Bond rx

IHC for MHC class I was performed for all lesions (initial discovery and validation cohorts) and for the 28 markers in the multiplexed IF panel (Supplementary Table S2) for lesion 3_2 using an automated staining system (Leica Bond RX) with 3,3’ Diaminobenzidine (DAB) detection (Leica Bond Polymer Refine Detection catalog# DS9800). Tissue sections were baked for 3 hours at 62°C in vertical slide orientation with subsequent deparaffinization performed on the Leica Bond RX. Antigen retrieval was conducted for 30 minutes using Leica Bond epitope retrieval solution 2 (ER2) (EDTA, pH 9.0) (catalog# AR9640) followed by incubation of the primary antibody at previously optimized concentrations for 30 minutes (a list of the primary antibodies used can be found in Supplementary Table S2) followed by incubation of the secondary antibody (Leica Bond Polymer Refine Detection catalog# DS9800). MHC class I staining was scored for the percentage of membrane-positive tumor cells within the entire tissue section in 5% increments (0% to 100%). Slides were scored blindly by Travis J. Hollmann and Maryam Pourmaleki. IHC staining for the 28 markers for lesion 3_2 was visually inspected alongside the multiplexed IF (Cell Dive) staining for the 28 markers for lesion 3_2 by Travis J. Hollmann and Maryam Pourmaleki to ensure accuracy of the multiplexed IF method.

FFPE tissue samples from biopsies of patients enrolled in the MEM-288 clinical trial were immunostained using the PerkinElmer OPAL ^™ 7-Color Automation IHC kit (Cat No. NEL821001KT) on the BOND RX autostainer (Leica Biosystems, Vista, CA). The OPAL 7-color kit uses tyramide signal amplification (TSA)-conjugated to individual fluorophores to detect various targets within the multiplex assay. Sections were baked at 65⁰C for one hour then transferred to the BOND RX (Leica Biosystems). All subsequent steps (e.g., deparaffinization, antigen retrieval) were performed using an automated OPAL IHC procedure (PerkinElmer). OPAL staining of each antigen occurred as follows: slides were blocked with PerkinElmer blocking buffer for 10 min then incubated with primary antibody at optimized concentrations followed by OPAL HRP polymer and one of the OPAL fluorophores. Primary antibodies used for mIF are listed in supplementary Table S1. Individual antibody complexes are stripped after each round of antigen detection. After the final stripping step, DAPI counterstain is applied to the multiplexed slide and is removed from BOND RX for coverslipping. Autofluorescence slides (negative control) were included, which use primary and secondary antibodies omitting the OPAL fluors and DAPI. All slides were imaged with the Vectra^®3 Automated Quantitative Pathology Imaging System.

FFPE TMAs were immunostained using the PerkinElmer OPAL TM 7-Color Automation IHC kit (Waltham, MA) on the BOND RX autostainer (Leica Biosystems, Vista, CA) with the following anti-human antibodies: CD3 (ThermoFisher, SP7, 1:400, Thermo Fisher Scientific Cat# MA5-14524, RRID:AB_10982026), CD8 (DAKO, C8/144B, 1:100, Agilent Cat# M7103, RRID:AB_2075537), TCF1/7 (CST, C63D9, 1:100, Cell Signaling Technology Cat# 2203, RRID:AB_2199302), CD103 (Abcam, SP301, 1:100, Cat# ab227697), CD69 (Abcam, EPR21814, 1:300, Cat# ab233396), CLEC9A (Abcam, EPR22324, 1:100, Cat#ab223188, RRID:AB_2884022), PCK (DAKO, M3515, 1:200, Agilent Cat# M3515, RRID:AB_2132885). DAPI was used to stain nuclei. Tissues were heated at 65°C for 2h then transferred to the BOND RX (Leica Biosystems) followed by automated deparaffinization and antigen retrieval using OPAL IHC procedure (PerkinElmer). As a negative control autofluorescence slides were included. Slides were scanned and imaged with the PerkinElmer Vectra®3 Automated Quantitative Pathology Imaging System. For quantitative image analysis multi-layer TIFF images were exported from InForm (PerkinElmer) and loaded into HALO (Indica Labs, New Mexico). Each fluorescent fluorophore was assigned to a dye color and positivity thresholds were determined per marker based on published nuclear or cytoplasmic staining patterns.

Immunostaining was performed on a Bond Rx (Leica) or Ventana discovery ultra (Roche) autostainer as per manufacturer’s instructions as previously described (Campton et al., 2015 (link); Pitarresi et al., 2016 (link)). Primary antibodies and dilutions used in this study were as follows: Ki-67 (Abcam; ab16667, 1:200), pH3-S10 (Millipore; 06–570, 1:250), FoxM1 (Santa Cruz; sc-502, 1:800), E2f3a (Millipore; 05–551, 1:100) and Myc-tag (Cell Signaling Technology; 2278, 1:100). EdU staining was performed following the manufacturer’s protocol (Life Technologies; C10337).

Human gingival tissue samples were collected as surgical waste from clinics using NYU School of Medicine IRB approved protocol (Pushalkar et al., 2014 (link)). Samples were preserved in 10% neutral buffered formalin, followed by dehydration and then embedded in paraffin. The paraffin block was sectioned at 5 μm thickness. Sections were deparaffinized and stained on a Leica BondRX autostainer, according to the manufacturers’ instructions. In brief, sections underwent epitope retrieval for 20 min at 100°C with Leica Biosystems ER1 solution (pH 6.0, AR9961) followed by a 30 min incubation at 22°C with anti-GPR91 (Abcam, Cambridge, United Kingdom) diluted 1:200 in Leica diluent (Leica, Cat ARD1001EA) and subsequent detection using the BOND Polymer Refine Detection System (Leica, DS9800). Sections were counter-stained with hematoxylin scanned on a Hamamatsu Nanozoomer HT whole slide scanner and imported into the NYU Omero image database for viewing and annotation.