SPP1+ Neutrophils Mediate Resistance to lmmune Checkpoint Blockade in BAP1-Inactivated Tumors

BAP1 inactivation, observed across multiple human cancers, is linked to immune checkpoint blockade (ICB) resistance and adverse clinical outcomes. The mechanisms underlying BAP1-associated ICB sensitivity could provide potential targets to enhance ICB efficacy. Here, we showed that BAP1 inactivation fosters an immunosuppressive tumor microenvironment (TME), marked by increased infiltration of M2-like macrophages and neutrophils. Single-cell transcriptomic analysis revealed an expansion of SPP1+ neutrophils in ICB-treated, BAP1-inactivated tumors. These SPP1+ neutrophils displayed a pro-tumorigenic phenotype and conferred resistance to anti-PD-1 therapy by engaging with cytotoxic T cells via PD-1/PD-L1 signaling. Notably, depletion of neutrophils, but not macrophages, restored sensitivity to ICB in BAP1-inactivated tumors. Mechanistically, BAP1 loss significantly increased CCL2 secretion, driving neutrophil SPP1+ polarization, delaying neutrophil apoptosis, and promoting ICB resistance. This resistance could be significantly mitigated by targeting the CCL2-CCR2 axis. These results underscore the role of BAP1 in modulating the immune landscape and suggest that targeting CCL2-CCR2-mediated neutrophil polarization may overcome ICB resistance in BAP1-inactivated tumors. Overall design: A single-cell suspension was prepared from the dissociation of tumors. For labeling, 40 µl of CD45 microbeads (Miltenyi #130052301) were added. After an incubation step (15 min at 4°C) the antigen-antibody reaction was stopped by adding 800 µl ice-cold PBS. Then, the cell suspension mixture is loaded onto a MACS Column, which is placed in the magnetic field of a MACS Separator. The magnetically labeled CD45+ cells were retained within the column. The unlabeled cells ran through; this cell fraction was thus depleted of CD45+ cells, the CD45-negative cells were in the eluent buffer. After removing the column from the magnetic field, the magnetically retained CD45+ cells were eluted as the positively selected cell fraction and used for subsequent experiments. The samples were stained with TrypanBlue and the cell viability was evaluated microscopically.Single-cell suspensions (2×105 cells/mL) with PBS (HyClone) were loaded onto microwell chips using the Singleron Matrix® Single Cell Processing System. Barcoding Beads are subsequently collected from the microwell chip, followed by reverse transcription of the mRNA captured by the Barcoding Beads and to obtain cDNA, and PCR amplification. The amplified cDNA is then fragmented and ligated with sequencing adapters. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPE® Single Cell RNA Library Kits (Singleron) [21]. Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina novaseq 6000 with 150 bp paired end reads. Raw reads from scRNA-seq were processed to generate gene expression matrixes using CeleScope (https://github.com/singleron-RD/CeleScope) v1.9.0 pipeline. Briefly, raw reads were first processed with CeleScope to remove low quality reads with Cutadapt v1.17 to trim poly-A tail and adapter sequences. Cell barcode and UMI were extracted. After that, we used STAR v2.6.1b to map reads to the reference genome GRCm38 (ensemble version 92 annotation). UMI counts and gene counts of each cell were acquired with featureCounts v2.0.1software and used to generate expression matrix files for subsequent analysis.