Abstract 325: Innovative approach in evaluating gap junction functionality using co-cultured spheroids

Abstract In recent years, significant advancements were made in colorectal cancer research, particularly in understanding the role of gap junctions in cancer development and progression. However, challenges persisted in effectively evaluating gap junction functionality within 3D structures without disrupting spheroids or relying on dye-addition methods to the media, which often had limitations. To address these issues, we developed an innovative method for microinjecting dye into a single cell within a multicellular spheroid, enabling real-time analysis of gap junction functionality in living cells. Building on this approach, we aimed to create a more physiologically relevant tumor microenvironment by co-culturing spheroids with fibroblast cells. This setup better replicated the complexity of in-vivo conditions. To further enhance our analysis, we fixed these spheroids at various time points and used immunohistochemistry (IHC) to evaluate dye penetration into the spheroid core. The experimental method involved co-culturing fibroblasts (CCD-112CoN) with colorectal cancer epithelial cells (Caco2, SW620, SW480, or HT-29) in a CelVivo Clinostar reactor to produce spheroids larger than 300 µm in diameter. These spheroids were then microinjected using an Eppendorf FemtoJet 4i Microinjector with a dye mixture containing Texas Red (high molecular weight, 40, 000 Da) and Lucifer Yellow (low molecular weight) to assess gap junction activity using a Leica confocal microscope. Additionally, we examined gap junction proteins using an immunofluorescence assay between co-cultured and single-cell cultured spheroids. The results indicated that microinjection was a reliable method for evaluating gap junction functionality within spheroids. Notably, different stages of colorectal cancer modulated dye absorption and transfer, with SW620 spheroids exhibiting lower transfer through cells, while HT-29 spheroids showed increased dye transfer between cells. Furthermore, co-cultured spheroids demonstrated enhanced dye transfer compared to single-cell-type spheroids of the same type. There was an increase in connexin 43 expression based on immunofluorescence in Caco2 with CCD-112CoN spheroids compared to Caco2 and CCD-112CoN single-cell cultures. In conclusion, to our knowledge, this study introduced a novel approach to measuring gap junction activity for spheroids. Furthermore, it highlighted the significant role fibroblasts may play in influencing gap junction dynamics in colorectal cancer cells within complex 3D structures. Citation Format: Tomas Lugo, Annelise Nguyen, Stephanie Myers. Innovative approach in evaluating gap junction functionality using co-cultured spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 325.