Abstract BACKGROUND Current treatment modalities, including surgical resection, radiosurgery and immune checkpoint inhibitors, have improved local control rates of brain metastases, but overall prognosis is still poor. AIM: To investigate the immune microenvironment in brain metastases for better understanding of treatment failures. METHODS We identified 42 patients with melanoma (M), breast (B) and lung (L) brain metastases and performed multiplex immunofluorescent staining for immune markers (CD4, CD8, CD45, PD1, PD-L1) and quantitative analysis with HALO software. NanoString nCounter mRNA gene expression assay was used for identification of immune pathways. CyTOF and single cell RNA sequencing were done on 12 different brain metastases and their matched peripheral blood samples. RESULTS The median CD8, CD4 and PD1 density (as a total number of cells) was 0.24%, 0.13%, and 0.07% for M, 0.24%, 0.51% and 0.08% for B, and 0.87%, 1.36%, 0.67% for L. CD4 and CD8+PD1+ expression was significantly different between all groups (p=0.01), CD4+PD1+ in M vs. L (p=0.03) and PD1 in B vs. L (p=0.03). No significant difference was found in terms of CD45 positive cells (M 2.13%, B, 1.99%, L 4.95%). PD-L1 expression was significantly different between histologies; the highest expression was present in breast metastases (M: 1.51%, B: 49.98%, L: 31.90%, p=0.0002). Spatial distribution revealed a characteristic pattern with a peritumoral border infiltrate in M, and predominant intratumoral distribution in B and L (M 0.2%, B 2.6%, L 3.6%, p=0.02). CyTOF clustering demonstrated that macrophages are the most prevalent immune population overall (83.08%), with an inhibitory phenotype and gene expression pattern present in different histologies (S100A9, CCL20, IFI16, MARCO). T-cells (predominantly CD4+, 3.6%), and B-cells (1.5%) comprise the remaining immune cell populations. CONCLUSION Neoadjuvant targeted treatment approaches may be needed to alter predominance of inhibitory macrophages and recruit activated T lymphocytes in brain metastases.