Pannoramic midi tissue imaging system

A whole slide scan was performed for each fluorescence-stained slide using a digital microscopy scanner Pannoramic MIDI tissue imaging system (3DHISTECH Ltd., Hungary). Because both tumor cells and normal epithelial cells have positive CK expression, it is hard to distinguish these two cell types in immunofluorescence staining. To exclude the normal epithelial cells in analysis, we applied Hematoxylin and Eosin (H&E) staining in the tissue sections after finishing the fluorescence scan. Images were analyzed by Indica Halo software (Indica Labs, UK). Two independent blinded pathologists performed histologic evaluation and supervised to split the tumor and stromal compartments by using Halo software. Cells were phenotyped into the following subsets: DC (CD11c⁺), macrophage (CD68⁺), tumor cell (CK⁺), and PD-L1⁺ subpopulations of these cells.
Immune cell infiltration was evaluated as the number of cells per slide, in the tumor compartment, stromal compartment, or total viable tissue area of the slides, respectively. To evaluate the spatial relationship between immune cells and tumor cells, the distance between each tumor cell and its nearest neighbor immune cells was measured.

mIHC staining was performed with the tyramide signal amplification (TSA) 6-color IHC kit (D110061-50T, WiSee Bio, China) according to the manufacturer’s instructions. Briefly, sections (3-μm thickness) obtained from paraffin-embedded samples were dewaxed, rehydrated, and subjected to 100 °C for antigen retrieval. Then, the sections were incubated with blocking antibody at room temperature for 10 min, treated with the first primary antibody for 30 min, horseradish peroxidase-conjugated (HRP) secondary antibody for 10 min, and a tyramide signal amplification for 10 min. After washing in TBST buffer, the slides went through citrate buffer antigen retrieval using a microwave set at 20% of maximum power for 15 min. The same process was repeated for the following five primary antibodies. Each slide was then treated with two drops of DAPI and manually coverslipped. Slides pictures were taken with Pannoramic MIDI tissue imaging system (3D HISTECH). Antibodies and reagents are listed in Supplementary Table S8. The distances between the interested cells were measured and quantitated with Halo Proximity Histoprogram.