Epigenetic and tumor microenvironmental factors controlling CDX2 in colon cancer during EMT and tumor budding
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:
Investigate the function of CDX2 in CRC cell migration, invasion, and tumor budding using CRISPR/Cas9 knockout and CRISPR/dCas9 synergistic activation mediator (SAM) approaches
Analyze the association between CDX2 loss, E-cadherin downregulation, and EMT in tumor buds using multiplex immunofluorescence (mIF) in CRC patient samples and patient-derived xenograft (PDX) models
Model tumor budding and invasive behavior of CDX2 knockout CRC cells using ex ovo chorioallantoic membrane (CAM) assays and digital pathology tools including QuPath
This project integrates molecular biology methodologies with digital pathology techniques, supported by an interdisciplinary team, with the ultimate goal of evaluating CDX2 as a potential therapeutic target to counteract CRC cell dissemination.
Methods
The study used CRISPR/Cas9 to knock out CDX2 in two colorectal cancer (CRC) cell lines (LS174T and CaCo2), and a CRISPR activation (SAM) system to reactivate CDX2 in CDX2-negative cell lines. Migration and invasion were assessed via wound healing and transwell assays. Multiplex immunofluorescence (mIF) was applied to a primary CRC patient sample and patient-derived xenograft (PDX) liver metastasis model to quantify CDX2 and E-cadherin expression in tumor buds versus adjacent tumor regions. An ex ovo chorioallantoic membrane (CAM) assay provided a 3D model of tumor invasiveness.
Results
CDX2 knockout significantly increased CRC cell migration and invasion, while re-expressing CDX2 reversed these effects. Reactivating endogenous CDX2 in CDX2-negative cell lines similarly reduced motility in a dose-dependent manner. In both the patient sample and PDX model, tumor buds showed markedly lower CDX2 and E-cadherin levels compared to the surrounding primary tumor, consistent with epithelial-mesenchymal transition (EMT). CDX2 knockout cells also displayed greater tissue infiltration and more frequent tumor budding in the CAM assay.
Taken together, the findings support CDX2 as a suppressor of CRC cell dissemination and tumor budding, with potential as a therapeutic target.
Members
Rina Mehmeti
Inti Zlobec
Mario Tschan
Publications
Bodmer, N., Uth, K., Mehmeti, R. et al CDX2 loss in colorectal cancer cells is associated with invasive properties and tumor budding. Sci Rep 15, 24113 (2025). https://doi.org/10.1038/s41598-025-07278-x
Collaboration