Epitope retrieval 2

Tissues were fixed in 10% normal buffer saline prior paraffin embedding. Four-micron tissue slices were deparaffinised and rehydrated. Epitope retrieval was performed at 60 °C for 20 min using Epitope Retrieval 2 (AR9640, Leica). Tissues were then stained with rabbit anti-CD46 at 0.078 μg/mL for 60 min (1:1000, ab108307, Abcam). Detection of primary antibody was performed using the Polymer Refine Detection Horseradish peroxidase for 8 min (DS9800, Leica). Between antibody or polymer incubation steps, tissues were washed twice with Wash buffer (AR9590, Leica). Tissues were counterstained with haematoxylin. Slides were processed using the Bond-Max (Leica).

Immunohistochemistry was done using 4 µ thick sections of FPPE tissue. Immunohistochemical stains for GFAP clone EP672Y (Cell Marque 258R-16, lot 32653, 1:25), MN1 (Proteintech 24697-1-AP, lot 00021048, 1:40) and IGF2 (Abcam; ab9574, lots GR31975-61, and GR31975-64, 1:500) were performed on the Leica Bond III using Leica Epitope Retrieval 1 (Leica; AR9961) for GFAP, and Epitope Retrieval 2 (Leica; AR9640) for MN1 and IGF2. Retrieval was performed for 20 min, followed by 15 min primary antibody incubation, and Bond Polymer Refine Detection (Leica; DS9800). Hematoxylin was used as a counterstain. Positive IGF2 staining was defined as dark granular perinuclear cytoplasmic staining.

To detect Gli1 and Col1a1 RNA in formalin-fixed, paraffin-embedded tissues, ISH was performed using the BOND RNAscope Detection Reagents kit (DS9790, Leica Biosystems, Buffalo Grove, IL, USA) according to the manufacturer’s instructions. Briefly, 20 ZZ probe pairs targeting the Gli1 and Col1a1 mRNA were designed and synthesized by Advanced Cell Diagnostics (catalog numbers 311008 and 537048). Tissue samples were incubated with Leica Epitope Retrieval 2 for 20 min at 95 °C, then pre-treated using Leica Protease at 40 °C for 20 min, then incubated with Gli1 or Col1a1 RNA probe at 40 °C for 240 mins. ACD AMP 1–6 was applied at 40 °C for 60–120 min for signal amplification before application of 3,3′-Diaminobenzidine (DAB).

All serologic measurements were performed at Imperial College NHS Trust Clinical Laboratories. Serum insulin, CRP, and IGF‐1 were analyzed using enzyme‐linked immunosorbent assays. Markers of apoptosis (M30), colonocyte proliferation (Ki‐67), and insulin signaling pathway activation (phospho‐mTOR) were assessed using immunohistochemical staining in the laboratory of Professor Robert Goldin, Imperial College, and Dr Naomi Guppy, University College London. All staining was performed using the Leica Bond III automated immunostaining platform on the Leica Bond Polymer Refine (Leica, DS9800) DAB polymer detection system. Ki‐67 (mouse monoclonal MIB‐1, Dako, cat. no. M7240) was applied at a dilution of 1/120 for 15 minutes at room temperature, following on‐board heat‐induced epitope retrieval (HIER) using Leica Epitope Retrieval 2 (ER2, pH9 retrieval solution; Leica, AR9640) for 20 minutes. M30 (mouse monoclonal M30 cytoDEATH, Roche, cat. no. 12 140 322 001) was applied at a dilution of 1/50 for 30 minutes at room temperature, following on‐board HIER using Leica Epitope Retrieval 1 (ER1, pH6 retrieval solution; Leica, AR9961) for 30 minutes. Phospho‐mTOR (rabbit monoclonal 49F9, Cell Signaling Technologies cat. no. 2976) was applied at a dilution of 1/50 for 20 minutes at room temperature, following on‐board HIER using Leica ER2 for 30 minutes.