Abstract 5930: Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method
Authors: McDonald James , Chan Leo Li-Ying , Reader Jocelyn , Mojica Sergio , Pierce Mackenzie 
Published: March 2024

Abstract
Ovarian cancer accounts for approximately 6% of all cancer death in women and has one of the highest mortality rates out of all gynecologic malignancies in the United States. Ovarian cancer can exhibit innate or acquired chemoresistance behavior, thus it is critical to identify novel therapeutic drug candidates. One of important characteristics of identifying potential chemotherapy drug candidate is the ability to induce apoptosis or programmed cell death in the target cancer cells. In order to measure apoptosis, various biomarkers are commonly employed such as Caspase 3, 7, 8 or 9, as well as Annexin V, which are all part of the cascade in apoptotic intrinsic pathway. However, typical fluorescent staining for image cytometry is not multiplexed that required multiple steps in order to determine the different type of apoptotic populations. In this work, we demonstrate a multiplexing apoptosis detection method to investigate the apoptotic effects of EP4A1, EP4A2, Paclitaxel, and Carboplatin on Kuramochi ovarian cancer cells. We employed the use of a high-throughput plate-based image cytometer (Celigo, Revvity Health Sciences Inc.) to image and analyze drug-treated Kuramochi cells stained with Annexin V-APC, Caspase 3/7 (488), and propidium iodide (PI) to assess the early- and late-stage apoptosis, as well as necrosis. We expected to see an increase in dual staining with the addition of EP4A to paclitaxel and carboplatin; however, the more interesting data was observed by separating single positive cell populations (Caspase, Annexin or PI only) from dual positive cell populations (Annexin + PI, Caspase + PI, Annexin +PI). The results show that treatment with EP4A lead to a time dependent increase in Caspase 3 cleavage but only in the single positive cell population. This trend was also observed in Annexin V single positive cell populations which is an indication of early apoptosis. The additive effect observed with the addition of EP4A was not seen in the dual positive cell populations. The high throughput nature of the image cytometry also allowed us to easily and quickly determine the optimal treatment conditions for this experiment. We were able to record multiple time points and not only capture proliferation, but also simultaneously collect data on caspase cleavage, Annexin-V staining and PI staining on multiple concentrations of compounds in monotherapy, dual therapy, and triple therapy combinations. Typically, it may be difficult to test multiple conditions in duplicate and triplicate using flow cytometry, while Celigo Image Cytometry may be used to obtain more robust data, and simultaneously acquire data of proliferation and multiplex staining with Annexin-V, PI and Caspase 3/7.
Citation Format: James McDonald, Leo Li-Ying Chan, Jocelyn Reader, Sergio Mojica, Mackenzie Pierce. Measuring apoptotic effects of EP4A1 and EP4A2 on Kuramochi with a high-throughput multiplex image cytometric method [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 5930.