Establishing a standardized murine orthotopic intra-rectal model for the study of colorectal adenocarcinoma.

Background: Orthotopic models offer a more accurate representation of colorectal cancer (CRC) compared to subcutaneous models. Despite promising results from the reported intra-rectal models, establishing a standardized method for CRC research remains challenging due to model variability, hindering comprehensive studies on CRC pathogenesis and treatment modalities, such as brachytherapy. This study aimed to establish a standardized workflow for an orthotopic intra-rectal animal model to induce the growth of colorectal adenocarcinoma in male and female mice. Methods: HT-29 colorectal adenocarcinoma cells were injected into the rectal mucosa of female (n=21) and male (n=26) non-obese diabetic severe combined immunodeficiency (NOD SCID) gamma (NSG) mice. Mice were placed on a 45° wedge elevating their pelvis for better visualization of the anus. Tumor growth and localization were monitored using a 7-T magnetic resonance imaging (MRI) scanner with rapid acquisition with relaxation echo (RARE) sequence at weeks 1, 2, and 3 post-cell instillation. Once tumors reached 5-8 mm in diameter, the mice were euthanized. Histopathology and immunohistochemical analyses confirmed the tumors' morphology, including necrosis, vascularity (CD-31) and apoptosis (cleaved caspase-3). Results: There was a 92% and 95% tumor growth success rate in male and female mice, respectively. Tumors grew to 5-8 mm in diameter within ~20 days. No significant difference in tumor size was observed between genders. Tumor morphology was consistent across cases. Most tumors exhibited a lack of central blood vessels, accompanied by varying degrees of necrosis and apoptosis, whereas external portions were highly vascularized. Conclusions: An orthotopic intra-rectal model was successfully developed. This model will be used in future studies to evaluate the efficacy of CRC treatments.