Viola Caretti

Neuronal Activity Promotes Glioma Growth through Neuroligin-3 Secretion

Active neurons exert a mitogenic effect on normal neural precursor and oligodendroglial precursor cells, the putative cellular origins of high-grade glioma (HGG). By using optogenetic control of cortical neuronal activity in a patient-derived pediatric glioblastoma xenograft model, we demonstrate that active neurons similarly promote HGG proliferation and growth in vivo. Conditioned medium from optogenetically stimulated cortical slices promoted proliferation of pediatric and adult patient-derived HGG cultures, indicating secretion of activity-regulated mitogen(s). The synaptic protein neuroligin-3 (NLGN3) was identified as the leading candidate mitogen, and soluble NLGN3 was sufficient and necessary to promote robust HGG cell proliferation. NLGN3 induced PI3K-mTOR pathway activity and feedforward expression of NLGN3 in glioma cells. NLGN3 expression levels in human HGG negatively correlated with patient overall survival. These findings indicate the important role of active neurons in the brain tumor microenvironment and identify secreted NLGN3 as an unexpected mechanism promoting neuronal activity-regulated cancer growth.

Monitoring of tumor growth and post-irradiation recurrence in a diffuse intrinsic pontine glioma mouse model.

Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy because of its diffuse infiltrative growth pattern. Translational research suffers from the lack of a representative DIPG animal model. Hence, human E98 glioma cells were stereotactically injected into the pons of nude mice. The E98 DIPG tumors presented a strikingly similar histhopathology to autopsy material of a DIPG patient, including diffuse and perivascular growth, brainstem‐ and supratentorial invasiveness and leptomeningeal growth. Magnetic resonance imaging (MRI) was effectively employed to image the E98 DIPG tumor. [18F] 3′‐deoxy‐3′‐[18F]fluorothymidine (FLT) positron emission tomography (PET) imaging was applied to assess the subcutaneous (s.c.) E98 tumor proliferation status but no orthotopic DIPG activity could be visualized. Next, E98 cells were cultured in vitro and engineered to express firefly luciferase and mCherry (E98‐Fluc‐mCherry). These cultured E98‐Fluc‐mCherry cells developed focal pontine glioma when injected into the pons directly. However, the diffuse E98 DIPG infiltrative phenotype was restored when cells were injected into the pons immediately after an intermediate s.c. passage. The diffuse E98‐Fluc‐mCherry model was subsequently used to test escalating doses of irradiation, applying the bioluminescent Fluc signal to monitor tumor recurrence over time. Altogether, we here describe an accurate DIPG mouse model that can be of clinical relevance for testing experimental therapeutics in vivo.

Crizotinib inhibits metabolic inactivation of gemcitabine in c-Met-driven pancreatic carcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclinical tests fail in these patients, emphasizing the need of improved preclinical models to test novel anticancer strategies. Here, we developed four orthotopic mouse models using primary human PDAC cells genetically engineered to express firefly- and Gaussia luciferase, simplifying the ability to monitor tumor growth and metastasis longitudinally in individual animals with MRI and high-frequency ultrasound. In these models, we conducted detailed histopathologic and immunohistochemical analyses on paraffin-embedded pancreatic tissues and metastatic lesions in liver, lungs, and lymph nodes. Genetic characteristics were compared with the originator tumor and primary tumor cells using array-based comparative genomic hybridization, using frozen specimens obtained by laser microdissection. Notably, the orthotopic human xenografts in these models recapitulated the phenotype of human PDACs, including hypovascular and hypoxic areas. Pursuing genomic and immunohistochemical evidence revealed an increased copy number and overexpression of c-Met in one of the models; we examined the preclinical efficacy of c-Met inhibitors in vitro and in vivo. In particular, we found that crizotinib decreased tumor dimension, prolonged survival, and increased blood and tissue concentrations of gemcitabine, synergizing with a cytidine deaminase-mediated mechanism of action. Together, these more readily imaged orthotopic PDAC models displayed genetic, histopathologic, and metastatic features similar to their human tumors of origin. Moreover, their use pointed to c-Met as a candidate therapeutic target in PDAC and highlighted crizotinib and gemcitabine as a synergistic combination of drugs warranting clinical evaluation for PDAC treatment.

WEE1 Kinase Inhibition Enhances the Radiation Response of Diffuse Intrinsic Pontine Gliomas

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric disease. Thus far, no therapeutic agent has proven beneficial in the treatment of this malignancy. Therefore, conventional DNA-damaging radiotherapy remains the standard treatment, providing transient neurologic improvement without improving the probability of overall survival. During radiotherapy, WEE1 kinase controls the G2 cell-cycle checkpoint, allowing for repair of irradiation (IR)-induced DNA damage. Here, we show that WEE1 kinase is one of the highest overexpressed kinases in primary DIPG tissues compared with matching non-neoplastic brain tissues. Inhibition of WEE1 by MK-1775 treatment of DIPG cells inhibited the IR-induced WEE1-mediated phosphorylation of CDC2, resulting in reduced G2–M arrest and decreased cell viability. Finally, we show that MK-1775 enhances the radiation response of E98-Fluc-mCherry DIPG mouse xenografts. Altogether, these results show that inhibition of WEE1 kinase in conjunction with radiotherapy holds potential as a therapeutic approach for the treatment of DIPG. Mol Cancer Ther; 12(2); 141–50. ©2012 AACR.

Subventricular spread of diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is the second most common malignant pediatric brain tumor and the leading cause of brain tumor death in childhood [1]. 80 % of DIPG tumors exhibit a specific mutation (H3K27M) in the genes encoding histone 3.1 or 3.3 [2, 3]. standard therapy consisting of local radiotherapy to a dosage of 54–60 Gy extends median survival from 5 months to ∼9 months; 5-year survival remains less than 1 % [1]. The practice of focal radio-therapy to the brainstem is based in part on a 1982 autopsy study reporting DIPG to be relatively localized to the pons and adjacent structures [4]. In contrast, other neuroimaging and autopsy studies have identified widespread disease including supratentorial extension and leptomeningeal spread [5, 6]. Here, we report an autopsy series of 16 patients evaluated from 2009–2014 at stanford (n = 10) and VU (n = 6) University Medical Centers [7]. patient characteristics are listed in Table S1. Consistent with previous reports [5, 6], we found widespread dissemination of DIPG with extension to midbrain and medulla in 63 %, cerebellum in 56 %, thalamus in 56 %, frontal cortex in 25 % and supratentorial leptomeninges in 25 % (Fig. 1). The spinal cord was not consistently examined, but metastases were found in two of three cases examined; both had clinical evidence of spinal cord spread. Fig. 1 Extent of spread in DIPG. a Neuroanatomical sites and frequency of tumor invasion. Numbers indicate the percentage of cases that exhibit tumor invasion at the indicated anatomical location. The size of the circles marking each anatomical site (color key ... A previously under-recognized pattern of subventricular spread was noted in 10/16 cases, with infiltration of the subventricular zone (SVZ) and tumor nodules in the frontal horns of the lateral ventricles. In three cases lateral ventricular disease was noted on pre-mortem MRI (Fig. 2a), but subclinical tumor invasion in the SVZ of the lateral ventricles was found in six additional cases; subventricular spread was found in the third ventricle of one additional case (Fig. 2). The observed pattern of ventricular/subventricular involvement could be due to direct invasion along the SVZ corridor, intraventricular cerebrospinal fluid (CSF) seeding of the SVZ, or an as yet undescribed mechanism. The postnatal SVZ is a neural stem cell niche in the human brain [8] and DIPG cells express an immunophenotype reminiscent of neural precursor cells (Fig. S1 and [9]). Whether DIPG cells exhibit a particular tropism for this niche remains to be explored. Fig. 2 Invasion of the subventricular zone in DIPG (a). MRI images illustrating enhancing lesions (T1 post gadolinium, left image) at the frontal horns of the lateral ventricles with associated edema (FLAIR, right image) in case SU-DIPG-XIII. b H&E ( ... Following standard brainstem radiotherapy, disease progression typically occurs locally in the brainstem. However, in three of sixteen cases the subventricular frontal lobe disease contributed substantially to morbidity and mortality and preceded pontine recurrence in two cases. As therapies improve and patients survive longer in the natural history of their cancer, new patterns of regional relapse often appear (e.g. sanctuary disease in childhood leukemia). Our data show subventricular tumor spread in the majority of patients, typically later in the course of their disease. Thus as future therapies evolve to control local disease, strategies including extended or whole brain irradiation may become crucial. The patterns of widespread dissemination, including leptomeningeal, direct extension and subventricular spread, suggest that the extent of the optimal radiation field should be re-examined.

Role of CYB5A in Pancreatic Cancer Prognosis and Autophagy Modulation

BACKGROUND Loss of 18q22.3 is a prognostic marker in pancreatic ductal adenocarcinoma (PDAC). This study investigated genes encoded by this cytoband. METHODS We studied mRNA/protein expression in radically resected (n = 130) and metastatic patients (n = 50). The role of CYB5A was tested in 11 PDAC cell lines and five primary cultures through retrovirus-mediated upregulation and small interfering RNA using wound-healing, invasion, annexin-V, electron microscopy, and autophagic assays, as well as autophagy genes and kinases arrays. CYB5A+ orthotopic models (n = 6 mice/group) were monitored by Firefly and Gaussia-luciferase bioluminescence, magnetic resonance imaging, and high-frequency ultrasound. Data were analyzed by t test, Fisher exact-test, log-rank test and Cox proportional hazards models. All statistical tests were two-sided. RESULTS Both resected and metastatic patients with low mRNA or protein expression of CYB5A had statistically significantly shorter survival (eg, median = 16.7 months, 95% confidence interval [CI] = 13.5 to 19.9; vs median = 24.8 months, 95% CI = 12.8 to 36.9; P = .02, two-sided log-rank test; n = 82 radically resected PDACs), and multivariable analyses confirmed prognostic relevance. Moreover, we characterized a novel function to CYB5A, autophagy induction, concomitant with reduced proliferation and migration/invasion of PDAC cells. Network analysis of proautophagic pathways suggested CYB5A interaction with TRAF6, which was confirmed by TRAF6 downregulation after CYB5A reconstitution (-69% in SU.86.86-CYB5A+; P = .005, two-sided t test). CYB5A silencing had opposite effects, restoring TRAF6 expression and wound healing. In vivo studies showed that CYB5A induced autophagy while inhibiting tumor growth/metastasis and increasing survival (median = 57 days, 95% CI = 52 to 61; vs median = 44 days, 95% CI = 21 to 57; P = .03, two-sided log-rank test). CONCLUSIONS These results define CYB5A as a novel prognostic factor for PDAC that exerts its tumor-suppressor function through autophagy induction and TRAF6 modulation.

Implementation of a multi-institutional diffuse intrinsic pontine glioma autopsy protocol and characterization of a primary cell culture

V. Caretti, M. H. A. Jansen, D. G. van Vuurden, T. Lagerweij, M. Bugiani, I. Horsman, H. Wessels, P. van der Valk, J. Cloos, D. P. Noske, W. P. Vandertop, P. Wesseling, T. Wurdinger, E. Hulleman and G. J. L. Kaspers (2013) Neuropathology and Applied Neurobiology39, 426–436

Convection enhanced delivery of carmustine to the murine brainstem: a feasibility study.

BACKGROUND Systemic delivery of therapeutic agents remains ineffective against diffuse intrinsic pontine glioma (DIPG), possibly due to an intact blood-brain-barrier (BBB) and to dose-limiting toxicity of systemic chemotherapeutic agents. Convection-enhanced delivery (CED) into the brainstem may provide an effective local delivery alternative for DIPG patients. NEW METHOD The aim of this study is to develop a method to perform CED into the murine brainstem and to test this method using the chemotherapeutic agent carmustine (BiCNU). To this end, a newly designed murine CED catheter was tested in vitro and in vivo. After determination of safety and distribution, mice bearing VUMC-DIPG-3 and E98FM-DIPG brainstem tumors were treated with carmustine dissolved in DW 5% or carmustine dissolved in 10% ethanol. RESULTS Our results show that CED into the murine brainstem is feasible and well tolerated by mice with and without brainstem tumors. CED of carmustine dissolved in 5% DW increased median survival of mice with VUMC-DIPG-3 and E98FM-DIPG tumors with 35% and 25% respectively. Dissolving carmustine in 10% ethanol further improved survival to 45% in mice with E98FM-DIPG tumors. COMPARISON WITH EXISTING METHODS Since genetically engineered and primary DIPG models are currently only available in mice, murine CED studies have clear advantages over CED studies in other animals. CONCLUSION CED in the murine brainstem can be performed safely, is well tolerated and can be used to study efficacy of chemotherapeutic agents orthotopically. These results set the foundation for more CED studies in murine DIPG models.

Bevacizumab Targeting Diffuse Intrinsic Pontine Glioma: Results of 89Zr-Bevacizumab PET Imaging in Brain Tumor Models

The role of the VEGF inhibitor bevacizumab in the treatment of diffuse intrinsic pontine glioma (DIPG) is unclear. We aim to study the biodistribution and uptake of zirconium-89 (89Zr)-labeled bevacizumab in DIPG mouse models. Human E98-FM, U251-FM glioma cells, and HSJD-DIPG-007-FLUC primary DIPG cells were injected into the subcutis, pons, or striatum of nude mice. Tumor growth was monitored by bioluminescence imaging (BLI) and visualized by MRI. Seventy-two to 96 hours after 89Zr-bevacizumab injections, mice were imaged by positron emission tomography (PET), and biodistribution was analyzed ex vivo. High VEGF expression in human DIPG was confirmed in a publically available mRNA database, but no significant 89Zr-bevacizumab uptake could be detected in xenografts located in the pons and striatum at an early or late stage of the disease. E98-FM, and to a lesser extent the U251-FM and HSJD-DIPG-007 subcutaneous tumors, showed high accumulation of 89Zr-bevacizumab. VEGF expression could not be demonstrated in the intracranial tumors by in situ hybridization (ISH) but was clearly present in the perinecrotic regions of subcutaneous E98-FM tumors. The poor uptake of 89Zr-bevacizumab in xenografts located in the brain suggests that VEGF targeting with bevacizumab has limited efficacy for diffuse infiltrative parts of glial brain tumors in mice. Translating these results to the clinic would imply that treatment with bevacizumab in patients with DIPG is only justified after targeting of VEGF has been demonstrated by 89Zr-bevacizumab immuno-PET. We aim to confirm this observation in a clinical PET study with patients with DIPG. Mol Cancer Ther; 15(9); 2166–74. ©2016 AACR.

Anatomical differences determine distribution of adenovirus after convection-enhanced delivery to the rat brain

Background Convection-enhanced delivery (CED) of adenoviruses offers the potential of widespread virus distribution in the brain. In CED, the volume of distribution (Vd) should be related to the volume of infusion (Vi) and not to dose, but when using adenoviruses contrasting results have been reported. As the characteristics of the infused tissue can affect convective delivery, this study was performed to determine the effects of the gray and white matter on CED of adenoviruses and similar sized super paramagnetic iron oxide nanoparticles (SPIO). Methodology/Principal Findings We convected AdGFP, an adenovirus vector expressing Green Fluorescent Protein, a virus sized SPIO or trypan blue in the gray and white matter of the striatum and external capsule of Wistar rats and towards orthotopic infiltrative brain tumors. The resulting Vds were compared to Vi and transgene expression to SPIO distribution. Results show that in the striatum Vd is not determined by the Vi but by the infused virus dose, suggesting diffusion, active transport or receptor saturation rather than convection. Distribution of virus and SPIO in the white matter is partly volume dependent, which is probably caused by preferential fluid pathways from the external capsule to the surrounding gray matter, as demonstrated by co-infusing trypan blue. Distant tumors were reached using the white matter tracts but tumor penetration was limited. Conclusions/Significance CED of adenoviruses in the rat brain and towards infiltrative tumors is feasible when regional anatomical differences are taken into account while SPIO infusion could be considered to validate proper catheter positioning and predict adenoviral distribution.