Abstract A069: High-resolution volumetric imaging of primary and metastatic high-grade serous ovarian cancer explants

Abstract

Abstract Introduction: As high-grade serous ovarian cancer (HGSOC) rarely shows symptoms, most patients present with metastases. Tumor heterogeneity between metastases may impede a patient’s response to therapy and thus impact survival. We aim to decipher the heterobiology of HGSOC lesions using high-resolution imaging of patient-derived explants (PDEs), to determine the differential treatment response between primary and metastatic tumors. Methods: PAX8, CK7, RAD51, γH2AX, Ki67, cPARP, and CD3 were chosen to enumerate tumor and T cells, and their activity. Antibodies were optimized in monolayer and spheroid cultures of HGSOC cell lines, COV318 and OVCAR4. CD3 was optimized in FFPE human tonsillitis tissue. Primary and metastatic HGSOC explants were cultured for 48h followed by 24h fixation. Several immunostaining and optical clearing protocols were tested on PDEs, which were imaged using a fully automated confocal high-content screening system. Results: Immunostaining and automated image analysis facilitated the identification of individual nuclei within PDEs without Refractive Index (RI) matching or optical clearing. However, high background impeded further image analysis, preventing the quantification of certain antibodies. Glycerol-based RI matching and ScaleS tissue clearing were implemented to try and overcome this. However, neither generated sufficiently high-quality images for analysis. A novel Clearing-enhanced 3D (Ce3D) immunostaining system recently developed for high-quality multiplex fluorescence microscopy of large tissue volumes was tested. While individual nuclei were recognized, as well as RAD51 foci and CK7 cytoskeleton staining, image analysis was still impeded due to high background. Conclusions: Protocols without RI matching and optical clearing can stain large, complex structures such as PDEs. However, high background impedes image analysis suggesting the need for optical clearing and RI matching. The inclusion of various RI matching and/or optical clearing steps failed to overcome high background staining. Further work is ongoing to refine immunostaining, RI matching, optical clearing, and mounting of PDEs to allow the quantification of all antibodies. Citation Format: Adele E. Connor, Joanne Lysaght, Donal J. Brennan, Jeremy C. Simpson, Antoinette S. Perry. High-resolution volumetric imaging of primary and metastatic high-grade serous ovarian cancer explants [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A069.