Abstract 1066: Colocalization of ovarian cancer-associated biomarkers in tumors, cancer cells and extracellular vesicles
Authors: Couvillon Anthony D. , Winn-Deen Emily S. , Thornton Shelby , Liu Shuhong , DeRan Michael , Pacula Maciej , Banerjee Sanchari , Gusenleitner Daniel , Bortolin Laura T. , Grosha Jonian , Santos-Heiman Timothy , Sangiuliano Gabrielle , Biette Kelly M. , Sedlak Christopher R. , Hamzeh Bilal , Byrne Delaney M. , Duff Peter A. , Ho Julie , Gao Dongxia , Jamieson Amy , Cuoco Lauren T. , King MacKenzie S. , Mattoon Dawn R. , Guettouche Toumy , McAlpine Jessica N. , Huntsman David 
Published: March 2024

Abstract
Introduction: We have previously shown that ovarian cancer can be detected with high specificity and sensitivity by interrogating colocalized membrane-associated cell-surface biomarkers on single tumor-associated extracellular vesicles (EVs). Our blood based Ovarian Cancer Test (OC Test) is composed of 5 cancer-associated biomarkers (BST-2, FOLR1, MUC-1, MUC-16, and sialylated Thomsen-nouveau antigen (sTn)) known to be overexpressed in ovarian cancer relative to healthy tissues. The test employs these 5 biomarkers in 3 combinations to capture and detect EVs from human plasma to distinguish high-grade serous ovarian carcinoma (HGSC) from both benign ovarian tumors and normal samples. Using super-resolution microscopy on ovarian cancer cell lines and EVs and multiplex immunohistochemistry of ovarian benign and tumor tissue, our study aimed to show that the 5 ovarian cancer biomarkers show 1) colocalization on ovarian cancer cells, 2) colocalization on the surface of EVs, and 3) colocalization on cancer cells within ovarian tumor biopsies.
Methods: Super-resolution microscopy was employed to visualize stained biomarkers on the cell surface of a collection of ovarian cancer cell lines as well as on the surface of extracellular vesicles released by these cell lines. 58 HGSC (17 Stage I, 30 Stage II, and 10 Stage III) and 17 benign ovarian tumor K2EDTA plasma samples and paired FFPE tissue were sourced from the Ovarian Cancer Research Program (OVCARE, Vancouver BC, Canada). A tumor microarray from identical tissue samples was stained with an Opal multiplex-IHC (mIHC) assay comprised of antibodies to BST2, MUC1, sTn, and pan-cytokeratin and a DAPI counterstain. Machine learning based image analysis was utilized to determine colocalization of the biomarkers at the cellular level within benign and tumor tissue. RNA sequencing was carried out to assess expression levels of the biomarkers in each FFPE tissue sample. OC Tests were run on each plasma sample.
Results: Super-resolution microscopy showed colocalization of OC Test combination biomarkers on ovarian cancer cells and EVs derived therefrom. Machine learning-based image analysis of tumor and benign tissue microarrays stained for BST2/MUC1/sTn using mIHC, showed strong evidence for colocalization of these biomarkers on single cells within ovarian tumor tissue, particularly in early-stage tumors, but not on benign tissue. RNA sequencing analysis showed good correlation of biomarker RNA expression levels and with the OC Test results.
Conclusions: The results presented here demonstrate that the OC Test biomarkers are colocalized on tumor tissue, ovarian cancer cells, and EVs from ovarian cancer cell lines. RNA and protein expression levels show good correlation with OC Test results. These results provide further evidence that colocalized biomarkers on EVs can be utilized for early detection of cancer.
Citation Format: Anthony D. Couvillon, Emily S. Winn-Deen, Shelby Thornton, Shuhong Liu, Michael DeRan, Maciej Pacula, Sanchari Banerjee, Daniel Gusenleitner, Laura T. Bortolin, Jonian Grosha, Timothy Santos-Heiman, Gabrielle Sangiuliano, Kelly M. Biette, Christopher R. Sedlak, Bilal Hamzeh, Delaney M. Byrne, Peter A. Duff, Julie Ho, Dongxia Gao, Amy Jamieson, Lauren T. Cuoco, MacKenzie S. King, Dawn R. Mattoon, Toumy Guettouche, Jessica N. McAlpine, David Huntsman. Colocalization of ovarian cancer-associated biomarkers in tumors, cancer cells and extracellular vesicles [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 1066.