knockout of Shcbp1 holds significant potential in enhancing the therapeutic response of Erdafitinib combined with PD-1 antibody treatment, offering promising prospects for future breast cancer therapies.

中文翻译：

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Shcbp1 的敲除通过调节 α-SMA 阳性癌症相关成纤维细胞使免疫治疗变得敏感

癌症相关成纤维细胞 (CAF) 在促进 T 细胞排斥方面发挥着至关重要的作用，对肿瘤免疫逃避和免疫治疗耐药性具有重大影响。因此，增强 T 细胞对实体瘤的浸润已成为一个关键的研究领域。我们通过 CRISPR/Cas9 基因编辑实现了Shcbp1 ( Shcbp1 ^−/− )的常规敲除，并将这些小鼠与自发性乳腺癌 MMTV-PyMT 小鼠杂交，产生了 PyMT Shcbp1 ^−/−小鼠。通过流式细胞术分析 (FCA) 检测不同的 CAF 亚型。我们使用天狼星红染色、免疫组织化学 (IHC) 和免疫荧光 (IF) 评估胶原蛋白和 CAF 水平。从 PyMT Shcbp1 ^+/+和 PyMT Shcbp1 ^−/−小鼠中分离出原代肿瘤细胞和 CAF 。我们分析了 CAF 的增殖、侵袭、迁移、凋亡和细胞周期。使用原代肿瘤细胞和 CAF 进行 Transwell 共培养实验，以评估 CAF 在增加肿瘤细胞对 Erdafitinib 敏感性中的作用。来自 PyMT Shcbp1 ^+/+和 PyMT Shcbp1 ^−/−小鼠的肿瘤被原位移植，以评估 Erdafitinib 和 PD-1 组合的治疗效果。使用 FCA 和 IF 评估这些肿瘤中的 CAF 和 T 细胞浸润。Shcbp1的敲除可显着减少肿瘤负荷，促进更长的生存期，并减少 MMTV-PyMT 中的 CAF。此外，敲除Shcbp1可增强Erdafitinib的敏感性，从而有效抑制CAF的增殖和侵袭，并诱导细胞凋亡。此外，它还会导致体外细胞周期停滞在 G2/M 期。同时，与Shcbp1 ^+/+ CAF 相比， Shcbp1 ^−/− CAF 改变了Shcbp1 ^−/−肿瘤细胞对 Erdafitinib的敏感性。重要的是，Shcbp1的敲除通过体内 CAF 调节增强 T 细胞浸润，从而提高了 Erdafitinib 与免疫检查点阻断疗法联合的有效性。我们的研究结果表明，敲除Shcbp1在增强Erdafitinib联合PD-1抗体治疗的治疗反应方面具有巨大潜力，为未来乳腺癌治疗提供了广阔的前景。