Inform software

Oropharynx squamous cell carcinoma tissue microarray (TMA) # 3 sections were provided by the Wisconsin Head and Neck Cancer SPORE. This TMA section contains 525 cores from 107 oropharynx squamous cell carcinoma, both HPV positive and HPV negative carcinoma; each sample is represented in triplicate 0.6 mm cores. The cancer cores include 171 primary, 207 lymph node metastatic, 6 distant metastatic, and 141 recurrent cancer cores. The tissue microarray (TMA) section was deparaffinized and blocked with 5% Goat serum. Antigens were retrieved in boiling 10 mM citrate buffer for 20 min. Tissues were then washed and stained overnight at 4 ˚C with anti-K17 (Abcam 109725), anti-CD8 (BioRad MCA351GT), anti-E-Cadherin (Abcam ab231303). Tissues were washed and stained with secondary antibodies conjugated with Alexa 488, Alexa 546 and Alexa 647. Tissues were washed and stained with Hoechst Dye before mounting in ProLong™ Diamond Antifade Mountant. The stained TMA was scanned by Vectra Automated Quantitative Pathology Imaging System at 20x objective. Scanned images were analyzed using inForm software (PerkinElmer). The software was trained using nine scanned images to distinguish tumor compartment (marked by positive E-Cadherin staining) and stromal compartment (marked by negative E-Cadherin staining). Each fluorescence channel was then analyzed within each compartment.

Antibodies used for multispectral imaging analysis are detailed in Key resources table. Staining of murine and human melanoma for multispectral imaging has previously been described (Mauldin et al., 2020 ). In brief, 4-μm thick sections were cut from formalin fixed paraffin embedded murine tumor specimens or human melanoma biopsies, and murine spleen and human lymph node biopsies was used as a positive control. Antigen retrieval was performed using AR9 buffer (PerkinElmer) according to the manufacturer instructions. OPAL Multiplex IHC Staining (PerkinElmer) was performed according to the manufacturer instructions. Slides were mounted using prolong diamond antifade (Life Technologies) and scanned at low magnification using the Vectra 3.0 system and software (PerkinElmer). Regions of interest were identified in Phenochart software, and high-powered magnification images were acquired with the Vectra 3.0 system (Akoya Biosciences). These images were spectrally unmixed using single stain positive controls and analyzed using the InForm software (PerkinElmer). Human specimen images were quantified using HALO software (Indica Labs, Albuquerque, NM) and murine specimens were quantified by eye.

H&E slides were viewed by a dermatopathologist to determine representative areas for multispectral image capture at 20× magnification using Mantra™ (PerkinElmer Images were analyzed using inform™ software (PerkinElmer) (Fig. 1A-G, Supplementary Fig. S2-5). Five representative areas were chosen by the dermatopathologist: (i) three areas with tumor and up to 50% stroma and (ii) two areas with tumor only (at least 90% tumor). These images were factored equally into the analysis for each patient. Mantra™ captures spectral information from a multiplexed panel of targets using a multispectral camera. For samples of small size, a minimum of two areas meeting the above criteria were required for inclusion. For spectral unmixing, examples of each fluorophore are taken from single stained slides for each antibody, as well as a representative autofluorescence spectrum from an unstained sample. Images from each of these single-stained and unstained slides were used to create a multispectral library in inform™. Intensity of each fluorescent target was extracted from the multispectral data using linear unmixing (47 (link)).