小鼠结肠癌细胞脑高转移亚株的筛选鉴定及其模型建立

Owing to remarkable life prolongation in mCRC patients, rising incidence of colorectal cancer and advancement in neuroimaging, the possibility for brain metastases to occur in advanced CRC patients is gradually shifting from an almost anecdotal event to a concrete clinical problem, which contributes to worsen prognosis. Our pilot studies have found that primary lesions in distal part of the colon have greater chance to develop brain metastasis; miRNAs are differentially expressed between colorectal cancers and matched brain metastatic carcinomas, which indicate particular anatomical and molecular mechanism of brain metastasis in CRC patients. Unfortunately, there has been little progress in this area, thus, establishment of stable and reliable animal model of CRC brain metastasis is essential. We try to establish a highly brain-metastatic sub-clone of mouse colon cancer cell line by double-cycling inoculation of tumor cell in vivo (subcutaneous tumor to lung metastasis; carotid artery tumor cell injection to brain metastasis), and then establish stable mouse model of colon cancer brain metastasis by this selected sub-clone. Metastatic features as mobility, adhesion and invasiveness are analyzed between primary cell line and selected sub-clones. Metastasis rate, time from inoculation to detectable metastasis and survival time are analyzed by pathological and radiological means. Qualitative and quantitative analysis of molecular markers related to metastasis (such as MMP, CD44, VEGF and miRNA profiles) are conducted by means of proteomics, tissue/ miRNA microarrays and other available molecular biological techniques. We hope this model will help to explore probable mechanisms of brain metastasis, with the final aim of developing adaptive treatment or prevention strategies of hematogenous metastasis of colorectal cancer.

结直肠癌发病率的增加、生存期的延长及影像学的进步使结直肠癌脑转移从偶然临床事件升级为重要的临床问题并成为阻碍患者疗效进一步提升的瓶颈。课题组前期研究发现，结直肠远端癌更易发生脑转移；原发与脑转移癌的miRNA表达谱存在显著差异；提示结直肠癌脑转移可能存在特殊解剖和分子机制。目前相关研究基本空白，故建立稳定的脑转移模型十分必要。本课题拟采用小鼠结肠癌细胞以皮下种植-肺转移及颈内动脉注射-脑转移双重体内循环传代筛选出脑高转移亚株，并以原位种植法建立稳定的脑转移模型。分析原代和各高转移细胞系迁移率、黏附性、侵袭性等特性；应用影像及病理学检查对模型的转移率、转移出现时间、存活期进行鉴定；应用组织芯片、蛋白组学及miRNA芯片技术对原发和转移瘤组织的转移相关标志物（如MMP、CD44、VEGF及miRNA谱等）表达进行定性定量研究，探寻其转移机制。进而为研究结直肠癌血行转移机制及防治策略提供新思路。

结直肠癌发病率的增加、生存期的延长及影像学的进步使结直肠癌脑转移从偶然临床事件升级为重要的临床问题并成为阻碍患者疗效进一步提升的瓶颈。目前相关研究基本空白，故建立稳定的脑转移模型十分必要。目前国内外尚无成功建立稳定可靠的结直肠癌脑转移动物模型的报道。本研究即以建立稳定可靠的结肠癌脑转移动物模型并筛选对脑组织具有高度亲和力的脑高转移结肠癌细胞系为目的。第一部分应用正常免疫小鼠（BALB/c）及鼠源性结肠癌细胞系（CT26），通过颈内动脉注射瘤细胞悬液的方法于国内外首次成功建立小鼠结肠癌脑转移实验模型并积累了模型相关基线数据。手术成功率达97%，颅外转移致死率4%，总并发症率在7%。建模方法安全可靠。病理微转移阳性率最早术后10天开始出现阳性，随时间逐步升高，第30天时达最高，为55.6%，此后则逐渐下降，至第50天时为0.0%。临床转移阳性率最早术后20天起出现阳性，随着时间推移逐步上升，至第50天时为50%。磁共振最早术后20天起出现阳性表现，为右侧单发转移。自第30天起出现多发转移。MR阳性率随时间推移逐渐升高，第50天阳性率达50%。最早术后第30天起出现阳性神经症状，其比例逐渐增加，至第50天比例为38.9%。磁共振对包括微转移的总体转移诊断敏感性为45.2%，特异性为100%。剔除微转移后对临床转移灶诊断的敏感性及特异性均为100%。神经症状对包括微转移的总体转移诊断敏感性为28.6%，特异性为100%。而剔除微转移后对临床转移灶诊断的敏感性为63.2%，特异性为100%。头颅磁共振成像可作为小鼠结肠癌脑转移实验模型转移监测和评估的无创手段。第二部分应用CT26小鼠结肠癌细胞悬液颈内动脉注射-脑转移体内连续筛选法（5轮），筛选出具有脑高转移能力的亚株CT26-B，其体外转移相关能力（增殖、黏附、侵袭、运动）及体内脑转移形成率(73.7% vs.47.3%,p<0.05）均显著高于原代细胞CT26，可用于更好地建立结肠癌脑转移实验模型。VEGF及MMP-9在CT26-B所致脑转移组织中的表达明显高于CT26，提示该两者可能在结肠癌脑转移过程中起到重要作用。通过本研究初步建立稳定的结肠癌脑转移实验模型。为探索结直肠癌脑转移机制提供研究工具。但尚需进一步研究验证CT26-B的其他靶器官转移潜力，进一步体内外筛选以改善其特异性并稳定其转移表型。