Fig 2: iNOS expression immunohistochemistry in TNBCRepresentative images of TNBC tumor with (A) negative, (B) low, (C) moderate, and (D) strong iNOS tumor epithelial staining.

Fig 3: Fc?RIIB deficiency suppresses the immunosuppressive activity and differentiation of MDSCs. (A) iNOS and Arg1 expression in WT and KO MDSCs from MC38 tumors were assessed using FCM; n = 5. (B, C) PD-L1 (B) and DCF-DA (C) expression in WT and KO MDSCs; n = 5. (D-F) Isolated MDSCs from MC38 tumors of WT and KO mice were cocultured with CFSE-labeled CD8+ T lymphocytes isolated from WT mice (1:2) for 3 days, the production of IFN-? (D), and GzmB (E) in CD8+ T cells, anti-CD3 and anti-CD28 induced proliferation (F) were measured by FCM; n = 4. Data are expressed as means ± SD. *P < 0.05, **P < 0.01, ns, no significant difference, by Mann-Whitney test.

Fig 4: iNOS expression is associated with decreased recurrence free survival, distant metastasis free survival and breast cancer survival in TNBC(A) Kaplan-Meier cumulative recurrence free survival curves for TNBC patients by iNOS status (n = 199). Patients with weak iNOS expression (n = 79) or moderate iNOS expression (n = 59) were significantly associated with decreased recurrence free survival compared to patients with negative iNOS expression (n = 27) (P=0.036 & P=0.007, log-rank test). Patients with strong iNOS expression (n = 34) showed a trend toward worse survival compared to patients with negative iNOS expression (n = 27), but did not reach significance (P=0.078, log-rank test). (B) Kaplan-Meier cumulative distant metastasis free survival curves for TNBC patients by iNOS status (n = 199). Patients with weak iNOS expression (n = 79) or moderate levels of iNOS expression (n = 59) were significantly associated with increased risk of distant metastasis compared to patients with negative iNOS expression (n = 27) (P=0.017 & P=0.002, log-rank test). Patients with strong iNOS expression (n = 34) showed a trend toward increased risk of distant metastasis compared to patients with negative iNOS expression (n = 27), but did not reach significance (P=0.052, log-rank test). (C) Kaplan-Meier cumulative breast cancer–specific survival curves for TNBC patients by iNOS status (n = 200). Patients with weak iNOS expression (n = 78) or moderate levels of iNOS expression (n = 59) were significantly associated with increased risk of death due to breast cancer compared to patients with negative iNOS expression (n = 28) (P=0.012 & P=0.007, log-rank test). Patients with strong iNOS expression (n = 35) showed a trend toward increased risk of distant metastasis compared to patients with negative iNOS expression (n = 28), but did not reach significance (P=0.093, log-rank test).

Fig 5: PLM-miRs reprogram inflamed BMDM to M2 phenotype in vitro. A) CLSM images indicating phenotypes of inflamed BMDMs after treated by PBS, LM-miRs or PLM-miRs. Scalar bar, 20 µm. B) MFI quantification of iNOS (M1) and CD206 (M2) in (A) (n = 3). C) Flow cytometry assay and D) statistical analyze of the polarization of inflamed BMDMs after treated by PBS, LM-miRs, or PLM-miRs (n = 3). M1 and M2 subtypes were indicated by CD86 and CD206, respectively. E) Real-time RT-qPCR analysis of the expression levels of M1 markers (IL-1ß, IL-6, iNOS) and M2 markers (IL-10, TGF-ß, Arg-1) in inflamed BMDMs after treated by PBS, LM-miRs, or PLM-miRs (n = 3). Results are presented as mean ± SD. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.