In vivo ³¹P magnetic resonance spectroscopic imaging (MRSI) for metabolic profiling of human breast cancer xenografts.

PURPOSE: To study cancer associated with abnormal metabolism of phospholipids, of which several have been proposed as biomarkers for malignancy or to monitor response to anticancer therapy. We explored 3D (31) P magnetic resonance spectroscopic imaging (MRSI) at high magnetic field for in vivo assessment of individual phospholipids in two patient-derived breast cancer xenografts representing good and poor prognosis (luminal- and basal-like tumors). MATERIALS AND METHODS: Metabolic profiles from luminal-like and basal-like xenograft tumors were obtained in vivo using 3D (31) P MRSI at 11.7T and from tissue extracts in vitro at 14.1T. Gene expression analysis was performed in order to support metabolic differences between the two xenografts. RESULTS: In vivo (31) P MR spectra were obtained in which the prominent resonances from phospholipid metabolites were detected at a high signal-to-noise ratio (SNR >7.5). Metabolic profiles obtained in vivo were in agreement with those obtained in vitro and could be used to discriminate between the two xenograft models, based on the levels of phosphocholine, phosphoethanolamine, glycerophosphocholine, and glycerophosphoethanolamine. The differences in phospholipid metabolite concentration could partly be explained by gene expression profiles. CONCLUSION: Noninvasive metabolic profiling by 3D (31) P MRSI can discriminate between subtypes of breast cancer based on different concentrations of choline- and ethanolamine-containing phospholipids.

Metabolic imaging of multiple x-nucleus resonances.

This study describes a technique for fast imaging of x-nuclei metabolites. Due to increased sensitivity and larger chemical shift dispersion at high magnetic fields, images of multiple metabolites can be obtained simultaneously by selective excitation of their resonances with a multifrequency selective radiofrequency pulse at any desired flip angle. This aim is achieved by combining a three-dimensional gradient echo imaging sequence with a Shinnar-LeRoux optimized excitation pulse. A proper choice of bandwidth, imaging matrix size, and field of view allows using the chemical shift dispersion of the different resonances to completely separate their images within one large field of view. The method of fast metabolic imaging is illustrated with (13)C measurements of a phantom containing a solution of (13)C labeled glucose, lactate, and sodium octanoate and by dynamic measurements of the (31)P metabolites phosphocreatine and ß-adenosine triphosphate in human femoral muscle in vivo, both at 7T. With dynamic selective (31)P imaging of the larger part of the upper leg, phosphocreatine signal intensity changes of specific muscles can be studied simultaneously by analyzing the sum of phosphocreatine signals within arbitrarily shaped regions of interest following the muscles' contours. This concept of dynamic metabolic imaging can be applied to other organs and further expanded to other MR-detectable nuclei and metabolites.

Clinical Validation of Siemens' Syngo.via Automatic Contouring System.

Purpose: The manual delineation of organs at risk is a process that requires a great deal of time both for the technician and for the physician. Availability of validated software tools assisted by artificial intelligence would be of great benefit, as it would significantly improve the radiation therapy workflow, reducing the time required for segmentation. The purpose of this article is to validate the deep learning-based autocontouring solution integrated in syngo.via RT Image Suite VB40 (Siemens Healthineers, Forchheim, Germany). Methods and Materials: For this purpose, we have used our own specific qualitative classification system, RANK, to evaluate more than 600 contours corresponding to 18 different automatically delineated organs at risk. Computed tomography data sets of 95 different patients were included: 30 patients with lung, 30 patients with breast, and 35 male patients with pelvic cancer. The automatically generated structures were reviewed in the Eclipse Contouring module independently by 3 observers: an expert physician, an expert technician, and a junior physician. Results: There is a statistically significant difference between the Dice coefficient associated with RANK 4 compared with the coefficient associated with RANKs 2 and 3 (P < .001). In total, 64% of the evaluated structures received the maximum score, 4. Only 1% of the structures were classified with the lowest score, 1. The time savings for breast, thorax, and pelvis were 87.6%, 93.5%, and 82.2%, respectively. Conclusions: Siemens' syngo.via RT Image Suite offers good autocontouring results and significant time savings.

Effects of targeting the VEGF and PDGF pathways in diffuse orthotopic glioma models.

Currently available compounds that interfere with VEGF-A signalling effectively inhibit angiogenesis in gliomas, but influence diffuse infiltrative growth to a much lesser extent. Development of a functional tumour vascular bed not only involves VEGF-A but also requires platelet-derived growth factor receptor-ß (PDGFRß), which induces maturation of tumour blood vessels. Therefore, we tested whether combined inhibition of VEGFR and PDGFRß increases therapeutic benefit in the orthotopic glioma xenograft models E98 and E473, both displaying the diffuse infiltrative growth that is characteristically observed in most human gliomas. We used bevacizumab and vandetanib as VEGF(R) inhibitors, and sunitinib to additionally target PDGFRß. We show that combination therapy of sunitinib and vandetanib does not improve therapeutic efficacy compared to treatment with sunitinib, vandetanib or bevacizumab alone. Furthermore, all compounds induced reduction of vessel leakage in compact E98 tumour areas, resulting in decreased detectability of these mostly infiltrative xenografts in Gd-DTPA-enhanced MRI scans. These data show that inhibition of VEGF signalling cannot be optimized by additional PDGFR inhibition and support the concept that diffuse infiltrative areas in gliomas are resistant to anti-angiogenic therapy.

Magnetic resonance imaging-based detection of glial brain tumors in mice after antiangiogenic treatment.

Proper delineation of gliomas using contrast-enhanced magnetic resonance imaging (CE-MRI) poses a problem in neuro-oncology. The blood brain barrier (BBB) in areas of diffuse-infiltrative growth may be intact, precluding extravasation and subsequent MR-based detection of the contrast agent gadolinium diethylenetriaminepenta-acetic acid (Gd-DTPA). Treatment with antiangiogenic compounds may further complicate tumor detection as such compounds can restore the BBB in angiogenic regions. The increasing number of clinical trials with antiangiogenic compounds for treatment of gliomas calls for the development of alternative imaging modalities. Here we investigated whether CE-MRI using ultrasmall particles of iron oxide (USPIO, Sinerem) as blood pool contrast agent has additional value for detection of glioma in the brain of nude mice. We compared conventional T1-weighted Gd-DTPA-enhanced MRI to T2*-weighted USPIO-enhanced MRI in mice carrying orthotopic U87 glioma, which were either or not treated with the antiangiogenic compound vandetanib (ZD6474, ZACTIMA). In untreated animals, vessel leakage within the tumor and a relatively high tumor blood volume resulted in good MRI visibility with Gd-DTPA- and USPIO-enhanced MRI, respectively. Consistent with previous findings, vandetanib treatment restored the BBB in the tumor vasculature, resulting in loss of tumor detectability in Gd-DTPA MRI. However, due to decreased blood volume, treated tumors could be readily detected in USPIO-enhanced MRI scans. Our findings suggest that Gd-DTPA MRI results in overestimation of the effect of antiangiogenic therapy of glioma and that USPIO-MRI provides an important complementary diagnostic tool to evaluate response to antiangiogenic therapy of these tumors.

Contrast enhanced susceptibility weighted imaging (CE-SWI) of the mouse brain using ultrasmall superparamagnetic ironoxide particles (USPIO).

Susceptibility weighted imaging (SWI) has been introduced as a novel approach to visualize the venous vasculature in the human brain. With SWI, small veins in the brain are depicted based on the susceptibility difference between deoxyhaemoglobin in the veins and surrounding tissue, which is further enhanced by the use of MR phase information. In this study we applied SWI in the mouse brain using an exogenous iron-based blood-pool contrast agent, with the aims of further enhancing the susceptibility effect and allowing the visualization of individual veins and arteries. Contrast enhanced (CE-) SWI of the brain was performed on healthy mice and mice carrying intracerebral glioma xenografts. This study demonstrates that detailed vascular information in the mouse brain can be obtained by using CE-SWI and is substantially enhanced compared to native SWI (i.e. without contrast agent). CE-SWI images of tumour-bearing mice were directly compared to histology, confirming that CE-SWI depicts the vessels supplying and draining the tumour. We propose that CE-SWI is a very promising tool for the characterization of tumour vasculature.

IDH1 R132H mutation generates a distinct phospholipid metabolite profile in glioma.

Many patients with glioma harbor specific mutations in the isocitrate dehydrogenase gene IDH1 that associate with a relatively better prognosis. IDH1-mutated tumors produce the oncometabolite 2-hydroxyglutarate. Because IDH1 also regulates several pathways leading to lipid synthesis, we hypothesized that IDH1-mutant tumors have an altered phospholipid metabolite profile that would impinge on tumor pathobiology. To investigate this hypothesis, we performed (31)P-MRS imaging in mouse xenograft models of four human gliomas, one of which harbored the IDH1-R132H mutation. (31)P-MR spectra from the IDH1-mutant tumor displayed a pattern distinct from that of the three IDH1 wild-type tumors, characterized by decreased levels of phosphoethanolamine and increased levels of glycerophosphocholine. This spectral profile was confirmed by ex vivo analysis of tumor extracts, and it was also observed in human surgical biopsies of IDH1-mutated tumors by (31)P high-resolution magic angle spinning spectroscopy. The specificity of this profile for the IDH1-R132H mutation was established by in vitro (31)P-NMR of extracts of cells overexpressing IDH1 or IDH1-R132H. Overall, our results provide evidence that the IDH1-R132H mutation alters phospholipid metabolism in gliomas involving phosphoethanolamine and glycerophosphocholine. These new noninvasive biomarkers can assist in the identification of the mutation and in research toward novel treatments that target aberrant metabolism in IDH1-mutant glioma.

Multivoxel ¹H MR spectroscopy is superior to contrast-enhanced MRI for response assessment after anti-angiogenic treatment of orthotopic human glioma xenografts and provides handles for metabolic targeting.

BACKGROUND: Anti-angiogenic treatment of glioblastoma characteristically results in therapy resistance and tumor progression via diffuse infiltration. Monitoring tumor progression in these patients is thwarted because therapy results in tumor invisibility in contrast-enhanced (CE) MRI. To address this problem, we examined whether tumor progression could be monitored by metabolic mapping using (1)H MR spectroscopic imaging (MRSI). METHODS: We treated groups of BALB/c nu/nu mice carrying different orthotopic diffuse-infiltrative glioblastoma xenografts with bevacizumab (anti-vascular endothelial growth factor [VEGF] antibody, n = 13), cabozantinib (combined VEGF receptor 2/c-Met tyrosine kinase inhibitor, n = 11), or placebo (n = 15) and compared CE-MRI with MRS-derived metabolic maps before, during, and after treatment. Metabolic maps and CE-MRIs were subsequently correlated to histology and immunohistochemistry. RESULTS: In vivo imaging of choline/n-acetyl aspartate ratios via multivoxel MRS is better able to evaluate response to therapy than CE-MRI. Lactate imaging revealed that diffuse infiltrative areas in glioblastoma xenografts did not present with excessive glycolysis. In contrast, glycolysis was observed in hypoxic areas in angiogenesis-dependent compact regions of glioma only, especially after anti-angiogenic treatment. CONCLUSION: Our data present MRSI as a powerful and feasible approach that is superior to CE-MRI and may provide handles for optimizing treatment of glioma. Furthermore, we show that glycolysis is more prominent in hypoxic areas than in areas of diffuse infiltrative growth. The Warburg hypothesis of persisting glycolysis in tumors under normoxic conditions may thus not be valid for diffuse glioma.

Microvascular cerebral blood volume changes in aging APP(swe)/PS1(dE9) AD mouse model: a voxel-wise approach.

Vascular disorders can either be cause or consequence in the pathophysiology of Alzheimer's disease (AD). To comprehensively characterize the occurrence of vascular impairment in a double transgenic mouse model for AD (APPswe/PS1dE9) during aging, we developed a new method to obtain microvascular relative cerebral blood volume (rCBV(micro)) maps from gradient echo MR imaging by histogram evaluation and we applied a voxel-wise approach to detect rCBV(micro) changes. With this methodology the development of cerebral microvascular impairments can be described in vivo with 0.16 mm isotropic resolution for the whole mouse brain. At 8 months, impaired rCBV(micro) appeared in some cortical regions and in the thalamus, which spreads over several sub-cortical areas and the hippocampus at 13 months. With a ROI-based approach, we further showed that hippocampal rCBV(micro) in 13-month-old wild-type and APP(swe)/PS1(dE9) mice correlates well with capillary density measured with immunohistochemical staining. However, no differences in capillary density were detected between genotypes. The rCBV(micro) values showed no significant correlation with amyloid-ß (Aß) plaque deposition, Aß at blood vessel walls and biochemically measured levels of Aß1₋40, Aß1₋42 oligomers and fibrillar forms. These results suggest that rCBV(micro) reduction is caused by an impaired vasoactivity of capillaries and arterioles, which is not directly correlated with the amount of Aß deposition in parenchyma nor blood vessel walls.

NMR at earth's magnetic field using para-hydrogen induced polarization.

A method to achieve NMR of dilute samples in the earth's magnetic field by applying para-hydrogen induced polarization is presented. Maximum achievable polarization enhancements were calculated by numerically simulating the experiment and compared to the experimental results and to the thermal equilibrium in the earth's magnetic field. Simultaneous 19F and 1H NMR detection on a sub-milliliter sample of a fluorinated alkyne at millimolar concentration (∼10(18) nuclear spins) was realized with just one single scan. A highly resolved spectrum with a signal/noise ratio higher than 50:1 was obtained without using an auxiliary magnet or any form of radio frequency shielding.

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. [(18) F] 3'-deoxy-3'-[(18) F]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.

Rapid and robust transgenic high-grade glioma mouse models for therapy intervention studies.

PURPOSE: To develop a transgenic mouse model of glioma that can be conveniently used for testing therapy intervention strategies. High-grade glioma is a devastating and uniformly fatal disease for which better therapy is urgently needed. Typical for high-grade glioma is that glioma cells infiltrate extensively into surrounding pivotal brain structures, thereby rendering current treatments largely ineffective. Evaluation of novel therapies requires the availability of appropriate glioma mouse models. EXPERIMENTAL DESIGN: High-grade gliomas were induced by stereotactic intracranial injection of lentiviral GFAP-Cre or CMV-Cre vectors into compound LoxP-conditional mice, resulting in K-Ras(v12) expression and loss of p16(Ink4a)/p19(Arf) with or without concomitant loss of p53 or Pten. RESULTS: Tumors reproduced many of the features that are characteristic for human high-grade gliomas, including invasiveness and blood-brain barrier functionality. Especially, CMV-Cre injection into p53;Ink4a/Arf;K-Ras(v12) mice resulted in high-grade glioma with a short tumor latency (2-3 weeks) and full penetrance. Early detection and follow-up was accomplished by noninvasive bioluminescence imaging, and the practical utility for therapy intervention was shown in a study with temozolomide. CONCLUSION: We have developed a realistic high-grade glioma model that can be used with almost the same convenience as traditional xenograft models, thus allowing its implementation at the forefront of preclinical evaluation of new treatments.