CCL5, CCR1 and CCR5 in murine glioblastoma: immune cell infiltration and survival rates are not dependent on individual expression of either CCR1 or CCR5

Glioblastoma multiforme (GBM) is the most malignant brain tumor. Microglia/macrophages are found within human GBM where they likely promote tumor progression. We report that CCL5, CCR1, and CCR5 are expressed in glioblastoma. Individual deletion of CCR1 or CCR5 had little to no effect on survival of tumor bearing mice, or numbers of glioblastoma-infiltrated microglia/macrophages or lymphocytes. CCL5 promoted in vitro migration of wild type, CCR1- or CCR5-deficient microglia/macrophages that was blocked by the dual CCR1/CCR5 antagonist, Met-CCL5. These data suggest that CCL5 functions within the glioblastoma microenvironment through CCR1 and CCR5 in a redundant manner.     

Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/microglial cells in rat and human gliomas

Allograft inflammatory factor-1 (AIF-1) is a Ca²⁺-binding peptide that constitutes a potential modulator of macrophage activation and function during the immune response of the brain. Peptides termed microglia response factor-1 or ionized calcium-binding adaptor molecule-1 have been reported to be identical with AIF-1. We have investigated the expression of AIF-1 in the rat C6 glioblastoma and 9L gliosarcoma tumor models and additionally assessed AIF-1 expression in a diverse range of human astrocytomas by immunohistochemistry. AIF-1 was expressed by activated microglial cells and a subset of infiltrating macrophages in areas of infiltrative tumor growth and in compact tumor areas in both rat and human gliomas. Double-labeling experiments in rats and humans characterized the nature and the functional status of AIF-1⁺ cells. AIF-1 expression was detected in cells expressing major histocompatibility complex class II molecules and in a subset of activated macrophages/microglial cells. All MRP-8⁺ cells coexpressed AIF-1. In humans, there was a strong correlation of AIF-1-expressing activated macrophages/microglial cells with tumor malignancy (P < 0.0001). These results suggest that AIF-1 defines a distinct subset of tumor-associated activated macrophages/ microglial cells. Received: 5 October 1998 / Revised: 15 June 1999, 6 August 1999, 28 February 2000 / Accepted: 28 February 2000     

VEGF as a modulator of the innate immune response in glioblastoma

VEGF is an important factor in tumor vascularization and used as target for anti‐angiogenic treatment strategies in glioma. In this study, we demonstrate for the first time that VEGF is a modulator of the innate immune response with suppressive effects on the immunologic and pro‐angiogenic function of microglia/macrophages in a glioblastoma rodent model. High level of VEGF led to threefold enlarged tumor volumes and a pronounced remodeling of the vascular structure along with a reduced infiltration of microglia/macrophages by approximately 50%. Remaining microglia/macrophages showed an enhanced rate of apoptosis as well as significant downregulation of the VEGF‐receptor, VEGFR2, and others such as CXCR4. Consequently, we determined a substantially impaired migration of these microglia/macrophages to VEGF and SDF1α in vitro. Furthermore, we observed an increased presentation of the surface molecules MHCI and MHCII on microglia/macrophages from VEGF‐overexpressing gliomas that are essential for activation of the adaptive immune system. In contrast, the expression of pro‐inflammatory and suppressive cytokines, associated with the innate immune response, were mainly downregulated. Remarkably, the abundance of VEGF provoked less accumulation of microglia/macrophages within the perivascular niche and concomitantly reduced the release of pro‐angiogenic factors, like VEGF, suggesting a possible regulatory feedback mechanism. Thus, the quantity of VEGF in the glioma microenvironment seems to be crucial for the participation of microglia/macrophages on tumor progression and should be considered for developing novel therapeutic approaches.     

小胶质细胞/巨噬细胞在胶质母细胞瘤中作用的研究进展

胶质母细胞瘤（GBM）是成人最常见的颅内恶性肿瘤,具有极强的侵袭性。此外,由于化疗药物难以通过血脑屏障和胶质母细胞瘤的耐药性,及对放疗敏感性较差等特点,故预后极差。其中肿瘤微环境的改变起到了至关重要的作用,在微环境中胶质瘤相关的小胶质细胞/巨噬细胞（GAMs）的作用正逐渐被重视。GAMs不仅有中枢系统的常驻小胶质细胞,还有来自外周的巨噬细胞。GAMs还有截然不同的两种极化类型,即抑制肿瘤生长的M1表型和促进肿瘤生长的M2表型。并且GAMs不单单和肿瘤细胞具有联系,还与微环境中其他非癌性脑细胞也有互动。该文将从GAMs的来源、极化、与肿瘤微环境中各种细胞间的相互影响阐述其在GBM中的作用,并且从靶向治疗的角度探讨其最新的研究进展。     

Expression of P2X4 receptor in rat C6 glioma by tumor-associated macrophages and activated microglia

Here we report an immunohistochemical analysis of P2X4 receptor (P2X4R), an ATP-gated ion channel, expression in rat C6 glioma model. Striking P2X4R expression was detected in compact and infiltrative tumor growth areas. Expression of P2X4R by perivascular cells was observed in normal control brain and in brain areas not affected by tumor growth. Double-immunolabeling revealed that P2X4R was co-expressed by ED1+, AIF-1+, and EMAP II+ tumor-infiltrating microglia/macrophages; whereas on GFAP+ and nestin+ astrocytes P2X4R was hardly seen. Our results indicate that P2X4R expression defines a distinct subset of tumor-associated macrophages and activated microglia in glioma.     

Hypoxia‐induced polypoid giant cancer cells in glioma promote the transformation of tumor‐associated macrophages to a tumor‐supportive phenotype

AimsPolypoid giant cancer cells (PGCCs) represent a unique subgroup of stem‐like cells, acting as a critical factor in promoting the recurrence of various solid tumors. The effect of PGCCs on the tumor malignancy of glioma and its immune microenvironment remains unclear.MethodsBioinformatic analysis was performed to investigate the relationship between M2 tumor‐associated macrophages (TAMs) infiltration and survival of glioblastoma (GBM) patients. The spatial location of M2 TAMs in GBM was also investigated using the Ivy Glioblastoma Atlas Project (Ivy GAP) database. PGCCs were quantified in glioma of different grades. CoCl₂ was used to induce PGCCs in cultures of A172 cells. PGCCs, and their progeny cells in cultures were further evaluated for morphological features, tumorsphere formation, and TAMs activation.ResultsThe magnitude of M2 TAMs infiltration is significantly correlated with poor survival in GBM patients. M2 TAMs were enriched in the perinecrotic zone (PNZ) of GBM and positively correlated with hypoxic levels. Increased PGCCs were detected in glioma specimens of higher grades. CoCl₂ induced hypoxia and the transformation of A172 cultures into PGCCs, producing the progeny cells, PGCCs‐Dau, through asymmetric division. PGCCs and PGCCs‐Dau possessed tumor stem cell‐like features, while PGCCs‐Dau enhanced the polarization of TAMs into an M2 phenotype with relevance to immunosuppression and malignancy in GBM.ConclusionsPGCCs promote malignancy and immune‐suppressive microenvironment in GBM. PGCCs or their progeny cells may be a potential therapeutic target for GBM.     

CNS macrophages and peripheral myeloid cells in brain tumours

Primary brain tumours (gliomas) initiate a strong host response and can contain large amounts of immune cells (myeloid cells) such as microglia and tumour-infiltrating macrophages. In gliomas the course of pathology is not only controlled by the genetic make-up of the tumour cells, but also depends on the interplay with myeloid cells in the tumour microenvironment. Especially malignant gliomas such as glioblastoma multiforme (GBM) are notoriously immune-suppressive and it is now evident that GBM cells manipulate myeloid cells to support tumour expansion. The protumorigenic effects of glioma-associated myeloid cells comprise a support for angiogenesis as well as tumour cell invasion, proliferation and survival. Different strategies for inhibiting the pathological functions of myeloid cells in gliomas are explored, and blocking the tropism of microglia/macrophages to gliomas or manipulating the signal transduction pathways for immune cell activation has been successful in pre-clinical models. Hence, myeloid cells are now emerging as a promising target for new adjuvant therapies for gliomas. However, it is also becoming evident that some myeloid-directed glioma therapies may only be beneficial for distinct subclasses of gliomas and that a more cell-type-specific manipulation of either microglia or macrophages may improve therapeutic outcomes.     

Neurofibromatosis-1 heterozygosity increases microglia in a spatially and temporally restricted pattern relevant to mouse optic glioma formation and growth

Whereas carcinogenesis requires the acquisition of driver mutations in progenitor cells, tumor growth and progression are heavily influenced by the local microenvironment. Previous studies from our laboratory have used Neurofibromatosis-1 (NF1) genetically engineered mice to characterize the role of stromal cells and signals to optic glioma formation and growth. Previously, we have shown that Nf1+/- microglia in the tumor microenvironment are critical cellular determinants of optic glioma proliferation. To define the role of microglia in tumor formation and maintenance further, we used CD11b-TK mice, in which resident brain microglia (CD11b+, CD68+, Iba1+, CD45low cells) can be ablated at specific times after ganciclovir administration. Ganciclovir-mediated microglia reduction reduced Nf1 optic glioma proliferation during both tumor maintenance and tumor development. We identified the developmental window during which microglia are increased in the Nf1+/- optic nerve and demonstrated that this accumulation reflected delayed microglia dispersion. The increase in microglia in the Nf1+/- optic nerve was associated with reduced expression of the chemokine receptor, CX3CR1, such that reduced Cx3cr1 expression in Cx3cr1-GFP heterozygous knockout mice led to a similar increase in optic nerve microglia. These results establish a critical role for microglia in the development and maintenance of Nf1 optic glioma.     

Blood vessels expressing CD90 in human and rat brain tumors

Blood vessels in brain tumors, particularly glioblastomas, have been shown to express CD90. CD90⁺ cells in and around blood vessels in cancers including brain tumors have been identified as endothelial cells, cancer stem cells, fibroblasts or pericytes. In this study, we aimed to determine the nature or type(s) of cells that express CD90 in human brain tumors as well as an experimental rat glioma model by double immunofluorescence staining. The majority of CD90⁺ cells in human glioblastoma tissue expressed CD31, CD34 and von Willebrand factor, suggesting that they were endothelial cells. Vasculatures in a metastatic brain tumor and meningioma also expressed CD90. CD90⁺ cells often formed glomeruloid structures, typical of angiogenesis in malignant tumors, not only in glioblastoma but also in metastatic tumors. Some cells in the middle and outer layers of the vasculatures expressed CD90. Similar results were obtained in the rat glioma model. There were cells expressing both α‐smooth muscle actin and CD90 in the middle layer of blood vessels, indicating that smooth muscle cells and/or pericytes may express CD90. CD90⁺ vasculatures were surrounded by tumor‐associated macrophages (TAMs). Thus, in addition to endothelial cells, some other types of cells, such as smooth muscle cells, pericytes and fibroblasts constituting the vasculature walls in brain tumors expressed CD90. Because CD90 has been shown to interact with integrins expressed by circulating monocytes, CD90 might be involved in angiogenesis through recruitment and functional regulation of TAMs in tumors. CD90⁺ vasculatures may also interact with tumor cells through interactions with integrins. Because CD90 was not expressed by vasculatures in normal brain tissue, it might be a possible therapeutic target to suppress angiogenesis and tumor growth.     

Distribution and characterization of microglia/macrophages in human brain tumors

The role of inflammatory reactions in brain tumors is still unclear. In particular, there is little information about the participation of the microglia/macrophage cell system. We therefore investigated 72 surgical biopsy samples of brain tumors (astrocytoma, glioblastoma, oligodendroglioma, ependymoma, medulloblastoma, cerebral lymphoma, gangliocytoma, neurocytoma and germinoma) and the brains of eight cases with malignant gliomas that came to autopsy, using immunohistochemical markers for the monocyte/macrophage lineage (Ki-M1P, HLA-DR, KP1, My4, My7, Ki-M1, Ki-M6, EBM 11). These markers allowed us to characterize four subtypes of the microglia/macrophage cell system: ramified microglia, ameboid microglia, perivascular microglia and brain macrophages. Among the different tumors, glioblastomas and anaplastic gliomas showed the largest number of mixed cell populations, which consisted of macrophages and ramified and ameboid microglia. In glial tumors of low malignancy fewer, predominantly ameboid, microglia were found. Neuronal tumors showed only a mild increase of microglia. Cerebral lymphomas contained macrophages diffusely distributed within the tumor center, while activated microglia were prominent at the border zone and in the adjacent brain tissue. The autopsy cases were used to study the morphometric distribution of microglia/macrophages. There was a significant increase of microglia/macrophages within the tumor, but no differences were seen between central and peripheral tumor areas. The non-neoplastic gray and white matter contained more microglial cells than controls. We conclude that the distribution pattern of ameboid and ramified microglial cells and macrophages is distinct in most of the investigated tumor types, underlining the complex immunological function of the microglia/macrophage cell system. Received: 24 July 1995 / Revised: 11 December 1995 / Accepted: 9 February 1996     

Reducing host aldose reductase activity promotes neuronal differentiation of transplanted neural stem cells at spinal cord injury sites and facilitates locomotion recovery

Neural stem cell(NSC)transplantation is a promising strategy for replacing lost neurons following spinal cord injury.However,the survival and differentiation of transplanted NSCs is limited,possibly owing to the neurotoxic inflammatory microenvironment.Because of the important role of glucose metabolism in M1/M2 polarization of microglia/macrophages,we hypothesized that altering the phenotype of microglia/macrophages by regulating the activity of aldose reductase(AR),a key enzyme in the polyol pathway of glucose metabolism,would provide a more beneficial microenvironment for NSC survival and differentiation.Here,we reveal that inhibition of host AR promoted the polarization of microglia/macrophages toward the M2 phenotype in lesioned spinal cord injuries.M2 macrophages promoted the differentiation of NSCs into neurons in vitro.Transplantation of NSCs into injured spinal cords either deficient in AR or treated with the AR inhibitor sorbinil promoted the survival and neuronal differentiation of NSCs at the injured spinal cord site and contributed to locomotor functional recovery.Our findings suggest that inhibition of host AR activity is beneficial in enhancing the survival and neuronal differentiation of transplanted NSCs and shows potential as a treatment of spinal cord injury.     

The flavonoid rutin and its aglycone quercetin modulate the microglia inflammatory profile improving antiglioma activity

Microglia cells are the immune effector in the Central Nervous System (CNS). However, studies have showed that they contribute more to glioma progression than to its elimination. Rutin and its aglycone quercetin are flavonoids present in many fruits as well as plants and have been demonstrated to bear anti-inflammatory, antioxidant and antitumor properties also to human glioblastoma cell lines. Previous studies also demonstrated that rutin, isolated from the Brazilian plant Dimorphandra mollis Bent., presents immunomodulatory effect on astrocytes and microglia. In this study, we investigate the antitumor and immunomodulatory properties of rutin and its aglycone quercetin on the viability of glioma cells alone and under direct and indirect interaction with microglia. Flavonoid treatment of rat C6 glioma cells induced inhibition of proliferation and migration, and also induced microglia chemotaxis that was associated to the up regulation of tumor necrosis factor (TNF) and the down regulation of Interleukin 10 (IL-10) at protein and mRNA expression levels, regulation of mRNA expression for chemokines CCL2, CCL5 and CX3CL1, and Heparin Binding Growth Factor (HDGF), Insulin-like growth factor (IGF) and Glial cell-derived neurotrophic factor (GDNF) growth factors. Treatment of human U251 and TG1 glioblastoma cells with both flavonoids also modulated negatively the expression of mRNA for IL-6 and IL-10 and positively the expression of mRNA for TNF characterizing changes to the immune regulatory profile. Treatment of microglia and C6 cells either in co-cultures or during indirect interaction, via conditioned media from glioma cells treated with flavonoids or via conditioned media from microglia treated with flavonoids reduced proliferation and migration of glioma cells. It also directed microglia towards an inflammatory profile with increased expression of mRNA for IL-1β, IL-6, IL-18 and decreased expression of mRNA for nitric oxide synthase 2 (NOS2) and prostaglandin-endoperoxide synthase 2 (PTGS2), arginase and transforming growth factor beta (TGF-β), as well as Insulin-like growth factor (IGF). Treatment of U251 cells with flavonoids also reduced tumorigenesis when the cells were xenotransplanted in rat brains, and directed microglia and also astrocytes in the microenvironment of tumor cell implantation as well as in the brain parenchyma to a not favorable molecular inflammatory profile to the glioma growth, as observed in cultures. Together these results demonstrate that the flavonoid rutin and its aglycone quercetin present antiglioma effects related to the property of modulating the microglial inflammatory profile and may be considered for molecular and preclinical studies as adjuvant molecules for treatment of gliomas.     

The role of microglia in central nervous system immunity and glioma immunology

The central nervous system (CNS) historically has been considered an immune-privileged organ, lacking a lymphatic system and shielded from the circulatory system by the blood-brain barrier. Microglia are an abundant portion of the CNS cell population, comprising 5% to 20% of the total glial cell population, and are as numerous as neurons. A crucial function of microglia is the ability to generate significant innate and adaptive immune responses. Microglia are involved in first line innate immunity of the CNS. Proper antigen presentation is critical in the generation of specific, durable responses by the adaptive immune system, and requires interaction between the T cell receptor and processed antigen peptide presented on major histocompatibility complex (MHC) molecules by the antigen presenting cells (APC). Microglia also have a large regulatory role in CNS immunity. Histopathologic studies of glioma tissue have consistently shown high levels of infiltrating microglia. Microglia are also localized diffusely throughout the tumor, rather than to the areas of necrosis, and phagocytosis of glioma cells or debris by microglia is not observed. Recent evidence indicates that glioma-infiltrating microglia/macrophages might be promoting tumor growth by facilitating immunosuppression of the tumor microenvironment. When activated, microglia can be potent immune effector cells, able to perform a broad range of functions, and they mediate both innate and adaptive responses during CNS injury and disease while remaining quiescent in the steady state. Their versatility in bridging the gap between the immune-privileged CNS and the peripheral immune system, in addition to their significant numbers in gliomas, makes them an attractive candidate in immunotherapy for gliomas. An enhanced understanding of microglia–glioma interaction may provide better methods to manipulate the glioma microenvironment to allow the generation of a specific and durable anti-glioma immunity. The role of microglia in CNS immunity is reviewed, with a focus on key advances made in glioma immunology.     

Reactive retinal microglia,neuronal survival,and the formation of retinal folds and detachments

Reactive microglia and macrophages are prevalent in damaged retinas. Accordingly, we investigate how the activation or ablation of microglia/macrophages influences the survival of neurons in the chick retina in vivo. We applied intraocular injections of interleukin 6 (IL6) to stimulate the reactivity of microglia/macrophages and clodronate‐liposomes to ablate microglia/macrophages. Activation of the microglia/macrophages with IL6 delays the death of retinal neurons from N‐methyl‐D‐aspartate (NMDA) ‐induced excitotoxicity. In addition, activation of microglia/macrophages combined with colchicine‐mediated retinal damage diminished the survival of ganglion cells. Application of IL6 after an excitotoxic insult greatly exacerbates the damage, and causes widespread retinal detachments and folds, accompanied by accumulation of microglia/macrophages in the subretinal space. Damage‐induced retinal folds and detachments were significantly reduced by the ablation of microglia/macrophages. We conclude that microglial reactivity is detrimental to the survival of ganglion cells in colchicine‐damaged retinas and detrimental to the survival of photoreceptors in retinal folds. In addition, we conclude that IL6‐treatment transiently protects amacrine and bipolar cells against an excitotoxic insult. We propose that suppressing reactivity of microglia/macrophages may be an effective means to lessen the damage and vision loss resulting from damage, in particular during retinal detachment injuries. GLIA 2015;63:313–327     

scRNA-seq and proteomics reveal the distinction of M2-like macrophages between primary and recurrent malignant glioma and its critical role in the recurrence

Aims

Tumor-associated macrophages (TAMs) in the immune microenvironment play an important role in the increased drug resistance and recurrence of malignant glioma, but the mechanism remains incompletely inventoried. The focus of this study was to investigate the distinctions of M2-like TAMs in the immune microenvironment between primary and recurrent malignant glioma and its influence in the recurrence.

Methods

We employed single-cell RNA sequencing to construct a single-cell atlas for a total of 23,010 individual cells from 6 patients with primary or recurrent malignant glioma and identified 5 cell types, including TAMs and malignant cells. Immunohistochemical techniques and proteomics analysis were performed to investigate the role of intercellular interaction between malignant cells and TAMs in the recurrence of malignant glioma.

Results

Six subgroups of TAMs were annotated and M2-like TAMs were found to increase in recurrent malignant glioma significantly. A pseudotime trajectory and a dynamic gene expression profiling during the recurrence of malignant glioma were reconstructed. Up-regulation of several cancer pathways and intercellular interaction-related genes are associated with the recurrence of malignant glioma. Moreover, the M2-like TAMs can activate the PI3K/Akt/HIF-1α/CA9 pathway in the malignant glioma cells via SPP1-CD44-mediated intercellular interaction. Interestingly, high expression of CA9 can trigger the immunosuppressive response in the malignant glioma, thus promoting the degree of malignancy and drug resistance.

Conclusion

Our study uncovers the distinction of M2-like TAMs between primary and recurrent glioma, which offers unparalleled insights into the immune microenvironment of primary and recurrent malignant glioma.