Targeted Delivery Of CXCR4-siRNA By ScFv Suppresses HER2~+ Breast Cancer Growth And Metastasis

【Background】Breast cancer is a malignant tumor disease which causes serious damage to women’s health. For most of patients with breast cancer, metastasis has been occurred when the disease detected. At present, the mechanism causing morbidity and metastasis of breast cancer is still not defined, and the treatment at metastasis advanced lesion is still a huge challenge. In spite of continuous improving of traditional treatment methods such as surgery, radiotherapy and chemotherapy, the morbidity and mortality rate of breast cancer have not been reduced significantly. Consequently, it is particularly urgent to understand deeply the morbidity and development mechanism of breast cancer and to seek for new type of effective treatment strategies.Researches have shown that chemokine CXCR4 is closely related to the growth and metastasis of breast cancer. And antagonism CXCR4 can inhibit the growth and metastasis of breast cancer. Long-term application of CXCR4 antagonists, however, will cause serious side effects on the immune system. That CXCR4 expression can prevent tumor growth and metastasis indicates that the strategy may become a potential anti-tumor treatment method. How to deliver si RNA into cells safely and effectively, however, is still a significant obstacle hindering clinical application of RNAi.HER2 in 20-35% invasive breast cancer patients is of high expression. And the coexpression rate of HER2 and CXCR4 of patients with breast cancer is high as well. Whether we can set HER2 as target, and take targeted delivery of CXCR4 si RNA for treatment? After being delivered to HER2 positive cells, whether si RNA can effectively intervene CXCR4 or not? Whether the interference of CXCR4 can play a role on inhibiting growth and metastasis of breast cancer? All these issues are to be explored. The research topic will carry out researches around this topic. 【Objective】To obtain a new type of fusion protein(e23s Fv-9R) to make it have ability of anti-HER2 single-chain antibody(e23s Fv) targeted HER2+ tumor cells simultaneously and the ability of nona-arginine(9R) carrying si RNA. To deliver CXCR4 si RNA in vivo by taking e23 s Fv-9R so as to observe the impact of CXCR4 inhibition on growth and metastasis of HER2+ breast cancer. And to bring a new therapy idea for breast cancer treatment. 【Methods】1. Established p QE30-e23 s Fv-9R expression plasmid by applying molecular cloning technique; 2. Expressed expression plasmid in E.coli. Obtained e23 s Fv-9R after purification and renaturation of expression product. Identified it through SDS-PAGE and Western Blot test; 3. Identified e23 s Fv-9R si RNA combining capacity by gel retardation assay and analyzed the ratio of molecules of e23 s Fv-9R being able to combine with si RNA through titration of e23 s Fv-9R binding to si RNA; 4. Performed CXCR4 staining by using breast cancer tissue microarray, and analyzed the relationship between CXCR4 and HER2; 5. Detected CXCR4 and HER2 expression in commonly used cells with q RT-PCR; 6. Identified HER2 antigen binding activity of e23 s Fv-9R through ELISA assay, flow cytometry and indirect fluorescent antibody assay; 7. Observed cellular internalization of e23 s Fv-9R carrying si RNA with confocal laser scanning microscopy; 8. Performed experimental analysis CXCR4 in vitro inhibition efficiency by q RT-PCR and Western Blot; 9. Detected the impact of CXCR4 interference on HER2+ tumor cells respectively by CCK-8 assay, colony formation assay, flow cytometry and transwell invasion assay; 10. Observed e23 s Fv-9R distribution in vivo by fluorescence imaging; 11. Detected inhibition efficiency of CXCR4 in vivo tissue by q RT-PCR, Western Blot and immunohistochemical experiment; 12. Detected the impact of CXCR4 down-regulation on proliferation and apoptosis of cancer cell through Ki-67 staining and TUNEL test; 13. Identified the impact of e23 s Fv-9R/CXCR4 si on growth of HER2+ breast cancer orthotopic transplantation tumor and tumor-bearing nude mice survival duration by adopting bioluminescence imaging, tumor volume measurement and survival analysis; 14. Observed the impact of e23 s Fv-9R/CXCR4 si on HER2+ breast cancer metastasis by adopting bioluminescence imaging; 15. Analyzed the expression of nude mice human housekeeping gene in lung by q RT-PCR analysis; 16. Analyzed and observed whether innate immunity or acute stress reaction on nude mice through quantitative ELISA assay and serological index analysis; 17. Observed the impact of repeat dosing and 5 times therapeutic dose on important tissues and organs of nude mice by HE staining. 【Results】1. The results of enzyme identification and sequencing results show that expression plasmid of p QE30-e23 s Fv-9R has been established successfully; 2. SDS-PAGE and Western Blot show that expression, purification and renaturation of fusion protein e23 s Fv-9R are successful, and are consistent with the size and prospection of protein; 3. The test results of gel retardation assay and titration of e23 s Fv-9R binding to si RNA show that e23 s Fv-9R can combine with si RNA, and one e23 s Fv-9R can combine with about 3 si RNA molecules 4. The result of breast cancer tissue microarray staining show that CXCR4 and HER2 expression have correlation; 5. According to q RT-PCR results combining with animals tumor formation in vivo, selected HER2+ BT-474 cells as positive cells for experiments, and HER2- MDA-MB-231 as control cells; 6. The results of ELISA assay, flow cytometry and indirect immunofluorescence assay show that e23 s Fv-9R has HER2 antigen binding activity, and can combine with HER2+ tumor cells delivering si RNA; 7. The result of confocal laser scanning microscopy shows that e23 s Fv-9R can deliver si RNA into HER2+ tumor cells; 8. The results of q RT-PCR and Western Blot show that e23 s Fv-9R/CXCR4 si can effectively intervene CXCR4 m RNA and protein expression; 9. The results of CCK-8 assay and colony formation assays show that CXCR4 interference will reduce proliferation and clone formation ability of HER2+ tumor cells. The result of flow cytometry detection of apoptosis indicates that CXCR4 interference will induce cell apoptosis of HER2+ tumor cells, and the result of transwell invasion assay indicates that CXCR4 interference will inhibit HER2+ tumor cell metastasis; 10. The result of fluorescence imaging shows that e23 s Fv-9R has good tumor targeting, and can accumulate for more than 36 h; 11. The results of q RT-PCR, Western Blot and immunohistochemical experiment show that e23 s Fv-9R/CXCR4 si can reduce CXCR4 m RNA and protein expression within the tissue. 12. The results of Ki-67 staining and TUNEL test show that CXCR4 interference can inhibit tumor proliferation and induce apoptosis of tumor cells; 13. The results of bioluminescence imaging of nude mice bearing breast tumor. tumor volume measurement and survival analysis show that e23 s Fv-9R/CXCR4 si can inhibit growth of HER2+ transplantation tumor and prolong survival time of nude mouse bearing the tumor; 14. Bioluminescence imaging of metastatic tumor show that e23 s Fv-9R/CXCR4 si can inhibit metastasis of HER2+ breast cancer 15. q RT-PCR analysis result of h HPRT in pulmonary metastasis shows that e23 s Fv-9R/CXCR4 si can inhibit metastasis of HER2+ breast cancer; 16. The results of quantitative ELISA and serological indexes detection show that e23 s Fv-9R/CXCR4 si dosing via intravenous injection will not cause innate immunity or acute stress reaction on nude mice; 17. No obvious pathological changes observed at HE staining of tissues and organs which indicates that repeated administration and 5 times therapeutic dose have good tolerance to the body. 【Conclusion】1. Fusion protein e23 s Fv-9R was obtained through the construction to make it have si RNA combining with HER2 antigen binding capacity, and able to delivery si RNA to HER2 positive breast cancer cells; 2. In vitro experiment, e23 s Fv-9R/CXCR4 si can reduce CXCR4 m RNA and protein expression level of HER2+ tumor cells, and CXCR4 interference can reduce proliferation and metastasis ability of HER2+ breast cancer cells, and induce apoptosis of tumor cells; 3. e23 s Fv-9R has good tumor targeting performance in vivo. e23 s Fv-9R/CXCR4 si can reduce CXCR4 m RNA and protein expression of HER2+ tumor tissue. And CXCR4 down-regulation can inhibit tumor proliferation and metastasis ability, and prolong the survival time of nude mice bearing the tumor; 4. Administration of e23 s Fv-9R/CXCR4 si via caudal vein was very well tolerated and did not cause obvious dose-dependent and treatment-related toxicity.