This study investigated the genomic mechanisms underlying acquired resistance (AR) to immune checkpoint inhibitors (ICIs) across tumor types, specifically examining how copy number variant (CNV)-driven subclonal evolution contributes to clinical relapses in melanoma patients. We analyzed longitudinal clinical melanoma samples from patients who experienced relapses on ICI therapy, complemented by isogenic human melanoma cell line models (M486) and murine melanoma tumor systems. Single-cell whole-genome sequencing (scWGS) was employed to resolve subclonal CNV patterns in AR tumors. scWGS datasets from isogenic human melanoma cell lines, patient-matched tumor samples, and murine melanoma models will be made available through dbGaP, enabling researchers to access high-resolution subclonal CNV profiles that define the genomic landscape of acquired ICI resistance evolution.
YER tumor samples were dissociated in NST-DAPI buffer using the gentleMACS Octo Dissociator (Miltenyi Biotec, '4c_nuclei' protocol). Nuclei were stained with DAPI and sorted on a BD FACSAria II to isolate diploid or aneuploid populations. Sorted nuclei were diluted and dispensed into 350v nanowell chips (Takara ICELL8 cx). Wells with single nuclei were selected using ICELL8 CellSelect software. scWGS libraries were prepared by lysing selected nuclei, followed by tagmentation using the Illumina Tagment DNA kit. Samples were barcoded with 72×72 indexed primers and PCR-amplified. Libraries were collected (Takara Collection Kit), purified with 1.8× Ampure XP beads (Beckman), QC'ed by Qubit and TapeStation, and sequenced on Illumina NovaSeq X Plus (~1 million reads per cell/nucleus).