肿瘤新抗原疫苗对EGFR-TKI耐药肺癌的作用及机制研究

Study on the mechanism of tumor neoantigen vaccine in treatment of EGFR-TKI resistant lung cancer

目的通过采用肿瘤新抗原疫苗对产生表皮细胞生长因子受体-酪酸激酶抑制剂(EGFR-TKI)耐药性的肺癌大鼠模型进行治疗,并进一步探究其机制。方法采用胸部X射线照射,剂量为3 Gy,每周3次,构建大鼠肺癌模型,对EGFR-TKI治疗2周后,大鼠肿瘤大小不再减小,视为耐药性大鼠肺癌模型建立成功。采用标准的固相合成肽化学和反相高效液相色谱法合成和纯化多肽制备特异性新抗原,将大鼠肺癌EGFR-TKI耐药模型随机分为模型组、传统化疗组[采用传统一线联合化疗方式,先ip培美曲塞10 mg/(kg·d),再ip顺铂0.6 mg/(kg∙d),连续用药10 d],新抗原组(采用sc特异性新抗原进行化疗,剂量3 mL/只,连续用药2次)。PET-CT检测各组大鼠的肿瘤大小,苏木素-伊红(HE)染色观察组织病变情况,流式细胞术检测各组大鼠外周血淋巴细胞CD4^(+)、CD8^(+)、CD127^(+)表达,酶联免疫吸附实验检测外周血γ干扰素(IFN-γ)、白细胞介素(IL)-2、IL-5水平。结果新抗原组肿瘤质量显著低于模型组和传统化疗组(P<0.05),同时PET-CT扫描新抗原组肿瘤病灶出现缩小趋势;HE染色中,相比于模型组和传统化疗组,新抗原组肺组织中炎性浸润现象呈现显著下降趋势,同时新生毛细血管的增加量和肺泡腔的孔径呈现上升趋势;相比于模型组和传统化疗组,新抗原组大鼠外周血淋巴细胞中CD4^(+)、CD4^(+)/CD127^(+)、CD8^(+)/CD127^(+)的表达情况呈现显著升高,CD8^(+)的表达则显著降低(P<0.05);相比于模型组和传统化疗组,新抗原组大鼠外周血中IL-2、IFN-γ、IL-5水平显著下降(P<0.05)。结论采用肿瘤新抗原疫苗对EGFR-TKI耐药后肺癌大鼠模型进行干预,能够有效地缩减肿瘤质量,对肺部组织炎性病变程度起到一定的缓解作用,能够减轻炎症浸润情况,通过升高外周血淋巴细胞内CD4^(+)、CD4^(+)/CD127^(+)、CD8^(+)/CD127^(+),减少CD8^(+)因子的表达,起到免疫抑制作用。另外,新抗原疫苗能够降低外周血IFN-γ、IL-2、IL-5炎性因子水平,减轻肺部炎性程度。

Objective To further explore the mechanism of tumor neoantigen vaccine to treat lung cancer rat models with epidermal growth factor receptor-tyrosate kinase inhibitor(EGFR-TKI)resistance.Methods The rat lung cancer model was established by chest X-ray irradiation at a dose of 3 Gy,3 times weekly.After EGFR-TKI treatment for 2 weeks,the tumor size of the rats did not decrease,which was regarded as the successful establishment of drug-resistant rat lung cancer model.Specific neoantigens are prepared by synthesizing and purifying peptides using standard solid-phase synthetic peptide chemistry and reversed-phase high performance liquid chromatography.The EGFR-TKI resistant rat lung cancer model was randomly divided into model group,conventional chemotherapy group[Traditional first-line combined chemotherapy,peritoneally injected pemetrexed 10 mg/(kg·d),followed by cisplatin 0.6 mg/(kg·d)for 10 consecutive days],neoantigen group(Specific neoantigen was injected subcutaneously for 2 times at a dose of 3 mL).The tumor size of rats in each group was detected by PET-CT,the histological changes were observed by hematoxylin-eosin(HE)staining,the expression of CD4^(+),CD8^(+),and CD127^(+)in peripheral blood lymphocytes of rats in each group was detected by flow cytometry,and the levels of interferon(IFN)-γ,interleukin(IL)-2,and IL-5 in peripheral blood were detected by enzyme linked immunosorbent assay.Results The tumor quality in the neoantigen group was significantly lower than that in the model group and traditional chemotherapy group(P<0.05),and the tumor lesions in the PET-CT scanning neoantigen group showed a trend of shrinkage.In HE staining,compared with the model group and traditional chemotherapy group,the inflammatory infiltration in the lung tissues of the neoantigen group showed a significant decreasing trend,while the increase of new capillaries and the aperture of alveolar cavity showed an increasing trend.Compared with model group and conventional chemotherapy group,the expression of CD4^(+),CD4^(+)/CD127^(+),and CD8^(+)/CD127^(+)in peripheral blood lymphocytes of neoantigen group was significantly increased,while the expression of CD8^(+)was significantly decreased(P<0.05).Compared with model group and conventional chemotherapy group,the levels of IL-2,IFN-γ,and IL-5 in peripheral blood of neoantigen group were significantly decreased(P<0.05).Conclusion The intervention of tumor neoantigen vaccine on EGFR-TKI resistant lung cancer rat model can effectively reduce the tumor quality,alleviate the degree of inflammatory lesions in lung tissue to a certain extent,and reduce inflammatory infiltration.By increasing CD4^(+),CD4^(+)/CD127^(+),and CD8^(+)/CD127^(+)in peripheral blood lymphocytes,and reducing the expression of CD8^(+)factors,play an immunosuppressive role.In addition,neoantigen vaccine can reduce the levels of IFN-γ,IL-2,IL-5 inflammatory factors in peripheral blood,and reduce the degree of lung inflammation.