# Targeting Colorectal Cancer Stem Cells Through Inhibition of the Fibroblast Growth Factor Receptor 4 Pathway with a Novel Antibody

## TL;DR

A new antibody targeting FGFR4 shows promise in fighting colorectal cancer by attacking resilient cancer stem cells.

## Contribution

A novel antibody targeting FGFR4 was developed to inhibit colorectal cancer stem cell growth.

## Key findings

- FGFR4 is a key regulator of colorectal cancer stem cell proliferation and tumorigenicity.
- The 3B6 antibody effectively inhibits FGFR4 signaling and reduces tumor growth in xenograft models.
- Modulation of epithelial–mesenchymal transition pathways was confirmed via Western blot analysis.

## Abstract

Metastatic colorectal cancer (CRC) is a severe disease, often resistant to current treatments due to a subpopulation of resilient tumor cells known as cancer stem cells (CSCs). The aim of this study was to find a new way to target these difficult-to-treat cells. Through genetic analysis, we discovered fibroblast growth factor receptor 4 (FGFR4) controlling the survival and growth of these cells. We then created a specific antibody to bind to this protein and inhibit tumor growth. The results demonstrate that this antibody could represent a novel and promising therapeutic approach for patients with metastatic CRC who currently lack effective treatment options.

Background/Objectives: The progression and dissemination of CRC are heavily influenced by a subpopulation of tumor cells known as CSCs. This study aimed to identify novel protein membrane antigens expressed by colorectal CSCs and the consequent development of targeted therapies based on monoclonal antibodies directed against the identified antigens. Methods: Integrated bioinformatics analyses were conducted using proprietary CSC gene expression profiles and public colon gene expression databases, leading to the identification of five plasma membrane proteins enriched in CSCs. Genetic immunization in rats was employed to generate monoclonal antibodies (mAbs) targeting these antigens. FGFR4 was prioritized due to its overexpression in colorectal tumors. Its function was characterized in vitro and in vivo through assays evaluating proliferation, colony formation, migration, and tumorigenicity. The anti-FGFR4 antibody 3B6 was selected based on its affinity and ability to inhibit FGFR4 signaling in CSCs. Its therapeutic potential was further assessed in xenograft models, and alterations in downstream signaling were analyzed via Western blot. Results: FGFR4 emerged as a key regulator of CRC CSC proliferation, migration, and tumorigenic capacity. The 3B6 antibody, a high-affinity FGFR4 binder, demonstrated robust in vitro inhibition of CSC features and significant antitumor effects in patient-derived xenograft models. Western blot analysis confirmed the modulation of FGFR4-driven signaling pathways, particularly those involved in epithelial–mesenchymal transition (EMT). Conclusions: This study successfully identified several CSC-selective membrane antigens that can become therapeutic targets in CRC. Among them, we focused on FGFR4 as a promising target and developed the anti-FGFR4 3B6 monoclonal antibody which offers potential for both diagnostic and therapeutic applications.