Paired tumor biopsies were obtained at baseline and week 7 (after at least two doses of GVAX + IPI) in 6 patients on Arm A. A pathologist selected samples containing >30% tumor cellularity for 10-plex multiplex IHC. Multiplex IHC was conducted on 4 micron thick formalin fixed paraffin embedded (FFPE) tissue sections mounted on slides as previously described(18 (link)). Iterative staining was achieved by washing and antibody stripping in heated citrate (HK080–9K BioGenex, Fremont, CA). Samples were restained sequentially with antibodies indicated (Supplementary Table S2 , Supplementary Figure S2.A ).
Whole-slide scans were obtained after each stain on Hamamatsu Scanner at 20x magnification. Digital image analysis consisted of three steps: 1) image coregistration using Cell Profiler 2 (Version 2.2.0, Broad Institute) with “coregistration 12 markers” pipeline, 2) visualization in ImageJ Version 2 (Java 1.8.0_172 (64-bit), NIH), and 3) quantitative image analysis of staining intensity was performed in three areas per FFPE tissue (≥6.25mm2) using Cell Profiler 2 with “cytometry 12 markers” pipeline and FlowFCS Express 6 Image Cytometry Version 6.06.022 (De Novo Software) (Supplementary Figure S2 ). Images were merged in Halo (version 2.0.1145.31, Indica labs) to identify immune subsets according to lineage markers described (Supplementary Table S3 ).
Whole-slide scans were obtained after each stain on Hamamatsu Scanner at 20x magnification. Digital image analysis consisted of three steps: 1) image coregistration using Cell Profiler 2 (Version 2.2.0, Broad Institute) with “coregistration 12 markers” pipeline, 2) visualization in ImageJ Version 2 (Java 1.8.0_172 (64-bit), NIH), and 3) quantitative image analysis of staining intensity was performed in three areas per FFPE tissue (≥6.25mm2) using Cell Profiler 2 with “cytometry 12 markers” pipeline and FlowFCS Express 6 Image Cytometry Version 6.06.022 (De Novo Software) (
