Cd326

In each of the four treatment groups, immune cells from four mice were used for single-cell RNA sequencing. Tumor tissues were isolated, minced with scalpels, and digested with Collagenase IV and Dnase I at 37°C for 20-30 minutes. After enzymatic digestion, immune cells were enriched using lymphocyte separation medium. The enriched cells were then subjected to magnetic bead separation (EasySep Mouse Streptavidin Rapidspheres Isolation Kit, Stem Cell, 19860) to remove the EpCAM⁺ cells (CD326 1:200, ThermoFisher Scientific, 13-5791-82). The EpCAM-depleted cells were stained with antibodies against CD45 and a viability dye. Live CD45⁺ cells (CD45 1:100, Biolegend, 103112), were sorted using MoFlo and then processed for droplet-based 3' end scRNA-seq by encapsulating sorted live CD45⁺ tumor-infiltrating immune cells into droplets via a 10× Genomics platform according to the manufacturer's instructions (10× Genomics). Paired-end RNA-seq was performed via an Illumina NovaSeq 6000 sequencing system.

Cells in both channels on chips were stained for DAPI (1 ug/mL) and Alexa Fluor-647 conjugated Phalloidin (both ThermoFisher Scientific), as well as cancer cell-specific marker EpCAM (CD326, Catalogue #53-8326-42, Thermofisher Scientific). Imaging was performed at 20× on a Zeiss 710 ELYRA PS.1 confocal microscope using an EC Plan-Neofluar10×/0.3 M27 objective (Zeiss, Oberkochen, Germany). Confocal z-sections were made throughout the cell depth (approximately 20 sections) using 5 μm step size with an image format of 2048 × 2048 yielding a pixel size of 0.415 × 0.415 μm (image size approximately 850 × 850 μm).