Fig 2: Arginine starvation induced immune cell infiltration in xenograft tumors. (A) Immunodeficient mice carrying CWR22Rv1 xenografts were fed either a control or arginine-free diet, and tumor growth was measured regularly. (n = 6, ***p < 0.001) (B) Tumor weight of xenografts. (n = 6, **p < 0.01) (C) Endpoint body weights of mice from both diets. (n = 3) (D-I) IHC staining for cell proliferation and immune cell infiltration markers. (D) Ki-67, (E) CD45, (F) F4/80, (G) CD11c, (H) NKp46, and (I) IFNα2.

Fig 3: Distance from tumor cells to the nearest dendritic cells and macrophages. (A) Representative multiplex multi-immunofluorescence image (case N14, before treatment) showing staining for CD11 (yellow), CD68 (green), PD-L1 (red), and CK (cyan). (B) Cellular phenotype map of image shown in A depicting the locations of CK+ tumor cells (cyan dots), PD-L1+ (red dots), CD11c+ dendritic cells (orange dots), and CD68+ macrophages (green dots). (C) Ray plot depicting the distance from each CK+ tumor cell to the nearest PD-L- dendritic cells. (D) Ray plot depicting the distance from each CK+ tumor cell to the nearest PD-L+ dendritic cells. (E) Ray plot depicting the distance from each CK+ tumor cell to the nearest PD-L- macrophages. (F) Ray plot depicting the distance from each CK+ tumor cell to the nearest PD-L+ macrophages. (G, H) Distances from tumor cells to the nearest PD-L1- and PD-L1+ dendritic cells (G) and macrophages (H) for all patients with available tumors at baseline and during treatment. (I, J) Kaplan–Meier curves showing overall survival (I) and progression-free survival (J) based on distance from tumor cells to the nearest PD-L1- macrophages during treatment. The tumors are ordered by the percentage of PD-L1+ dendritic cells or macrophages, from highest to lowest. Cutoff: (I) 83.454 µm; (J) 83.454 µm. On-treatment, after 40 Gy radiation. p = 0.05, statistically significant.

Fig 4: Effects of M1 and APS1 on expression of (A) macrophage markers; (B) inflammatory markers; (C) M1‐ and M2-activated macrophages and (D) immunohistochemistry (IHC) analysis of F4/80 and CD11c in adipose tissues of high-fat diet (HFD)-induced obese mice. Relative quantification of gene expressions was performed using the comparative Ct method with normalization to GAPDH. Results were expressed as fold difference relative to a relevant control sample. Data are expressed as the mean ± SEM of 8–10 mice per group. Labeled means without a common superscript letter differ, p < 0.05.

Fig 5: Sample stratification based on immune marker assessment. (A) Correlation plot (Spearman’s correlation) of immune cell marker expression in the analysis sample set. The highest correlation was found for CD8a, CD11c, CD68 and CD16 (r > 0.55). n = 84. (B) Heatmap showing protein expression levels of CD8a, CD11c, CD68 and CD16. Hierarchical clustering of analytes and samples with Euclidean distance and complete linkage. Data are normalized to total protein, centered on median of all samples and log2-transformed. (C) Representative Western blot mimics of CD8a, CD11c, CD68 and CD16 (grayscale maps generated from DigiWest data). For graphical representation, background-subtracted raw data from representative hot and cold samples were used. n = 3. The uncropped blots are shown in Supplementary File S1. (D) Representative images of CD11c, CD8, CD68 and CD16 immunohistochemical staining in hot and cold samples. Scale bar, 50 µm. (E) Mean positive pixel values of 5 representative 10× sections per available FFPE sample for hot and cold carcinomas classified by DigiWest. CD8a: cold n = 8, hot n = 17; CD68: cold n = 7, hot n = 13; CD16: cold = 9, hot n = 14; CD11c: cold n = 10, hot n = 17. Mann–Whitney U test, ** p < 0.01; * p < 0.05. (F) Distributions of responder, ER, PR and Her2 statuses as percentages, stratified by infiltration status. Chi-square-test, * p < 0.05; ns indicates no significant difference. (G) Kaplan–Meier analyses of event-free survival in patients stratified by infiltration status. p = 0.0298, log-rank test.