Intratumoral Immune Cell Profiling by Flow Cytometry

For flow cytometry on intratumoral immune cell populations, tumors were processed as described previously (54 (link)). Briefly, tumors were chopped using a sterile scalpel until 2–3 mm in size, then placed in digestion media containing collagenase I (0.05% wt/vol; MilliporeSigma), DNase type IV (30 U/mL; MilliporeSigma), and hyaluronidase type V (0.01% wt/vol; MilliporeSigma). Mechanical dissociation using the gentleMACS Octo Dissociator (Miltenyi Biotec) was performed followed by a 40-minute incubation at 37°C. Tumor samples were mechanically dissociated again, then passed through a 70 μm filter. RBC lysis (BioLegend) was performed on tumor cell suspension following the manufacturer’s recommendations. For the ATX inhibitor plus anti–PD-1 flow studies, 344SQ tumor samples were stained with a 34-color panel and acquired using a Cytek Aurora. Antibodies and dilutions are listed in Supplemental Table 2. Additional details can be found in Supplemental Methods.