Vectra Polaris 20X images of human rectal cancer (CRC1) from: A SIMPLI (Single-cell Identification from MultiPLexed Images) approach for spatially resolved tissue phenotyping at single-cell resolution.

Two 4 µm thick serial sections were cut from CRC1 FFPE block using a microtome. The first slide was dewaxed and rehydrated before carrying out HIER with Antigen Retrieval Reagent-Basic (R&D Systems). The tissue was then blocked and incubated with the anti-CD3 antibody (Dako, Supplementary Table 2) followed by horseradish peroxidase (HRP) conjugated anti-rabbit antibody (Dako) and stained with 3,3' diaminobenzidine (DAB) substrate (Abcam) and haematoxylin. Areas with CD3⁺ infiltration in the proximity of the tumour invasive margin were identified by a clinical pathologist (M. R-J.) The second slide was stained with a panel of six antibodies (CD8, PD1, Ki67, PDL1, CD68, GzB, Supplementary Table 2), Opal fluorophores and 4’,6-diamidino-2-phenylindole (DAPI) on a Ventana Discovery Ultra automated staining platform (Roche). Expected expression and cellular localisation of each marker as well as fluorophore brightness were used to minimise fluorescence spillage upon antibody-Opal pairing. Following a one-hour incubation at a 60°C, the slide was subjected to an automated staining protocol on an autostainer. The protocol involved deparaffinisation (EZ-Prep solution, Roche), HIER (DISC. CC1 solution, Roche) and seven sequential rounds of: one hour incubation with the primary antibody, 12 minutes incubation with the HRP-conjugated secondary antibody (DISC. Omnimap anti-Ms HRP RUO or DISC. Omnimap anti-Rb HRP RUO, Roche) and 16 minute incubation with the Opal reactive fluorophore (Akoya Biosciences). For the last round of staining, the slide was incubated with Opal TSA-DIG reagent (Akoya Biosciences) for 12 minutes followed by Opal 780 reactive fluorophore for our hour (Akoya Biosciences). A denaturation step (100°C for 8 minutes) was introduced between each staining round in order to remove the primary and secondary antibodies from the previous cycle without disrupting the fluorescent signal. The slide was counterstained with DAPI (Akoya Biosciences) and coverslipped using ProLong Gold antifade mounting media (Thermo Fisher Scientific). The Vectra Polaris automated quantitative pathology imaging system (Akoya Biosciences) was used to scan the labelled slide. Six fields of view, within the area selected by the pathologist, were scanned at 20x and 40x magnification using appropriate exposure times and loaded into inForm{Kramer, 2018 #23} for spectral unmixing and autofluorescence isolation using the spectral libraries. After spectral unmixing and merging of six 20x fields of view for a total of >5mm² ROI (Table 2), one single-tiff image was extracted for each marker and its intensity was rescaled from 0 to 1 with custom R scripts.