The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer‐permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T‐cell‐directed therapies fail to elicit effective anti‐tumor immune responses. Here, we seek to evaluate the applicability of radio‐immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti‐cancer activity. Radiotherapy‐induced immune modulation resulted in an increase in cytotoxic T‐cell numbers and prevented the induction of lymphocyte‐mediated immune suppression. Radio‐immunotherapy led to significantly improved tumor control with prolonged median survival in experimental breast‐to‐brain metastasis. However, long‐term efficacy was not observed. Recurrent brain metastases showed accumulation of blood‐borne PD‐L1+ myeloid cells after radio‐immunotherapy indicating the establishment of an immune suppressive environment to counteract re‐activated T‐cell responses. This finding was further supported by transcriptional analyses indicating a crucial role for monocyte‐derived macrophages in mediating immune suppression and regulating T‐cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio‐immunotherapy in brain metastases.

Diabetes is associated with platelet hyper-reactivity and enhanced risk of thrombosis development. Here we compared protein expression in platelets from healthy donors and diabetic patients to identify differentially expressed proteins and their possible function in platelet activation. Mass spectrometry analyses identified cyclin Y (CCNY) in platelets and its reduced expression in platelets from diabetic patients, a phenomenon that could be attributed to the increased activity of calpains. To determine the role of CCNY in platelets, mice globally lacking the protein were studied. CCNY-/- mice demonstrated lower numbers of circulating platelets but platelet responsiveness to thrombin and a thromboxane A2 analogue were comparable with that of wild-type mice, as was agonist-induced α and dense granule secretion. CCNY-deficient platelets demonstrated enhanced adhesion to fibronectin and collagen as well as an attenuated spreading and clot retraction, indicating an alteration in “outside in” integrin signalling. This phenotype was accompanied by a significant reduction in the agonist-induced tyrosine phosphorylation of β3 integrin. Taken together we have shown that CCNY is present in anucleated platelets where it is involved in the regulation of integrin-mediated outside in signalling associated with thrombin stimulation.

Macrophages represent a highly plastic cell type,indispensable for tissue and organ homeostasis, as well as innate immunity. Basic and translational research attributed tumor-promoting functions to macrophages, and their presence is often associated to poor patient prognosis and therapy resistance. While brain-resident macrophages, the so-called microglia (MG), represent the major immune cell type in the parenchyma under normal conditions, primary and metastatic brain tumors induce the recruitment of different immune cell types from the periphery, including monocyte-derived macrophages (MDM). Controversy remained about the redundancy of disease-associated molecular signatures and functions. The identification of markers that reliably distinguish brain-resident from blood-borne tumor-associated macrophages (TAMs) allowed the interrogation of molecular traits of different TAM populations in mouse and human brain tumors. Using RNA-Seq, we demonstrated that TAMs rapidly acquire disease-associated transcriptional programs upon initial tumor infiltration, while gene expression remained stable during different stages of BrM progression. Across different BrM models, disease-associated transcriptional changes revealed lineage-specific, non-redundant functions of TAM populations, which was further reflected by cell type-specific occupation of different niches within the BrM microenvironment. Furthermore, we observed dose- and cell type-specific immune modulatory effects of whole brain radiotherapy on myeloid cells in BrM leading to a transient loss of disease-associated transcriptional programs predominately in blood-borne myeloid populations. This effect can at least in part be attributed to a replenishment of the recruited macrophage pool. This observation was further supported by scRNA-Seq analyses revealing higher heterogeneity of TAM-MDM compared to TAM-MG under treatment-naïve conditions and in response to radiotherapy. Together, our results point towards the phenotypic plasticity of TAMs, especially MDMs, and the contribution of each compartment in instigating cancer-associated inflammation or the establishment of an immuno-suppressive TME. While TAM-MG exert functions related to pro-inflammatory responses, TAM-MDM are rather involved in tissue repair and regulation of adaptive immune cell functions.