Anti-tumor effect of CTL on colon cancer xenograft in nude mice after blockingout CTLA-4 with CRSIPR/Cas9 technology
10.3872/j.issn.1007-385x.2020.03.002
- VernacularTitle:以CRSIPR/Cas9技术删除CTLA-4后CTL抗裸鼠结肠癌移植瘤的效果
- Author:
SHI Long
1
;
GENG Songsong
2
;
CAI Ziqi
2
;
HAN Jinsheng
3
;
ZHAO Zhilong
4
;
ZHANG Wei
5
;
SONG Hongtao
6
;
MENG Tongyu
7
,
8
;
CAI Jianhui
9
,
10
Author Information
1. Department of Vascular Surgery, the Second Hospital of Hebei Medical University
2. Nongfuyu (Hebei) Biomedical Co., Ltd
3. Department of Gastroenteroabdominal Hernia, Cangzhou Hospital of Traditional Chinese and Western Medicine
4. Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Jinzhou Medical University
5. The Second Department of General Surgery, Handan Central Hospital
6. Department of Peripheral Vascular Disease, the Second Hospital of Shijiazhuang
7. Department of Gynecology, Shijiazhuang People'
8. s Hospital
9. General Surgery &
10. Oncology Department, the Hebei General Hospital
- Publication Type:Journal Article
- From:
Chinese Journal of Cancer Biotherapy
2020;27(3):221-227
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To investigate the anti-tumor effect of CTL cells on colon cancer xenograft in nude mice after knocking out the immune check point CTLA-4 by CRISPR/Cas9 technology. Methods: A specific small guide RNA (sgRNA) for CTLA-4 was designed to construct sgRNA/Cas9 plasmid, which was then transfected into CTL using a lentiviral vector to obtain CTL cells with CTLA-4 deletion (CTLA-4 KO CTL). The transfection efficiency of the plasmid and the deletion efficiency of CTLA-4 were verified. BALB/c nude mice were randomly divided into two groups to prophylactically inoculate CTLA-4 KO CTL (experimental group) or CTL (control group); 3 days later, the animals of two groups were inoculated with colon cancer cell line LS174-T to observe the tumor formation rate and tumor formation time. After constructing colon cancer xenograft model in nude mice, the animals were randomly divided into two groups, respectively treated with CTLA-4 KO CTL (experimental group) and CTL (control group) cells to observe the tumor growth volume and survival time of mice. The serum levels of TNF-α and IFN-γ in nude mice were detected. Results: sgRNAwas designed and CRSIPR/Cas9 system with lentivirus as vector was successfully constructed. CTL cells were transfected with the established CRSIPR/ Cas9 system, and the highest transfection efficiency was up to (28.80±0.62)%. After transfection, the deletion efficiency of CTLA-4 was detected by Flow cytometry. The CTLA-4 expression of CTLA-4 KO CTL group was significantly lower than that of CTL group [(0.91±0.25)% vs (42.70±2.72)%, P<0.05]. In prophylactic assay, the formation rate of colon cancer xenografts in the experimental group was significantly lower than that in the control group(33.33%vs100%,P<0.05). In treatment assay, the tumor volume in the experimental group was significantly inhibited compared with the control group ([503±23.9] vs [911.2±51.4] mm3, P<0.05), and the survivaltimeoftheexperimentalgroupwassignificantlyprolonged (mediansurvivaltime:78dvs42d,P<0.05); Moreover, the secretion levels of serumTNF-α([268.93±17.04]pg/mlvs[148.26±20.07]pg/ml,P<0.05) and IFN-γ(315.38±18.67 pg/ml vs 202.92±29.32 pg/ml, P<0.05) in the experimental group were significantly higher than those in the control group. Conclusions: The lentiviral vector CRSIPR/Cas9 system is an effective gene editing method; its successful deletion of CTLA-4 in CTL cells can significantly inhibit the tumor formation rate of colon cancer xenografts in nude mice and enhance the anti-tumor effect of CTLon colon cancer xenografts.
