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Background Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Whilst the role of the efflux transporters are well established in GBM, the expression and function of uptake transporters, such as the organic anion transporting polypeptide (OATP) family, are not well understood. OATPs possess broad substrate specificity that includes anti-cancer agents; therefore, we sought to investigate the expression of four OATP isoforms in human GBM cell types using patient tumor tissue. Methods We used fluorescent immunohistochemical labeling of paraffin-embedded surgically resected tissues and single-cell image analysis methods to explore the expression of the OATP isoforms in different tumor cell types through co-labeling with cell-type specific markers, such as IBA1 (pan-myeloid), GFAP (tumor cell), PDGFRβ (stromal cell), and UEA-1-lectin (endothelial). Results We found significant over-expression of all the OATP isoforms (OATP1A2, 2B1, 1C1 and 4A1) in GBM tumor sections when compared to non-neoplastic brain. A single-cell image analysis revealed that OATPs were significantly upregulated throughout the tumor parenchyma, with significantly higher expression found on lectin-positive blood vessels and IBA1-positive myeloid cells in GBM compared to non-tumor brain tissue. Qualitative analysis of the four OATP isoforms demonstrated greater expression of OATP4A1 in peri-necrotic regions of GBM tissue, which correlated with hypoxia-related markers within the Ivy GAP RNAseq dataset. Conclusion Here, we demonstrate, for the first time, the protein expression of four OATPs in human GBM tissue, including upregulation within the tumor microenvironment by myeloid cells and tumor vasculature, and isoform-specific upregulation within hypoxic niches.

Summary Microglia are the primary innate immune effectors of the central nervous system. Although numerous protocols have been developed to isolate fetal mouse microglia, the isolation of adult mouse microglia has proven more difficult. Here, we present a simple, widely accessible protocol to isolate pure microglia cultures from 4- to 14-month-old mouse brains using their adherent properties in vitro. These isolated microglia recapitulate the adherent properties of adult human microglia and present a more suitable model for studying age-related diseases. For complete details on the use and execution of this protocol in adult human microglia, please refer to Rustenhoven et al. (2016)., Graphical abstract, Highlights • An optimized protocol for the isolation of pure adult mouse microglia • Utilization of the adherent properties of adult mouse microglia for isolation • Isolated microglial cultures suitable for a range of functional assays, Microglia are the primary innate immune effectors of the central nervous system. Although numerous protocols have been developed to isolate fetal mouse microglia, the isolation of adult mouse microglia has proven more difficult. Here, we present a simple, widely accessible protocol to isolate pure microglia cultures from 4- to 14-month-old mouse brains using their adherent properties in vitro. These isolated microglia recapitulate the adherent properties of adult human microglia and present a more suitable model for studying age-related diseases.

Background Microglia and tumor-associated macrophages (TAMs) constitute up to half of the total tumor mass of glioblastomas. Despite these myeloid populations being ontogenetically distinct, they have been largely conflated. Recent single-cell transcriptomic studies have identified genes that distinguish microglia from TAMs. Here we investigated whether the translated proteins of genes enriched in microglial or TAM populations can be used to differentiate these myeloid cells in immunohistochemically stained human glioblastoma tissue. Methods Tissue sections from resected low-grade, meningioma, and glioblastoma (grade IV) tumors and epilepsy tissues were immunofluorescently triple-labeled for Iba1 (pan-myeloid marker), CD14 or CD163 (preferential TAM markers), and either P2RY12 or TMEM119 (microglial-specific markers). Using a single-cell-based image analysis pipeline, we quantified the abundance of each marker within single myeloid cells, allowing the identification and analysis of myeloid populations. Results P2RY12 and TMEM119 successfully discriminated microglia from TAMs in glioblastoma. In contrast, CD14 and CD163 expression were not restricted to invading TAMs and were upregulated by tumor microglia. Notably, a higher ratio of microglia to TAMs significantly correlated with increased patient survival. Conclusions We demonstrate the validity of previously defined microglial-specific genes P2RY12 and TMEM119 as robust discriminators of microglia and TAMs at the protein level in human tissue. Moreover, our data suggest that a higher proportion of microglia may be beneficial for patient survival in glioblastoma. Accordingly, this tissue-based method for myeloid population differentiation could serve as a useful prognostic tool.

BACKGROUND Glioblastoma multiforme (GBM) is the most common primary brain tumour that affects adults. This aggressive tumour is invariably fatal, carrying a rapid progression and a dismal median survival period of only 15 months despite multimodal treatment approaches. Central to GBM pathogenesis is the immunosuppressive profile of these tumours. The two cell types that are highly abundant in these tumours and play critical roles in the immunosuppressive niche are the brain’s resident microglia and their peripheral counterparts - tumour associated macrophages (TAMs). Despite microglia and TAMs being ontogenetically distinct, these cells have largely been grouped together in research owing to the previous lack of cell-specific markers. Recent evidence has suggested that although TAMs may hold a predominantly pro-tumoral role, microglia may adopt a more anti-tumoral phenotype. Therefore, the differentiation of these two cell types is critical in elucidating the potentially characteristic roles of these two cell types in GBM pathogenesis. MATERIAL AND METHODS Tissue sections from resected low- and high-grade glioma tumours, along with epilepsy tissue (control), were used for immunohistochemistry (IHC) staining of macrophage pan-makers (Iba1, CD45, PU.1) and microglial-specific markers (TMEM119, P2RY12). Marker co-localisation was then used to differentiate microglia from TAMs. We further investigated a wider subset of cell-specific markers using multicolour flow cytometry and immunocytochemical staining of isolated cells from patient tissue samples. RESULTS Immunofluorescent staining of glioma and epilepsy tissue revealed two clear populations of cells; one population displayed long processes and co-labelling for both pan- and microglial-specific markers, whilst the other population displayed an amoeboid phenotype with only pan-maker staining. Preliminary analysis comparing microglia/TAM populations in low-grade, high-grade and epilepsy tissue suggests a clear difference in the proportions of these cells. CONCLUSION Our work complements RNA-Seq studies, showing that TMEM119 and P2RY12, alongside other markers, can indeed identify two distinct myeloid cell populations within glioma tissue. This provides a strong basis for further study where we aim to elucidate the respective roles of microglia and TAMs within tumours. Ultimately, this may hold the potential for differential targeting of these cells using immunotherapies.