A Preliminary Study Of The CRISPR/Cas9-FAP Lentiviral Particles For Breast Cancer Therapy Mediated By Ultrasound Targeted Microbubble Destruction Technology

Objective:1.To observe the biological function of FAP gene silencing in CAFs on 4T1 breast cancer cell in vivo and vitro.2.To explore the feasibility of CRISPR/Cas9-FAP delivery system mediated by UTMD.Methods:1.CAFs were isolated from fresh 4T1 breast cancer tissues of mice and identified by immunofluorescence.The lentiviral vector solution(sgRNA/Cas9)and microbubbles lentiviral vector solution(MBs-sgRNA/Cas9)were prepared after the design of targeting sequence(sgRNA)for FAP.2.CAFs were transfected by the prepared CRISPR/Cas9 lentiviral vector.The G1 was the control group(CAFs).The CAFs of G2 were transfected only by Cas9 lentivirus(CAFs-NC-Cas9).The CAFs of G3 were transfected by sgRNA and Cas9 lentivirus(CAFs-sgRNA-Cas9).QPCR analyzed the FAP mRNA expression.The proliferation of 4T1 cells was evaluated by flow cytometry after co-cultured with those CAFs in vitro.3.The transplanted tumor model of Bale/c nude mice was established and treated by intravenous injection.The G1 was treated by normal saline(NC).The G2 was injected by lentiviral vector solution(sgRNA-Cas9).The G3 was injected by microbubbles lentiviral vector solution and mediated by UTMD(UTMD-sgRNA-Cas9).The volume of tumor was recorded regularly.UE assessed the stiffness of breast lesions and IVIS detected the distribution of lentivirus in mice.At the end of the experiment,the tumor was stripped,photographed and weighed.Results:1.CAFs cells were identified successfully.2.The FAP-targeted CRISPR/Cas9 system(CAFs-sgRNA-Cas9)downregulated FAP expression on CAFs(P<0.05),decreased 4T1 proliferation in vitro(P<0.05).3.The tumor growth of UTMD-sgRNA-Cas9 group and sgRNA-Cas9 group was slower than that of the control group in vivo.The tumor volume and weight of mice in UTMD-sgRNA-Cas9 group and sgRNA-Cas9 group were significantly lower than those in control group(sgRNA-Cas9 vs NC:1232.54 ±495.42mm3 vs 2035.64 ±446.68mm3,p=0.0274;0.84±0.30g vs 1.53 ± 0.50g,P=0.0295)(UTMD-sgRNA-Cas9 vs NC:1058.65±767.64mm3 vs 2035.64 ±446.68mm3,P=0.0393;0.75 ±0.22g vs 1.53±0.50g,p=0.0129).Moreover,in UTMD-sgRNA-Cas9 treated mice,the visualization of the distribution of MBs and lentiviral vector showed that MBs-based carrier system enhanced delivery efficiency and decreased the stiffness of tumors(P<0.05).But there was no significant difference in tumor stiffness between the sgRNA-Cas9 and the NC group(P>0.05).Conclusions:1.These data provided the proof that the gene therapy tools targeting the tumor microenvironment mediated by CRISPR/Cas9 is an effective approach for 4T1 breast carcinoma therapy,even epithelial-derived solid tumors.2.UTMD technology developed an efficient gene delivery platform for breast cancer.