甲磺酸阿帕替尼联合特瑞普利单抗对三阴性乳腺癌小鼠的免疫治疗效果

Immunotherapy effects of apatinib mesylate combined with toripalimab on triple-negative breast cancer mice

目的 探讨在三阴性乳腺癌(TNBC)免疫治疗中甲磺酸阿帕替尼联合程序性死亡受体-1(PD-1)单克隆抗体——特瑞普利单抗的应用及效果。方法 在雌性BALB/C小鼠40只中,随机选取8只不接种肿瘤作为空白组,余32只接种4T1细胞建立TNBC模型,随机分为特瑞普利组(n=8,第3、6、9天通过尾静脉注射10 mg/kg的特瑞普利单抗)、阿帕替尼组(n=8,每天灌胃60 mg/kg的甲磺酸阿帕替尼,连续给药21 d)、联合组(n=8,甲磺酸阿帕替尼、特瑞普利单抗给药方法剂量同各单药组)及模型组(n=8,同时点灌胃和尾静脉注射与给药组同体积的生理盐水)。监测小鼠肿瘤体积变化;采用苏木精-伊红(HE)染色法分析各组小鼠原位瘤及肝/肺转移灶的病理学改变;采用流式细胞术分析各组小鼠肿瘤组织中CD8^(+)T淋巴细胞、髓源性抑制细胞(MDSCs)、M2型肿瘤相关巨噬细胞(M2-TAMs)、调节性T细胞(Tregs)标志物的表达。结果 (1)原位瘤:给药三组小鼠原位瘤体积均小于模型组(P<0.01),联合组小于阿帕替尼组、特瑞普利组(P<0.01);以模型组为基准,阿帕替尼组、特瑞普利组和联合组的肿瘤抑制率分别增高(20.3±0.9)%、(24.5±1.2)%和(52.9±0.8)%,联合组最高(F=997.528,P<0.01);病理可见联合组原位瘤细胞凋亡现象最显著。(2)肺/肝转移病灶:病理显示,联合组肺转移病灶面积最小、肝转移病灶数量最少。(3)免疫细胞:与其他三组相比,联合组肿瘤组织内免疫促进细胞(CD8^(+)T细胞)比例显著提高(P<0.05),免疫抑制细胞(MDSCs、M2-TAMs和Tregs细胞)比例显著降低(P<0.05)。结论 甲磺酸阿帕替尼与特瑞普利单抗联用治疗TNBC小鼠,可能通过提高瘤内免疫促进细胞比例、降低免疫抑制细胞比例,实现抑制TNBC小鼠原位瘤生长与肺/肝转移病灶形成,明显改善免疫疗效的作用。

Objective To investigate the application and effects of apatinib mesylate combined with programmed death receptor-1(PD-1) monoclonal antibody(mAb)—toripalimab in immunotherapy for triple negative breast cancer(TNBC).Methods Among 40 female BALB/C mice,8 mice were randomly selected as the blank group without tumor inoculation,and the remaining 32 mice were inoculated with 4T1 cells to establish TNBC models.They were randomly divided into toripalimab group(n=8,administered 10 mg/kg of toripalimab through the tail vein on the 3rd,6th,and 9th day),apatinib group(n=8,received 60 mg/kg of apatinib mesylate by gavage daily for 21 consecutive days),combination group(n=8,administration route and dose of apatinib mesylate and toripalimab were the same as those of each single drug group),and model group(n=8,administered normal saline with same volume as administration groups by gavage and tail vein injection at the same time points).The changes of tumor weight and tumor volume in mice were monitored,the pathological changes of tumor in situ and liver/lung metastases in each group were analyzed by HE method,and the markers expressions of CD8^(+)T lymphocytes,myeloid-derived suppressor cells(MDSCs),M2 tumor-associated macrophages(M2-TAMs) and regulatory T cells(Tregs) in the tumor tissues of mice in each group were analyzed by flow cytometry.Results(1) In situ tumors:the volume of in situ tumors in all three administration groups of mice was smaller than that in model group(P<0.01),while that was smaller in combined group compared with apatinib group and toripalimab group(P<0.01).Based on model group,the tumor inhibition rates of apatinib group,toripalimab group and combination group increased by(20.3±0.9)%,(24.5±1.2)% and(52.9±0.8)%,respectively,with the highest in combination group(F=997.528,P<0.01).Pathology showed that the apoptosis phenomenon of in situ tumor cells in combination group was the most significant.(2) Lung/liver metastases:the pathology showed that combination group had the smallest area of lung metastases and the fewest number of liver metastases.(3) Immune cells:compared with the other three groups,the proportion of immune promoting cells(CD8^(+)T cells) in the tumor tissue of combination group significantly increased(P<0.05),while the proportion of immunosuppressive cells(MDSCs,M2-TAMs,and Tregs cells) significantly reduced(P<0.05).Conclusion The combination of apatinib mesylate and toripalimab in the treatment of TNBC mice may inhibit the growth of in situ tumor and the formation of lung/liver metastasis formation,and significantly improve the immune efficacy by increasing the proportion of intratumoral immune-promoting cells and reducing the proportion of immunosuppressive cells.