Fig. 6

REG + aPD1 relieves the immunosuppressive tumor microenvironment and induces sustained suppression of tumor growth and metastasis. a Effects on tumor vasculature analyzed by DCE-MRI and CD31 staining. Longitudinal effects on vascularization are shown by relative amplitude changes versus baseline (day 4). Black arrow indicates treatment stop. b Effects on Treg cells. Quantification of immunostaining for FoxP3⁺ Treg cells and determination of the ratio of CD8⁺ cytotoxic T cells to Treg cells (see Supplementary Figure S4b for quantification of CD8⁺ cells). c Effects on macrophages and polarization. Quantification of total macrophages (F4/80), CD206 (M2) and iNOS (M1). Mean values ± SD (n = 6–7) and individual values (dots) are shown. *p < 0.05; **p < 0.01; ***p < 0.001. d Representative DCE-MRI and immunofluorescence images. Left panel: T1-weighted MR images with overlayed amplitude parameter maps from DCE-MRI analyses (scale bar indicates arbitrary units); panels 2–8: images of co-stainings as indicated by the antigen labels. Yellow staining indicates overlapping signals. Nuclei are DAPI stained (blue). Scale bars: 100 µm. e Mechanisms of sustained tumor growth inhibition by REG + aPD1. In untreated tumors, monocytes are recruited which differentiate to M2 macrophages. In addition, VEGFA promotes tumor angiogenesis, proliferation of Treg cells, and upregulation of PD1 on cytotoxic T cells. Together this creates an immunosuppressive tumor microenvironment that inhibits cytotoxic T cell activity, thus driving tumor growth and liver metastasis. Via inhibition of VEGFR and CSF1R signaling, REG reduces tumor vascularization and reduces the number of intratumoral macrophages and Treg cells. Blockade of PD1 by aPD1, which is likely enhanced by REG-mediated prevention of PD1 expression, leads to the activation of cytotoxic T cells, increases IFNγ expression, and induces tumor cell death. Importantly, REG and aPD1 synergize to induce sustained M1 polarization resulting in durable suppression of tumor growth and liver metastasis