肺腺癌通过BCAR1生成高侵袭性循环肿瘤细胞及免疫逃避的研究

There are two critical steps in the metastasis of lung adenocarcinoma(LAC): ① In order to evade anoikis, LAC will induce epithelial-mesenchymal transition (EMT) to generate M-circulating tumor cells which are highly invasive, and ② The platelets are supposed to be activated to help M-CTCs survive from the killing of natural killer cells. We had found that the high expression of BCAR1 in lung cancer predicts poor prognosis, and are significantly related to the increasing of M-CTCs and decreasing of NK activity. Additionally, we had found BCAR1\Crk complex can induce EMT and activate platelets via the activation of p38MAPK signaling cascade. We postulated BCAR1\Crk\p38MAPK cascade enhances the occurrence of M-CTCs and immune evasion. We aimed to verify the abovementioned hypothesis via the clinical observation and studies in vivo and vitro. Importantly, we had obtained the McAb of BCAR1 which can block the formation of BCAR1\Crk complex effectively. We will block the BCAR1 signaling cascade simultaneously in cancer cells and platelets by using intracellular BCAR1 antibody so as to avoid the occurrence of M-CTCs and immune evasion. This project is supposed to elucidate the mechanism of tumor recurrence or metastasis. In LAC cases, we should test and screen the expression of BCAR1 in tumor tissue and peripheral blood. Intracellular BCAR1 antibody is supposed to prevent the tumor metastasis.

肺腺癌(LAC)转移存在两个关键环节：通过上皮间质转化(EMT)生成间质型循环肿瘤细胞（M-CTCs）逃避失巢凋亡；活化血小板使M-CTCs逃避NK细胞杀伤。我们发现LAC中BCAR1高表达预后差；癌组织及外周血BCAR1高表达分别与M-CTCs增多及NK失活密切相关；BCAR1\Crk复合物激活p38MAPK诱发EMT及活化血小板。我们拟通过临床观察、细胞及动物等多层面研究证实如下假设：“BCAR1\Crk\p38MAPK信号轴诱发EMT及活化血小板，从而催生M-CTCs及促使免疫逃避”。我们已获得BCAR1单抗可阻断BCAR1\Crk复合物形成;在此基础上拟通过细胞内抗体同时抑制癌细胞及血小板中BCAR1信号通路，从而预防M-CTCs形成及免疫逃避。本项目将阐明LAC复发转移机制,并有重要临床价值：LAC患者应检测癌组织及监测外周血BCAR1表达，BCAR1胞内抗体有望预防肿瘤转移。

肺腺癌是肺癌中最常见的亚型，肺腺癌发病趋于年轻化。全球每年约500,000人死于肺腺癌。深入研究肺癌的转移机制非常重要。本研究通过二代循环肿瘤细胞CTC检测、Crisp/cas9技术、细胞实验、动物实验等多技术手段，从分子、细胞、组织以及动物等层面，完成了BCAR1促进循环肿瘤细胞(CTC)形成及免疫逃避相关的研究。.早期肺腺癌组织中BCAR1、PD-L1高表达提示预后差，是影响患者总体生存时间的独立预后因素。肺癌组织中BCAR1与PD-L1表达显著正相关。肺癌患者外周血中出现BCAR1（+） CTC更易发生EMT。术前出现BCAR1（++）CTC是早期肺腺癌患者预后不良的独立因素。复发3例术后BCAR1（+）-CTCs显著增高。45例病情稳定的患者中，19例术后持续监测CTCs，其中12例总CTCs及BCAR1（+）-CTCs均显著降低或保持稳定；7例总CTCs升高，其中2例BCAR1（+）-CTCs保持稳定，1例降低，4例BCAR1（+）-CTCs增高者继续密切随访。动态监测BCAR1（+）-CTCs 有助于预测早期肺腺癌复发或转移。成功构建H1975、H1299人肺腺癌细胞BCAR1敲除（BCAR1-KO）稳定株。将BCAR1-KO后，H1975、H1299的失巢凋亡显著增高、细胞侵袭功能降低、增殖显著降低；H1975克隆形成显著降低；间质型标志物如N-Cadherin表达显著降低、Vimentin表达显著降低；H1975、H1299细胞的PD-L1表达显著降低。成功构建A549肺腺癌细胞BCAR1高表达（BCAR1-OE稳定株）。将BCAR1-OE后，细胞侵袭功能、细胞增殖、克隆形成能力均显著增加，N-Cadherin表达显著增加。将BCAR1-KO后，H1975、H1299腹腔荷瘤转移能力均显著降低；H1975的皮下荷瘤能力显著降低；H1975腹腔荷瘤后鼠外周血CTC数量降低。.首次揭示了肺腺癌通过BCAR1生成高侵袭性循环肿瘤细胞及免疫逃避的机制。主要有以下三方面的应用前景：（1）BCAR1（+）CTC检测可能应用于肺癌的早诊及筛查；（2）BCAR1（+）CTC有望为肺癌的治疗的疗效评价提供依据，成为肺癌精准治疗的重要靶标（3）BCAR1有望成为肺癌免疫治疗的靶标。本研究发表SCI论文3篇，指导研究生2人。