Abstract PO5-18-07: A Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits TNBC Lung Metastases

Abstract

Abstract Introduction: A promising novel target for breast cancer is secreted frizzled-related protein 2 (SFRP2). SFRP2 protects tumors against apoptosis, promotes T-cell exhaustion, and induces angiogenesis. SFRP2 is expressed in breast cancer but has not been specifically evaluated in triple negative breast cancer. We previously developed a humanized monoclonal antibody to SFRP2, (hSFRP2 mAb) that inhibits primary breast cancer growth in mice without toxicity. The purpose of this study is to establish the presence of SFRP2 in human TNBC, evaluate the efficacy of hSFRP2 mAb in metastatic TNBC in vivo, and in a cell line resistant to chemotherapy. Methods: Immunohistochemistry (IHC) with SFRP2 antibody on a human TNBC tissue microarray (TMA) containing 88 FFPE cores. After IHC with primary antibody to SFRP2, the TMA was scanned and staining intensity was quantified using spatial analysis. Results for SFRP2 staining were analyzed as previously described for analyses of estrogen and progesterone receptor positivity; categories are: 0-absence of staining; low positive (0-10%), and positive >10%. Development of a doxorubicin (dox)-resistant cell line and induction of apoptosis with hSFRP2 mAb. MDA-MB-231 cells were treated with 4nM dox and increased 2nM at a time until the cells were resistant to 10µM dox after 12 months. WT and dox-resistant cells were treated for 4 hours with either hSFRP2 mAb (10uM,) or IgG control (10uM) (n=6). Apoptosis was detected with Promokine Apoptosis/Necrosis/Healthy cells kit. Cells were imaged using the EVOS FLc and counted using Imagej software. Differences between treated and control were compared with two-tailed T-test. hSFRP2 mAb treatment of mice with metastatic TNBC. PY8119 and E0771 TNBC murine cells (5 × 105/100 μL) were injected iv into 6–8-week-old C57BL/6 female mice. The next day the mice were treated with IgG1 control 8 mg/kg iv or hSFRP2 mAb 8 mg/kg iv every 3 days. After four weeks, the lungs were resected, and surface metastases were counted. Differences between treated and control were compared with Poisson distributions. FFPE sections of lungs were stained with ApopTag kit. Three metastases per lung were counted at 20 X, and the difference between IgG1 and hSFRP2 mAb groups compared with two-tailed T-test. Results: SFRP2 protein is present in human TNBC. Out of 88 cores, 83 cores (94%) were positive, 4 cores (4.5%) were minimally positive, and 1 core (1.1%) was negative for SFRP2. Development of a doxorubicin (dox)-resistant cell line and induction of apoptosis with hSFRP2 mAb treatment in vitro. The number of apoptotic cells in IgG1 control-treated MDA-MB-231 cells was 11.5 ± 3.7, and 76.6 ± 5.0 for hSFRP2 mAb-treated tumors (p< 0.001, n=9). The mean number of apoptotic MDA-MB-231 dox-resistant cells in IgG1 control was 16.7 ± 6.0, and 68.4 ± 6.4 for hSFRP2 mAb-treated tumors, (p< 0.001, n=9). hSFRP2 mAb inhibits the growth of metastatic TNBC in vivo and increases apoptosis. For E0771, the number of surface metastases was 8.9 ±2.8 in the IgG1 control group and 4.9 ± 1.1 in the hSFRP2 mAb treated group (p< 0.05, n=15). The number of apoptotic cells/HPF was 9.8 ± 1.8 for the IgG1 control group and 21.0 ± 4.6 for the hSFRP2 mAb group (n=15, p< .01). For Py8119 tumors there were 6.4 ± 1.0 metastases in the IgG1 control group and 3.6 ± 0.9 in the hSFRP2 mAb group (p< 0.05, n=11). The number of apoptotic cells/HPF was 6 ± 1.8 for the IgG1 control group and 14 ±1.3 for the hSFRP2 mAb group (n=10, p< 0.001). Conclusions: SFRP2 protein is abundantly expressed in human TNBC. A humanized monoclonal antibody to SFRP2 reduces TNBC lung metastases growth in two in vivo models and increases tumor apoptosis. TNBC resistant to doxorubicin remains sensitive to apoptosis induced by the hSFRP2 mAb. Citation Format: Lillian Hsu, Patrick Nasarre, Julie Siegel, Eleanor Hilliard, Rupak Mukherjee, Nancy Klauber-Demore. A Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein 2 Inhibits TNBC Lung Metastases [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-18-07.