46 TKI Resistance is Associated with Immune Dysfunction in ccRCC

Abstract

Abstract Background Antiangiogenic tyrosine kinase inhibitors (TKIs) have become a cornerstone in the management of clear cell renal cell carcinoma (ccRCC) as monotherapy or in combination with immune checkpoint blockades (ICB). However, TKIs produce limited long-term durability even in combination with ICBs, indicating that prolonged TKI treatment may foster an immunosuppressive microenvironment contributing to therapy resistance. We aimed to identify these escape mechanisms and propose strategies to overcome them. Methods In this study, we performed Xenium in situ transcriptomic profiling with a 5,000-gene panel on tissue microarrays comprising treatment-naïve or sunitinib-treated tumors from RCC patients. The Renca murine RCC model was used to evaluate therapeutic responses in vivo. Results From more than 220,000 profiled cells, we identified and contrasted the major cell types and subtypes present in sunitinib‑treated versus untreated RCC tumors. In sunitinib-treated tumors, both tumor and immune cells displayed a highly inflammatory state, marked by upregulation of type I and type II interferon response pathways. As an anti-angiogenic agent, sunitinib inhibits multiple receptor tyrosine kinases (RTKs) and reduces tumor vascularization. Here we observed that sunitinib treatment leads to a marked reduction in endothelial cells (ECs, 13.39% to 1.70%) and cancer‑associated fibroblasts (CAFs, 14.65% to 6.42%), confirming a massive collapse of the vascular and perivascular niche. However, the inflammatory subsets of ECs and CAFs were less susceptible to sunitinib treatment. We identified two major tumor cell populations that adapt in distinct ways to sunitinib exposure and the resulting vascular collapse. The dominant population showed strong hypoxic and glycolytic signatures and transitioned toward a mesenchymal‑like phenotype. In contrast, a smaller subset lacked canonical RCC markers and instead expressed high levels of the transcription factor DUX4, which drives embryonic genome activation programs while suppressing MHC class I expression, promoting a stem‑cell–like state. Although sunitinib did not alter the overall abundance of tumor-associated macrophages (TAMs), it markedly shifted their spatial distribution: in treatment-naïve tumors, TAMs were sequestered by endothelial cells, whereas in sunitinib-treated tumors, they were released and engaged to interaction with T cells. We further identified two TAM subsets—interferon-primed TAMs (IFN-TAMs) and proliferating TAMs (Prolif-TAMs)—that were expanded in sunitinib-treated tumors and associated with poorer progression-free survival. IFN-TAMs exhibited strong IFN-γ signaling activity and high expression of the immune checkpoint ligands PD-L1 and PD-L2. Prolif-TAMs were defined by elevated proliferation markers and by increased expression of tumor-promoting proteins, such as CSF1R, MSR1, VSIG4, and TREM2. Notably, both IFN- and Prolif-TAMs expressed high levels of the A2A adenosine receptor (A2AR). In sunitinib‑treated tumors, IFN‑TAMs displayed strengthened communication with multiple immune populations, including proliferating T cells and cytotoxic T lymphocytes (CTLs). Interactions mediated by Th2 cytokines (IL‑4 and IL‑13) and their receptors were also elevated after treatment, consistent with enhanced M2‑polarizing signals. In contrast, Prolif TAMs maintained stable interactions with tumor cells—but not immune cells—regardless of treatment status, highlighting a tumor‑focused rather than immune‑focused communication pattern. Both proliferating T cells and CTLs expressed PD‑1 and ENTPD1 (CD39), enabling the generation of immunosuppressive adenosine and engagement of A2AR. Together, these findings point to a reciprocal immunosuppressive circuit between T cells and TAMs mediated through the PD‑1–PD‑L1/L2 axis and the ENTPD1–adenosine–A2AR pathway. To counteract this immunosuppressive network, we found that PD‑1 blockade acts synergistically with axitinib to amplify antitumor activity in our animal model. Likewise, inhibiting A2AR signaling with CPI‑444 diminished the expression of tumor‑promoting factors and further strengthened the therapeutic efficacy of axitinib in our model system. Conclusions These results show that sunitinib treatment induces an immunosuppressive tumor microenvironment driven by macrophage reprogramming and altered spatial organization. Targeting these macrophage subsets markedly enhances the therapeutic efficacy of TKIs. DOD CDMRP Funding yes