Aim

The homeobox gene CDX2 is a transcription factor epigenetically regulated and involved in intestinal development and differentiation. Tumor buds (TBs) represent single cells or clusters of up to 4 cells detached from the main primary tumor and often found at the invasive front. Studies suggest that CDX2 loss is a poor prognostic factor in colorectal cancer (CRC) and is often found in TBs. It has been reported that tumor budding and epithelial-mesenchymal transition (EMT) are related. We hypothesize that the downregulation of CDX2 contributes to increased migration, invasion, and tumor budding of CRC cells. To test our hypothesis, our project aims to achieve the following objectives:

1. Identify novel epigenetic regulators of CDX2; 2. Analyze the influence of colon cancer-associated fibroblasts (CAF) on CDX2 expression in 2D and 3D cell cultures; 3. Investigate a possible function of CDX2 in EMT and tumor budding of CRC cells

This project inherently involves the integration of both molecular biology methodologies and digital pathology techniques. To assess cell migration, invasion, and tumor budding capacity of CDX2 knockout (KO) cell lines, we are employing transwell migration/invasion assays, chick chorioallantoic membrane (CAM) assays, and the U-Cup Bioreactor System. Additionally, we are using multiplex immunofluorescence (mIF) to analyze the function of CDX2 in tumor budding in CRC patient samples and patient-derived xenograft models. To facilitate the analysis, we are using QuPath, open-source software for digital pathology image analysis, and applying algorithms for tumor bud detection on tissue images, all thanks to the support of our interdisciplinary team. The use of mIF allows us to analyze a wide range of markers in our region of interest (ROIs), enabling us to obtain a more comprehensive understanding of the data.