Additional file 1 of A drug screening to identify novel combinatorial strategies for boosting cancer immunotherapy efficacy
Creators: name: Zhang, Zongliang
name: Wang, Guoqing
name: Zhong, Kunhong
name: Chen, Yongdong
name: Yang, Nian
name: Lu, Qizhong
name: Yuan, Boyang
name: Wang, Zeng
name: Li, Hexian
name: Guo, Liping
name: Zhang, Ruyuan
name: Wu, Zhiguo
name: Zheng, Meijun
name: Zhao, Shasha
name: Tang, Xin
name: Shao, Bin
name: Tong, Aiping
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Fig. S1. Production of B7-H3 CAR T Cells. (A) Schematic representation of the B7-H3 CAR vector including a J42-scFv, linker, hinge, CD8 transmembrane domain, intracellular signaling domain of 4-1BB, and CD3-z, P2A, and mCherry. (B and C) Representative image of B7-H3 CAR expression in human T cells, which was detected using mCherry and analyzed using flow cytometry. (D) Flow cytometry analysis of the expression of B7-H3 in MDA-MB-231 and HCT116 cells. Cells were incubated with B7-H3-PE (red) or its corresponding isotype control (blue). (E) 51Cr-release assay to measure the cytotoxicity of B7-H3 CAR T cells against MDA-MB-231 and HCT116 cells at different E:T ratios. All error bars represent SD. T tests were used to determine statistical significance of the differences in (E). *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001. Fig. S2. BML284/PPP/JK184 inhibited tumor cells and enhanced B7-H3 CAR T cells antitumor activity. (A) IC50 of BML284/PPP/JK184 in MDA-MB-231 and HCT116 cells. (B) Cells were treated with BML284/PPP/JK184 (1 µM) for 24h, then flow cytometry analysis of the expression of B7-H3 in MDA-MB-231 and HCT116 cells. (C) T cells were treated with different concentrations of BML284/PPP/JK184. (D, E and F) The expression levels of CD25, CD69, PD1 and LAG3 were detected by FACS after T cells and B7-H3 CAR T cells were treated with BML284/PPP/JK184 (1 µM) for 24h. T tests were used to determine statistical significance of the differences in (F). **P &lt; 0.01, ***P &lt; 0.001, ns not significant. (G) Diagram showing the residual tumor cells estimated from the crystal violet staining after the tumor cells were inhibited by CAR T cells or CAR T cells combined with BML284/PPP/JK184. Fig. S3. Identification of JK184 as a Hedgehog inhibitor. (A) KEGG analysis highlighted the breast cancer, Hedgehog signaling pathway, and apoptosis alternations in JK184-treated MDA-MB-231 cells versus nontreated MDA-MB-231 cells (WT). Each group comprised three replicates. (B) Heatmap showing the expression of hedgehog signaling target-related genes (fold change) in WT and JK184-treated MDA-MB-231 cells. (C) Real-time PCR results confirming the regulation of hedgehog signaling by the target genes SMO, PTCH1, GLI1, GLI2, GLI3 and SUFU. Each sample group comprised three replicates. Fig. S4. Mouse body weight was calculated in multiple mouse models. Body weight for each mouse was evaluated every 3 days in the MDA-MB-231 tumor-bearing mouse model (A), HCT116 tumor-bearing mouse model (B), 4T1 tumor-bearing mouse model (C), MC38 tumor-bearing mouse model (PD1 combined with JK184) (D) and MC38 tumor-bearing mouse model (CTLA4 combined with JK184) (E). Fig. S5. JK184 skews macrophages to an M1-like phenotype in the tumor microenvironment. Flow cytometry assays to analyze the macrophages of the TME in PD1 combined with JK184-treated MC38 tumors. CD11b+ CD86+ macrophagess and CD11b+ CD206+ macrophages represent M1-like macrophages (M1) and M2-like macrophages (M2) respectivelay. Representative images of (A) M1, (B) M2 and (C) the ratio of M1/M2. T tests were used to determine statistical significance of the differences in (C). **P &lt; 0.01, ****P &lt; 0.0001, ns not significant. Table S1. The antibodies for flow cytometry. Table S2. Primer nucleotide sequences for qRT-PCR.