Diverticulitis samples served as inflammatory, non-cancer controls.
De-identified clinical data were provided by the CDP. Additional polyps with normal controls were stained on proprietary TMAs (US Biomax). IF scoring IF staining was performed on TMAs to detect human TLR4 (Novus Biologicals). Pan-cytokeratin was used as a counterstain to highlight intestinal epithelium (Abcam), and DAPI to counterstain nuclei. TLR4 detection was enhanced using conjugated Tyramide #see more randurls[1|1|,|CHEM1|]# with the fluorochrome Alexa Fluor 488 (Invitrogen). Pan-cytokeratin was detected using an anti-rabbit secondary antibody conjugated with Alexa Fluor 647 (Invitrogen). Stained slides were scanned Temsirolimus (Olympus VS120) and viewed using OlyVIA 2.4. A Leica TCS-SP5 Confocal was used for triple IF images. Staining patterns, intensity quantification, and extent TLR4 by surface area were determined by two senior GI pathologists (PAB and MTG) masked to diagnoses. A training subset was independently interpreted and inter-observer variation was determined. Moderate agreement was
noted for the stromal score (weighted κ = 0.58 [95%CI 0.28-0.89]); moderate-to-strong agreement was observed for epithelium (weighted κ = 0.68 [95%CI 0.39-0.97]). Disagreement between scoring was settled by consensus. TLR4 signal intensity was scored in the stroma and epithelium. The signal intensity was scored as 0, no TLR4 staining; 1+, low intensity; 2+, moderate intensity; or 3+, high intensity. The extent of surface P-type ATPase area with TLR4 was scored on a scale of 0–3 (0: no staining; 1+: present, but <20%; 2+: 20–50%; and 3+: >50%). A TLR4 positivity score was calculated by multiplying staining intensity and surface area data by tissue compartment (range: 0–9) [7, 12, 13]. To qualify TMA observations, IHC was performed on normal colon, adenomas, and CRCs for TLR4 (Novus Biologicals), smooth muscle actin (α-SMA, Abcam), vimentin (Cell Signaling), and CD68 (Dako) on curls from tissue blocks. Secondary antibody conjugated with
horseradish peroxidase was used prior to incubation with the substrate 3,3′-diaminobenzidine. Samples were counterstained with hematoxylin and scored (pathologist MTP). Approval by the university’s Institutional Review Board was obtained. Data analysis Gene expression data Analysis included quality control assessments of processed data. Differential expression discovery was performed using linear models and empirical Bayes methods (t-tests and ANOVA) via R statistical language [14]. Survival analyses were conducted using Cox proportional hazards, with results corrected for multiple comparisons using false discovery rate procedures [15]. Results were assessed for biological relevance.