Subcutaneous deuterated substrate administration in mice: An alternative to tail vein infusion.

PURPOSE: Tail-vein catheterization and subsequent in-magnet infusion is a common route of administration of deuterium (2 H)-labeled substrates in small-animal deuterium (D) MR studies. With mice, because of the tail vein's small diameter, this procedure is challenging. It requires considerable personnel training and practice, is prone to failure, and may preclude serial studies. Motivated by the need for an alternative, the time courses for common small-molecule deuterated substrates and downstream metabolites in brain following subcutaneous infusion were determined in mice and are presented herein. METHODS: Three 2 H-labeled substrates-[6,6-2 H2 ]glucose, [2 H3 ]acetate, and [3,4,4,4-2 H4 ]beta-hydroxybutyrate-and 2 H2 O were administered to mice in-magnet via subcutaneous catheter. Brain time courses of the substrates and downstream metabolites (and semi-heavy water) were determined via single-voxel DMRS. RESULTS: Subcutaneous catheter placement and substrate administration was readily accomplished with limited personnel training. Substrates reached pseudo-steady state in brain within ∼30-40 min of bolus infusion. Time constants characterizing the appearance in brain of deuterated substrates or semi-heavy water following 2 H2 O administration were similar (∼15 min). CONCLUSION: Administration of deuterated substrates via subcutaneous catheter for in vivo DMRS experiments with mice is robust, requires limited personnel training, and enables substantial dosing. It is suitable for metabolic studies where pseudo-steady state substrate administration/accumulation is sufficient. It is particularly advantageous for serial longitudinal studies over an extended period because it avoids inevitable damage to the tail vein following multiple catheterizations.

Comparison of hyperpolarized 13 C and non-hyperpolarized deuterium MRI approaches for imaging cerebral glucose metabolism at 4.7 T.

PURPOSE: The purpose of this study was to directly compare two isotopic metabolic imaging approaches, hyperpolarized (HP) 13 C MRI and deuterium metabolic imaging (DMI), for imaging specific closely related segments of cerebral glucose metabolism at 4.7 T. METHODS: Comparative HP-13 C and DMI neuroimaging experiments were conducted consecutively in normal rats during the same scanning session. Localized conversions of [1-13 C]pyruvate and [6,6-2 H2 ]glucose to their respective downstream metabolic products were measured by spectroscopic imaging, using an identical 2D-CSI sequence with parameters optimized for the respective experiments. To facilitate direct comparison, a pair of substantially equivalent 2.5-cm double-tuned X/1 H RF surface coils was developed. For improved results, multidimensional low-rank reconstruction was applied to denoise the raw DMI data. RESULTS: Localized conversion of HP [1-13 C]pyruvate to [1-13 C]lactate, and [6,6-2 H2 ]glucose to [3,3-2 H2 ]lactate and Glx-d (glutamate and glutamine), was detected in rat brain by spectroscopic imaging at 4.7 T. The SNR and spatial resolution of HP-13 C MRI was superior to DMI but limited to a short time window, whereas the lengthy DMI acquisition yielded maps of not only lactate, but also Glx production, albeit with relatively poor spectral discrimination between metabolites at this field strength. Across the individual rats, there was an apparent inverse correlation between cerebral production of HP [1-13 C]lactate and Glx-d, along with a trend toward increased [3,3-2 H2 ]lactate. CONCLUSION: The HP-13 C MRI and DMI methods are both feasible at 4.7 T and have significant potential for metabolic imaging of specific segments of glucose metabolism.

15 N-carnitine, a novel endogenous hyperpolarized MRI probe with long signal lifetime.

PURPOSE: The purpose of this study was to investigate hyperpolarization and in vivo imaging of [15 N]carnitine, a novel endogenous MRI probe with long signal lifetime. METHODS: L-[15 N]carnitine-d9 was hyperpolarized by the method of dynamic nuclear polarization followed by rapid dissolution. The T1 signal lifetimes were estimated in aqueous solution and in vivo following intravenous injection in rats, using a custom-built dual-tuned 15 N/1 H RF coil at 4.7 T. 15 N chemical shift imaging and 15 N fast spin-echo images of rat abdomen were acquired 3 minutes after [15 N]carnitine injection. RESULTS: Estimated T1 times of [15 N]carnitine at 4.7 T were 210 seconds (in H2 O) and 160 seconds (in vivo), with an estimated polarization level of 10%. Remarkably, the [15 N]carnitine coherence was detectable in rat abdomen for 5 minutes after injection for the nonlocalized acquisition. No downstream metabolites were detected on localized or nonlocalized 15 N spectra. Diffuse liver enhancement was detected on 15 N fast spin-echo imaging 3 minutes after injection, with mean hepatic SNR of 18 ± 5 at a spatial resolution of 4 × 4 mm. CONCLUSION: This study showed the feasibility of hyperpolarizing and imaging the biodistribution of HP [15 N]carnitine.

Longitudinal preclinical magnetic resonance imaging of diffuse tumor burden in intramedullary myeloma following bortezomib therapy.

Multiple myeloma (MM) is a largely incurable, debilitating hematologic malignancy of terminally differentiated plasma cells in the bone marrow (BM). Identification of therapeutic response is critical for improving outcomes and minimizing costs and off-target toxicities. To assess changes in BM environmental factors and therapy efficacy, there is a need for noninvasive, nonionizing, longitudinal, preclinical methods. Here, we demonstrate the feasibility of preclinical magnetic resonance imaging (MRI) for longitudinal imaging of diffuse tumor burden in a syngeneic, immunocompetent model of intramedullary MM. C57Bl/KaLwRij mice were implanted intravenously with 5TGM1-GFP tumors and treated with a proteasome inhibitor, bortezomib, or vehicle control. MRI was performed weekly with a Helmholtz radiofrequency coil placed on the hind leg. Mean normalized T1-weighted signal intensities and T2 relaxation times were quantified for each animal following manual delineation of BM regions in the femur and tibia. Finally, tumor burden was quantified for each tissue using hematoxylin and eosin staining. Changes in T2 relaxation times correlated strongly to cell density and overall tumor burden in the BM. Median T2 relaxation times and regional T1-weighted contrast uptake were shown to be most relevant in identifying posttherapy disease stage in this model of intramedullary MM. In summary, our results highlighted potential preclinical MRI markers for assessing tumor burden and BM heterogeneity following bortezomib therapy, and demonstrated the application of longitudinal imaging with preclinical MRI in an immunocompetent, intramedullary setting.

Can anti-vascular endothelial growth factor antibody reverse radiation necrosis? A preclinical investigation.

Anti-vascular endothelial growth factor (anti-VEGF) antibodies are a promising new treatment for late time-to-onset radiation-induced necrosis (RN). We sought to evaluate and validate the response to anti-VEGF antibody in a mouse model of RN. Mice were irradiated with the Leksell Gamma Knife Perfexion™ and then treated with anti-VEGF antibody, beginning at post-irradiation (PIR) week 8. RN progression was monitored via anatomic and diffusion MRI from weeks 4-12 PIR. Standard histology, using haematoxylin and eosin (H&E), and immunohistochemistry staining were used to validate the response to treatment. After treatment, both post-contrast T1-weighted and T2-weighted image-derived lesion volumes decreased (P < 0.001), while the lesion volumes for the control group increased. The abnormally high apparent diffusion coefficient (ADC) for RN also returned to the ADC range for normal brain following treatment (P < 0.001). However, typical RN pathology was still present histologically. Large areas of focal calcification were observed in ~50% of treated mouse brains. Additionally, VEGF and hypoxia-inducible factor 1-alpha (HIF-1α) were continually upregulated in both the anti-VEGF and control groups. Despite improvements observed radiographically following anti-VEGF treatment, lesions were not completely resolved histologically. The subsequent calcification and the continued upregulation of VEGF and HIF-1α merit further preclinical/clinical investigation.

O2 -sensitive MRI distinguishes brain tumor versus radiation necrosis in murine models.

PURPOSE: The goal of this study was to quantify the relationship between the (1) H longitudinal relaxation rate constant, R1 , and oxygen (O2 ) concentration (relaxivity, r1 ) in tissue and to quantify O2 -driven changes in R1 (ΔR1 ) during a breathing gas challenge in normal brain, radiation-induced lesions, and tumor lesions. METHODS: R1 data were collected in control-state mice (n = 4) during three different breathing gas (and thus tissue O2 ) conditions. In parallel experiments, pO2 was measured in the thalamus of control-state mice (n = 4) under the same breathing gas conditions using an O2 -sensitive microprobe. The relaxivity of tissue O2 was calculated using the R1 and pO2 data. R1 data were collected in control-state (n = 4) mice, a glioma model (n = 7), and a radiation necrosis model (n = 6) during two breathing gas (thus tissue O2 ) conditions. R1 and ΔR1 were calculated for each cohort. RESULTS: O2 r1 in the brain was 9 × 10(-4) ± 3 × 10(-4) mm Hg(-1) · s(-1) at 4.7T. R1 and ΔR1 measurements distinguished radiation necrosis from tumor (P< 0.03 and P< 0.01, respectively). CONCLUSION: The relaxivity of O2 in the brain is determined. R1 and ΔR1 measurements differentiate tumor lesions from radiation necrosis lesions in the mouse models. These pathologies are difficult to distinguish by traditional imaging techniques; O2 -driven changes in R1 holds promise in this regard. Magn Reson Med 75:2442-2447, 2016. © 2015 Wiley Periodicals, Inc.

Wide-field two-dimensional multifocal optical-resolution photoacoustic-computed microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technique that directly images optical absorption in tissue at high spatial resolution. To date, the majority of OR-PAM systems are based on single-focused optical excitation and ultrasonic detection, limiting the wide-field imaging speed. While 1D multifocal OR-PAM (1D-MFOR-PAM) has been developed, the potential of microlens and transducer arrays has not been fully realized. Here we present the development of 2D multifocal optical-resolution photoacoustic-computed microscopy (2D-MFOR-PACM), using a 2D microlens array and a full-ring ultrasonic transducer array. The 10 mm×10 mm microlens array generates 1800 optical foci within the focal plane of the 512-element transducer array, and raster scanning the microlens array yields optical-resolution photoacoustic images. The system has improved the in-plane resolution of a full-ring transducer array from ≥100 to 29 µm and achieved an imaging time of 36 s over a 10 mm×10 mm field of view. In comparison, the 1D-MFOR-PAM would take more than 4 min to image over the same field of view. The imaging capability of the system was demonstrated on phantoms and animals both ex vivo and in vivo.

Evaluation of gliovascular functions of AQP4 readthrough isoforms.

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to WT and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

Evaluation of gliovascular functions of Aqp4 readthrough isoforms.

Aquaporin-4 (AQP4) is a water channel protein that links astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, that preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel mouse AQP4 line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wildtype (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to wildtype, and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. Increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two were unchanged. However, at 100% readthrough, AQP4x localization and formation of higher-order complexes was disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for increased endothelial cell vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

Quantitative analysis of tumor burden in mouse lung via MRI.

Lung cancer is the leading cause of cancer death in the United States. Despite recent advances in screening protocols, the majority of patients still present with advanced or disseminated disease. Preclinical rodent models provide a unique opportunity to test novel therapeutic drugs for targeting lung cancer. Respiratory-gated MRI is a key tool for quantitatively measuring lung-tumor burden and monitoring the time-course progression of individual tumors in mouse models of primary and metastatic lung cancer. However, quantitative analysis of lung-tumor burden in mice by MRI presents significant challenges. Herein, a method for measuring tumor burden based upon average lung-image intensity is described and validated. The method requires accurate lung segmentation; its efficiency and throughput would be greatly aided by the ability to automatically segment the lungs. A technique for automated lung segmentation in the presence of varying tumor burden levels is presented. The method includes development of a new, two-dimensional parametric model of the mouse lungs and a multi-faceted cost function to optimally fit the model parameters to each image. Results demonstrate a strong correlation (0.93), comparable with that of fully manual expert segmentation, between the automated method's tumor-burden metric and the tumor burden measured by lung weight.

Distinguishing Tumor Admixed in a Radiation Necrosis (RN) Background: 1H and 2H MR With a Novel Mouse Brain-Tumor/RN Model.

Purpose: Distinguishing radiation necrosis (RN) from recurrent tumor remains a vexing clinical problem with important health-care consequences for neuro-oncology patients. Here, mouse models of pure tumor, pure RN, and admixed RN/tumor are employed to evaluate hydrogen (1H) and deuterium (2H) magnetic resonance methods for distinguishing RN vs. tumor. Furthermore, proof-of-principle, range-finding deuterium (2H) metabolic magnetic resonance is employed to assess glycolytic signatures distinguishing RN vs. tumor. Materials and Methods: A pipeline of common quantitative 1H MRI contrasts, including an improved magnetization transfer ratio (MTR) sequence, and 2H magnetic resonance spectroscopy (MRS) following administration of 2H-labeled glucose, was applied to C57BL/6 mouse models of the following: (i) late time-to-onset RN, occurring 4-5 weeks post focal 50-Gy (50% isodose) Gamma Knife irradiation to the left cerebral hemisphere, (ii) glioblastoma, growing ~18-24 days post implantation of 50,000 mouse GL261 tumor cells into the left cerebral hemisphere, and (iii) mixed model, with GL261 tumor growing within a region of radiation necrosis (1H MRI only). Control C57BL/6 mice were also examined by 2H metabolic magnetic resonance. Results: Differences in quantitative 1H MRI parametric values of R1, R2, ADC, and MTR comparing pure tumor vs. pure RN were all highly statistically significant. Differences in these parameter values and DCEAUC for tumor vs. RN in the mixed model (tumor growing in an RN background) are also all significant, demonstrating that these contrasts-in particular, MTR-can effectively distinguish tumor vs. RN. Additionally, quantitative 2H MRS showed a highly statistically significant dominance of aerobic glycolysis (glucose ➔ lactate; fermentation, Warburg effect) in the tumor vs. oxidative respiration (glucose ➔ TCA cycle) in the RN and control brain. Conclusions: These findings, employing a pipeline of quantitative 1H MRI contrasts and 2H MRS following administration of 2H-labeled glucose, suggest a pathway for substantially improving the discrimination of tumor vs. RN in the clinic.

Selective Cell Size MRI Differentiates Brain Tumors from Radiation Necrosis.

Brain metastasis is a common characteristic of late-stage lung cancers. High doses of targeted radiotherapy can control tumor growth in the brain but can also result in radiotherapy-induced necrosis. Current methods are limited for distinguishing whether new parenchymal lesions following radiotherapy are recurrent tumors or radiotherapy-induced necrosis, but the clinical management of these two classes of lesions differs significantly. Here, we developed, validated, and evaluated a new MRI technique termed selective size imaging using filters via diffusion times (SSIFT) to differentiate brain tumors from radiotherapy necrosis in the brain. This approach generates a signal filter that leverages diffusion time dependence to establish a cell size-weighted map. Computer simulations in silico, cultured cancer cells in vitro, and animals with brain tumors in vivo were used to comprehensively validate the specificity of SSIFT for detecting typical large cancer cells and the ability to differentiate brain tumors from radiotherapy necrosis. SSIFT was also implemented in patients with metastatic brain cancer and radiotherapy necrosis. SSIFT showed high correlation with mean cell sizes in the relevant range of less than 20 µm. The specificity of SSIFT for brain tumors and reduced contrast in other brain etiologies allowed SSIFT to differentiate brain tumors from peritumoral edema and radiotherapy necrosis. In conclusion, this new, cell size-based MRI method provides a unique contrast to differentiate brain tumors from other pathologies in the brain. SIGNIFICANCE: This work introduces and provides preclinical validation of a new diffusion MRI method that exploits intrinsic differences in cell sizes to distinguish brain tumors and radiotherapy necrosis.

Irradiation-Modulated Murine Brain Microenvironment Enhances GL261-Tumor Growth and Inhibits Anti-PD-L1 Immunotherapy.

PURPOSE: Clinical evidence suggests radiation induces changes in the brain microenvironment that affect subsequent response to treatment. This study investigates the effect of previous radiation, delivered six weeks prior to orthotopic tumor implantation, on subsequent tumor growth and therapeutic response to anti-PD-L1 therapy in an intracranial mouse model, termed the Radiation Induced Immunosuppressive Microenvironment (RI2M) model. METHOD AND MATERIALS: C57Bl/6 mice received focal (hemispheric) single-fraction, 30-Gy radiation using the Leksell GammaKnife® Perfexion™, a dose that does not produce frank/gross radiation necrosis. Non-irradiated GL261 glioblastoma tumor cells were implanted six weeks later into the irradiated hemisphere. Lesion volume was measured longitudinally by in vivo MRI. In a separate experiment, tumors were implanted into either previously irradiated (30 Gy) or non-irradiated mouse brain, mice were treated with anti-PD-L1 antibody, and Kaplan-Meier survival curves were constructed. Mouse brains were assessed by conventional hematoxylin and eosin (H&E) staining, IBA-1 staining, which detects activated microglia and macrophages, and fluorescence-activated cell sorting (FACS) analysis. RESULTS: Tumors in previously irradiated brain display aggressive, invasive growth, characterized by viable tumor and large regions of hemorrhage and necrosis. Mice challenged intracranially with GL261 six weeks after prior intracranial irradiation are unresponsive to anti-PD-L1 therapy. K-M curves demonstrate a statistically significant difference in survival for tumor-bearing mice treated with anti-PD-L1 antibody between RI2M vs. non-irradiated mice. The most prominent immunologic change in the post-irradiated brain parenchyma is an increased frequency of activated microglia. CONCLUSIONS: The RI2M model focuses on the persisting (weeks-to-months) impact of radiation applied to normal, control-state brain on the growth characteristics and immunotherapy response of subsequently implanted tumor. GL261 tumors growing in the RI2M grew markedly more aggressively, with tumor cells admixed with regions of hemorrhage and necrosis, and showed a dramatic loss of response to anti-PD-L1 therapy compared to tumors in non-irradiated brain. IHC and FACS analyses demonstrate increased frequency of activated microglia, which correlates with loss of sensitivity to checkpoint immunotherapy. Given that standard-of-care for primary brain tumor following resection includes concurrent radiation and chemotherapy, these striking observations strongly motivate detailed assessment of the late effects of the RI2M on tumor growth and therapeutic efficacy.

Magnetic resonance imaging of hypoxic injury to the murine placenta.

We assessed the use of magnetic resonance imaging (MRI) to define placental hypoxic injury associated with fetal growth restriction. On embryonic day 18.5 (E18.5) we utilized dynamic contrast-enhanced (DCE)-MRI on a 4.7-tesla small animal scanner to examine the uptake and distribution of gadolinium-based contrast agent. Quantitative DCE parameter analysis was performed for the placenta and fetal kidneys of three groups of pregnant C57BL/6 mice: 1) mice that were exposed to Fi(O(2)) = 12% between E15.5 and E18.5, 2) mice in normoxia with food restriction similar to the intake of hypoxic mice between E15.5 and E18.5, and 3) mice in normoxia that were fed ad libitum. After imaging, we assessed fetoplacental weight, placental histology, and gene expression. We found that dams exposed to hypoxia exhibited fetal growth restriction (weight reduction by 28% and 14%, respectively, P < 0.05) with an increased placental-to-fetal ratio. By using MRI-based assessment of placental contrast agent kinetics, referenced to maternal paraspinous muscle, we found decreased placental clearance of contrast media in hypoxic mice, compared with either control group (61%, P < 0.05). This was accompanied by diminished contrast accumulation in the hypoxic fetal kidneys (23%, P < 0.05), reflecting reduced transplacental gadolinium transport. These changes were associated with increased expression of placental Phlda2 and Gcm1 transcripts. Exposure to hypoxia near the end of mouse pregnancy reduces placental perfusion and clearance of contrast. MRI-based DCE imaging provides a novel tool for dynamic, in vivo assessment of placental function.

Immunodeficient mouse strains display marked variability in growth of human melanoma lung metastases.

PURPOSE: Immunodeficient mice serve as critical hosts for transplantation of xenogeneic cells for in vivo analysis of various biological processes. Because investigators typically select one or two immunodeficient mouse strains as recipients, no comprehensive study has been published documenting differences in human tumor engraftment. Taking advantage of the increased metastatic potential of RhoC-expressing human (A375) melanoma cells, we evaluate four immunodeficient mouse strains: severe combined immunodeficiency (scid), nonobese diabetic (NOD)-scid, NOD-scid beta2m(null), and NOD-scid IL2Rgamma(null) as xenograft tumor recipients. EXPERIMENTAL DESIGN: Bioluminescence, magnetic resonance imaging, and histopathology were used to monitor serial tumor growth. Natural killer (NK) cell function was examined in each mouse strain using standard (51)Chromium release assays. RESULTS: Melanoma metastases growth is delayed and variable in scid and NOD-scid mice. In contrast, NOD-scid beta2m(null) and NOD-scid IL2Rgamma(null) mice show rapid tumor engraftment, although tumor growth is variable in NOD-scid beta2m(null) mice. NK cells were detected in all strains except NOD-scid IL2Rgamma(null), and in vitro activated scid, NOD-scid, and NOD-scid beta2m(null) NK cells kill human melanoma lines and primary melanoma cells. Expression of human NKG2D ligands MHC class I chain-related A and B molecules renders melanoma susceptible to murine NK cell-mediated cytotoxicity and killing is inhibited by antibody blockade of murine NKG2D. CONCLUSIONS: Murine NKG2D recognition of MICA/B is an important receptor-ligand interaction used by NK cells in immunodeficient strains to limit engraftment of human tumors. The absolute NK deficiency in NOD-scid IL2Rgamma(null) animals makes this strain an excellent recipient of melanoma and potentially other human malignancies.

Test-Retest Performance of a 1-Hour Multiparametric MR Image Acquisition Pipeline With Orthotopic Triple-Negative Breast Cancer Patient-Derived Tumor Xenografts.

Preclinical imaging is critical in the development of translational strategies to detect diseases and monitor response to therapy. The National Cancer Institute Co-Clinical Imaging Resource Program was launched, in part, to develop best practices in preclinical imaging. In this context, the objective of this work was to develop a 1-hour, multiparametric magnetic resonance image-acquisition pipeline with triple-negative breast cancer patient-derived xenografts (PDXs). The 1-hour, image-acquisition pipeline includes T1- and T2-weighted scans, quantitative T1, T2, and apparent diffusion coefficient (ADC) parameter maps, and dynamic contrast-enhanced (DCE) time-course images. Quality-control measures used phantoms. The triple-negative breast cancer PDXs used for this study averaged 174 ± 73 µL in volume, with region of interest-averaged T1, T2, and ADC values of 1.9 ± 0.2 seconds, 62 ± 3 milliseconds, and 0.71 ± 0.06 µm2/ms (mean ± SD), respectively. Specific focus was on assessing the within-subject test-retest coefficient-of-variation (CVWS) for each of the magnetic resonance imaging metrics. Determination of PDX volume via manually drawn regions of interest is highly robust, with ∼1% CVWS. Determination of T2 is also robust with a ∼3% CVWS. Measurements of T1 and ADC are less robust with CVWS values in the 6%-11% range. Preliminary DCE test-retest time-course determinations, as quantified by area under the curve and Ktrans from 2-compartment exchange (extended Tofts) modeling, suggest that DCE is the least robust protocol, with ∼30%-40% CVWS.

Late Effects of Radiation Prime the Brain Microenvironment for Accelerated Tumor Growth.

PURPOSE: Glioblastoma (GBM) remains incurable, despite state-of-the-art treatment involving surgical resection, chemotherapy, and radiation. GBM invariably recurs as a highly invasive and aggressive phenotype, with the majority of recurrences within the radiation therapy treatment field. Although a large body of literature reporting on primary GBM exists, comprehensive studies of how prior irradiation alters recurrent tumor growth are lacking. An animal model that replicates the delayed effects of radiation therapy on the brain microenvironment, and its impact on the development of recurrent GBM, would be a significant advance. METHODS AND MATERIALS: Cohorts of mice received a single fraction of 0, 20, 30, or 40 Gy Gamma Knife irradiation. Naïve, nonirradiated mouse GBM tumor cells were implanted into the ipsilateral hemisphere 6 weeks postirradiation. Tumor growth was measured by magnetic resonance imaging, and animal survival was assessed by monitoring weight loss. Magnetic resonance imaging results were supported by hemotoxylin and eosin histology. RESULTS: Tumorous lesions generated from orthotopic implantation of nonirradiated mouse GBM tumor cells into irradiated mouse brain grew far more aggressively and invasively than implantation of these same cells into nonirradiated brain. Lesions in irradiated brain tissue were significantly larger, more necrotic, and more vascular than those in control animals with increased invasiveness of tumor cells in the periphery, consistent with the histologic features commonly observed in recurrent high-grade tumors in patients. CONCLUSIONS: Irradiation of normal brain primes the targeted cellular microenvironment for aggressive tumor growth when naïve (not previously irradiated) cancer cells are subsequently introduced. The resultant growth pattern is similar to the highly aggressive pattern of tumor regrowth observed clinically after therapeutic radiation therapy. The mouse model offers an avenue for determining the cellular and molecular basis for the aggressiveness of recurrent GBM.

Discriminating radiation injury from recurrent tumor with [18F]PARPi and amino acid PET in mouse models.

BACKGROUND: Radiation injury can be indistinguishable from recurrent tumor on standard imaging. Current protocols for this differential diagnosis require one or more follow-up imaging studies, long dynamic acquisitions, or complex image post-processing; despite much research, the inability to confidently distinguish between these two entities continues to pose a significant dilemma for the treating clinician. Using mouse models of both glioblastoma and radiation necrosis, we tested the potential of poly(ADP-ribose) polymerase (PARP)-targeted PET imaging with [18F]PARPi to better discriminate radiation injury from tumor. RESULTS: In mice with experimental radiation necrosis, lesion uptake on [18F]PARPi-PET was similar to contralateral uptake (1.02 ± 0.26 lesion/contralateral %IA/ccmax ratio), while [18F]FET-PET clearly delineated the contrast-enhancing region on MR (2.12 ± 0.16 lesion/contralateral %IA/ccmax ratio). In mice with focal intracranial U251 xenografts, tumor visualization on PARPi-PET was superior to FET-PET, and lesion-to-contralateral activity ratios (max/max, p = 0.034) were higher on PARPi-PET than on FET-PET. CONCLUSIONS: A murine model of radiation necrosis does not demonstrate [18F]PARPi avidity, and [18F]PARPi-PET is better than [18F]FET-PET in distinguishing radiation injury from brain tumor. [18F]PARPi-PET can be used for discrimination between recurrent tumor and radiation injury within a single, static imaging session, which may be of value to resolve a common dilemma in neuro-oncology.

Inhibitors of HIF-1α and CXCR4 Mitigate the Development of Radiation Necrosis in Mouse Brain.

PURPOSE: There is mounting evidence that, in addition to angiogenesis, hypoxia-induced inflammation via the hypoxia-inducible factor 1α (HIF-1α)-CXC chemokine receptor 4 (CXCR4) pathway may contribute to the pathogenesis of late-onset, irradiation-induced necrosis. This study investigates the mitigative efficacy of an HIF-1α inhibitor, topotecan, and a CXCR4 antagonist, AMD3100, on the development of radiation necrosis (RN) in an intracranial mouse model. METHODS AND MATERIALS: Mice received a single-fraction, 50-Gy dose of hemispheric irradiation from the Leksell Gamma Knife Perfexion and were then treated with either topotecan, an HIF-1α inhibitor, from 1 to 12 weeks after irradiation, or AMD3100, a CXCR4 antagonist, from 4 to 12 weeks after irradiation. The onset and progression of RN were monitored longitudinally via noninvasive, in vivo magnetic resonance imaging (MRI) from 4 to 12 weeks after irradiation. Conventional hematoxylin-eosin staining and immunohistochemistry staining were performed to evaluate the treatment response. RESULTS: The progression of brain RN was significantly mitigated for mice treated with either topotecan or AMD3100 compared with control animals. MRI-derived lesion volumes were significantly smaller for both of the treated groups, and histologic findings correlated well with the MRI data. By hematoxylin-eosin staining, both treated groups demonstrated reduced irradiation-induced tissue damage compared with controls. Furthermore, immunohistochemistry results revealed that expression levels of vascular endothelial growth factor, CXC chemokine ligand 12, CD68, CD3, and tumor necrosis factor α in the lesion area were significantly lower in treated (topotecan or AMD3100) brains versus control brains, while ionized calcium-binding adapter molecule 1 (Iba1) and HIF-1α expression was similar, though somewhat reduced. CXCR4 expression was reduced only in topotecan-treated mice, while interleukin 6 expression was unaffected by either topotecan or AMD3100. CONCLUSIONS: By reducing inflammation, both topotecan and AMD3100 can, independently, mitigate the development of RN in the mouse brain. When combined with first-line, antiangiogenic treatment, anti-inflammation therapy may provide an adjuvant therapeutic strategy for clinical, postirradiation management of tumors, with additional benefits in the mitigation of RN development.

Assessment of myocardial blood flow using 15O-water and 1-11C-acetate in rats with small-animal PET.

UNLABELLED: This feasibility study was undertaken to determine whether myocardial blood flow (MBF, mL/g/min) could be quantified noninvasively in small rodents using microPET and 15O-water or 1-11C-acetate. METHODS: MBF was measured in 18 healthy rats using PET and 15O-water (MBF-W) under different interventions and compared with direct measurements obtained with microspheres (MBF-M). Subsequently, MBF was estimated in 24 rats at rest using 1-11C-acetate (MBF-Ace) and compared with measurements obtained with 15O-water. Using factor analysis, images were processed to obtain 1 blood and 1 myocardial time-activity curve per tracer per study. MBF-W was calculated using a well-validated 1-compartment kinetic model. MBF-Ace was estimated using a simple 1-compartment model to estimate net tracer uptake, K1 (K1 (mL/g/min) = MBF.E; E = first-pass myocardial extraction of 1-11C-acetate) and washout (k2 (min(-1))) along with F(BM) (spillover correction) after fixing F(MM) (partial-volume correction) to values obtained from 15O-water modeling. K1 values were converted to MBF values using a first-pass myocardial extraction/flow relationship measured in rats (E = 1.0-0.74.exp(-1.13/MBF)). RESULTS: In the first study, MBF-W correlated well with MBF-M (y = 0.74x + 0.96; n = 18, r = 0.91, P < 0.0001). However, the slope was different than unity, P < 0.05). Refitting of the data after forcing the intercept to be zero resulted in a nonbias correlation between MBF-W and MBF-M (y = 0.95x + 0.0; n = 18, r = 0.86, P < 0.0001) demonstrating that the underestimation of the slope could be attributed to the overestimation of MBF-W for 2 MBF-M values lower than 1.50 mL/g/min. In the second study, MBF-Ace values correlated well with MBF-W with no underestimation of MBF (y = 0.91x + 0.35; n = 24, r = 0.87, P < 0.0001). CONCLUSION: MBF can be quantified by PET using (15)O-water or 1-11C-acetate in healthy rats. Future studies are needed to determine the accuracy of the methods in low-flow states and to develop an approach for a partial-volume correction when 1-11C-acetate is used.