EpCAM抗体偶联纳米磁珠阳性分离实体瘤外周血循环肿瘤细胞方法的建立

目的 建立EpCAM抗体偶联纳米磁珠阳性分离上皮来源的实体瘤外周血CTC的方法(简称"磁珠分离法"),探讨该方法分离CTC的灵敏度和应用意义.方法 用7.5 ml健康志愿者外周血裂解红细胞分别倍比稀释加入5、10、20、50、100个乳腺癌细胞系MCF7、食管癌细胞系KYSE70、胰腺癌细胞系BxPC-3及胃癌细胞系9811P,通过磁珠分离法富集癌细胞,荧光显微镜下检测体外加入癌细胞的回收率.然后,用磁珠分离法富集未治疗的30例食管癌(Ⅰ期+Ⅱ期6例、Ⅲ期+Ⅳ期24例)、35例乳腺癌(Ⅰ期+Ⅱ期15例、Ⅲ期+Ⅳ期20例)、30例胰腺癌(Ⅰ期+Ⅱ期5例、Ⅲ期+Ⅳ期25例)、33例胃癌患者(Ⅰ期+Ⅱ期13例、Ⅲ期+Ⅳ期20例)和30例慢性胃炎及30名健康志愿者的CTC,同时结合IF和HE染色鉴定各组的CTC.另外,用FISH分别检测分析食管癌、乳腺癌、胰腺癌、胃癌患者200个CTC中8号和20号染色体拷贝数水平.结果 用4,6-二眯-2苯吲哚(DAPI)染色食管癌、乳腺癌、胰腺癌、胃癌等4种癌细胞系并加入健康志愿者7.5 ml外周血的组内试验中,荧光显微镜下计数4种癌细胞的组内的平均回收率分别为87%、87%、86%和88%;5种稀释梯度的细胞数在4种癌细胞系的组间的平均回收率分别是88%、85%、87%、88%和87%.在107的外周血白细胞中能富集到1个癌细胞.采用磁珠分离法从食管癌、乳腺癌、胰腺癌及胃癌患者中检出≥2个CTC的阳性率分别为50%(15/30)、63%(22/35)、70%(21/30)及61%(20/33),健康志愿者和炎症患者均未检出CTC.食管癌、乳腺癌、胰腺癌组分别与健康对照组和炎症组比较,经精确概率法分析,差异均有统计学意义(P值均为0.000).80%的食道癌、75%的乳腺癌、65%的胰腺癌和59%的胃癌患者CTC中均发生8号和20号染色体非整倍体改变.结论 建立的磁珠分离法对富集分离实体瘤外周血CTC具有较好的分选灵敏度和准确性.食道癌、乳腺癌、胰腺癌和胃癌患者中的CTC多发生8号和20号染色体非整倍体改变.

Abstract：

Objective To establish an isolation method for solid GTC in peripheral blood using EpCAM antibody-linked nanobeads and evaluate the sensitivity of the method and its application significance. Methods Five, ten, twenty, fifty and one hundred MCF7 (breast cancer), KYSE70 (esophageal cancer), BxPC-3 (pancreatic cancer) and 9811P (stomach cancer) cells were added into 7. 5 ml erythrocyte lysed peripheral blood obtained from healthy volunteers respectively. EpCAM antibodylinked nanobeads were used to enrich cancer cells. The recovery rates of the in vitro added cancer cells were evaluated by fluorescence microscopy. Then, the untreated thirty cases of esophageal cancer (six cases at stage Ⅰ and Ⅱ, twenty-four cases at stage Ⅲ and Ⅳ), thirty-five cases of breast cancer (fifteen cases at stage Ⅰ and Ⅱ , twenty cases at stage Ⅲ and Ⅳ), thirty cases of pancreatic cancer (five cases at stage Ⅰ and Ⅱ , twenty-five cases at stage Ⅲ and Ⅳ), thirty-three gastric cancer (thirteen cases for stage Ⅰ and Ⅱ ,twenty cases at stage Ⅲ and Ⅳ) were enrolled to enrich the peripheral blood CTC. Thirty healthy volunteers and thirty gastritis patients served as two groups of control. Meanwhile the enriched CTC was identified by IF and HE staining. FISH was used to analyze the copy number of chromosome 8 and chromosome 20 in two hundred esophageal cancer, breast cancer, pancreatic caner and gastric cancer CTC. Results After DAPI staining and mixing with 7.5 ml peripheral blood from healthy donors, the average cell recovery rates of KYSE70, MCF7, BxPC-3 and 9811P cells evaluated under fluorescence microscope were 87%, 87%, 86% and 88% (within group), and the recovery rates of 5 gradient dilution levels were 88%, 85%, 87%, 88% and 87% (intergroup). With a high sensitivity, this method was able to isolate one cancer cell in 107 white blood cells of peripheral blood. The positive rates of more than 2 CTC in the peripheral blood detected by this method were 50% (15/30) of esophageal cancer, 63% (22/35) of breast cancer, 70% (21/30) of pancreatic cancer and 61% (20/33) gastric cancer patients respectively,but no CTC was detected in the peripheral blood of healthy volunteers and gastritis patients (P = 0. 000).The aneusomy of chromosome 8 and chromosome 20 were found in 80% esophageal cancer, 75% breast cancer, 65% pancreatic cancer and 59% gastric cancer. Conclusions The CTC isolation technique with EpCAM antibody-linked nanobeads is sensitive and accurate. The aneusomy of chromosome 8 and 20 is frequent in CTC from esophageal cancer, breast cancer, pancreatic cancer and gastric cancer.

A new isolation method for peripheral blood circulating solid tumor cells with EpCAM antibody linked nanobeads

Objective To establish an isolation method for solid GTC in peripheral blood using EpCAM antibody-linked nanobeads and evaluate the sensitivity of the method and its application significance. Methods Five, ten, twenty, fifty and one hundred MCF7 (breast cancer), KYSE70 (esophageal cancer), BxPC-3 (pancreatic cancer) and 9811P (stomach cancer) cells were added into 7. 5 ml erythrocyte lysed peripheral blood obtained from healthy volunteers respectively. EpCAM antibodylinked nanobeads were used to enrich cancer cells. The recovery rates of the in vitro added cancer cells were evaluated by fluorescence microscopy. Then, the untreated thirty cases of esophageal cancer (six cases at stage Ⅰ and Ⅱ, twenty-four cases at stage Ⅲ and Ⅳ), thirty-five cases of breast cancer (fifteen cases at stage Ⅰ and Ⅱ , twenty cases at stage Ⅲ and Ⅳ), thirty cases of pancreatic cancer (five cases at stage Ⅰ and Ⅱ , twenty-five cases at stage Ⅲ and Ⅳ), thirty-three gastric cancer (thirteen cases for stage Ⅰ and Ⅱ ,twenty cases at stage Ⅲ and Ⅳ) were enrolled to enrich the peripheral blood CTC. Thirty healthy volunteers and thirty gastritis patients served as two groups of control. Meanwhile the enriched CTC was identified by IF and HE staining. FISH was used to analyze the copy number of chromosome 8 and chromosome 20 in two hundred esophageal cancer, breast cancer, pancreatic caner and gastric cancer CTC. Results After DAPI staining and mixing with 7.5 ml peripheral blood from healthy donors, the average cell recovery rates of KYSE70, MCF7, BxPC-3 and 9811P cells evaluated under fluorescence microscope were 87%, 87%, 86% and 88% (within group), and the recovery rates of 5 gradient dilution levels were 88%, 85%, 87%, 88% and 87% (intergroup). With a high sensitivity, this method was able to isolate one cancer cell in 107 white blood cells of peripheral blood. The positive rates of more than 2 CTC in the peripheral blood detected by this method were 50% (15/30) of esophageal cancer, 63% (22/35) of breast cancer, 70% (21/30) of pancreatic cancer and 61% (20/33) gastric cancer patients respectively,but no CTC was detected in the peripheral blood of healthy volunteers and gastritis patients (P = 0. 000).The aneusomy of chromosome 8 and chromosome 20 were found in 80% esophageal cancer, 75% breast cancer, 65% pancreatic cancer and 59% gastric cancer. Conclusions The CTC isolation technique with EpCAM antibody-linked nanobeads is sensitive and accurate. The aneusomy of chromosome 8 and 20 is frequent in CTC from esophageal cancer, breast cancer, pancreatic cancer and gastric cancer.