Apatinib remodels the immunosuppressive tumor ecosystem of gastric cancer enhancing anti-PD-1 immunotherapy
Description
Apatinib has been shown to clinically enhance anti-PD-1 immunotherapy for advanced gastric cancer (GC). However, the complexity of GC immunosuppression remains a challenge for precision immunotherapy. Here, we profiled the transcriptomes of 34,182 single cells from GC patient-derived xenografts of humanized mouse models treated with vehicle, nivolumab, or nivolumab plus apatinib. Notably, excessive expression of CXCL5 in CellCycle malignant epithelium, induced by anti-PD-1 immunotherapy and blocked by combined apatinib treatment, was found to be a key driver of tumor-associated neutrophil (TAN) recruitment in tumor microenvironment through the CXCL5/CXCR2 axis. We further revealed that the protumor TAN signature was associated with anti-PD-1 immunotherapy-related progressive disease and poor cancer prognosis. Molecular and functional analyses in cell-derived xenograft models directly confirmed the positive in vivo therapeutic effect of targeting CXCL5/CXCR2 axis during anti-PD-1 immunotherapy. Altogether, our study elucidates GC immunosuppressive landscape in anti-PD-1 immunotherapy and highlights potential targets for overcoming checkpoint immunotherapy resistance.
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Five- to six-week-old immunodeficient NCG mice were intravenously injected with human CD34+ hematopoietic stem cells (hCD34+ HSCs), and in parallel, a cohort of NCG mice was implanted with the same gastric adenocarcinoma patient-derived xenografts for rederivation. At 8 to 20 weeks post-engraftment of the HSCs, the mice were bled for assessment of humanized chimerism in the periphery, along with “warm” PDX implants at week 15. Humanized mice with GC-PDXs were randomized into the vehicle, nivolumab, and nivolumab plus apatinib groups for subsequent dosing and analysis at week 17-20. To investigate multiple anti-PD-1-based immunotherapy-induced transcriptomic remodeling in the multicellular ecosystem of GC, we performed scRNA-seq on tumors collected from humanized GC-PDX models treated with nivolumab, nivolumab plus apatinib, or vehicle control using the 10X Genomics Chromium platform. All fresh samples obtained immediately after resection of PDX tissues were weighed. The PDX tissues with the largest and smallest weights in each group were excluded in the subsequent single-cell RNA sequencing study. After additional sampling by puncture for liquid nitrogen cryopreservation and formalin fixation, PDX tissues from the same group were pooled and rinsed with PBS, minced into small cubic pieces (approximately 1 mm in diameter) on ice, and dissociated with a digestive enzyme mixture including 10 ml prewarmed RPMI-1640 (ThermoFisher Scientific), 2 mg/ml dispase (Roche), 1 mg/ml type IV collagenase (Sigma) and 10 U/µl DNase I (Roche) for 30 minutes at 37°C. The reaction was terminated with RPMI-1640 supplemented with 10% FBS. Cell suspensions were filtered using a 70 μm filter and then centrifuged at 500 rpm for 6 min at 4°C to pellet dead cells and red blood cells. The cells were washed twice and resuspended in PBS with 0.5% bovine serum albumin (BSA, Sigma). According to the manufacturer's protocol, single-cell RNA-seq libraries were generated from cell suspensions using a Chromium Single Cell 5' Library & Gel Bead Kit and finally sequenced using an Illumina sequencer (Novaseq6000) with a sequencing depth of at least 100,000 reads per cell with a paired-end 150 bp (PE150) reading strategy (performed by CapitalBio Technology, Beijing).