Additional file 2 of Ginseng-derived nanoparticles reprogram macrophages to regulate arginase-1 release for ameliorating T cell exhaustion in tumor microenvironment
Creators: name: Lv, Yan
name: Li, Mengyuan
name: Weng, Ling
name: Huang, Haoying
name: Mao, Yujie
name: Yang, Danchen Aaron
name: Wei, Qingyun
name: Zhao, Mengmeng
name: Wei, Qin
name: Rui, Ke
name: Han, Xuan
name: Fan, Weiwei
name: Cai, Xueting
name: Cao, Peng
name: Cao, Meng
Publisher: name: figshare
Year: 2024
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 2: Figure S1. ARG1 was mainly derived from tumor-associated macrophages. a The expression of ARG1 in colorectal cancer patients was analyzed using the GSE4107 dataset. b Arginase1+ cells, macrophages (F4/80+) were detected in fixed colon tumor serial sections using immunofluorescence. Bar=20 μm. c ARG1 expression in tumor cells (CD45- cells) and immune cells (CD45+ cells) determined by flow cytometry. Orange represents isotype control, blue reflects ARG1 expression of CD45- cells, and red reflects ARG1 expression of CD45+ cells. d Frequency of main immune cells infiltrated in MC38 tumor. e Pie chart of the proportion of Macrophages, MDSC, DCs cells, T cells, NK cells, and other immune cells in mouse MC38 tumor environment. f The proportion of ARG1 expression in the main immune cell. Figure S2. GDNPs decreased ARG1 expression in M2-like macrophages. a ARG1/GAPDH ratios. Figure S3. GDNPs could effectively inhibit the growth of MC38 colon tumor in mice. a The tumor size of Vehicle and GDNPs group was photographed. b Tumor weights of two groups. c Body weights of two groups. All results represent the mean ± SEM (n=5). Two-way ANOVA (c) and Student’s t test (b) were used to compare results of different experimental groups for statistically significant difference (***P &lt; 0.001). Figure S4. GDNPs could regulate the expression of immune checkpoints and transcription factors. a Representative histograms of ICOS, PD-1, TIGIT, TIM3 on CD4+ and CD8+ T cells in M2, M2+GDNPs, M2+nor-NOHA and M2+L-Arg groups. b Representative histograms of Emoes, Tox, and T-bet in CD4+ and CD8+ T cells in M2, M2+ GDNPs, M2+ nor-NOHA and M2+ L-Arg groups. The results represent three independent experiments as the mean ± SEM. Figure S5. GDNPs promoted the immune effector function of CD8 Teff. a The proportion of immune cells in MC38 colon cancer of mice was analyzed by single cell sequencing. b GO enrichment analysis was performed in CD8 Teff (G VS V). c GO enrichment analysis was performed in CD8 Tex (G VS V). d GO enrichment analysis was performed in naive T (G VS V). e Ucell score was performed for gene sets with M2 polarization characteristics in macrophages. Wilcox (e) was used to compare results of different experimental groups for statistically significant difference  (****P &lt; 0.0001). Figure S6. L-Arginine promoted T cell activation and proliferation. a L-Arginine depletion prevented IFN-γ secretion by splenocytes. Splenocytes were activated with αCD3/CD28 in the presence of media with L-Arginine levels of 0, 5, 50, 100, 1000 μM. After 48 h, IFN-γ was determined by ELISA in the culture supernatants. b, c, d T cells proliferation assay. Splenocytes were cultured in medium with and without L-Arginine. The proliferation of CD4+ and CD8+ T cells was detected by Flow cytometry. The results represent three independent experiments as the mean± SEM. Student’s t test (a, c, d) were used to compare results of different experimental groups for statistically significant difference (*P &lt; 0.05, **P &lt; 0.01). Figure S7. Rapamycin inhibited mTOR activity in GDNPs-acting T cells a, b, c Rapamycin (10 nM) was added or not added to M2+GDNPs supernatants to incubate splenocytes. Representative histograms and quantification showing the levels of p-4EBP1, p-S6 in CD4+ and CD8+ T cells. The results represent three independent experiments as the mean ± SEM. Student’s t test (b, c) were used to compare results of different experimental groups for statistically significant difference (**P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001). Figure S8. Inhibition of mTOR activity ineffectively restored the anti-tumor effect of GDNPs. a The tumor size of M2+G, M2+G+R, M2+L and M2+L+R four groups was photographed (n=4). b, c Flow cytometry was used to detect the expression of p-4EBP1 and p-S6 in T cells from four groups. d, e The transcription factor T- bet in T cells from four groups was detected by flow cytometry. One-way ANOVA (b, c, d, e) was used to compare results of different experimental groups for statistically significant difference (*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001). Figure S9. Representative gating strategies for flow cytometry. Figure S10. Unprocessed original images of gels and western blots.