Phenoimager ht

All multiplex IF-stained (mIF) TMAs were scanned using the PhenoImager HT (Akoya Biosciences) at 20x magnification. Images were obtained through tile scanning using 7-color whole-slide unmixing filters. These filters included DAPI + Opal 570/690, Opal 480/620/780, and Opal 520. To ensure accurate signal specificity of the obtained images the synthetic Opal library in the image processing software InForm version 2.4.11 (Akoya Biosciences) was used for spectral unmixing. Obtained tiles were subsequently stitched together with QuPath version 0.3.2^{85 (link)} using a QuPath script available in GitHub.

Formalin‐fixed and paraffin‐embedded normal colonic and polyp tissues from paediatric patients sectioned to 4 µm were used for histological evaluation. Haematoxylin and eosin staining was performed on each polyp sample. For IF staining, tissue slides were deparaffinized, rehydrated and processed to antigen retrieval. Primary antibodies for anti‐EpCAM (1:100, Invitrogen, Life Technologies, #14‐9326‐82), anti‐TGFBI (1:150, Invitrogen, #MA5‐32736) and anti‐LAMC2 (1:750, Invitrogen, #PA5‐109901) were used. The slides were then incubated with secondary antibodies for 10 min at room temperature and counterstained for nuclei with DAPI (2 µg/mL, Invitrogen, #D1306) for 15 min. The images were scanned with a laser scanning confocal microscope (Carl Zeiss Meditec AG). For mIHC analysis, primary antibodies against CD3 (1:5, MXB Biotechnologies, #MAB‐0740), CD19 (1:2000, Cell Signaling Technology, #90176), EpCAM (1:2000, Abcam, #ab223582), PECAM1 (1:5, MXB Biotechnologies, #MAB‐0720), CD11C (1:2000, Cell Signaling Technology, #45581) and α‐SMA (1:2000, Boster Biological Technology, #BM0002) were used. Multiplex IF staining was performed using the Opal Polaris 7 Color IHC Detection Kit (Akoya Bioscience, #NEL871001KT), and multispectral images were scanned with PhenoImager HT (Akoya Bioscience).

Slides were imaged on the PhenoImager™ HT (AKOYA Biosciences). Further analysis of the slides was performed using inForm® Software v2.6.0 (AKOYA Biosciences). The whole slides were first scanned in an unmixed view, then representative ROIs were selected for acquisition under guidance of a pathologist. These ROIs were then rescanned to achieve full spectral unmixing. A representative subset of these unmixed ROIs was then used to train tissue and cell segmentation. Next a unique algorithm was created using a machine learning technique, in which the operator selects positive and negative cell examples for each marker. These algorithms were then batch applied across a greater number of ROIs selected for inclusion in further analysis. The RStudio plugin, phenoptrReports, was used to extract phenotype counts from the resulting data tables.

Multispectral immunofluorescence staining was performed on 4um tumor FFPE sections: CD3 (cat# 85061S), CD8 (cat# ACI3160A), PD1 (cat# ab137132), PanCK (cat# sc-81714), phosphor-p38 (cat# 4511S), CD68 (cat# 76437S), CD163 (cat# CM353AK), CD11c (cat# 45581S), and DAPI. Automated staining of tissues was performed on the Leica Bond RX. For staining, Akoya Bioscience’s Opal 6-Plex Manual Detection Kit was used according to the manufacturer’s instructions (cat# NEL861001KT). Imaging was performed at 20X on the PhenoImager^™ HT (Akoya Biosciences). InForm^® (v2.4.6) and Phenochart^™ (v1.0) (Akoya Biosciences, Inc.) analysis software was used for whole slide scanning and regions of interest (ROI) spectral unmixing. Multi-channel composite TIFF files were exported for further data analysis.