Cytof system

All the kidney samples were processed in the same batch. CyTOF analysis was performed by PLTTech, Inc. (Hangzhou, China) according to a published protocol [12 (link)]. The kidney tissue was dissociated into a single-cell suspension with a mixture of DNase I, collagenase IV, and hyaluronidase (Sigma-Aldrich). Immune cells were enriched using Percoll density gradient media (Sigma-Aldrich), and erythrocytes were fully removed using ACK Lysing Buffer (Sigma-Aldrich). Qualified samples were blocked and stained with a mixed panel of surface antibodies, followed by overnight fixation. After fixing with Fix & Perm Buffer (Fluidigm), the cells were incubated in an intracellular antibody mix. The signals of the stained cells were detected using a CyTOF system (Fluidigm). The types of immune cells were identified via nonlinear dimensionality reduction (t-SNE), followed by density clustering.

Eight fresh early stage NSCLC samples were obtained from the Zhujiang Hospital, Southern Medical University. The detailed clinical characteristics of the samples were shown in Supplementary Table 1. After washing by the RPMI 1640 medium, the fresh lung tumor samples were then dissociated into single cells under Deoxyribonuclease and Collagenase type IV exposure. ACK Lysing Buffer (PLT) was used to remove the erythrocyte, the amounts of viable and dead cells were then counted to provide a preliminary estimate of the sampling efficiency. Cell-ID™ Cisplatin-194Pt (Fluidigm) was used to specifically identify dead cells; next, qualified samples were blocked on ice for 20 minutes. Without removing the blocking solution, samples were incubated with a surface antibody mix (Maxpar^® Antibody Labeling Kit, Fluidigm) for 30 minutes on ice. With Maxpar^® Fix and Perm Buffer, an eventual 500μM DNA intercalator (Cell-ID™ Intercalator-Ir, Fluidigm) were incubated with the washed 200uL re-suspended cells per sample overnight at 4°C. Subsequently, intracellular staining was performed. After washing, pre-fixing, and 30 minutes of co-incubation with intracellular antibody mix on ice, cells were then rinsed and subsequently obtained in the CyTOF system (Helios, Fluidigm) to detect the signals. For the detailed procedures, please refer to the reported protocol (34 (link)).

Immune cells were collected from KPC tumors using the method detailed in Section Tumor Immune Microenvironment Assay using Flow Cytometry. Immune cells were labeled using antibodies with metal tags. The mixed antibody panel consisted of 42 antibodies conjugated with different metals (Table S1, Supporting Information). The metal signals were detected to evaluate the expression of the conjugated target molecule using the CyTOF system (Helios, Fluidigm, San Francisco, CA, USA).