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High-resolution imaging of sentinel lymph nodes (SLN) from melanoma patients is a crucial approach to specify staging and determine individuals requiring adjuvant treatment. Current histologic SLN analysis has the substantial drawback that only a small portion of the node is sampled while most of the tissue is discarded which might explain the high false-negative rate of SLN diagnosis. Therefore, we developed an algorithm-enhanced light sheet fluorescence microscopy (LSFM) approach to three-dimensionally reconstruct the entire SLN with the power to identify single tumor cells. We comprehensively quantified total tumor volume while simultaneously visualizing cellular and anatomical hallmarks of the associated SLN architecture. In a first-in-human prospective study (21 SLN from 11 melanoma patients), LSFM not only identified all metastases seen histologically, but additionally detected metastases not recognized by routine histology. Thus, our 3-D digital pathology approach can increase sensitivity and accuracy of SLN-metastasis detection and potentially alleviate the need for conventional histopathological assessment in the future. Contact: Professor Dr. Joachim Klode University Hospital Essen Department of Dermatology Hufelandstraße 55, 45147 Essen, Germany Phone.: +49 201 723-1812 Fax: +49 201 723-5424 E-Mail: joachim.klode@uk-essen.de Professor Dr. Matthias Gunzer University Hospital Essen Institute for Experimental Immunology and Imaging Hufelandstraße 55, 45147 Essen, Germany Phone: +49 201 183 6640 Fax: +49 201 183 6642 E-Mail: matthias.gunzer@uni-due.de

The manifestation of brain metastases in patients with advanced melanoma is a common event that limits patient's survival and quality of life. The immunosuppressive properties of the brain parenchyma are very different compared to the rest of the body, making it plausible that the current success of cancer immunotherapies is specifically limited here. In melanoma brain metastases, the reciprocal interplay between immunosuppressive mediators such as indoleamine 2, 3-dioxygenase (IDO) or programmed cell death-ligand 1 (PD-L1) in the context of neoplastic transformation are far from being understood. Therefore, we analyzed the immunoreactive infiltrate (CD45, CD3, CD8, Forkhead box P3 [FoxP3], CD11c, CD23, CD123, CD68, Allograft Inflammatory factor 1[AIF-1]) and PD-L1 with respect to IDO expression and localization in melanoma brain metastases but also in matched metastases at extracranial sites to correlate intra- and interpatient data with therapy response and survival. Comparative tissue analysis identified macrophages/microglia as the major source of IDO expression in melanoma brain metastases. In contrast to the tumor infiltrating lymphocytes, melanoma cells per se exhibited low IDO expression levels paralleled by cell surface presentation of PD-L1 in intracranial metastases. Absolute numbers and pattern of IDO-expressing cells in metastases of the brain correlated with recruitment and localization of CD8⁺ T cells, implicating dynamic impact on the regulation of T cell function in the brain parenchyma. However, paired analysis of matched intra- and extracranial metastases identified significantly lower fractions of cytotoxic CD8⁺ T cells in intracranial metastases while all other immune cell populations remain unchanged. In line with the already established clinical benefit for PD-L1 expression in extracranial melanoma metastases, Kaplan-Meier analyses correlated PD-L1 expression in brain metastases with favorable outcome in advanced melanoma patients undergoing immune checkpoint therapy. In summary, our data provide new insights into the landscape of immunosuppressive factors in melanoma brain metastases that may be useful in the implication of novel therapeutic strategies for patients undergoing cancer immunotherapy. OA Förderung 2020

Background A high neutrophil-to-lymphocyte ratio in the circulation and high frequencies of tumor-associated neutrophils (TAN) in malignant tissue are associated with poor outcome and tumor progression in patients with cancer. It is hypothesized that immunosuppressive neutrophil activity (aka PMN-MDSC activity) contributes to this effect. In addition, this MDSC activity represents a major resistance mechanism in different types of immunotherapy. The exact cellular identity of human PMN-MDSC is still under debate. Improved immunomonitoring and functional characterization of MDSC is needed in order to exploit these cells as novel biomarkers and targets for combination immunotherapy. Methods In this study, we sought to identify the neutrophil subset that contained the highest T cell suppressive activity. To this end, we purified different subsets of circulating neutrophils by FACS and performed multi-parameter immunofluorescence together with digital pathology on 2-D and 3-D tumor tissue samples. Results We found that a population of circulating, mature, arginase I+ neutrophils that co-purified with mononuclear cells in density gradients, most potently suppressed T cell function in multiple in vitro assays. These PMN-MDSC were also superior to monocytic MDSC in T cell suppression. Using a novel technology of tissue whole mount labelling, clearing and imaging we derived 3-D spatial maps of neutrophil – T cell interaction in human tumors. We found that T cells, which were conjugated to arginase I+, myeloperoxidase+ TAN, had significantly reduced expression of proliferation and cytotoxicity markers. In patients, frequent conjugation of T cells to those PMN-MDSC was associated with poor prognosis. In contrast to circulating PMN-MDSC, tissue PMN-MDSC expressed high amounts of LOX-1 (oxidized low density lipoprotein receptor 1) and a high intratumoral frequency of LOX-1+ PMN-MDSC was associated with poor survival. Conclusions We identified and characterized PMN-MDSC activity in human cancer patients. Our findings will facilitate and improve MDSC immunomonitoring and MDSC tarteting in combination therapies. Ethics Approval Use of patient material was approved by the Ethics committee of the Medical Faculty of the University of Duisburg-Essen kein CA