CAFs对乳腺癌细胞生物学特性的影响及机制探讨

目的：通过乳腺癌间质成纤维细胞（ carcinoma-associated fibroblasts，CAFs）与乳腺癌细胞系MDA-MB-231共培养的体外细胞实验及裸鼠接种的在体动物实验，观察CAFs对乳腺癌细胞增殖、凋亡、侵袭和转移活性的影响，并探讨其作用机制。方法：体外实验：分离培养浸润性导管癌组织中CAFs和正常成纤维细胞（ normal fibroblasts，NFs），然后分别与乳腺癌细胞MDA-MB-231体外共培养，采用MTT法、流式细胞仪、Ma-trigel人工模拟基底膜法分别检测乳腺癌细胞的增殖、凋亡、细胞黏附和侵袭能力。动物在体实验：选择乳腺癌细胞系MDA-MB-231、CAFs和NFs，结合生理盐水（NS）、基质细胞衍生因子-1（SDF-1）及其配体拮抗剂AMD3100，组成不同的组别并接种于裸鼠（共6组）。观察肿瘤的大小，有无淋巴结、肺、肝脏转移。留取血标本及肿瘤组织行SDF-1表达水平的检测。结果：MDA-MB-231与CAFs和NFs共培养后乳腺癌细胞的增殖活性显著增强，其中CAFs的作用较NFs更强（P＝0.011）；CAFs组的黏附能力（34.70±4.84个/视野）明显强于NFs组（20.16±3.09个/视野），P＝0.000；而CAFs组的侵袭性（89.0±4.62个/视野）也明显强于NFs组（81.6±6.08个/视野，P＝0.045）。CAFs组中的MDA-MB-231的早期凋亡率（2.9±2.4）较NFs组（5.0±4.2）明显降低（P＝0.026）；MDA231﹢CAFs ﹢NS 组的种植肿瘤平均体积最大（9.092±2.662cm3， P＝0.000）；此外，该组共有4只（66.6％），MDA231﹢NS组有2只（33.3％）存在腋窝淋巴结转移，未见肝肺转移灶。在MDA231﹢CAFs﹢NS组中，血标本 SDF -1值（75.25±16.23pg/ml）、肿瘤组织标本中 SDF -1mRNA值（11.686±8.926）、组织中SDF-1蛋白表达水平（1.006±0.327）均为最高，与其他各组相比均有统计学差异（ P＝0.000）。结论：CAFs可影响乳腺癌肿瘤细胞的生物学特性，具有促进肿瘤细胞增殖，增强其黏附、侵袭及转移能力。其机制可能是通过乳腺癌间质成纤维细胞分泌SDF-1与其特定的受体CXCR4结合这一信号通路来实现的。

Effects and mechamism of the breast cancer associated fibroblasts on breast cancer cells biology

Objective：To explore the effects and mechanism of the breast cancer stromal fibroblasts on the growth, apoptosis,invasion and metastasis of breast cancer cells by the co-culture in vitro and the inoculation of nude mice in vivo. Methods：In vitro：CAFs and NFs were separated from the same patient with infiltrating ductal carcinoma and co-cultured with breast cancer cell line MDA-MB-231 in vitro. The growth,adherence,invasion and apoptosis of breast cancer cells were detected by using the method of MTT,Matrigel membrane or flow cytometer respectively. In vivo：MDA -MB -231 cells,CAFs,or NFs,combined with normal solution（ NS ）,SDF -1 or its ligand blocker （AMD3100）were inoculated into nude mice（six groups）. The tumor size and metastasis of lymph node,lung and liver were observed. Blood SDF-1 levels of nude mice and expression of SDF-1 mRNA and protein in tumor specimens were investigated. Results：The growth of MDA-MB-231 cells were enhanced by co-culturing with CAFs or NFs. CAFs played a more significant effect on breast cells than NFs（P=0. 011）. Breast cancer cells co-cultured with CAFs had an obviously stronger adherence（34. 70 ± 4. 84 adhered cells/field）than those with NFs（20. 16 ± 3. 09 ad-hered cells/field）,P=0. 000. Breast cancer cells co-cultured with CAFs also had a significantly stronger invasion （89. 0 ± 4. 62 cells/field）than those with NFs（81. 6 ± 6. 08 cells/field）,P=0. 045. In flow cytometer assay,MDA-MB-231cells were found less early apoptosis in CAFs group（2. 9 ± 2. 4）than those in NFs group（5. 0 ± 4. 2）,P=0. 026. In group of MDA-MB-231 ﹢CAFs﹢NS（group 6）,the average tumor volume（9. 092 ± 2. 662 cm3 ）was greater than any other groups significantly（P=0. 000）. In addition,lymph node metastases were found in four mice in group 6（66. 6%）,and 2 in group of MDA-MB-231﹢NS（group 1）（33. 3%）. The metastatic tumor was not found in lungs and livers of all nude mice. Blood SDF-1 level（75. 25 ± 16. 23pg/ml）,expression level of SDF-1 mRNA （11. 686 ± 8. 926）and SDF-1 protein（1. 006 ± 0. 327）of tumor tissue in group 6 were all higher than the other five groups significantly（P=0. 000）. Conclusion：CAFs can promote growth,adherence,invasion and lymph node metas-tasis of breast cancer cells,which mechanism is probably related with SDF-1 secreted by CAFs and SDF-1/ CX-CR4 signal pathway.