CD13 expression affects glioma patient survival and influences key functions of human glioblastoma cell lines in vitro.

CD13 (APN) is an Alanyl-Aminopeptidase with diverse functions. The role of CD13 for gliomas is still unknown. In this study, data of glioma patients obtained by TCGA and CGGA databases were used to evaluate the survival rate and prognostic value of CD13 expression level. Protein expression of CD13 was confirmed by immunofluorescence staining of fresh patient tissues. Eight human glioblastoma cell lines were studied by RT-PCR, Western Blot, immunofluorescence staining and flow cytometry to define CD13 expression. Cell lines with different CD13 expression status were treated with a CD13 inhibitor, bestatin, and examined by MTT, scratch and colony formation assaysas well as by apoptosis assay and Western Blots. Bioinformatics analysis indicated that patients with high expression of CD13 had poor survival and prognosis. Additionally, CD13 protein expression was positively associated with clinical malignant characteristics. Investigated glioblastoma cell lines showed distinct expression levels and subcellular localization of CD13 with intracellular enrichment. Bestatin treatment reduced proliferation, migration and colony formation of glioma cells in a CD13-dependent manner while apoptosis was increased. In summary, CD13 has an impact on glioma patient survival and is important for the main function of specific glioma cells.

Microglia/macrophages express alternative proangiogenic factors depending on granulocyte content in human glioblastoma.

Myeloid cells are an inherent part of the microenvironment of glioblastoma multiforme (GBM). There is growing evidence for their participation in mechanisms of tumor escape, especially in the development of resistance following initially promising anti-VEGF/VEGFR treatment. Thus, we sought to define the capability of myeloid cells to contribute to the expression of proangiogenic molecules in human GBM. We investigated GBM specimens in comparison with anaplastic astrocytoma (WHO grade III) and epilepsy patient samples freshly obtained from surgery. Flow cytometric analyses revealed two distinct CD11b+ CD45+ cell populations in GBM tissues, which were identified as microglia/macrophages and granulocytes. Due to varied granulocyte influx, GBM samples were subdivided into groups with low (GBM-lPMNL) and high (GBM-hPMNL) numbers of granulocytes (polymorphonuclear leukocytes; PMNL), which were related to activation of the microglia/macrophage population. Microglia/macrophages of the GBM-lPMNL group were similar to those of astrocytoma specimens, but those of GBM-hPMNL tissues revealed an altered phenotype by expressing high levels of CD163, TIE2, HIF1α, VEGF, CXCL2 and CD13. Although microglia/macrophages represented the main source of alternative proangiogenic factors, additionally granulocytes participated by production of IL8 and CD13. Moreover, microglia/macrophages of the GBM-hPMNL specimens were highly associated with tumor blood vessels, accompanied by remodeling of the vascular structure. Our data emphasize that tumor-infiltrating myeloid cells might play a crucial role for limited efficacy of anti-angiogenic therapy bypassing VEGF-mediated pathways through expression of alternative proangiogenic factors. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Regulation of the Receptor Tyrosine Kinase AXL in Response to Therapy and Its Role in Therapy Resistance in Glioblastoma.

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM.

Microglia as target for anti-inflammatory approaches to prevent secondary brain injury after subarachnoid hemorrhage (SAH).

BACKGROUND: Microglia-driven cerebral spreading inflammation is a key contributor to secondary brain injury after SAH. Genetic depletion or deactivation of microglia has been shown to ameliorate neuronal cell death. Therefore, clinically feasible anti-inflammatory approaches counteracting microglia accumulation or activation are interesting targets for SAH treatment. Here, we tested two different methods of interference with microglia-driven cerebral inflammation in a murine SAH model: (i) inflammatory preconditioning and (ii) pharmacological deactivation. METHODS: 7T-MRI-controlled SAH was induced by endovascular perforation in four groups of C57Bl/6 mice: (i) Sham-operation, (ii) SAH naïve, (iii) SAH followed by inflammatory preconditioning (LPS intraperitoneally), and (iv) SAH followed by pharmacological microglia deactivation (colony-stimulating factor-1 receptor-antagonist PLX3397 intraperitoneally). Microglia accumulation and neuronal cell death (immuno-fluorescence), as well as activation status (RT-PCR for inflammation-associated molecules from isolated microglia) were recorded at day 4 and 14. Toll-like receptor4 (TLR4) status was analyzed using FACS. RESULTS: Following SAH, significant cerebral spreading inflammation occurred. Microglia accumulation and pro-inflammatory gene expression were accompanied by neuronal cell death with a maximum on day 14 after SAH. Inflammatory preconditioning as well as PLX3397-treatment resulted in significantly reduced microglia accumulation and activation as well as neuronal cell death. TLR4 surface expression in preconditioned animals was diminished as a sign for receptor activation and internalization. CONCLUSIONS: Microglia-driven cerebral spreading inflammation following SAH contributes to secondary brain injury. Two microglia-focused treatment strategies, (i) inflammatory preconditioning with LPS and (ii) pharmacological deactivation with PLX3397, led to significant reduction of neuronal cell death. Increased internalization of inflammation-driving TLR4 after preconditioning leaves less receptor molecules on the cell surface, providing a probable explanation for significantly reduced microglia activation. Our findings support microglia-focused treatment strategies to overcome secondary brain injury after SAH. Delayed inflammation onset provides a valuable clinical window of opportunity.

The CXCL2/IL8/CXCR2 Pathway Is Relevant for Brain Tumor Malignancy and Endothelial Cell Function.

We aimed to evaluate the angiogenic capacity of CXCL2 and IL8 affecting human endothelial cells to clarify their potential role in glioblastoma (GBM) angiogenesis. Human GBM samples and controls were stained for proangiogenic factors. Survival curves and molecule correlations were obtained from the TCGA (The Cancer Genome Atlas) database. Moreover, proliferative, migratory and angiogenic activity of peripheral (HUVEC) and brain specific (HBMEC) primary human endothelial cells were investigated including blockage of CXCR2 signaling with SB225502. Gene expression analyses of angiogenic molecules from endothelial cells were performed. Overexpression of VEGF and CXCL2 was observed in GBM patients and associated with a survival disadvantage. Molecules of the VEGF pathway correlated but no relation for CXCR1/2 and CXCL2/IL8 was found. Interestingly, receptors of endothelial cells were not induced by addition of proangiogenic factors in vitro. Proliferation and migration of HUVEC were increased by VEGF, CXCL2 as well as IL8. Their sprouting was enhanced through VEGF and CXCL2, while IL8 showed no effect. In contrast, brain endothelial cells reacted to all proangiogenic molecules. Additionally, treatment with a CXCR2 antagonist led to reduced chemokinesis and sprouting of endothelial cells. We demonstrate the impact of CXCR2 signaling on endothelial cells supporting an impact of this pathway in angiogenesis of glioblastoma.

Tumor-Associated Microglia/Macrophages as a Predictor for Survival in Glioblastoma and Temozolomide-Induced Changes in CXCR2 Signaling with New Resistance Overcoming Strategy by Combination Therapy.

Tumor recurrence is the main challenge in glioblastoma (GBM) treatment. Gold standard therapy temozolomide (TMZ) is known to induce upregulation of IL8/CXCL2/CXCR2 signaling that promotes tumor progression and angiogenesis. Our aim was to verify the alterations on this signaling pathway in human GBM recurrence and to investigate the impact of TMZ in particular. Furthermore, a combi-therapy of TMZ and CXCR2 antagonization was established to assess the efficacy and tolerability. First, we analyzed 76 matched primary and recurrent GBM samples with regard to various histological aspects with a focus on the role of TMZ treatment and the assessment of predictors of overall survival (OS). Second, the combi-therapy with TMZ and CXCR2-antagonization was evaluated in a syngeneic mouse tumor model with in-depth immunohistological investigations and subsequent gene expression analyses. We observed a significantly decreased infiltration of tumor-associated microglia/macrophages (TAM) in recurrent tumors, while a high TAM infiltration in primary tumors was associated with a reduced OS. Additionally, more patients expressed IL8 in recurrent tumors and TMZ therapy maintained CXCL2 expression. In mice, enhanced anti-tumoral effects were observed after combi-therapy. In conclusion, high TAM infiltration predicts a survival disadvantage, supporting findings of the tumor-promoting phenotype of TAMs. Furthermore, the combination therapy seemed to be promising to overcome CXCR2-mediated resistance.

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.

EphB4 mediates resistance to antiangiogenic therapy in experimental glioma.

INTRODUCTION: Alterations in vascular morphogenesis are hallmarks of antiangiogenesis-resistant tumor vessels. Vascular morphogenesis is regulated by ephrinB2-EphB4 system which may induce different biological effects depending on the oncological and molecular contexts. It was the aim of the current study to characterize the influence of EphB4 on tumor microcirculation after antiangiogenic treatment using different SF126 glioma models. MATERIALS AND METHODS: Using an ecotropic transfection system, empty vector (pLXSN) or EphB4 (EphB4OE) overexpressing Phoenix-ECO cells were coimplanted with SF126 glioma cells subcutaneously (dorsal skinfold chamber, DSC) and orthotopically (cranial window, CW). Tumor volume was assessed by MRI. Intravital microscopy (IVM) allowed microcirculatory analysis (total {TVD} and functional vessel density {FVD}, diameter {D}, and permeability index {PI}) before and after antiangiogenic treatment (Sunitinib: DSC: 40 mg/kg BW, 6 days; CW: 80 mg/kg BW, 4 days). Immunohistochemistry included Pecam-Desmin, Ki67, TUNEL, and Caspase 3 stainings. RESULTS: EphB4OE induced large and treatment-resistant tumor vessels (FVD: Control/Su: 110 ± 23 cm/cm2 vs. EphB4OE/Su: 103 ± 42 cm/cm2). Maintenance of pericyte-endothelial cell interactions (Control: 80 ± 12 vs. Control/Su: 47 ± 26%; EphB4OE: 88 ± 9 vs. EphB4OE/Su: 74 ± 25%) and reduced antiproliferative (Control: 637 ± 80 vs. Control/Su: 110 ± 22; EphB4OE: 298 ± 108 vs. EphB4OE/Su: 213 ± 80) and proapoptotic responses (Control: 196 ± 25 vs. Control / Su: 404 ± 60; EphB4OE: 183 ± 20 vs. EphB4OE/Su: 270 ± 66) were observed under EphB4 overexpression. CONCLUSION: EphB4 overexpression leads to vascular resistance by altering vascular morphogenesis, pericyte coverage, and cellular proliferation/apoptosis in experimental SF126 glioma models.

EphrinB2 Activation Enhances Vascular Repair Mechanisms and Reduces Brain Swelling After Mild Cerebral Ischemia.

OBJECTIVE: Cerebral edema caused by the disruption of the blood-brain barrier is a major complication after stroke. Therefore, strategies to accelerate and enhance neurovascular recovery after stroke are of prime interest. Our main aim was to study the role of ephrinB2/EphB4 signaling in mediating the vascular repair and in blood-brain barrier restoration after mild cerebral ischemia occlusion/reperfusion. APPROACH AND RESULTS: Here, we show that the guidance molecule ephrinB2 plays a key role in neurovascular protection and blood-brain barrier restoration after stroke. In a focal stroke model, we characterize the stroke-induced damage to cerebral blood vessels and their subsequent endogenous repair on a cellular, molecular, and functional level. EphrinB2 and its tyrosine kinase receptor EphB4 are upregulated early after stroke by endothelial cells and perivascular support cells, in parallel to their reassembly during neurovascular recovery. Using both retroviral and pharmacological approaches, we show that the inhibition of ephrinB2/EphB4 signaling suppresses post-middle cerebral artery occlusion neurovascular repair mechanisms resulting in an aggravation of brain swelling. In contrast, the activation of ephrinB2 after brain ischemia leads to an increased pericyte recruitment and increased endothelial-pericyte interaction, resulting in an accelerated neurovascular repair after ischemia. CONCLUSIONS: We show that reducing swelling could result in improved outcome because of reduction in damaged brain tissue. We also identify a novel role for ephrinB2/EphB4 signaling in the maintenance of the neurovascular homeostasis and provide a novel therapeutic approach in reducing brain swelling after stroke.

Myoblast-mediated gene therapy improves functional collateralization in chronic cerebral hypoperfusion.

BACKGROUND AND PURPOSE: Direct extracranial-intracranial bypass surgery for treatment of cerebral hemodynamic compromise remains hindered by complications but alternative simple and safe indirect revascularization procedures, such as an encephalomyosynangiosis (EMS), lack hemodynamic efficiency. Here, the myoblast-mediated transfer of angiogenic genes presents an approach for induction of therapeutic collateralization. In this study, we tested the effect of myoblast-mediated delivery of vascular endothelial growth factor-A (VEGF) to the muscle/brain interface of an EMS in a model of chronic cerebral hypoperfusion. METHODS: Permanent unilateral internal carotid artery-occlusion was performed in adult C57/BL6 mice with or without (no EMS) surgical grafting of an EMS followed by implantation of monoclonal mouse myoblasts expressing either VEGF164 or an empty vector (EV). Cerebral hemodynamic impairment, transpial collateralization, angiogenesis, mural cell investment, microvascular permeability, and cortical infarction after ipsilateral stroke were assessed by real-time laser speckle blood flow imaging, 2- and 3-dimensional immunofluorescence and MRI. RESULTS: VEGF-expressing myoblasts improved hemodynamic rescue by day 14 (no EMS 37±21%, EV 42±9%, VEGF 48±12%; P<0.05 for VEGF versus no EMS and versus EV), together with the EMS take rate (VEGF 60%, EV 18.2%; P<0.05) and angiogenesis of mature cortical microvessels below the EMS (P<0.05 for VEGF versus EV). Importantly, functional and morphological results were paralleled by a 25% reduction of cortical infarction after experimental stroke on the side of the EMS. CONCLUSIONS: Myoblast-mediated VEGF supplementation at the target site of an EMS could help overcome the clinical dilemma of poor surgical revascularization results and provide protection from ischemic stroke.

Cerebral hemodynamic reserve and vascular remodeling in C57/BL6 mice are influenced by age.

BACKGROUND AND PURPOSE: Age is the most important risk factor for ischemic stroke. Recent experiments evidenced an age-associated rarefaction of the native collateral vasculature. The purpose of this study was to assess in what way age and arteriogenesis influence cortical perfusion and recovery of hemodynamic impairment in aged and young C57/BL6 mice. METHODS: After model establishment of chronic cerebral hypoperfusion in the C57/BL6 strain, sustained hemodynamic impairment was induced by permanent unilateral internal carotid artery occlusion in animals aged 4 to 6 weeks, 12 weeks, and 18 months. Functional and morphological outcome was assessed by laser speckle imaging before and during acetazolamide challenge on Days 0, 3, 7, and 14 and latex/carbon black angiography and immunohistochemistry on Day 21. RESULTS: Although internal carotid artery occlusion did not result in a reduction of baseline perfusion, it led to significant hemodynamic impairment in all age groups. Furthermore, baseline perfusion in sham and cerebrovascular reactivity after internal carotid artery occlusion were significantly lower in animals aged 18 months (468±57 Flux; 20.8%±17%) compared with mice aged 4 to 6 weeks (568±120 Flux; 30.3%±17%) and 12 weeks (591±72 Flux; 34.2%±12%) from the beginning until Day 7 of the monitoring period. Functional outcome was in line with a 27% reduction of native leptomeningeal anastomoses in aged mice and only limited collateral outgrowth compared with young animals. Strikingly, all age groups reached spontaneous functional compensation by Day 14. CONCLUSIONS: Next to limited collateral remodeling, our results suggest that a hampered cerebrovascular response with age could intensify the risk for hemodynamic stroke in the elderly.

Vascular Endothelial Growth Factor Augments the Tolerance Towards Cerebral Stroke by Enhancing Neurovascular Repair Mechanism.

The breakdown of the blood-brain barrier (BBB) is a critical event in the development of secondary brain injury after stroke. Among the cellular hallmarks in the acute phase after stroke are a downregulation of tight-junction molecules and the loss of microvascular pericyte coverage and endothelial sealing. Thus, a rapid repair of blood vessel integrity and re-stabilization of the BBB is considered an important strategy to reduce secondary brain damage. However, the mechanisms underlying BBB disruption remain poorly understood. Especially, the role of VEGF in this context remains inconclusive. With the conditional and reversible VEGF expression systems, we studied the time windows of deleterious and beneficial VEGF actions on blood vessel integrity in mice. Using genetic systems for gain of function and loss of function experiments, we activated and inhibited VEGF signaling prior and simultaneously to ischemic stroke onset. In both scenarios, VEGF seems to play a vital role in containing the stroke-induced damage after cerebral ischemia. We report that the transgenic overexpression of VEGF (GOF) prior to the stroke stabilizes the vasculature and prevents blood-brain barrier disruption in young and aged animals after stroke. Whereas inhibition of signals for endogenous VEGF (LOF) prior to stroke results in bigger infarction with massive brain swelling and enhanced BBB permeability, furthermore, activating or blocking VEGF signaling after ischemic stroke onset had comparable effects on BBB repair and cerebral edema. VEGF can function as an anti-permeability factor, and a VEGF-based therapy in the context of stroke prevention and recovery has an enormous potential.

Establishment and Validation of CyberKnife Irradiation in a Syngeneic Glioblastoma Mouse Model.

CyberKnife stereotactic radiosurgery (CK-SRS) precisely delivers radiation to intracranial tumors. However, the underlying radiobiological mechanisms at high single doses are not yet fully understood. Here, we established and evaluated the early radiobiological effects of CK-SRS treatment at a single dose of 20 Gy after 15 days of tumor growth in a syngeneic glioblastoma-mouse model. Exact positioning was ensured using a custom-made, non-invasive, and trackable frame. One superimposed target volume for the CK-SRS planning was created from the fused tumor volumes obtained from MRIs prior to irradiation. Dose calculation and delivery were planned using a single-reference CT scan. Six days after irradiation, tumor volumes were measured using MRI scans, and radiobiological effects were assessed using immunofluorescence staining. We found that CK-SRS treatment reduced tumor volume by approximately 75%, impaired cell proliferation, diminished tumor vasculature, and increased immune response. The accuracy of the delivered dose was demonstrated by staining of DNA double-strand breaks in accordance with the planned dose distribution. Overall, we confirmed that our proposed setup enables the precise irradiation of intracranial tumors in mice using only one reference CT and superimposed MRI volumes. Thus, our proposed mouse model for reproducible CK-SRS can be used to investigate radiobiological effects and develop novel therapeutic approaches.

The CXCR2/CXCL2 signalling pathway - An alternative therapeutic approach in high-grade glioma.

OBJECTIVE: Besides VEGF, alternative signalling via CXCR2 and its ligands CXCL2/CXCL8 is a crucial part of angiogenesis in glioblastoma. Our aim was to understand the role of CXCR2 for glioma biology and elucidate the therapeutic potential of its specific inhibition. METHODS: GL261 glioma cells were implanted intracranially in syngeneic mice. The 14 or 7 days of local or systemic treatment with CXCR2-antagonist (SB225002) was initiated early on the day of tumour cell implantation or delayed after 14 days of tumour growth. Glioma volume was verified using MRI before and after treatment. Immunofluorescence staining was used to investigate tumour progression, angiogenesis and microglial behaviour. Furthermore, in vitro assays and gene expression analyses of glioma and endothelial cells were performed to validate inhibitor activity. RESULTS: CXCR2-blocking led to significantly reduced glioma volumes of around 50% after early and delayed local treatments. The treated tumours were comparable with controls regarding invasiveness, proliferation and apoptotic cell activity. Furthermore, no differences in CXCR2/CXCL2 expression were observed. However, immunostaining revealed reduction in vessel density and accumulation of microglia/macrophages, whereas interaction of these myeloid cells with tumour vessels was enhanced. In vitro analyses of the CXCR2-antagonist showed its direct impact on proliferation of glioma and endothelial cells if used at higher concentrations. In addition, expression of CXCR2/CXCL2 signalling genes was increased in both cell types by SB225002, but VEGF-relevant genes were unaffected. CONCLUSION: The CXCR2-antagonist inhibited glioma growth during tumour initiation and progression, whereas treatment was well-tolerated by the recipients. Thus, the CXCR2/CXCL2 signalling represents a promising therapeutic target in glioma.

Balanced single-vector co-delivery of VEGF/PDGF-BB improves functional collateralization in chronic cerebral ischemia.

The myoblast-mediated delivery of angiogenic genes represents a cell-based approach for targeted induction of therapeutic collateralization. Here, we tested the superiority of myoblast-mediated co-delivery of vascular endothelial growth factor-A (VEGF) together with platelet-derived growth factor-BB (PDGF-BB) on transpial collateralization of an indirect encephalomyosynangiosis (EMS) in a model of chronic cerebral ischemia. Mouse myoblasts expressing a reporter gene alone (empty vector), VEGF, PDGF-BB or VEGF and PDGF-BB through a single bi-cistronic vector (VIP) were implanted into the temporalis muscle of an EMS following permanent ipsilateral internal carotid artery occlusion in adult, male C57BL/6N mice. Over 84 days, myoblast engraftment and gene product expression, hemodynamic impairment, transpial collateralization, angiogenesis, pericyte recruitment and post-ischemic neuroprotection were assessed. By day 42, animals that received PDGF-BB in combination with VEGF (VIP) showed superior hemodynamic recovery, EMS collateralization and ischemic protection with improved pericyte recruitment around the parenchymal vessels and EMS collaterals. Also, supplementation of PDGF-BB resulted in a striking astrocytic activation with intrinsic VEGF mobilization in the cortex below the EMS. Our findings suggest that EMS surgery together with myoblast-mediated co-delivery of VEGF/PDGF-BB may have the potential to serve as a novel treatment strategy for augmentation of collateral flow in the chronically hypoperfused brain.

CCR2 of Tumor Microenvironmental Cells Is a Relevant Modulator of Glioma Biology.

Glioblastoma multiforme (GBM) shows a high influx of tumor-associated macrophages (TAMs). The CCR2/CCL2 pathway is considered a relevant signal for the recruitment of TAMs and has been suggested as a therapeutic target in malignant gliomas. We found that TAMs of human GBM specimens and of a syngeneic glioma model express CCR2 to varying extents. Using a Ccr2-deficient strain for glioma inoculation revealed a 30% reduction of TAMs intratumorally. This diminished immune cell infiltration occurred with augmented tumor volumes likely based on increased cell proliferation. Remaining TAMs in Ccr2-/- mice showed comparable surface marker expression patterns in comparison to wildtype mice, but expression levels of inflammatory transcription factors (Stat3, Irf7, Cox2) and cytokines (Ifnß, Il1ß, Il12α) were considerably affected. Furthermore, we demonstrated an impact on blood vessel integrity, while vascularization of tumors appeared similar between mouse strains. The higher stability and attenuated leakiness of the tumor vasculature imply improved sustenance of glioma tissue in Ccr2-/- mice. Additionally, despite TAMs residing in the perivascular niche in Ccr2-/- mice, their pro-angiogenic activity was reduced by the downregulation of Vegf. In conclusion, lacking CCR2 solely on tumor microenvironmental cells leads to enhanced tumor progression, whereby high numbers of TAMs infiltrate gliomas independently of the CCR2/CCL2 signal.

Targeting the extradomain A of fibronectin allows identification of vascular resistance to antiangiogenic therapy in experimental glioma.

INTRODUCTION: Clinical application of antiangiogenic therapy lacks direct visualization of therapy efficacy and vascular resistance. We aimed to establish molecular imaging during treatment with sunitinib using the fibronectin extradomain A specific small immunoprotein(SIP)-F8 in glioma. METHODS: Biodistribution analysis of F8-SIP-Alexa-555 was performed in SF126-glioma bearing or control mice (n = 23 and 7, respectively). Intravital microscopy(IVM) was performed on a microvascular level after 7 days (n = 5 per group) and subsequently after 6 days of sunitinib treatment (n = 4) or without (n = 2).Additionally, near infrared fluorescence(NIRF) imaging was established with F8-SIP-Alexa-750 allowing non-invasive imaging with and without antiangiogenic treatment in orthotopic tumors (n = 38 divided in 4 groups). MRI was used to determine tumor size and served as a reference for NIRF imaging. RESULTS: F8-SIP demonstrated a time and hemodynamic dependent tumor specific accumulation. A significantly higher vascular accumulation occurred with antiangiogenic treatment compared to untreated tumors enabling visualization of resistant tumor vessels by F8-SIP-mediated NIRF imaging. In orthotopic tumors, sunitinib reduced F8-SIP-Alexa-750 enrichment volume but not fluorescence intensity indicative of F8-SIP accumulation in fewer vessels. CONCLUSION: F8-SIP is highly tumor specific with time and hemodynamic dependent biodistribution. The higher vascular accumulation to remaining vessels enables molecular imaging and targeting of therapy resistant tumor vessels.

Intravascular Inflammation Triggers Intracerebral Activated Microglia and Contributes to Secondary Brain Injury After Experimental Subarachnoid Hemorrhage (eSAH).

Activation of innate immunity contributes to secondary brain injury after experimental subarachnoid hemorrhage (eSAH). Microglia accumulation and activation within the brain has recently been shown to induce neuronal cell death after eSAH. In isolated mouse brain capillaries after eSAH, we show a significantly increased gene expression for intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Hence, we hypothesized that extracerebral intravascular inflammatory processes might initiate the previously reported microglia accumulation within the brain tissue. We therefore induced eSAH in knockout mice for ICAM-1 (ICAM-1-/-) and P-selectin glycoprotein ligand-1 (PSGL-1-/-) to find a significant decrease in neutrophil-endothelial interaction within the first 7 days after the bleeding in a chronic cranial window model. This inhibition of neutrophil recruitment to the endothelium results in significantly ameliorated microglia accumulation and neuronal cell death in knockout animals in comparison to controls. Our results suggest an outside-in activation of the CNS innate immune system at the vessel/brain interface following eSAH. Microglia cells, as part of the brain's innate immune system, are triggered by an inflammatory reaction in the microvasculature after eSAH, thus contributing to neuronal cell death. This finding offers a whole range of new research targets, as well as possible therapy options for patients suffering from eSAH.