Additional file 1 of A nano-innate immune system activator for cancer therapy in a 4T1 tumor-bearing mouse model

Additional file 1: Figure S1. TEM image of nude MSN without PEGylation and Fcfragments. Figure S2. Representative size distribution profiles of MSN-SH,MSN-COOH, MSN-Fc, and NISA. Figure S3. Surfaceelement components of the nanoparticles were detected using X-ray photoelectronspectroscopy (XPS) assay. The changes of surface N1s confirmed the sequentialmodification and loading of the functional molecules and IgG3 Fc. FigureS4. Fc conjugation efficiency along with increased feeding amount. Data areexpressed as means ± SD (n = 3). Figure S5. Examination of the left IgG3 Fc in the supernatantof MSN-Fc using SDS-PAGE. Figure S6. IgG3 Fc retention on NISA in PBScontaining 10% FBS at 37 ℃. Data are expressed as means ± SD (n = 3). FigureS7. Colloid stability of NISA in PBS at 4 ℃ for 7 days. Data are expressedas means ± SD (n = 3). Figure S8. Fcγ receptors on the cell membrane ofmacrophages (RAW264.7) and dendritic cells (DC2.4). Fcγ receptors wasidentified using anti-CD64/FcγR rabbit polyclonal antibody (1:500, SinoBiological, Beijing, China) and Alexa Fluor 488-labeled goat anti-rabbit IgG secondaryantibody (1:1000, Abcam, Shanghai, China) for RAW264.7 or Alexa Fluor 647-labeledgoat anti-rabbit IgG secondary antibody (1:1000, ThermoFisher, Shanghai, China)for DC2.4. Figure S9. Examination of ERK activation in the cells treatedwith free Fc or MSN-Fc. (A) Western blot assay of p-ERK expression inRAW264.7 and DC2.4 cells. Statistical assay (B, C) of p-ERK expressionin panel A. Data are expressed as means ± SD (n = 3). Figure S10.MSN-Fc treatment induced the formation of more pseudopodiums in RAW264.7 andDC2.4 cells. The cell pseudopodiums were observed through the immunofluorescentstaining of F-actin using acti-stain 670 phalloidin. Notedthat TNF-α (green color) in the cells was stained with rabbitanti-TNF-α antibody (Abcam, Hong Kong),and Alexa Fluor 546 donkey anti-rabbit IgG (Thermo Fisher, Waltham)and observed at Ex 556 nm and Em 573 nm. FigureS11. Blood clearance curve of free Fc (iFluor 647 labeled). Thestandard curve between iFluor 647 fluorescence intensity and corresponding Fcconcentration was used to determine the Fc content in blood. Data are expressedas means ± SD (n = 5). Figure S12. Quantification of nanoparticle content in tumors.The dose of MSN-Fc (A) and NISA (B) in the tumors was determinedusing the established standard curve between the iFluor 647 fluorescenceintensity and the corresponding nanoparticle (MSN-Fc or NISA) concentrations.#1, #2, and #3 were three separate tumor samples. 6 h after injection, 1.2% ofthe injected MSN-Fc was obtained at the tumor site. In contrast, dramaticallyincreased proportion of the injected NISA (2.2%) was found in the tumor. FigureS13. Individual tumor growth curves of the mice in each group. Figure S14.The antitumor effect of Fc, Fc + fMLP and MSN-Fc in orthotopic 4T1-bearingmice. (A) Tumor growth curve (n = 5, mean ± SEM).S. a, NISA versus MSN-Fc. b, NISA versus Saline, Fc, and Fc + fMLP. c, NISA + fMLPversus all other groups. (B) Mouse body weight (n = 5, mean ± SD). Thegroups of saline, NISA, and NISA + fMLP indicated with dotted blue lines werealso displayed in Fig. 5 in the main text. Figure S15. On day 9 (24 hafter the final injection), 3 mice from each group were sacrificed, and themajor organs (heart, liver, spleen, lung, and kidney) of the mice were removedand processed for paraffin sections and histopathological examination (H&Estaining). Figure S16. Macrophagepolarization analysis. (A) Macrophages are defined as F4/80+cell. M1-like cells are F4/80+CD11c+CD206−,whereas M2-like cells are F4/80+CD11c−CD206+cells. (B) Statistical assay of the M1- and M2-like cell proportion intotal macrophages. Data are expressed as mean ± SD. n = 3. ***p < 0.001. Figure S17. Analysisof mature DCs in the tumors after various treatments. (A) Mature DCswere identified as LY75+CD11c+MHCII+.(B) Statistical assay of the mature DC proportions. Data areexpressed as mean ± SD (n = 4) ***p < 0.001. FigureS18. Examination of NK cells in tumors after theindicated treatments. (A) Representative flow cytometry profiles of NK cells in tumorsafter the indicated treatment. NK cells were marked with goat anti-mouseNKp46/NCR1 antibody (FITC) (R&D, Minneapolis, MN). (B) Statisticalassay of the NK cell percentage. Data are expressed as mean ± SD. n = 3. *p < 0.05, **p < 0.01. Table S1. Survival analysis of themice with various treatments.