PURPOSE: The treatment options for metastatic soft tissue sarcomas (STSs) are limited. In most cases, immunotherapy with immune checkpoint inhibitors has not been successful so far. Macrophages dominate the immune landscape of STSs; thus, combinatorial strategies aiming at both tumor-infiltrating lymphocytes and macrophages may represent a particularly relevant treatment approach for metastatic or recurrent STSs. METHODS: In this cohort study, 66 patients who underwent surgery for STSs were enrolled. Tumor cells and tumor-infiltrating immune cells were analyzed using flow cytometry and immunohistochemistry. In cell suspensions obtained from surgical resections, human T cells were activated by superparamagnetic polymer beads and cultured at a concentration of 0.3 × 106/µl in the absence or presence of therapeutic monoclonal antibodies (anti-PD-1, anti-CD47, and anti-PD-1 + anti-CD47). Supernatants from cell suspensions were analyzed using multiplex Luminex cytokine bead-based immunoassays. RESULTS: The most profound response to anti-CD47 therapy was observed in an undifferentiated pleiomorphic sarcoma which also displayed high expression of CD47 in the tumor microenvironment. Both anti-PD-1 and anti-CD47 therapies drastically increased the production of pro-inflammatory cytokines in the tumor microenvironment of STSs, but co-administration of both agents did not further increase cytokine secretion. Furthermore, all patient samples treated with a combination of both anti-PD-1 and anti-CD47 antibodies showed a dramatic reduction in cytokine secretion. CONCLUSION: Our findings suggest that anti-PD-1 and anti-CD47 therapies do not enhance each other, and the combined application of anti-PD-1 and anti-CD47 agents in vitro limits rather than potentiates their efficacy.

3rd Department of Surgery 1st Faculty of Medicine Charles University and University Hospital Motol 5 Uvalu 84 150 06 Prague Czech Republic

Chemokine Research Group Institute of Infection Immunity and Inflammation College of Medical Veterinary and Life Sciences University of Glasgow Glasgow G12 8TT UK

Department of Immunology 2nd Faculty of Medicine Charles University and University Hospital Motol 5 Uvalu 84 150 06 Prague 5 Czech Republic

Department of Immunology Institute for Clinical and Experimental Medicine Videnska 1958 140 21 Prague Czech Republic

Department of Pathology and Molecular Medicine 2nd Faculty of Medicine Charles University and University Hospital Motol 5 Uvalu 84 150 06 Prague Czech Republic

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A comprehensive analysis of CD47 expression in various histological subtypes of soft tissue sarcoma: exploring novel opportunities for macrophage-directed treatments