Abstract B059: Developing novel surface plasmon resonance fluorescence imaging (SPFI) for near single molecule quantitation of immune cell surface-bound MUC16- a novel diagnostic assay for ovarian cancer

Abstract The ovarian tumor marker, MUC16 (CA125) is a ligand of the immunosuppressive receptor Siglec-9. Previously, we have demonstrated that MUC16-Siglec-9 binding suppresses the cytolytic responses of NK cells, contributing to the immunosuppressive ovarian tumor microenvironment. Binding to Siglec-9 captures MUC16 on the surface of circulating immune cells. This observation led us to propose that MUC16 binding to circulating immune cells could serve as a diagnostic marker for early detection and diagnostic monitoring of high grade serous ovarian cancer (HGSOC). The binding of MUC16 to circulating immune cells is a low incidence event and as a result conventional tools (e.g., fluorescence microscopy and flow cytometry) lack the sensitivity required to quantify immune cell MUC16 binding, a requirement for the development of a diagnostic assay. To overcome this technological challenge, we established an academia-industry collaboration to exploit the near single molecule detection sensitivity of plasmonic gold nanoparticles (PNP) for detection of the low levels of MUC16 on immune cells. PNP functionalized with anti-CA125 antibody bind to immune cells that have captured MUC16 on their surface. Darkfield imaging allows for sensitive detection of the mucin at near single molecule level. Imaging software is used for automated detection and quantification of the MUC16-bound PNP on the immune cells. Additionally, the imaging system developed by our group also incorporates four channels for fluorescence. The fluorescence detection is used to identify the phenotype of the immune cells that capture MUC16. Our technology thus provides an assessment of not only the amount of MUC16 on the circulating immune cells but also allows for characterization of the immune subsets (NK, T, B, monocytes, etc.). The two tiers of information gained through these assessments can be incorporated into a novel diagnostic algorithm for HGSOC. Siglec-9 expression on immune cell subsets varies based on pathological states and hence the immune cells on which MUC16 is captured can serve as a discriminator between HGSOC and other diseases. Our analysis of HGSOC patients has shown increased binding of MUC16 to NK and T cells compared to postmenopausal healthy donors. Furthermore, we demonstrate that increase in immune cell-bound MUC16 precedes elevation of serum CA125 by 2-3 months in patients with HGSOC. Longitudinal analysis of blood samples from six HGSOC patients conducted over a period of 1-3 years consistently shows presence of MUC16 on the surface of immune cells. In these samples, MUC16 binding to T cells correlates to the progression of the cancer. Overall, our results demonstrate that MUC16 bound to immune cells is an entirely new biomarker and is not dependent on the level of CA125 in the blood. Data from the analysis of the clinical samples will be presented along with description of the surface plasmon resonance-fluorescence imaging system and the algorithms used for automated counting of MUC16 molecules captured on immune cells. Citation Format: Gloria Marquina. Ospina, Antonio Casado, Peter Hansen, Petra Krauledat, Roberta Lloyd. Fritz-Klaus, Tom Mullins, Megan Harrison, Rock Pulak, Manish S. Patankar. Developing novel surface plasmon resonance fluorescence imaging (SPFI) for near single molecule quantitation of immune cell surface-bound MUC16- a novel diagnostic assay for ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B059.