Supplemental Material for: Unexpected Limited Immune Potentiation of Common Multikinase Inhibitors Combined with Immune Checkpoint Blockade in HCC: Insights from Murine Models

Antiangiogenic activity was assessed by CD31⁺ microvessel density (MVD). Transcriptomic remodeling was analyzed by bulk RNA sequencing with gene set variation analysis and principal component analysis. Tumor-infiltrating lymphocytes (TILs) were evaluated for proliferation (Ki67 expression) and interferon-γ (IFN-γ) production.

Antitumor efficacy with or without anti–PD-1 was assessed<i> in vivo</i>.</p><p dir="ltr"><b>Results:</b><br>All MKIs demonstrated broadly <b>comparable antiangiogenic activity to DC101</b> (mean CD31⁺MVD reduction vs. control: DC101, 27.6%; MKIs: 23.4–42%). <b>Despite this, selective VEGFR2 blockade induced minimal immune perturbation, </b>whereas MKIs induced <b>divergent transcriptomic remodeling</b> across immune, metabolic, stress-response, and proliferation-related pathways.

Notably, low-dose cabozantinib and lenvatinib showed reductions in both immune-activating and immune-suppressive gene signatures, <b>suggesting potentially opposing immunologic effects.</b><b> </b><b>Functionally, neither DC101 nor MKIs enhanced proliferation or IFN-γ production of tumor-infiltrating T cells. Moreover, MKIs did not improve the antitumor efficacy of anti–PD-1 therapy.</b></p><p dir="ltr"><b>Conclusion:</b><br>Despite similar antiangiogenic effects, MKIs induce broader transcriptomic remodeling without enhancing anti–PD-1 efficacy.

These findings suggest that VEGFR-targeting MKIs can induce opposing immune effects beyond VEGF–VEGFR blockade, potentially limiting net enhancement of antitumor immunity.</p>