Abstract 5196: Large scale in vitro expansion of primary tumors from metastasis cancer patients with different mutations for preclinical drug screening

The use of patient derived primary tumors (PDPT) is becoming a powerful vitro tool in candidate cancer drug screening for safety and efficacy. Compared to 2D cell lines, the PDPTs can 1) better recapitulate human tumor microenvironment, 2) predict drug responses in humans, 3) be used in high throughput drug screening platforms to test dose responses and drug-drug interactions, and 4) ultimately, be used in precision medicine. However, adoption of PDPTs has been hindered by challenges related to in vitro tumor cell growth and expansion without inducing phenotypic changes. Here, we present a novel system of in vitro PDPT expansion method and biobanking of tumoroids that can be used in high throughput drug screening for metastasis disease. We have combined a specialized matrix coated plates with that of microwell technology to scale-up in vitro tumor cell growth and expansion. In this study we introduced two assay systems for screening of drugs/compounds: a) expanded colorectal cancer (CRC) tumoroids as a stand-alone test system and b) reconstructed complex 3D tumor containing tissues with matrix, fibroblasts, endothelial cells, and immune cells that mimics the tumor microenvironment (TME). To examine the utility of both testing platforms, single tumoroids or tumoroids embedded in 3D tissues were exposed to different doses of 5 chemotherapeutic drugs Cisplatin, Doxorubicin, Oxaliplatin, Fluorouracil, and Cetuximab. Treatment was initiated on day 10 of the culture period. Thereafter, tumoroids were dosed with drugs every other day for 11 days. After each treatment cycle, tumoroids or tumoroid containing tissues were fixed for H&E and live/dead cell staining. The results showed that: 1) large scale expansion of PDPT is achievable, 2) PDPT can be cultured with multiple cell types to simulate the TME, 3) histological evaluation of the tumor models showed a glandular-like tumoroid structure, 4) immunohistochemical staining revealed epithelial origin of the CRC (CK20+), and 5) live-dead cell staining of tumoroids showed differential drug responses in a dose and time dependent manner. Additionally, a by-standard cell death effect was noted for some of the drugs. In summary, large-scale in vitro tumoroid expansion is critical to scale up metastasis cancer drug screening. The developed assay systems are more physiological, cost effective, less time-consuming, better predictive of drug responses in humans, can lead to precision medicine, and reduce animal use for experiments. Citation Format: Dylan Bryda, Megan Groves, Anthony Tolcher, Alex Armento, Seyoum Ayehunie. Large scale in vitro expansion of primary tumors from metastasis cancer patients with different mutations for preclinical drug screening [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 5196.