Secreted frizzled-related protein 2 monoclonal antibody-mediated IFN-ϒ reprograms tumor-associated macrophages to suppress triple negative breast cancer.

PURPOSE: We hypothesize that SFRP2 is a promising target for Triple Negative Breast Cancer (TNBC). EXPERIMENTAL DESIGN: 1. Multiplex immunohistochemistry (IHC) was performed on human TNBC to identify SFRP2 localization in the tumor microenvironment. 2. Tumor associated macrophages (TAMs) were isolated from E0771.LMB breast tumors. TAMs were treated with hSFRP2 mAb (10 µM) or control (10 µM) for 1 h and analyzed by western blot and qRT-PCR for IFN-ϒ. 3 SFRP2 and IFN-ϒ mRNA expression levels were analyzed from the Cancer Genome Atlas (tCGA) for breast cancer patients using least squares-linear regression analysis. 4. PY8119 or E0771.LMB TNBC cells were injected i.v. into mice, and mice were treated with either IgG1 or hSFRP2 mAb every 3 days. Lung metastases were counted after 4 weeks and analyzed by IHC for M1/M2 ratio. 5. MDA-MB-231 TNBC cells were injected into the mammary fat pad, and when tumors were established, mice were treated with IGg1 or hSFRP2 mAb every 3 days i.v. for 79 days and tumor volumes were compared. 6. Wild-type (WT) MDA-MB-231 and doxorubicin-resistant MDA-MB-231 cells were treated with hSFRP2 mAb and apoptosis was compared. RESULTS: 1) Multiplex IHC on human breast tumors showed that SFRP2 localized to tumor cells (87%), TAMs (90%), and tumor-infiltrating lymphocytes (TILs) (96%) in the microenvironment. 2) TAMs treated with hSFRP2 mAb had an increase in IFN-ϒ mRNA by 2.35 ± 0.08-fold (n = 3, p = 0.02) and protein levels by1.9-fold compared to control. 3). Analysis of 1075 breast cancer patients from TCGA database revealed a significant negative association between SFRP2 mRNA and IFN-ϒ expression (p < 0.0001). 4) hSFRP2 mAb reduced lung metastases in EO771.LMB (n = 15, p < 0.05) and PY8119 (n = 11, p < 0.05) mice with an increase the M1/M2 ratio in lungs (n = 3, p = 0.02). 5) hSFRP2 mAb inhibited MDA-MB-231 growth in vivo by 61% percent (n = 9, p < 0.001). 6) hSFRP2 mAb promoted apoptosis in doxorubicin-resistant cells (n = 6, p < 0.0001). CONCLUSIONS: SFRP2 localizes to tumor, TAMs and TILs in the tumor microenvironment and is negatively associated INF-ƴ in human tumors. hSFRP2 mAb reduces primary and metastatic TNBC growth, increases INF-ƴ from TAMS, boosts the M1/M2 ratio in lung metastases, and induces apoptosis in doxorubicin-resistant cells.