目标：PD-1/PD-L1免疫检查点治疗对于某些表达PD-L1的三阴性乳腺癌患者有效，但是其反应率仍然不令人满意。本研究旨在探索乳腺癌抗PD-1治疗的耐药机制以及克服PD-1治疗耐药的策略。 方法：通过构建BT-549R5人类三阴性乳腺癌耐药细胞系和4T1R3小鼠乳腺癌耐药细胞系，并应用全基因shRNA文库筛选，得到候选的与抗药性相关的分子，并进行细胞学实验证实。使用Western blot方法检测4T1R3组中TAM家族的Tyro3、Axl和MerTK的表达。通过T细胞杀伤实验观察CDK9下调对T细胞杀伤BT-549R5细胞的影响，通过小鼠肿瘤形成实验观察Tyro3和CDK9下调对抗PD-1治疗转移性乳腺肿瘤的影响。 结果：成功构建了对PD-1治疗耐药的乳腺癌细胞系和动物模型。4T1R3细胞中高表达Tyro3、Axl和MerTK。整个基因组测序显示，BT-549R5细胞中高表达Tyro3和CDK9。T细胞杀伤实验显示，BT-549R5细胞在CDK9下调组和对照组中的存活率随T细胞数目的增加逐渐降低，但是在CDK9下调组中，BT-549R5细胞的存活率迅速降低。小鼠肿瘤形成实验显示，在抗PD-1治疗下，4T1R3细胞组移植瘤的生长速度与4T1细胞组相比快速增长（P<0.05），且4T1R3组的肿瘤体积大于4T1组在20天时。然而，在CDK9敲低4T1R3细胞组和Tyro3敲低4T1R3细胞组中，肿瘤生长速度与4T1细胞组相似，并且20天时的肿瘤体积显著低于4T1R3细胞组（P<0.05）。 结论：Tyro3和CDK9与乳腺癌抗PD-1治疗的耐药性相关。抑制Tyro3和CDK9的表达可以逆转乳腺癌治疗的耐药性。

Objective: PD-1/PD-L1 immune checkpoint treatment is effective for some triple-negative breast cancer populations with PD-L1 expression, but the response rate is still not satisfactory. This study aims to explore the mechanism of drug resistance to breast cancer anti-PD-1 therapies and the strategies for overcoming the resistance to PD-1therapies. Methods: By constructing a human triple-negative breast cancer drug-resistant cell line called BT-549R5 and a mouse breast cancer drug-resistant cell line called 4T1R3, and applying the whole-gene shRNA library screening, candidate drug resistance-associated molecules were obtained and verified by cytological experiments. The expression of Tyro3, Axl and MerTK of the TAM family in the 4T1R3 group was tested using the Western blot method. The down-regulation of CDK9 on the effect of T cells killing the BT-549R5 cells was observed through T cell killing tests, while the down-regulation of Tyro3 and CDK9 on the effect of anti-PD-1 therapies for transplanted breast tumors was observed in mouse tumor formation experiments. Results: The cell lines and animal models of breast cancer resistant to PD-1 treatment were successfully constructed. Tyro3, Axl and MerTK were highly expressed in 4T1R3 cells. Whole genome sequencing showed that Tyro3 and CDK9 were highly expressed in BT-549R5 cells. T cell killing experiment showed that the survival rate of BT-549R5 cells in the CDK9 down-regulated group and the control group decreased gradually with the increase of T cells, but the survival rate of BT-549R5 cells in the CDK9 down-regulated group decreased rapidly. Tumor formation experiment in mice showed that under anti-PD-1 treatment, the transplanted tumor in the 4T1R3 cell group grew rapidly compared with the 4T1 cell group (P<0.05), and the tumor volume of the 4T1R3 group was larger than that of the 4T1 group on Day 20. Nevertheless, the tumor growth rates in the CDK9-knockdown 4T1R3 cell group and the Tyro3-knockdown 4T1R3 cell group were similar to that of the 4T1 cell group, and the tumor volumes at day 20 were signiference lower than that of 4T1R3 cell group(P<0.05). Conclusions: Tyro3 and CDK9 are associated with the drug resistance to anti-PD-1 therapies for breast cancer. Inhibiting the expression of Tyro3 and CDK9 can reverse the drug resistance to breast cancer treatment.