Abstract 2912: SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment
Authors: Hsu Lillian , Nasarre Patrick , Mukherjee Rupak , Hilliard Eleanor , Romeo Martin , O'Quinn Elizabeth , Christians Whitney , DeMore Nancy Klauber 
Published: March 2024

Abstract
Introduction: A promising target for breast cancer is secreted frizzled-related protein 2 (SFRP2), and a humanized monoclonal antibody to SFRP2 (hSFRP2 mAb) inhibits primary triple negative breast cancer (TNBC) in vivo. CD38 is a known cause of PD-1 inhibitor resistance in some tumors, and in metastatic osteosarcoma hSFRP2 mAb overcomes PD-1 mAb resistance by reducing CD38 in tumor infiltrating lymphocytes (TILs). Could this phenomenon be replicated in TNBC? This is unknown as the expression of CD38 in TNBC has not been studied. This study will establish the presence of SFRP2 and CD38 in the TNBC microenvironment.
Methods: Immunohistochemistry (IHC) was performed on an 88 core human TNBC tissue microarray (TMA) with antibodies to SFRP2 and CD38. Spatial analysis software was used to determine percent positivity. Each core was categorized using the scoring system for the estrogen receptor in breast cancer (&lt;1% negative, 1-10% low positive, and &gt;10% positive). Multiplex IHC was performed on 4 human triple TNBCs sections. Tissues were stained with antibodies to CD38, CD68 (macrophage), CD3 (T-cell), CD19 (B-cell), CytoKeratin (tumor), and SFRP2. Percent positivity of each marker was analyzed with a software package. For flow cytometry of tumor associated macrophages (TAMs), 1 million EO771 cells were injected into the mammary fat pad of female C57/BL6 mice. After 4 weeks tumors were extracted and a cell suspension was incubated in a mastermix with antibodies to SFRP2, CD38, PD1, CD11B and F480. Cells were fixed and conjugated to a fluorochrome secondary antibody. TAM gated cells were analyzed for the presence of SFRP2 and CD38.
Results: For the TNBC TMA, 83 cores were positive and 4 cores were low positive for SFRP2, 1 not evaluable; 87 cores were positive and 1 low positive for CD38. For multiplex IHC there was a total of 1,753,544 individual cells in all 4 human TNBC tumors that were stained. 94% of cells stained positive for SFRP2, and 65% stained positive for CD38. Of the cells that were positive for SFRP2, 70% were also positive for CD38, 15% were positive for CD68, 1% were positive for CD19, 77% were positive for CytoKeratin, and 10% were positive for CD3. Of the cells that were CD38 positive, 99% were also positive for SFRP2, 15% were positive for CD68, 1% were positive for CD19, 81% were positive for CytoKeratin, and 9% were positive for CD3. Flow cytometry analysis of TAMs confirmed the presence of SFRP2, CD38 on TAMs. TAMs that were negative for PD1 and CD38 (pro-inflammatory) had a 56% reduction in SFRP2 compared to TAMs that were double positive for PD1 and CD38 (anti-inflammatory).
Conclusion: SFRP2 and CD38 are prevalent in human TNBC and colocalize in the TNBC microenvironment in tumor cells, TAMs and TILs, but not B-cells. The presence and co-localization of SFRP2 and CD38 in TAMs was confirmed with flow cytometry. This confirms that SFRP2 is a therapeutic target in TNBC and provides a mechanistic rational for combination therapy with PD-1 inhibitor.
Citation Format: Lillian Hsu, Patrick Nasarre, Rupak Mukherjee, Eleanor Hilliard, Martin Romeo, Elizabeth O'Quinn, Whitney Christians, Nancy Klauber DeMore. SFRP2 and CD38 colocalize in triple negative breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2912.