Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an <i>In-Situ</i> Hybridisation Assay for Therapeutic Studies

<div><p>Ulcerative colitis is a chronic inflammatory disease affecting the colon and is characterized by epithelial damage and barrier dysfunction. Upregulation of the tight junction protein claudin-2 by cytokines is hypothesized to contribute to the dysregulation of the epithelial barrier. New therapeutic agents which block the action of cytokines are being investigated in patients with ulcerative colitis. In order to understand the potential of these therapies, it is important to have reliable assays that can assess downstream endpoints that reflect drug mechanism of action. The aim of the current study was therefore to establish & validate an assay to reproducibly assess the expression and distribution of claudin-2 in human colon biopsy samples. Initially, the potential to measure claudin-2 protein by immunohistochemistry (IHC) was investigated. To identify suitable reagents to develop an IHC assay, pre-established criteria were used to screen five commercial antibodies by Western blotting, immunofluorescence and immunohistochemistry on claudin-2 positive and negative cells and healthy and ulcerative colitis colon tissue. Despite some of these antibodies specifically detecting claudin-2 using some of these techniques, none of the antibodies showed the expected specific staining pattern in formalin fixed human colon samples. As an alternative method to detect claudin-2 expression and distribution in formalin fixed biopsy sections, an <i>in situ</i> hybridization assay was developed. This assay underwent a novel tiered approach of validation to establish that it was fit-for-purpose, and suitable for clinical deployment. In addition, to understand the possible relationship of claudin-2 in the context of disease severity, expression was compared to the Geboes score. Overall, the microscopical Geboes score correlated with the claudin-2 biomarker score for samples that retained crypt morphology; samples with the highest Geboes score were not specifically distinguished, probably due to crypt destruction. In summary, we have applied a strategy for identifying target-specific antibodies in formalin fixed biopsy samples and highlighted that (published) antibodies may not correctly identify the intended antigen in tissues fixed using this method. Furthermore, we have developed and, for the first time, validated an <i>in situ</i> hybridization assay for detection of claudin-2 mRNA, suitable for use as a supportative method in clinical trials. Using our validated assay, we have demonstrated that increased claudin-2 expression correlates with the severity of ulcerative colitis, where crypt destruction is not seen.</p></div

Effect of prolonged time intervals (> 1 day), following biopsy section pre-treatment, on ISH signal outcome.

<p>Samples were analysed in the same analytical run. (A) shows the variation in claudin-2 (Cldn-2) ISH signal by (eye) & by (auto) computer algorithm on sequential days (from 1 to 4) after slide pre-treatment step. Minimal variability is observed over the time period.</p

Assay variability for the Claudin-2 ISH method using FFPE cell controls.

<p>Three analysts each performed 2 independent analytical runs using positive (HT29) (A and B) and negative (CHO K1) (C and D) Formalin Fixed Paraffin Embedded (FFPE) cell lines. Cells were stained with claudin-2 (Cldn-2) probe (VA1-12003-01) (A and C) or Sense probe (VA1-14363-01) (B and D) from Panomics. Variability within & between analytical batches (performed by a single analyst) was minimal. Moreover, no analyst bias was observed across the 3 analysts’ performance. Data presented are ISH scores assigned by a single trained analyst.</p

Comparative analysis of claudin-2 protein and mRNA localisation in adjacent sections from affected UC colon.

<p>(A) H&E staining, (B) IHC claudin-2 staining and (C) ISH claudin-2 staining. The localisation of claudin-2 protein (IHC staining) correlates to observed mRNA (ISH staining) pattern. The scale bars represent 50 μm.</p

Outcome of the five commercial antibodies in the screening process.

<p>Outcome of the five commercial antibodies in the screening process.</p

Heat-map (with sample frequency) showing the association between the Claudin-2 ISH score and a microscopical histopathological activity index.

<p>ISH scores and the activity index evaluations by the scoring pathologist were measured in the same biopsy sections from in 47 (affected and unaffected) UC biopsy sections used in the validation of the assay.</p

Investigating the potential variability of biomarker signal for adjacent and non-adjacent sequential biopsy sections.

<p>One micro-tomed 4 μM tissue section was generated (per datapoint) and ISH stained for claudin-2 mRNA distribution. Variability in the claudin-2 signal was investigated in (A) adjacent sections (3–6 novel sections per tissue) generated from a pancolitic (completely) active subject, and active & non-active UC material from two further subjects and in (B) the mean of two (adjacent) sections taken from four non-adjacent (≥ 24 μm distance apart).sites within a tissue biopsy, generated from 3 subjects who had non, low & moderate activity. In total, nine biopsies were analysed, and signal variability appears to be minimal within a specific biopsy.</p

Western blot analysis to demonstrate the specificity of the 32–5600 (12H2) antibody to recognise Claudin-2 protein transcript.

<p>40 <b>μ</b>g of each cell lysate and 40 ng recombinant human GST tagged claudin-2 was loaded. Lane order is as follows: (A & I) magic marker, (B) HT29 cell lysate, (C) T84 cell lysate, (D & F) empty lane, (E) recombinant GST-Claudin-2 protein, (G) CHO K1 (overexpressing GFP-CLDN2 construct) cell lysate and (H) CHO K1 (untransfected control) cell lysate. 12H2 clearly detects recombinant GST-CLDN2 protein (lane E, 52kDa). CHO-K1 cells do not express CLDN2, as seen in lane H, however upon transient transfection of CHO-K1 cells with a GFP-CLDN2 construct (53kDa), the antibody detects the overexpressed construct. 12H2 is able to detect endogenous levels of Claudin-2 protein (MW ~20kDa) in the HT29 & T84 cell lysates (B & C respectively), as confirmed by mass spectrometry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162076#pone.0162076.s002" target="_blank">S2 Fig</a>).</p

Representative grades of ISH claudin-2 staining in UC colon biopsies as disease severity increases.

<p>The level of disease severity was determined, by a trained pathologist, as (A) 0, none (B) 1, low, (C) 2, slight, (D) 3, moderate, (E) 4, marked and (F) 5, severe. The scale bars represent 50 μm.</p

Lead candidate antibody staining patterns for claudin-2 in cell models.

<p>Fig 2 shows claudin-2 staining, using (A) the lead candidate antibody (12H2) or (B) isotype control in diseased UC. Panels (C-E) show the claudin-2 staining patterns in ICC FFPE cell pellets using 12H2. Specifically, (C) CLDN2 overexpressing CHO K1 cells, (D) untransfected CHO K1 cells and (E) T84 cells. The scale bars represent 50 <b>μm</b>.</p