GSM8773106: SS95 CAR-T, IP D9 D30, VDJ; Mus musculus; OTHER
Lungs were isolated and placed in GentleMACS C Tubes with 6 ml of complete RPMI supplemented with 2 mg/ml collagenase IV (Worthington) and 80 U/ml DNAse I (Worthington). Lungs were minced using the “m_impTumor_01_01” program on a GentleMACS Octo Dissociator and incubated in a 37°C shaker at 120 rpm for 30 min. Cells were filtered through a 100 μm filter, lysed with ACK lysing buffer (Gibco), and resuspended as single cell suspensions. Lung single cell suspensions were cryopreserved in complete RPMI supplemented with 10% FBS and 10% DMSO. Cryopreserved lung tumor cell suspensions and CAR-T infusion products were thawed, washed, and labeled with TotalSeq-C anti-mouse Hashtags (Biolegend) according to the manufacturer's protocol. Following hashing, equal numbers of live cells were pooled from 3 mice in each treatment group to form 4 pools: D9 ROR1 CAR, D9 cJun.ROR1 CAR, D30 ROR1 CAR, D30 cJun.ROR1 CAR. For analysis of T cells, CD8+ T cells were isolated from each pool using the Mouse CD8a Positive Selection Kit II (Stem Cell). For analysis of the TME, each pool was stained Live/Dead Fixable Aqua Dead Cell stain kit (Invitrogen) and live CD45-PECy7- cells were sorted. For analysis of CAR-T infusion products, CAR-T cells were used directly after hashing. Libraries from CAR-T infusion product, T cell, and TME samples were prepared using the 10X Genomics Chromium GEM-X Single Cell 5' Kit as per the manufacturer's instructions and were sequenced on an Illumina NextSeq 2000 according to the standard 10X Genomics protocol.