GSM8322781: SMAC mimetic single arm; Mus musculus; OTHER
15 days post treatment start, explanted Pan02 tumors were minced into small ~1mm³ pieces and single cell suspensions were prepared using the Tumor Dissociation Kit, mouse (Miltenyi, 130-096-730) and the gentleMACS Octo Dissociator (Miltenyi, 130-096-427) following manufacturer's protocol using the programs “37_m_TDK_2” and “m_impTumor_01”. Red blood cells were lysed by incubating resuspended pellets for 1-2 min on ice in 1 ml ACK lysing buffer (Thermo Fisher, A10492-01). 7 dissociated tumors per group were used for analysis by flow cytometry and 3/group were additionally enriched for tumor infiltrating leukocytes with CD45 (TIL) MicroBeads, mouse (Miltenyi, 130-110-618) using the MultiMACS X Separator (Miltenyi) followed by single cell labelling for CITE-seq analysis.Single cell libraries were generated using the BD Rhapsody system. Enriched CD45+ TILs from 3 tumors/group were stained for multiplexing and with AbSeq Ab-Oligos according to manufacturer's protocol (BD Doc ID: 214419 Rev 2.0) with slight modifications. In detail, cells were labelled with the Mouse Immune Single-Cell Multiplexing Kit, (BD, 633793), followed by equal pooling of 3 samples each (resulting in 4 pooled samples), Fc blocking with TruStain FcX PLUS antibody (Biolegend, 156604) and staining with oligo-conjugated Ab-Seq antibody mix. Single cell capture and cDNA synthesis was performed with the BD Rhapsody Single-Cell Analysis System and the Cartridge Reagent Kit (BD, 633731), the Cartridge Kit (BD, 33733) and the cDNA Kit (BD, 633773) according to manufacturer's protocol (BD Doc ID: 210966 Rev. 1.0). Libraries were generated with the Targeted Amplification Kit (BD, 633774) and the Mouse Immune Response Panel (BD, 633753) according to manufacturer's protocol (Doc ID: 214508 Rev. 3.0). Pooling of libraries of each cartridge was calculated following instructions given in BDs' “SEQ Calculator v1.2.xlsm” and libraries were sequenced (paired-end) on the Illumina platform.