Abstract LB249: Development of 3D organoid raft cultures of colon cancer as a model to screen the therapeutic efficacy of PRIMA-1Met (APR-246)

Abstract

Abstract Background: Cancer is among the leading causes of death worldwide. Hence, new drugs and faster screening models are needed. However, costly experiments for determining the therapeutic efficacy of drugs in animals before they reach clinical trials make the process difficult. To address this issue, we developed an alternative strategy to screen drugs against human tumors. 3D tumor organoids are termed Organoid Raft Cultures (ORCs) due to the strategy involved in culturing tumors ex vivo on collagen beds with grid supports to maintain their morphological structure and molecular makeup. Methodology: After receiving informed consent of patients and IRB approval at the University of Alabama at Birmingham, patients with histologically confirmed colon cancers were recruited. Portions of collected colon tumors were then implanted into the flanks of mice to generate Patient-Derived Xenografts (PDXs). Once their volumes reached 2000 mm2, tumors were excised, and ORCs were generated. This approach addresses two concerns: 1) how well are the morphological features retained when the tumors are grown ex vivo, and 2): can we accomplish faster screening of the efficacy of drugs. In the present study, we used a tumor in which we identified, by DNA-sequencing, a TP53 missense mutation at amino acid R248Q (a change from arginine to glutamine). We tested PRIMA-1Met (APR-246), an agent that reactivates mutant p53 by causing a conformational change and restores its function as a tumor suppressor. Excised xenograft tumor tissue was cut into small pieces and seeded on mucosa-stromal equivalent consisting of buffered collagen and fibroblasts in 24-well plates and allowed to grow for 2-3 days. Next, the tissue assembly was detached and placed on raft shaped stainless steel grids in 6-well plates containing ORC media. These cultures, termed ORCs, were then divided into groups of APR-246 treated and untreated. After 7 days, tissues were formalin fixed, and slides of 5-micron thickness were cut for Hematoxylin and eosin (H&E), p53 and TUNEL staining. Results: H&E staining of PDXs and ORCs showed the normal glandular morphology of colon cancers. This conventional characteristic of adenocarcinoma was retained along with other morphologic features, indicating the usefulness of ORCs. Additionally, comparisons of p53 and TUNEL staining of untreated vs APR-246-treated ORCs showed that lack of nuclear accumulation of mutant p53 in this p53 mutant tissue and induced apoptosis by APR-246 treatment, indicating the efficacy of the drug and the usefulness of the ORCs as alternative screening model. Conclusion: ORCs are economical and will be useful for screening new drugs/therapies ex vivo, allowing faster drug screening and thus moving new agents to clinical trials. This work was supported in part by the ELKUS foundation. (*Equal contributing first coauthors) Citation Format: Sanjib Nilam Banerjee, Prachi Bajpai, Amr Elkholy, Dianne W. Moore, Hyung Gyoon Kim, Sumit Agarwal, Michael Behring, Sameer Al Diffalha, Upender Manne. Development of 3D organoid raft cultures of colon cancer as a model to screen the therapeutic efficacy of PRIMA-1Met (APR-246) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB249.