Abstract 6136: Generation and characterization of colorectal cancer cell lines from the Buffalo rat
Authors: Muñoz Nina M. , Dupuis Crystal J. , Dixon Katherine A. , Williams Malea L. , Maldonado Kiersten L. , Rashid Asif , Tam Alda L. 
Published: September 2020

Introduction: Rat models of metastatic colorectal cancer (mCRC) to the liver are ideal for the development of transarterial therapies. To date, only one model of CRC cells grown in vitro and implanted into the liver of syngeneic rats is available. Yet, the small size of the WAG-Rij rat limits the use of the CC-531/WAG-Rij model for transarterial interventions. This work was aimed at generating an analogous model on the Buffalo rat, strain that has successfully been used for transarterial interventions in primary liver cancer.

Materials and Methods: To induce CRC, 5 6-week old Buffalo rats were treated with Azoxymethane (AOM) and Dextran Sodium Sulfate (DSS). To assess tumor progression, the rats were regularly examined by colonoscopy as well as to T1- and T2-MR imaging from w20 up to w63. Standard procedures of tumor cell isolation and in vitro expansion were used. Once the polyclonal lines were established, expression of E-cadherin, Ceacam5, Mucin1, Vimentin1, and S100a4 was evaluated by flow cytometry (FACS) or qPCR. Additionally, the gene regions of APC (nt. 3049 to 3697) and β-catenin (nt. 371 to 1104) were analyzed by Sanger sequencing. Lastly, 23 adult Buffalo rats received implantations of 0.4-1 × 107 cells in the liver only or in the liver as well as subcutaneously.

Results: Colonoscopies, MRIs, and histopathology demonstrated that all 5 rats treated with AOM/DSS developed colorectal neoplasms by w22. Tumors were harvested at various time points, at weeks 24, 32, 45, and 63. 2 polyclonal cell lines derived from invasive adenocarcinomas of a female (18L002) and a male (18L004) were successfully established and have proliferated in vitro &gt;1year. FACS and qPCR results showed that both lines express the epithelial markers E-cadherin, Ceacam5, and Mucin1. Interestingly, despite high E-cadherin expression in both lines (&gt;90% by FACS), qPCR data revealed that both lines also express the mesenchymal markers Vimentin and S100a4, suggestive of a trans-differentiated phenotype. DNA sequences of regions of the APC and β-catenin genes frequently mutated by AOM treatment appeared to be intact in both lines. MRIs of the 23 rats that received implants showed that 4 seem to have allowed for short-term growth (~14d) of the 18L004 cells in the liver before being re-absorbed. Histopathological analyses showed evidence of a serosal reaction at the site of implantation in those cases.

Conclusions: Two polyclonal CRC lines with phenotypical characteristics of trans-differentiated cells were successfully generated from AOM/DSS-treated Buffalo rats. In spite of continued in vitro growth for over a year, neither line was tumorigenic in vivo, potentially due to the lack of mutations in key regions of the APC and β-catenin genes. Forced expression of mutant genes known to drive CRC progression such as β-catenin, Kras, or P53 could potentially increase in vivo tumorigenicity and allow for the establishment of a rat model of mCRC suitable for the study of transarterial therapies.

Citation Format: Nina M. Muñoz, Crystal J. Dupuis, Katherine A. Dixon, Malea L. Williams, Kiersten L. Maldonado, Asif Rashid, Alda L. Tam. Generation and characterization of colorectal cancer cell lines from the Buffalo rat [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6136.