Spatially segregated APOE⁺ macrophages restrict immunotherapy efficacy in clear cell renal cell carcinoma

<b>Background:</b> Immunotherapy has revolutionized cancer treatment and holds great potential for them, including metastatic clear cell renal cell carcinoma (ccRCC). However, immune resistance remains a major obstacle, limiting its efficacy and durability. Understanding the mechanisms of immune tolerance in the tumor microenvironment (TME) is pivotal for overcoming these challenges and enhancing therapeutic outcomes. <b>Methods:</b> Over 2000 samples, including a real-world cohort of 230 advanced ccRCC patients treated with immune checkpoint blockade (ICB) were analyzed. Single-cell RNA sequencing data from 13 tumor regions were categorized into ICB-exposed, ICB-resistant, and ICB-responsive groups. Multiple robust algorithms and multiplex immunofluorescence were used to explore TME composition and macrophage heterogeneity. Spatial communication dynamics were further investigated. <i>In vitro</i> experiments were performed to evaluate the impact of SPP1 on 786-O and 769-P cells. Co-culture experiments with THP-1-derived macrophages, followed by Western blot, flow cytometry, and functional assays, were performed to investigate SPP1-mediated macrophage polarization and its impact on tumor progression. <b>Results:</b> The results revealed an elevated presence of Apolipoprotein E (APOE)⁺ macrophages in ICB-resistant ccRCC. Notably, higher APOE⁺ macrophage proportion indicated shorter prognosis and worse response to ICB (<i>P</i> < 0.001). Elevated expression of CCAAT Enhancer Binding Protein Delta (CEBPD) was markedly linked to several immunosuppressive pathways, hindering T cell recruitment, promoting exhaustion, ultimately diminishing poorer prognosis and worse ICB efficacy. Meanwhile, upregulated Secreted Phosphoprotein 1 (SPP1) significantly enhances the proliferation, clonal formation, and migration of ccRCC cells. Tumor-derived SPP1. Additionally, SPP1 signaling from malignant cells appeared to recruit APOE⁺ macrophages to tumor margins, and promotes macrophage polarization into APOE⁺ M2-like macrophages. In the vicinity of the tumor, these APOE⁺ macrophages shape immunosuppressive TME by releasing abundant TGF-β signals, limiting anti-tumor effector T cells activity in ICB-resistant tumors, and contributing to tumor progression. <b>Conclusion:</b> This study reveals the critical role of APOE⁺ macrophages in promoting immune suppression and resistance to ICB therapy in ccRCC. By promoting T cell exhaustion and immunosuppressive signaling, particularly via localized TGF-β, these spatially segregated macrophages undermine treatment efficacy. Targeting APOE⁺ macrophages, especially in conjunction with ICB, presents a promising strategy to overcome immune resistance and enhance outcomes for ccRCC patients.