Dynamic stroma reorganization drives blood vessel dysmorphia during glioma growth

Glioma growth and progression are characterized by abundant development of blood vessels that are highly aberrant and poorly functional, with detrimental consequences for drug delivery efficacy. The mechanisms driving this vessel dysmorphia during tumor progression are poorly understood. Using longitudinal intravital imaging in a mouse glioma model, we identify that dynamic sprouting and functional morphogenesis of a highly branched vessel network characterize the initial tumor growth, dramatically changing to vessel expansion, leakage, and loss of branching complexity in the later stages. This vascular phenotype transition was accompanied by recruitment of predominantly pro-inflammatory M1-like macrophages in the early stages, followed by in situ repolarization to M2-like macrophages, which produced VEGF-A and relocate to perivascular areas. A similar enrichment and perivascular accumulation of M2 versus M1 macrophages correlated with vessel dilation and malignancy in human glioma samples of different WHO malignancy grade. Targeting macrophages using anti-CSF1 treatment restored normal blood vessel patterning and function. Combination treatment with chemotherapy showed survival benefit, suggesting that targeting macrophages as the key driver of blood vessel dysmorphia in glioma progression presents opportunities to improve efficacy of chemotherapeutic agents. We propose that vessel dysfunction is not simply a general feature of tumor vessel formation, but rather an emergent property resulting from a dynamic and functional reorganization of the tumor stroma and its angiogenic influences

Effect of surgical experience and spine subspecialty on the reliability of the {AO} Spine Upper Cervical Injury Classification System

OBJECTIVE The objective of this paper was to determine the interobserver reliability and intraobserver reproducibility of the AO Spine Upper Cervical Injury Classification System based on surgeon experience (< 5 years, 5–10 years, 10–20 years, and > 20 years) and surgical subspecialty (orthopedic spine surgery, neurosurgery, and "other" surgery). METHODS A total of 11,601 assessments of upper cervical spine injuries were evaluated based on the AO Spine Upper Cervical Injury Classification System. Reliability and reproducibility scores were obtained twice, with a 3-week time interval. Descriptive statistics were utilized to examine the percentage of accurately classified injuries, and Pearson’s chi-square or Fisher’s exact test was used to screen for potentially relevant differences between study participants. Kappa coefficients (κ) determined the interobserver reliability and intraobserver reproducibility. RESULTS The intraobserver reproducibility was substantial for surgeon experience level (< 5 years: 0.74 vs 5–10 years: 0.69 vs 10–20 years: 0.69 vs > 20 years: 0.70) and surgical subspecialty (orthopedic spine: 0.71 vs neurosurgery: 0.69 vs other: 0.68). Furthermore, the interobserver reliability was substantial for all surgical experience groups on assessment 1 (< 5 years: 0.67 vs 5–10 years: 0.62 vs 10–20 years: 0.61 vs > 20 years: 0.62), and only surgeons with > 20 years of experience did not have substantial reliability on assessment 2 (< 5 years: 0.62 vs 5–10 years: 0.61 vs 10–20 years: 0.61 vs > 20 years: 0.59). Orthopedic spine surgeons and neurosurgeons had substantial intraobserver reproducibility on both assessment 1 (0.64 vs 0.63) and assessment 2 (0.62 vs 0.63), while other surgeons had moderate reliability on assessment 1 (0.43) and fair reliability on assessment 2 (0.36). CONCLUSIONS The international reliability and reproducibility scores for the AO Spine Upper Cervical Injury Classification System demonstrated substantial intraobserver reproducibility and interobserver reliability regardless of surgical experience and spine subspecialty. These results support the global application of this classification system

Local and systemic immune interactions in malignant gliomas

Glioblastoma (GBM), the most frequent primary intrinsic brain tumor, is without any doubt one of the most devastating diseases known to mankind. GBM are currently being treated with neurosurgical resection followed by radio- and chemotherapy. However, despite this treatment, prognosis for these patients is grim with a median survival of only 15 months and less than 20% 3-year survival rates. Already at diagnosis, GBM cells are infiltrating beyond the visible tumor margins, making complete resection impossible. A therapy-resistant subpopulation of these remaining cells eventually leads to tumor recurrence. In the past 2 decades, immunotherapy has gained interest as a possible fourth treatment strategy. Immunotherapy is theoretically appealing, firstly because tumor-infiltrating cytotoxic immune cells could target all invasive cancer cells without damaging surrounding normal tissue. Secondly, when immunological memory develops, responses can be long-lasting without the need of persistent administration of therapy. For GBM, these concepts have been shown in preclinical animal models with several vaccination strategies. However, until now no randomized immunotherapy trials have been able to shown survival benefit in humans. In this research project we aimed to get more insight in the complex interactions between the immune system and GBM, in particular the differences in immune profiles between the local tumor micro-environment and the systemic / peripheral compartment. We hypothesize that better understanding on how the immune system interacts with a developing GBM is essential to develop effective immunotherapy, or at least to understand why successful preclinical therapies don't work in the clinical setting. For this research we used tumor tissue and blood samples from glioma patients, as well as the orthotopic murine GL261 malignant glioma model in which malignant brain tumors are induced by stereotactic injection of GL261 cells. In the first part of the research (Chapter 3 - Research paper 1), we focused on the immune checkpoint molecule PD-1 as a contemporary paradigm target for current immunotherapy in several cancers. PD-1 is a surface protein present on activated T-lymphocytes that, after binding with its ligand PD-L1, leads to a negative feedback signal. This pathway is being used by many cancers to suppress infiltrating lymphocytes, and blockade of PD-1 has been shown to reactivate anti-tumor T cells and prolong survival in advanced melanomas and other cancers. Using flow cytometry, we show a strong upregulation of PD-1 on tumor-infiltrating CD4+, CD8+ and regulatory T cells. This upregulation was not restricted to GBM, but also present in lower grade 2 and 3 gliomas. Moreover, we showed that PD-1 expression on circulating T-lymphocytes is not different between patients and healthy volunteers. In the GL261 model we found a similar upregulation on brain-infiltrating T cells, and markedly prolonged survival with a CD8+ driven T cell response after treatment with a PD-1 blocking antibody. This research provides further evidence for trials with PD-1 blocking strategies to treat GBM. In the second part of the research (Chapter 4 - Research paper 2), we wondered whether local and systemic immune profiles in human GBM could be related to specific biological subgroups of GBM. We studied a large cohort of patients with histologically proven GBM, which were treated with standard therapy and experimental immunotherapy. Tumor samples were subclassified according to DNA methylation profiles into 4 subgroups and a rest group with unclassifiable tumors. With immunohistochemistry, we quantified tumor-infiltrating CD3+ T cells and found these to be significantly more numerous in tumors belonging to the mesenchymal subgroup. On the other hand, IDH tumors had the lowest CD3+ T cell infiltration. This T cell infiltration contrasted with overall survival, which was significantly longer in IDH tumors than mesenchymal tumors. In a multivariate analysis, DNA methylation-based stratification of these tumors remained an independent variable for survival. We also characterized CD68+ myeloid cell infiltration, but found this not to be related to any methylation subgroups or survival. With flow cytometry, we also assessed T cell populations in the blood before and after radiochemotherapy in these patients. We found an overall reduction in lymphocyte count after radiochemotherapy, but relative increase in CD8+ T and CD8+ PD-1+ T cells in all subgroups except IDH tumors. Together, these finding support the hypothesis that histologically identical GBM can be classified into relevant biological subgroups, related to survival and T cell infiltration. Subclassification seems useful in trials as it is plausible that the efficacy of new therapies could be restricted to one or several subgroups. In the final part of the research (Chapter 5 - Research paper 3), we investigated the effects of subcutaneous implantations of living syngeneic GL261 cells in the murine GL261 malignant glioma model. We see living tumor cell vaccination as the theoretical most basic type of vaccination strategy, able to provide us with more insight in differences of immunity based on specific tumor location and timing of vaccination. The interest in heterotopic living tumor cell implantations originates from the rarity of extracranial metastasis of GBM, despite its local aggressiveness and frequent systemic dissemination of individual tumor cells. We found that subcutaneously implanted GL261 cells are rejected in > 80% of mice, while intracerebral implantations induce lethal tumors in all mice. Furthermore, 79% of mice that rejected subcutaneous tumors were protected against subsequent intracranial tumor implantation (prophylactic model). Immunologically, the brains of prophylactically vaccinated mice were noticed to have an early CD3+ CD8+ T cell influx and decreased regulatory T cells compared to control mice. Interestingly, unlike in control mice, an initial high PD-1 expression in prophylactically vaccinated mice decreased towards basal PD-1 expression 4 weeks after intracerebral tumor implantation. Despite the high protection rate in the prophylactic model, subcutaneous GL261 implantations did not induce any survival benefit in mice with established cerebral tumors, nor did they induce subcutaneous tumors. These findings confirm that the capability of the immune system to control and reject tumor cells depends on the implantation site, and that location-based prophylactic vaccination is possible with living unmodified tumor cells. However, in the presence of an established brain tumor, peripheral living tumor cell implantation isn't capable anymore to induce a clinically beneficial immunity, nor does it seem to induce palpable tumors. As there seem to be different immunological answers to subcutaneous GL261 implantations in the prophylactic and established tumor models, the local subcutaneous microenvironment after GL261 implantation in these different models is currently further investigated with pathology and immunohistochemistry.nrpages: 150status: publishe

Dendritic cell vaccination for glioblastoma multiforme: review with focus on predictive factors for treatment response

Joost Dejaegher,1 Stefaan Van Gool,2 Steven De Vleeschouwer1 1Department of Neurosciences, 2Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium Abstract: Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain cancer. Since median overall survival with multimodal standard therapy is only 15 months, there is a clear need for additional effective and long-lasting treatments. Dendritic cell (DC) vaccination is an experimental immunotherapy being tested in several Phase I and Phase II clinical trials. In these trials, safety and feasibility have been proven, and promising clinical results have been reported. On the other hand, it is becoming clear that not every GBM patient will benefit from this highly personalized treatment. Defining the subgroup of patients likely to respond to DC vaccination will position this option correctly amongst other new GBM treatment modalities, and pave the way to incorporation in standard therapy. This review provides an overview of GBM treatment options and focuses on the currently known prognostic and predictive factors for response to DC vaccination. In this way, it will provide the clinician with the theoretical background to refer patients who might benefit from this treatment. Keywords: immunotherapy, personalized medicine, brain tumor, stratificatio

Advantages of HCO3 solution with low sodium concentration over standard lactate solutions for acute peritoneal dialysis

The aim of this study was to identify the advantages of a bicarbonate solution with a low sodium concentration. Twelve children (3 days - 6 years) with acute renal failure (ARF), positive fluid balance, and lactate acidosis (>40 mg/dL) were treated by automated peritoneal dialysis (APD) with frequent exchanges of small fill volumes of a hypertonic solution. For Day I we used PD1/PD4 Dianeal (3.86%) (Baxter). After 24 hours we switched to a HCO3 solution: 38 mmol/L, Na 128 mmol/L. As the control group, we studied retrospectively the last 12 children of the previous period who were treated with APD. The age distribution was 4 days to 4 years. No significant differences were found between the groups for serum creatinine, blood urea nitrogen, and fluid overload (Day I to Day 4). Although the values for lactate and Na were not different before the start of the study (Day I) and after 24 hours of Dianeal (Day 2), they were significantly lower in the study group on Day 4 [HCO3 53 (23 - 83), Na 148 (137 - 136) mEq/L] than in the control group [lactate 148 (137 - 156), Na 154 (142 - 165) mEq/L]. A low sodium concentration results in higher sodium extraction, which is important for patients with fluid overload. Low sodium concentrations in APD are needed because the peritoneal membrane ''sieves'' the sodium during short dwells. HCO3 dialysis is a logical choice for patients with lactate acidosis, resulting in a significant lower serum lactate and increase of BE after 48 hours of treatment