肿瘤相关成纤维细胞促进原代胰腺癌细胞生长及人源性异种移植瘤模型体内成瘤

目的优化人原代胰腺导管癌(PDAC)细胞及肿瘤相关成纤维细胞(CAFs)的分离及培养方法,验证CAFs对原代PDAC细胞的体外生长以及其对高度免疫缺陷模型NOG小鼠体内成瘤的影响,以探索人源性异种移植瘤(PDX)模型构建的新方法。方法首先原代培养手术切除的PDAC组织。以原代PDAC细胞单独培养为对照,观察CAFs和原代PDAC细胞共培养对PDAC细胞传代生长活性的影响。最后在NOG小鼠肩胛部注射混合培养的原代PDAC细胞和CAFs,观察PDX模型建模情况。结果原代PDAC细胞在体外单独培养传代少,均在5代及以内停止生长;与CAFs共培养可促进原代PDAC细胞生长活性,可稳定传至10代以上。NOG小鼠体内注射混合细胞(原代PDAC细胞+CAFs)建模2~3周即可形成肿瘤,而注射CAFs无肿瘤形成。混合细胞法建模有13例(92.9%)成瘤,单一细胞法有9例(64.3%)成瘤,可能因为样本量少,二者成瘤率比较差异无统计学意义(P=0.167);并且肿瘤细胞注射后2、4、6、8周时混合细胞法建模的肿瘤体积均明显大于对应观察时间点单一细胞法建模的肿瘤体积(P<0.01)。结论CAFs对原代PDAC细胞生长传代具有促进作用,原代PDAC细胞与CAFs共培养法可显著提高PDAC原代培养稳定传代,原代PDAC细胞与CAFs混合培养的细胞注射可提高NOG小鼠PDX建模成功率。

Cancer associated fibroblasts promote growth of primarily cultured pancreatic ductal adenocarcinoma cells in vitro and tumor formation in patient-derived tumor xenograft model

Objective To optimize the culture method of human primary pancreatic ductal adenocarcinoma(PDAC)cells and cancer associated fibroblasts(CAFs)and investigate the effect of CAFs on the growth of primary PDAC cells in vitro and tumor formation in patient-derived xenograft(PDX)model.Methods The PDAC specimens were collected and primarily cultured.In order to observe the effect of CAFs on the growth of primary PDAC cells in vitro,the CAFs were co-cultured with primary PDAC cells consistently and the alone cultured primary PDAC cells served as the control.Then,these cells were injected into the shoulder blades of NOG mice in order to develop the PDX model.Results When the primary PDAC cells separated from the CAFs,the proliferation capacity of the primary PDAC decreased rapidly in the passage culture in vitro,and the most cells were terminated within 5 generations.By contrast,when the CAFs co-cultured with the primary PDAC cells,the proliferation capacity of primary PDAC cells were preserved,which could be stably transferred to at least 10 generations.The tumors of NOG mice were detected during 2–3 weeks after injecting the mixed cells(primary PDAC plus CAFs),while had no tumor formation after injecting CAFs alone.The rate of tumor was 92.9%(13 cases)in the primary PDAC plus CAFs group,which was higher than that of the CAFs alone group(64.3%,9 cases),but there was no statistical difference because of the small sample size.The volume of tumor in the primary PDAC plus CAFs group at 2,4,6,and 8 weeks after the tumor cells injection was significantly larger than that in the CAFs alone group at the corresponding time point,the differences were statistically significant(P<0.01).Conclusions The CAFs could promote the growth of primary PDAC cells in vitro.This new method of co-culture CAFs with primary PDAC could improve the success rate of primary PDAC cells culture and improve the success rate of PDX model in NOG mice.