Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.

The tumor suppressor protein p53 is mutated in close to 50% of human tumors and is dysregulated in many others, for instance by silencing or loss of the p14ARF gene. Under steady-state conditions, the two E3 ligases MDM2/MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 in tumors with wild-type (WT) p53 has therefore been considered a therapeutic strategy. Moreover, studies indicate that p53 reactivation may synergize with radiation and increase tumor immunogenicity. In vivo studies of most MDM2 inhibitors have utilized immunodeficient xenograft mouse models, preventing detailed studies of action of these molecules on the immune response. The mouse melanoma cell line B16-F10 carries functional, wild type p53 but does not express the MDM2 regulator p19ARF. In this study, we tested a p53-MDM2 protein-protein interaction inhibitor, the small molecule Navtemadlin, which is currently being tested in phase-II clinical trials. Using mass spectrometry-based proteomics and imaging-flowcytometry, we identified specific protein expression patterns following Navtemadlin treatment of B16-F10 melanoma cells compared to p53 CRISPR inactivated control cells. In vitro, Navtemadlin induced a significant, p53 dependent, growth arrest but little apoptosis in B16-F10 cells. When combined with radiotherapy, Navtemadlin showed synergistic effects and increased apoptosis. In vivo, Navtemadlin treatment significantly reduced the growth of B16-F10 melanoma cells implanted in C57Bl/6 mice. Our data highlight the utility of a syngeneic B16-F10 p53+/+ mouse melanoma model for assessing existing and novel p53-MDM2/4 inhibitors and in identifying new combination therapies that can efficiently eliminate tumors in vivo.

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<p>Complete list of proteins.</p>

<p>Navtemadlin treatment does not lead to bone marrow suppression.</p>

<p>Images radiation + Navtemadlin combination.</p>

<p>B16-F10 +/+ cells treated with increasing Navtemadlin concentrations over time.</p>

<p>Navtemadlin treatment does not affect tumor infiltrating lymphocytes.</p>