title: GSM9137132: Feature-2025-05-23-2-2; Homo sapiens; OTHER
library_construction_protocol: Fresh tumor samples were obtained from discarded surgical resection specimens collected from consented patients under a Cooperative Human Tissue Network-CHTN IRB approved protocol.  Tumor samples were cut into 1-2mm3 Patient-Derived Tumor Fragments (PDTFs) and cryopreserved in 1ml CryoStor CS10). Cryopreserved PDTFs from five distinct renal cell carcinoma patient tumors were rapidly thawed in a 37°C water bath and immediately placed in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS). Following thawing, individual tumor fragments were cultured for 48 hours as previously detailed73. For each donor, a total of 16 fragments per treatment condition were allocated to each of the four experimental arms: Isotype Control: anti-Respiratory Syncytial Virus (RSV) IgG1 (10 nM) and anti-Hen Egg Lysozyme (HEL) IgG4 (10 µg/mL), anti-PD-1 Monotherapy: anti-PD-1 (10 µg/mL; Cat: SIM0010) and anti-RSV-IgG1 (10 nM), MiTE144 Monotherapy: MITE144 (1 0nM) and anti-HEL-IgG4 (10 µg/mL), Combination Therapy: MITE144 (10 nM) and anti-PD-1 (10 µg/mL). Upon completion of the 48-hour culture period, 90ul of supernatants were collected and frozen for secreted protein analysis. Next, all fragments from the same donor and treatment arm were pooled and digested into single cell suspensions. Immune cells were enriched from the digested fragments using CD45 magnetic-activated cell sorting (MACS). The resulting CD45-enriched cell fractions were subsequently stained with a custom Immunai CITE-seq antibody panel (see STAR methods) and subjected to fluorescence-activated cell sorting (FACS) to isolate viable CD45 cells. Single-cell gene expression (GEX) and feature (CITE-seq) libraries were prepared from the sorted cells using the 10x Genomics Chromium Next GEM Single Cell 5' Kit v2. Libraries were sequenced on a Novaseq X Plus sequencer using a 300 cycle kit.