Time course analysis of human colorectal cancer liver metastasis models under Sacituzumab Govitecan (SG) treatment or untargeted control
Time course analysis of human colorectal cancer liver metastasis models under Sacituzumab Govitecan (SG) treatment or untargeted control
Tumors were enzymatically dissociated with a tumor dissociation kit (Miltenyi) following the manufacturer's instructions. In short, tumor fragments were incubated together with an enzyme cocktail in a gentleMACS Octo Dissociator with heaters (Miltenyi) using the default programs 37C_h_TDK_1 (for human tissue). Afterwards, the sample was strained to obtain single cells and frozen until processed for single-cell sequencing. For sample multiplexing, single cells were incubated with hashing antibodies (Total-Seq-B, BioLegend, 1 µg antibody) and additional cell sorting antibodies (anti-human EpCAM, Miltenyi; anti-mouse CD45, BioLegend) according to the manufacturer's protocol. In short, single cells were thawed and washed in FACS buffer. Cells were resuspended in 50-100 µl with Fc Blocking Reagent Mouse (Miltenyi) in FACS buffer (ratio 1:50) and incubated with the antibody cocktail at 4°C for 30 min in the dark. Then, samples were washed three times in FACS buffer before resuspending in 600 µl FACS buffer with ZombieNIR (BioLegend) for live tumor cell isolation (EpCAM-positive, CD45-negative, CD31-negative).  Patient-derived tumor organoids were injected into the flank of mice. Treatment with either the TROP2-directed ADC Sacituzumab Govitecan (SG) or untargeted ADC (containing the payload SN-38) was started when the tumor size had reached 100 mm3.  Tumors were harvested at indicated time points. Reads were mapped to the human reference genome assembly refdata-gex-GRCh38-2020-A and demultiplexed into their original samples using the cellranger multi pipeline with default settings of the 10X Genomics Cell Ranger (v9.0.1). Hashtag oligo-labelled samples were pooled (up to 12,000 cells each) and processed for single-cell RNA-sequencing using the 10X Chromium Next GEM Single Cell Reagent Kit 3.1 according to the manufacturer's instructions (Dual Index protocol). First, cells are resuspended in a master mix and loaded onto a chip. After encapsulation, cells were lysed inside their GEMs. After cell lysis inside of GEMs, mRNA is reverse transcribed to cDNA with droplet-specific barcodes. After GEM breakdown, the library is being prepared according to the protocol including cDNA amplification by PCR in 11 cycles. 