Fig 2: Immune responses assessment in vivo(A–C) Groups of 4T1-bearing mice were given tail vein injections three times with various formulations and sacrificed at day 15 for analysis. Six groups from left to right are PBS, PEG-PLGA, R837, FA-NPs, NPs+ NIR, and FA-NPs +NIR. (A) Representative flow cytometry plots illustrated CD4, CD8+T cells in peripheral blood; (B) Qualification of CD4, CD8+T cells according to (A), Data are represented as mean ± SEM; (C) Representative flow cytometry plots of CD80+ or CD86+ among CD11c+ DCs extracted from the popliteal LNs (tumor sentinel lymph nodes).(D) Qualification of CD80+ or CD86+ expressions gated by CD11c + cells according to (C), data are represented as mean ± SEM.(E) TUNEL staining of tumor slices in each group.(F) Qualification according to (E).(G) Representative immunofluorescence staining for CD8+ (Green) of tumor sections obtained after aforementioned treatments, while the nucleus was stained with DAPI (blue), data are represented as mean ± SEM.(H) Cytokine levels in sera from mice isolated at 24 h post different treatments, data are represented as mean ± SEM.(I) Tumor growth curves of different groups of mice (5 mice per group) with s.c. inoculation of secondary 4T1. Data are represented as mean ± SEM.(J) Morbidity-free survival of different groups of mice with secondary 4T1 tumors after various treatments indicated to eliminate their primary tumors (10 mice per group). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bars, 50 μm.

Fig 3: M1 macrophages secrete CCL7 in an autocrine manner, creating a positive feedback loop that enhances M1 cell polarization(A and B) Flow cytometry was used to detect the expression levels and mean fluorescence intensity (MFI) of CD86 (M1) in macrophages induced by LPS, with quantitative statistics performed (n = 3).(C) RT-qPCR showed the mRNA expression of M1 polarization markers (iNOS, TNF-α, IL-1β, and IL-6) in macrophages after induction with LPS (n = 3).(D) The expression fold changes of chemokines in control versus LPS-stimulated macrophages are presented in heatmaps (n = 3).(E) Enrichment bubble map of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of differentially expressed genes.(F) The expression fold changes of CCL7, CXCL2, and CXCL3 are presented in volcano plots.(G) RT-qPCR was employed to measure the mRNA expression of CCL7, CXCL2, and CXCL3 in macrophages induced with varying LPS concentrations (n = 3).(H) The expression of the CCL7 protein after M1 polarization macrophages, and a relative quantitative analysis was performed (n = 3).(I and K) RT-qPCR determined the effects of rmCCL7, rmCXCL2, and rmCXCL3 on the polarization markers of M1 (iNOS, TNF-α, and IL-6) and M2 (CD206) macrophages (n = 3).All the above LPS intervention time was 24 h. ns denotes not significant. Data are expressed as the mean ± SD. Statistical analyses were conducted using unpaired t tests (B and H) and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (C, G, I, J, and K). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 4: CCL7 promotes M1 macrophage polarization and proliferation(A and B) CCK-8 detected macrophage activity under different concentrations and times of rmCCL7 stimulation (n = 5).(C) The RNA-seq results of rmCCL7 affecting the polarization of macrophages were presented in heatmaps; Nos2 is iNOS and Mrc1 is CD206 (n = 3).(D) Using RT-qPCR, the influence of different concentrations of rmCCL7 on M1 and M2 macrophage polarization was investigated along with a relative quantitative analysis (n = 3).(E and F) Changes in the expression of iNOS and CD206 in macrophages treated with rmCCL7, LPS, and IL-4 (M2) were studied, and a relative quantitative analysis was conducted (n = 3).(G and H) Flow cytometry was utilized to examine the expression of CD86 in macrophages under the effect of rmCCL7, and the quantity analysis of its MFI was documented (n = 3).(I) Morphological differences in macrophages after 24 h of treatment with rmCCL7, LPS, and IL-4. Scale bars, 20 and 10 μm.ns indicates not significant. Data are presented as the mean ± SD. Statistical analyses were performed using unpaired t tests (H) and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests (A, B, D, and F). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 5: r-DKK1 blocked TNF-α-induced MHC II+ and CD86+ expression of dendritic cells(A) Monocyte-derived dendritic cells were incubated in rDKK1(100 ng/mL), rTNF-α (10 ng/mL), and (rDKK1 + rTNF-α). Cells harvested at 48 h post-incubation were used to determine the percentage of MHC II+ and CD86+ cells by flow cytometry. Representative flow cytometry dot plots generated 48 h post-incubation showed the analysis of MHC II+ and CD86+ as indicated (A).(B and C) The percentage (B and C) of MHC II+ and CD86+ in the different experimental conditions are shown in the bar graphs. Results are presented as mean (±SEM) and are representative of triplicate experiments. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data (∗p < 0.05, ∗∗p < 0.01; and “ns” indicates non-significant.