Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an In-Situ Hybridisation Assay for...

Area of Science:

• Gastroenterology
• Molecular Biology
• Biomarker Development

Background:

• Ulcerative colitis (UC) involves colon epithelial damage and barrier dysfunction.
• Cytokine-induced claudin-2 upregulation is a suspected contributor to UC's epithelial barrier issues.
• Targeting cytokines is a focus for new UC therapies, necessitating reliable assays.

Purpose of the Study:

• To establish and validate an assay for assessing claudin-2 expression and distribution in human colon biopsies.
• To evaluate the utility of claudin-2 as a biomarker in UC.
• To support the development and clinical deployment of novel UC therapeutics.

Main Methods:

• Screened five commercial antibodies for claudin-2 detection using Western blotting, immunofluorescence, and immunohistochemistry (IHC).
• Developed and validated an in situ hybridization (ISH) assay for claudin-2 mRNA in formalin-fixed colon biopsies.
• Correlated claudin-2 expression with the Geboes score to assess disease severity.

Main Results:

• Commercial antibodies failed to provide specific claudin-2 staining in formalin-fixed human colon samples.
• A novel, tiered validation approach established a fit-for-purpose ISH assay for claudin-2 mRNA.
• Increased claudin-2 expression correlated with UC severity, particularly in samples without crypt destruction.

Conclusions:

• Standard antibody-based IHC methods are unreliable for claudin-2 in formalin-fixed tissues.
• The validated ISH assay is suitable for clinical trials and detecting claudin-2 mRNA in UC.
• Claudin-2 serves as a relevant biomarker for UC severity, supporting the investigation of targeted therapies.