Anti-tumor Effect of CAR-NK Cells Targeting Prostate Cancer

doi:10.13523/j.cb.2302030

China Biotechnology

2023, Vol. 43

Issue (6): 1-11 DOI: 10.13523/j.cb.2302030

Anti-tumor Effect of CAR-NK Cells Targeting Prostate Cancer

SU Ling-yu¹,ZOU Jin-tao¹,NIU An-na¹,ZHANG Wei²,ZHANG Xiao-peng^1,^**(

),CHEN Wei^1,^**(

)

1 Academy of Military Medical Sciences, Beijing 100071,China
2 Nanhu Laboratory,Jiaxing 314001,China

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Abstract

Objective: To investigate the anti-tumor efficacy of chimeric antigen receptor-nature killing cells (CAR-NK cells) against prostate stem cell antigen (PSCA) via DAP12 co-stimulation signal. Methods: CAR-NK cells were generated via lentiviral transfection and the expression of CAR in NK cells was evaluated using flow cytometry. The anti-tumor activity of CAR-NK cells was further assessed in both cellular and animal models. Results: The prostate cancer cell DU145 expressed PSCA with a positive rate of about 98.50%. Flow cytometry revealed that (64.07 ± 3.01)% of CAR-NK cells were positive. Furthermore, the killing rate of CAR-NK cells was approximately 1.5 times higher than that of control NK cells. ELISA tests showed that CAR-NK cells released significantly higher levels of TNF-α, IFN-γ, CD107α, Granzyme B, and Perforin-1, when compared to control NK cells in the process of killing DU145 cells. Additionally, animal experiments show that CAR-NK cells had a better inhibiting effect in the growth of DU145 tumor cells in mice compared with control NK cells as significant difference was found between the two groups (P<0.000 1). Conclusion: The findings suggested that PSCA-targeting CAR-NK cells with DAP12 had a greater anti-prostate tumor effect, providing a potential cellular therapeutic option for the treatment of prostate cancer.

Key words： CAR-NK PSCA DAP12 Prostate cancer

Received: 17 February 2023 Published: 04 July 2023

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	Ling-yu SU
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	An-na NIU
	Wei ZHANG
	Xiao-peng ZHANG
	Wei CHEN

Cite this article:

SU Ling-yu, ZOU Jin-tao, NIU An-na, ZHANG Wei, ZHANG Xiao-peng, CHEN Wei. Anti-tumor Effect of CAR-NK Cells Targeting Prostate Cancer. China Biotechnology, 2023, 43(6): 1-11.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2302030 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I6/1

Fig.1 Design and construction of CAR plasmid based on DAP12 signal domain (a) Schematic diagram of PSCA-CAR structure (b) Agarose gel electrophoresis analysis of CAR fragment (c) Agarose gel electrophoresis analysis of CAR plasmid (d) Sanger sequencing result

Fig.2 Titers of lentivirus detected by flow cytometry (a) Fluorescence microscopy images of 293T cells packaging lentivirus (b) Histogram of flow cytometry results (c) The GFP expression of 293T cells detected by flow cytometry

Fig.3 The CAR expression of NK cells detected by flow cytometry (a) Negative control (b) Control-NK cells (c) CAR-NK cells (d) Histogram of flow cytometry results

Fig. 4 PSCA expression of DU145 cells detected by flow cytometry (a) A549 cells (b) DU145 cells (c) Histogram of flow cytometry results

Fig.5 Killing activity of CAR-NK cells against DU145 cells (a)-(c) Lysis rate evaluated by RTCA assay. (a) Cell index; (b) Lysis rate continuously monitored (c),(d) Lysis rate at 4 h (c) and 8 h (d) (e) Lysis rate determined by LDH assay(E:T=10:1) (f) Lysis rate measured by luciferase assay

Fig.6 Concentration of cytokines determined by ELISA (a) IFN-γ (b) CD107α (c) TNF-α (d) Perforin-1 (e) Granzyme B

Fig.7 Evaluation of tumor burden in mice (a) Tumor burden detected by bioluminescence assay (b) Line chart of bioluminescence imaging results (c) Line chart of tumor volume detected by vernier caliper (d) Images of tumor samples from sacrificed mice (e) Scatter diagram of tumor weight

Fig.8 Infiltration of CAR-NK and NK cells into DU145 tumors detected by flow cytometry (a) Negative control (b) NK cells (c) CAR-NK cells (d) Histogram of flow cytometry results (n=5)


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