Myeloid cell subpopulations compensate each other for Ccr2-deficiency in glioblastoma.

AIMS: Glioblastomas are high-grade brain tumours that are characterised by the accumulation of brain-resident microglia and peripheral macrophages. Recruitment of these myeloid cells can be facilitated by CCR2/CCL2 signalling. Besides the well-known CCR2+ macrophages, we have identified microglia expressing CCR2 in glioma tissues. Thus, we investigated how Ccr2-deficiency of one of the myeloid cell populations affects the other population and tumour biology. METHODS: We generated four chimeric groups to analyse single and combined Ccr2-deficiency of microglia and macrophages. On day 21 after tumour cell implantation (GL261), we conducted flow cytometry, immunofluorescence and real-time polymerase chain reaction analyses. Tumour volume and metabolism were determined by magnetic resonance imaging and magnetic resonance spectroscopy. Moreover, in vitro studies were performed with primary microglia and bone marrow-derived macrophages. RESULTS: We demonstrated reduced infiltration of macrophages and microglia depending on the lack of Ccr2. However, the total number of myeloid cells remained constant except for the animals with dual Ccr2-knockout. Both microglia and macrophages with Ccr2-deficiency showed impaired expression of proinflammatory molecules and altered phagocytic activity. Despite the altered immunologic phenotype caused by Ccr2-deficiency, glioma progression and metabolism were hardly affected. Alterations were detected solely in apoptosis and proliferation of tumours from animals with specific Ccr2-deficient microglia, whereas vessel stability was increased in mice with Ccr2-knockout in both cell populations. CONCLUSION: These results indicate that microglia and macrophages provide a homoeostatic balance within glioma tissue and compensate for the lack of the corresponding counterpart. Moreover, we identified that the CCR2/CCL2 axis is involved in the immunologic function of microglia and macrophages beyond its relevance for migration.

How to Target Spinal Metastasis in Experimental Research: An Overview of Currently Used Experimental Mouse Models and Future Prospects.

The spine is one of the organs that is most affected by metastasis in cancer patients. Since the control of primary tumor is continuously improving, treatment of metastases is becoming one of the major challenges to prevent cancer-related death. Due to the anatomical proximity to the spinal cord, local spread of metastasis can directly cause neurological deficits, severely limiting the patient's quality of life. To investigate the underlying mechanisms and to develop new therapies, preclinical models are required which represent the complexity of the multistep cascade of metastasis. Current research of metastasis focuses on the formation of the premetastatic niche, tumor cell dormancy and the influence and regulating function of the immune system. To unveil whether these influence the organotropism to the spine, spinal models are irreplaceable. Mouse models are one of the most suitable models in oncologic research. Therefore, this review provides an overview of currently used mouse models of spinal metastasis. Furthermore, it discusses technical aspects clarifying to what extend these models can picture key steps of the metastatic process. Finally, it addresses proposals to develop better mouse models in the future and could serve as both basis and stimulus for researchers and clinicians working in this field.

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.

Impact of 68Ga-DOTATOC PET/MRI on robotic radiosurgery treatment planning in meningioma patients: first experiences in a single institution.

OBJECTIVEFor stereotactic radiosurgery (SRS) planning, precise contouring of tumor boundaries and organs at risk is of utmost importance. Correct interpretation of standard neuroimaging (i.e., CT and MRI) can be challenging after previous surgeries or in cases of skull base lesions with complex shapes. The aim of this study was to evaluate the impact of 68Ga-DOTATOC PET/MRI on treatment planning for image-guided SRS by CyberKnife.METHODSThe authors retrospectively identified 11 meningioma treatments in 10 patients who received a 68Ga-DOTATOC PET/MRI prior to SRS. The planning target volume (PTV) used for the patients' treatment was defined as the reference standard. This was contoured by a treating radiosurgeon (RS0) using fused planning CT and PET/MRI data sets. The same tumors were then contoured by another experienced radiosurgeon (RS1) and by a less-experienced radiosurgeon (RS2), both blinded to PET data sets. A comparison of target volumes with focus on volume-based metrics and distance to critical structures was performed. RS1 and RS2 also filled in a questionnaire analyzing the confidence level and the subjective need for the implementation of PET data sets for contouring.RESULTSAnalysis showed a subjective personal preference for PET/MRI in all cases for both radiosurgeons, particularly in proximity to critical structures. The analysis of the planning volumes per physician showed significantly smaller RS2-PTV in comparison to RS1-PTV and to RS0-PTV, whereas the median volumes were comparable between RS1-PTV and RS2-PTV (median: RS0: 4.3 cm3 [IQR 3.4-6.5 cm3] and RS1: 4.5 cm3 [IQR 2.7-6 cm3] vs RS2: 2.6 cm3 [IQR 2-5 cm3]; p = 0.003). This was also reflected in the best spatial congruency between the 2 experienced physicians (RS0 and RS1). The percentage of the left-out volume contoured by RS1 and RS2 compared to RS0 with PET/MRI demonstrated a relevant left-out-volume portion in both cases with greater extent for the less-experienced radiosurgeon (RS2) (RS1: 19.1% [IQR 8.5%-22%] vs RS2: 40.2% [IQR 34.2%-53%]). No significant differences were detected regarding investigated critical structures.CONCLUSIONSThis study demonstrated a relevant impact of PET/MRI on target volume delineation of meningiomas. The extent was highly dependent on the experience of the treating physician. This preliminary study supports the relevance of 68Ga-DOTATOC PET/MRI as a tool for radiosurgical treatment planning of meningiomas.

Mean global DNA methylation serves as independent prognostic marker in IDH-wildtype glioblastoma.

BACKGROUND: The IDH-wildtype glioblastoma (GBM) patients have a devastating prognosis. Here, we analyzed the potential prognostic value of global DNA methylation of the tumors. METHODS: DNA methylation of 492 primary samples and 31 relapsed samples, each treated with combination therapy, and of 148 primary samples treated with radiation alone were compared with patient survival. We determined the mean methylation values and estimated the immune cell infiltration from the methylation data. Moreover, the mean global DNA methylation of 23 GBM cell lines was profiled and correlated to their cellular radiosensitivity as measured by colony formation assay. RESULTS: High mean DNA methylation levels correlated with improved survival, which was independent from known risk factors (MGMT promoter methylation, age, extent of resection; P = 0.009) and methylation subgroups. Notably, this correlation was also independent of immune cell infiltration, as higher number of immune cells indeed was associated with significantly better OS but lower mean methylation. Radiosensitive GBM cell lines had a significantly higher mean methylation than resistant lines (P = 0.007), and improved OS of patients treated with radiotherapy alone was also associated with higher DNA methylation (P = 0.002). Furthermore, specimens of relapsed GBM revealed a significantly lower mean DNA methylation compared to the matching primary tumor samples (P = 0.041). CONCLUSIONS: Our results indicate that mean global DNA methylation is independently associated with outcome in glioblastoma. The data also suggest that a higher DNA methylation is associated with better radiotherapy response and less aggressive phenotype, both of which presumably contribute to the observed correlation with OS.

Preoperatively-determined Red Distribution Width (RDW) predicts prolonged length of stay after single-level spinal fusion in elderly patients.

Introduction: Elderly patients receiving lumbar fusion surgeries present with a higher risk profile, which necessitates a robust predictor of postoperative outcomes. The Red Distribution Width (RDW) is a preoperative routinely determined parameter that reflects the degree of heterogeneity of red blood cells. Thereby, RDW is associated with frailty in hospital-admitted patients. Research question: This study aims to elucidate the potential of RDW as a frailty biomarker predictive of prolonged hospital stays following elective mono-segmental fusion surgery in elderly patients. Material and methods: In this retrospective study, we included all patients with age over 75 years that were treated via lumbar single-level spinal fusion from 2015 to 2022 at our tertiary medical center. Prolonged length of stay (pLOS) was defined as a length ≥ the 3rd quartile of LOS of all included patients. Classical correlation analysis, Receiver-operating characteristic (ROC) and new machine learning algorithms) were used. Results: A total of 208 patients were included in the present study. The median age was 77 (IQR 75-80) years. The median LOS of the patients was 6 (IQR 5-8) days. The data shows a significant positive correlation between RDW and LOS. RDW is significantly enhanced in the pLOS group. New machine learning approaches with the imputation of multiple variables can enhance the performance to an AUC of 71%. Discussion and conclusion: RDW may serve as a predictor for a pLOS in elderly. These results are compelling because the determination of this frailty biomarker is routinely performed at hospital admission. An improved prognostication of LOS could enable healthcare systems to distribute constrained hospital resources efficiently, fostering evidence-based decision-making processes.

DNA methylation subclasses predict the benefit from gross total tumor resection in IDH-wildtype glioblastoma patients.

BACKGROUND: DNA methylation-based tumor classification allows an enhanced distinction into subgroups of glioblastoma. However, the clinical benefit of DNA methylation-based stratification of glioblastomas remains inconclusive. METHODS: Multicentric cohort study including 430 patients with newly diagnosed glioblastoma subjected to global DNA methylation profiling. Outcome measures included overall survival (OS), progression-free survival (PFS), prognostic relevance of EOR and MGMT promoter methylation status as well as a surgical benefit for recurrent glioblastoma. RESULTS: 345 patients (80.2%) fulfilled the inclusion criteria and 305 patients received combined adjuvant therapy. DNA methylation subclasses RTK I, RTK II, and mesenchymal (MES) revealed no significant survival differences (RTK I: Ref.; RTK II: HR 0.9 [95% CI, 0.64-1.28]; p = 0.56; MES: 0.69 [0.47-1.02]; p = 0.06). Patients with RTK I (GTR/near GTR: Ref.; PR: HR 2.87 [95% CI, 1.36-6.08]; p < 0.01) or RTK II (GTR/near GTR: Ref.; PR: HR 5.09 [95% CI, 2.80-9.26]; p < 0.01) tumors who underwent gross-total resection (GTR) or near GTR had a longer OS and PFS than partially resected patients. The MES subclass showed no survival benefit for a maximized EOR (GTR/near GTR: Ref.; PR: HR 1.45 [95% CI, 0.68-3.09]; p = 0.33). Therapy response predictive value of MGMT promoter methylation was evident for RTK I (HR 0.37 [95% CI, 0.19-0.71]; p < 0.01) and RTK II (HR 0.56 [95% CI, 0.34-0.91]; p = 0.02) but not the MES subclass (HR 0.52 [95% CI, 0.27-1.02]; p = 0.06). For local recurrence (n = 112), re-resection conveyed a progression-to-overall survival (POS) benefit (p < 0.01), which was evident in RTK I (p = 0.03) and RTK II (p < 0.01) tumors, but not in MES tumors (p = 0.33). CONCLUSION: We demonstrate a survival benefit from maximized EOR for newly diagnosed and recurrent glioblastomas of the RTK I and RTK II but not the MES subclass. Hence, it needs to be debated whether the MES subclass should be treated with maximal surgical resection, especially when located in eloquent areas and at time of recurrence.

Combining TMZ and SB225002 induces changes of CXCR2 and VEGFR signalling in primary human endothelial cells in vitro.

Standard of care therapy for glioblastoma (GBM) consisting of surgical removal, temozolomide (TMZ) and radiotherapy fails to cure the disease and median survival is limited to 15 months. Therapeutic approaches targeting vascular endothelial growth factor (VEGF)­mediated angiogenesis, one of the major drivers of tumour growth, have not prolonged patient survival as reported in clinical studies. Apart from VEGFR signalling, proangiogenic C­X­C motif chemokine receptor 2 (CXCR2) is of special interest as its ligands C­X­C motif chemokine ligand 2 (CXCL2) and interleukin­8 (IL8) are upregulated and associated with reduced survival in GBM patients. As CXCR2 is also expressed by endothelial cells, the aim of the present study was to elucidate the effect of combination therapy on gene and protein expression of primary human endothelial cells (HUVECs). To mimic the GBM specific CXCL2/IL8 oversupply environment [referred to as stimulation (STIM)], HUVECs were treated with a cocktail of CXCL2/IL8 and/or TMZ and/or CXCR2­antagonist SB225002 (SB). In brief, six treatment conditions were utilized: i) Control, ii) STIM (CXCL2/IL8), iii) TMZ + SB, iv) STIM + TMZ, v) STIM + SB, vi) STIM + TMZ + SB followed by either RNA­isolation and RT­qPCR for BAX, BCL2, vascular endothelial growth receptor (VEGFR)1/2, VEGF, CXCR1/2, CXCL2 and IL8 or immunofluorescence staining for VEGFR2 and CXCR2. SB and TMZ led to morphological changes of HUVECs and downregulated antiapoptotic BCL2 in vitro. In addition, gene expression of the alternative proangiogenic CXCL2/IL8/CXCR2 signalling pathway was significantly altered by the combination therapy, while the VEGF/VEGFR1/2 axis was only mildly affected. Furthermore, VEGFR2 and CXCR2 gene and protein expression regulation differed. VEGFR2 was not altered at the gene expression level, while combination therapy with TMZ and SB led to a 74% upregulation of VEGFR2 at the protein level. By contrast, CXCR2 was upregulated 5­fold by the combination therapy at the gene expression level and downregulated by 72.5% at the protein expression level. The present study provided first insights into the molecular changes of two major proangiogenic pathways in primary endothelial cells during treatment with TMZ and SB. Different gene and protein expression levels of the proangiogenic receptors CXCR2 and VEGFR2 in vitro must be taken into consideration in future studies.

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.

Image-Guided Robotic Radiosurgery for Treatment of Recurrent Grade II and III Meningiomas. A Single-Center Study.

OBJECTIVE: Stereotactic radiosurgery (SRS) has been increasingly applied for malignant meningiomas as an alternative to conventionally fractioned radiation therapy. We performed a retrospective analysis of an institutional patient cohort with malignant meningiomas treated by image-guided SRS. METHODS: All patients with atypical or anaplastic meningiomas who were treated by SRS using CyberKnife (CK) were identified. Local failure and regional and/or distant recurrences were evaluated together with toxicity and overall survival. RESULTS: We identified 127 treated lesions (105 atypical and 22 anaplastic) in 35 patients. The mean time interval between the last surgery and subsequent CK-SRS was 30.8 ± 24.5 months. Most lesions (83.5%) were treated using single-fraction CK-SRS. The median planning target volume of all 127 lesions was 1.71 cm3 (range, 0.06-22.5 cm3). The median follow-up period was 23 months (range, 2.1-60.3 months). The estimated local control rates were 97%, 77%, and 67% at 12, 36, and 60 months, respectively, in atypical meningiomas and 66% each at 12 and 24 months in anaplastic meningiomas. The regional progression-free survival was 93%, 73%, and 59% at 12, 36, and 60 months, respectively, in atypical lesions and 93% and 46% at 12 and 24 months in anaplastic lesions. The estimated distant tumor progression-free interval in atypical lesions was 80%, 44%, and 44% at 12, 36, and 60 months, respectively, and 49% and 24% at 12 and 24 months, respectively, in anaplastic lesions. Age was identified as a risk factor for local failure. CONCLUSIONS: Although the real boundaries of efficacy of SRS have to be further evaluated in a prospective trial, it seems that aggressive treatment by high-dose single or multisession SRS of recurring malignant meningiomas provides satisfactory local control rates.