Fcs express 6 image cytometry

Tissue biopsies were obtained at baseline and at the end of the third cycle of therapy (after 2 doses of CY/nivolumab/GVAX and 1 dose of nivolumab/CRS-207 in Arm A and 2 doses of CY/GVAX and 1 dose of CRS-207 in Arm B). A blinded study pathologist selected biopsies containing >30% tumor cellularity for multiplex immunohistochemistry (IHC). Chromogenic sequential IHC was conducted on 5-micron formalin-fixed paraffin-embedded tissue sections as previously described (13 (link)). Three 12-plex multiplex IHC panels containing markers of lymphoid, myeloid, and T cell function were stained in the indicated order and condition shown in Supplementary Table 1. Whole slide scanning was conducted by Aperio ImageScope (Leica), image processing by CellProfiler Version 2.1.1, and visualization by ImageJ Version 1.48. Image cytometry data analysis was performed by FCS Express 6 Image Cytometry Version 6.03.0011 (De Novo Software, CA, USA) based on gating strategies previously reported (13 (link)). 16 immune cell lineages including lymphoid and myeloid cell populations were quantitatively evaluated by image cytometry according to lineage selective markers shown in Supplementary Table 2.

Formalin fixed paraffin embedded baked TMA sections were transferred to a BOND RX (Leica Biosystems) and staining performed using an automated OPAL-IHC system (PerkinElmer). Briefly, slides were treated with the PerkinElmer blocking buffer for 10 min and incubated with the specific primary antibodies, followed by OPAL-HRP polymer and one OPAL fluorophore. Individual antibody complexes were stripped after each round of detection and DAPI applied as the last staining. Auto-fluorescence slides (negative control) included primary and secondary antibodies, omitting the OPAL fluorophores. Slides were imaged with a Vectra®3 Automated Quantitative Pathology Imaging System. Cells resembling MDSC were identified by imaging as M-MDSC (Pan-cytokeratin^neg HLA-DR^neg/low CD14⁺ CD15^neg) or PMN-MDSC (Pan-cytokeratin^neg HLA-DR^neg/low CD15⁺ CD14^neg). Multi-layer TIFF images were exported from InForm (PerkinElmer) into HALO (Indica Labs) for quantitative analysis. Each fluorophore was assigned to a dye color and positivity thresholds determined visually per marker based on intensity thresholds normalized for exposure (counts/2 bit depth x exposure time x gain x binning area). Cell segmentation results from each core were analyzed using FCS Express 6 Image Cytometry (De Novo software).