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. 2023 Jan 12;18(2):585–596. doi: 10.1016/j.stemcr.2022.12.012

Figure 4.

Figure 4

Evaluation of antitumor activity of CAR-Ms (H9 derived) in vitro

(A) Killing of CHLA-20 neuroblastoma cells by CAR-Ms. CHLA-20-AkaLuc-GFP cells were mono-cultured or co-cultured with macrophages at different E:T ratios for 20–24 h. Statistics of CHLA-20 cell survival results are represented as mean ± SD. Student’s t test; ∗p < 0.05; ns, not significant; n > 4 independent experiments.

(B) Killing of WM266-4 melanoma cells by CAR-Ms. WM266-4-AkaLuc-GFP melanoma cells were mono-cultured or co-cultured with macrophages at different E:T ratios for 20–24 h. Statistics of WM266-4 cell survival. Results are mean ± SD. Student’s t test; ∗p < 0.05; ns, not significant; n > 4 independent experiments.

(C) Representative flow cytometry plots show CHLA-20 cells co-cultured with WT-Ms and CAR-Ms. CHLA-20 cells are labeled by GFP. WT-Ms and CAR-Ms are labeled by SIRPA immunostaining.

(D) Representative images show phagocytosis of CHLA-20 cells by CAR-Ms. CHLA-20-AkaLuc-GFP cells were co-cultured with macrophages for 6 h (E:T = 3:1). Green arrows indicate CHLA-20 cells, red arrows indicate WT-Ms, and yellow arrows indicate phagocytosis of CAR-Ms.

(E) Killing of CHLA-20 neuroblastoma cells by CAR-Ms with or without treatment. CHLA-20-AkaLuc-GFP cells were mono-cultured or co-cultured with macrophages at E:T ratio = 3:1 for 20–24 h. Statistics of CHLA-20 cell survival results are represented as mean ± SD. Student’s t test; ∗p < 0.05; n = 3 independent experiments.

(F) Representative histograms show flow cytometry analysis of GD2 expression in different cells. AEC is aretrial endothelial cells, SMC is smooth muscle cells.

(G) CAR-Ms were co-cultured with AEC-NOS3-NanoLuc-2A-tdTomato, SMC-MYH11-NanoLuc-2A-tdTomato, K562-AkaLuc-GFP, Raji-AkaLuc-GFP, and CHLA-20 cells (E:T = 3:1) for 20 h. Target cell survival was measured by luciferase assay. Results are represented as mean ± SD. Student’s t test; ∗p < 0.05; n = 3 independent experiments.

(H) Secretome analysis of macrophages. WT-Ms and CAR-Ms were mono-cultured or co-cultured with CHLA-20 for 20 h. Cell culture media were collected for secretome analysis. Results are mean ± SD. Student’s t test; ∗p < 0.05; n = 3 independent experiments.