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Colorectal cancer (CRC) remains one of the deadliest cancers worldwide due to poor outcomes upon metastatic spread. Given the drastic impact on mortality, a better understanding of the CRC metastatic process could yield significant improvements to overall survival. Here, we investigate the influences of biomechanical forces on early metastatic spread (i.e., tumor cell intravasation events) in an organ-on-chip (OOC) model. Our microfluidic CRC OOC model consists of two channels separated by a porous membrane: a top channel representing normal and cancerous colon epithelial tissue, and a bottom channel consisting of endothelial cells in a tube-like structure representing the vasculature. Vacuum channels flank the sides of the model to apply forces in a cyclic manner to mimic peristalsis-like motions and provide further in vivo resemblance. In our prior work, we demonstrated that peristaltic forces promoted tumor cell invasion within the CRC OOC. Specifically using on-chip confocal imaging, we revealed that cyclic strain leads to an increase in the number of tumor cells capable of intravasating from the epithelial channel into the vascular channel. Additionally, we showed dynamic sensitivity of tumor cell invasion in response to intermittent stretching conditions, leading us to hypothesize mechanical stress-induced phenotypic switching. Therefore, using single-cell RNA sequencing, we investigated gene expression changes of tumor cells in the presence or absence of peristaltic forces and found alterations in stemness, adhesion and metabolism-related genes. Using median fluorescence intensity (MFI) from flow cytometry data, we have revealed differences in stem-like properties (e.g., CD44, CD133, LGR5) of tumor cells with and without the influence of peristaltic forces both prior to and after intravasation. Additionally, effluent-based analysis of the OOC has shown cytokine and metabolism changes due to peristaltic forces. Ongoing work involves using flow cytometry to further quantify changes in adhesion and metabolism as well further validation in patient derived organoids-on-a-chip. Taken together, OOCs provide a physiologically relevant model to study early CRC intravasation events, revealing phenotypic changes in response to mechanical stimuli that influence tumor cell stemness and invasiveness. Citation Format: Curran A. Shah, Carly Strelez, Yonatan Amzaleg, Einar B. Gunnarsson, Jasmine Foo, Min Yu, Shannon M. Mumenthaler. Peristalsis based modulation of colorectal cancer cells leads to changes in stemness and invasiveness in an organ on chip model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1323.