Non-invasive assessment of telomere maintenance mechanisms in brain tumors.

Telomere maintenance is a universal hallmark of cancer. Most tumors including low-grade oligodendrogliomas use telomerase reverse transcriptase (TERT) expression for telomere maintenance while astrocytomas use the alternative lengthening of telomeres (ALT) pathway. Although TERT and ALT are hallmarks of tumor proliferation and attractive therapeutic targets, translational methods of imaging TERT and ALT are lacking. Here we show that TERT and ALT are associated with unique 1H-magnetic resonance spectroscopy (MRS)-detectable metabolic signatures in genetically-engineered and patient-derived glioma models and patient biopsies. Importantly, we have leveraged this information to mechanistically validate hyperpolarized [1-13C]-alanine flux to pyruvate as an imaging biomarker of ALT status and hyperpolarized [1-13C]-alanine flux to lactate as an imaging biomarker of TERT status in low-grade gliomas. Collectively, we have identified metabolic biomarkers of TERT and ALT status that provide a way of integrating critical oncogenic information into non-invasive imaging modalities that can improve tumor diagnosis and treatment response monitoring.

Preclinical assessment of clinically streamlined, 3D-printed, biocompatible single- and two-stage tissue scaffolds for ear reconstruction.

Auricular reconstruction is a technically demanding procedure requiring significant surgical expertise, as the current gold standard involves hand carving of the costal cartilage into an auricular framework and re-implantation of the tissue. 3D-printing presents a powerful tool that can reduce technical demands associated with the procedure. Our group compared clinical, radiological, histological, and biomechanical outcomes in single- and two-stage 3D-printed auricular tissue scaffolds in an athymic rodent model. Briefly, an external anatomic envelope of a human auricle was created using DICOM computed tomography (CT) images and modified in design to create a two-stage, lock-in-key base and elevating platform. Single- and two-stage scaffolds were 3D-printed by laser sintering poly-L-caprolactone (PCL) then implanted subcutaneously in five athymic rats each. Rats were monitored for ulcer formation, site infection, and scaffold distortion weekly, and scaffolds were explanted at 8 weeks with analysis using microCT and histologic staining. Nonlinear finite element analysis was performed to determine areas of high strain in relation to ulcer formation. Scaffolds demonstrated precise anatomic appearance and maintenance of integrity of both anterior and posterior auricular surfaces and scaffold projection, with no statistically significant differences in complications noted between the single- and two-staged implantation. While minor superficial ulcers occurred most commonly at the lateral and superior helix coincident with finite element predictions of high skin strains, evidence of robust tissue ingrowth and angiogenesis was visible grossly and histologically. This promising preclinical small animal model supports future initiatives for making clinically viable options for an ear tissue scaffold.

Quality assessment tests for tumorigenicity of human iPS cell-derived cartilage.

Articular cartilage damage does not heal spontaneously and causes joint dysfunction. The implantation of induced pluripotent stem cell (iPSC)-derived cartilage (iPS-Cart) is one candidate treatment to regenerate the damaged cartilage. However, concerns of tumorigenicity are associated with iPS-Cart, because the iPSC reprogramming process and long culture time for cartilage induction could increase the chance of malignancy. We evaluated the tumorigenic risks of iPS-Cart using HeLa cells as the reference. Spike tests revealed that contamination with 100 HeLa cells in 150 mg of iPS-Cart accelerated the cell growth rate. On the other hand, 150 mg of iPS-Cart without HeLa cells reached growth arrest and senescence after culture, suggesting less than 100 tumorigenic cells, assuming they behave like HeLa cells, contaminated iPS-Cart. The implantation of 10,000 or fewer HeLa cells into joint surface defects in the knee joint of nude rat did not cause tumor formation. These in vitro and in vivo studies collectively suggest that the implantation of 15 g or less iPS-Cart in the knee joint does not risk tumor formation if assuming that the tumorigenic cells in iPS-Cart are equivalent to HeLa cells and that nude rat knee joints are comparable to human knee joints in terms of tumorigenicity. However, considering the limited immunodeficiency of nude rats, the clinical amount of iPS-Cart for implantation needs to be determined cautiously.

Comparative Behavioral Assessment of Lewis and Nude Rats after Peripheral Nerve Injury.

Cell therapy has shown potential in the field of peripheral nerve repair, and research using rodents is a critical and essential step toward clinical development of this approach. Traditionally, most experimental peripheral nerve injuries are conducted in inbred Lewis or outbred Sprague-Dawley strains. However, transplantation of xenogeneic cells such as human-derived cells typically triggers rejection in these animals. An alternative approach is to use immunodeficient animals, such as athymic nude rats. The lack of functional T cells in these animals renders them more accommodating to foreign cells from a different host. Currently, no literature exists regarding sensorimotor behavioral assessment of nude rats after peripheral nerve injury. To this end, we compared the functional recovery during a 6-wk period of behavioral testing of Lewis and nude rats after unilateral sciatic nerve crushing injury. Three sensorimotor behavioral assessments were performed weekly: a ladder rungwalking task to assess slip ratio and cross duration, von Frey nociception testing to determine the paw withdrawal threshold thus monitoring the regaining of sensory function, and sciatic functional index evaluation to monitor the recovery of integrated motor function. Both strains demonstrated significant sensory and motor deficits in the first week after injury, with a slight regain of sensory function, reduced slip ratio, and increased sciatic functional index starting at 2 wk. No significance difference existed between nude and Lewis rats in their recovery courses. We conclude that nude rats are a suitable model for behavioral training and assessment for cell transplantation studies in peripheral nerve injury and repair.

MRI assessment of treatment delivery for interstitial photodynamic therapy of high-grade glioma in a preclinical model.

BACKGROUND: High-grade gliomas are primary brain tumors that have shown increasing incidence and unfavorable outcomes. Local control is crucial to the management of this pathology. Photodynamic therapy (PDT), based on the light-induced activation of a photosensitizer (PS), achieves local treatment by inducing selective lesions in tumor tissue. OBJECTIVES: Previous studies have reported the outcomes of PDT for glioblastoma via immunohistological data. Our study aimed to evaluate MRI findings, including diffusion, and perfusion sequences, compared with immunohistological data from the same population to address the efficiency of light fractionation. MATERIALS AND METHODS: Twenty-six "nude" rats grafted with human U87 cells into the right putamen underwent PDT. After PS precursor (5-ALA) intake, an optical fiber was introduced into the tumor. The rats were randomized into the following groups: those without illumination and those that received two or five fractions of light. Treatment effects were assessed with early high-field MRI to measure the volume of necrosis and edema using diffusion and perfusion sequences; the MRI results were compared with immunohistology results, including necrosis and apoptosis markers. RESULTS: Elevated diffusion values were observed on MRI in the centers of the tumors of the treated animals, especially in the 5-fraction group (P < 0.01). Perfusion was decreased around the treatment site, especially in the 5-fraction group (P = 0.024). The MRI findings were consistent with previously published histological data. The median volume of necrosis was significantly different between the sham group and treated groups, 0 mm3 versus 2.67 mm3 , P < 0.001. The same trend was previously observed in histology data when grading the absence or presence of necrosis and when the presence of necrosis was significantly more predominant for the treated group than for the untreated group (P < 001). Additionally, cell death represented by apoptosis marker data (TUNEL method) was significantly higher in the 5-fraction group than in the 2-fraction group (P = 0.01). CONCLUSION: Diffusion and perfusion MRI revealed histological lesions. Interstitial PDT (iPDT) induced specific lesions in the tumor tissue, which were observed with MRI and confirmed by histopathological analysis. Thus, MRI may provide a non-invasive and reliable tool to assess treatment outcomes after PDT. Lasers Surg. Med. 50:460-468, 2018. © 2017 Wiley Periodicals, Inc.

Quantitative assessment of the effects of water proton concentration and water T1 changes on amide proton transfer (APT) and nuclear overhauser enhancement (NOE) MRI: The origin of the APT imaging signal in brain tumor.

PURPOSE: To quantify pure chemical exchange-dependent saturation transfer (CEST) related amide proton transfer (APT) and nuclear Overhauser enhancement (NOE) signals in a rat glioma model and to investigate the mixed effects of water content and water T1 on APT and NOE imaging signals. METHODS: Eleven U87 tumor-bearing rats were scanned at 4.7 T. A relatively accurate mathematical approach, based on extrapolated semisolid magnetization-transfer reference signals, was used to remove the concurrent effects of direct water saturation and semisolid magnetization-transfer. Pure APT and NOE signals, in addition to the commonly used magnetization-transfer-ratio asymmetry at 3.5 ppm, MTRasym (3.5ppm), were assessed. RESULTS: The measured APT signal intensity of the tumor (11.06%, much larger than the value reported in the literature) was the major contributor (approximately 80.6%) to the MTRasym (3.5ppm) contrast between the tumor and the contralateral brain region. Both the water content ([water proton]) and water T1 (T1w ) were increased in the tumor, but there were no significant correlations among APT, NOE, or MTRasym (3.5ppm) signals and T1w /[water proton]. CONCLUSION: The effect of increasing T1w on the CEST signal in the tumor was mostly eliminated by the effect of increasing water content, and the observed APT-weighted hyperintensity in the tumor should be dominated by the increased amide proton concentration. Magn Reson Med 77:855-863, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Assessment of Glioma Response to Radiotherapy Using Multiple MRI Biomarkers with Manual and Semiautomated Segmentation Algorithms.

BACKGROUND AND PURPOSE: Multimodality magnetic resonance imaging (MRI) can provide complementary information in the assessment of brain tumors. We aimed to segment tumor in amide proton transfer-weighted (APTw) images and to investigate multiparametric MRI biomarkers for the assessment of glioma response to radiotherapy. For tumor extraction, we evaluated a semiautomated segmentation method based on region of interest (ROI) results by comparing it with the manual segmentation method. METHODS: Thirteen nude rats injected with U87 tumor cells were irradiated by an 8-Gy radiation dose. All MRI scans were performed on a 4.7-T animal scanner preradiation, and at day 1, day 4, and day 8 postradiation. Two experts performed manual and semiautomated methods to extract tumor ROIs on APTw images. Multimodality MRI signals of the tumors, including structural (T2 and T1 ), functional (apparent diffusion coefficient and blood flow), and molecular (APTw and magnetization transfer ratio or MTR), were calculated and compared quantitatively. RESULTS: The semiautomated method provided more reliable tumor extraction results on APTw images than the manual segmentation, in less time. A considerable increase in the ADC intensities of the tumor was observed during the postradiation. A steady decrease in the blood flow values and in the APTw signal intensities were found after radiotherapy. CONCLUSIONS: The semiautomated method of tumor extraction showed greater efficiency and stability than the manual method. Apparent diffusion coefficient, blood flow, and APTw are all useful biomarkers in assessing glioma response to radiotherapy.

NEMA NU 4-Optimized Reconstructions for Therapy Assessment in Cancer Research with the Inveon Small Animal PET/CT System.

PURPOSE: We compared conventional filtered back-projection (FBP), two-dimensional-ordered subsets expectation maximization (OSEM) and maximum a posteriori (MAP) NEMA NU 4-optimized reconstructions for therapy assessment. PROCEDURES: Varying reconstruction settings were used to determine the parameters for optimal image quality with two NEMA NU 4 phantom acquisitions. Subsequently, data from two experiments in which nude rats bearing subcutaneous tumors had received a dual PI3K/mTOR inhibitor were reconstructed with the NEMA NU 4-optimized parameters. Mann-Whitney tests were used to compare mean standardized uptake value (SUV(mean)) variations among groups. RESULTS: All NEMA NU 4-optimized reconstructions showed the same 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) kinetic patterns and detected a significant difference in SUV(mean) relative to day 0 between controls and treated groups for all time points with comparable p values. CONCLUSION: In the framework of therapy assessment in rats bearing subcutaneous tumors, all algorithms available on the Inveon system performed equally.

Label-free microscopic assessment of glioblastoma biopsy specimens prior to biobanking [corrected].

Glioblastoma is the most common primary brain tumor with a median 12- to 15-month patient survival. Improving patient survival involves better understanding the biological mechanisms of glioblastoma tumorigenesis and seeking targeted molecular therapies. Central to furthering these advances is the collection and storage of surgical biopsies (biobanking) for research. This paper addresses an imaging modality, confocal reflectance microscopy (CRM), for safely screening glioblastoma biopsy samples prior to biobanking to increase the quality of tissue provided for research and clinical trials. These data indicate that CRM can immediately identify cellularity of tissue biopsies from animal models of glioblastoma. When screening fresh human biopsy samples, CRM can differentiate a cellular glioblastoma biopsy from a necrotic biopsy without altering DNA, RNA, or protein expression of sampled tissue. These data illustrate CRM's potential for rapidly and safely screening clinical biopsy samples prior to biobanking, which demonstrates its potential as an effective screening technique that can improve the quality of tissue biobanked for patients with glioblastoma.

Assessment of intervertebral disc degeneration based on quantitative magnetic resonance imaging analysis: an in vivo study.

STUDY DESIGN: Animal experimental study. OBJECTIVE: To evaluate a novel quantitative imaging technique for assessing disc degeneration. SUMMARY OF BACKGROUND DATA: T2-relaxation time (T2-RT) measurements have been used to assess disc degeneration quanti-tatively. T2 values correlate with the water content of intervertebral disc tissue and thereby allow for the indirect measurement of nucleus pulposus (NP) hydration. METHODS: We developed an algorithm to subtract out magnetic resonance imaging (MRI) voxels not representing NP tissue on the basis of T2-RT values. Filtered NP voxels were used to measure nuclear size by their amount and nuclear hydration by their mean T2-RT. This technique was applied to 24 rat-tail intervertebral discs (IVDs), which had been punctured with an 18-gauge needle according to different techniques to induce varying degrees of degeneration. NP voxel count and average T2-RT were used as parameters to assess the degeneration process at 1 and 3 months postpuncture. NP voxel counts were evaluated against radiograph disc height measurements and qualitative MRI studies on the basis of the Pfirrmann grading system. Tails were collected for histology to correlate NP voxel counts to histological disc degeneration grades and to NP cross-sectional area measurements. RESULTS: NP voxel count measurements showed strong correlations to qualitative MRI analyses (R = 0.79, P < 0.0001), histological degeneration grades (R = 0.902, P < 0.0001), and histological NP cross-sectional area measurements (R = 0.887, P < 0.0001).In contrast to NP voxel counts, the mean T2-RT for each punctured group remained constant between months 1 and 3. The mean T2-RTs for the punctured groups did not show a statistically significant difference from those of healthy IVDs (63.55 ms ± 5.88 ms mo 1 and 62.61 ms ± 5.02 ms) at either time point. CONCLUSION: The NP voxel count proved to be a valid parameter to assess disc degeneration quantitatively in a needle puncture model. The mean NP T2-RT does not change significantly in needle-puncture-induced degenerated IVDs. IVDs can be segmented into different tissue components according to their innate T2-RT.

Quantitative multiparametric MRI assessment of glioma response to radiotherapy in a rat model.

BACKGROUND: The inability of structural MRI to accurately measure tumor response to therapy complicates care management for patients with gliomas. The purpose of this study was to assess the potential of several noninvasive functional and molecular MRI biomarkers for the assessment of glioma response to radiotherapy. METHODS: Fourteen U87 tumor-bearing rats were irradiated using a small-animal radiation research platform (40 or 20 Gy), and 6 rats were used as controls. MRI was performed on a 4.7 T animal scanner, preradiation treatment, as well as at 3, 6, 9, and 14 days postradiation. Image features of the tumors, as well as tumor volumes and animal survival, were quantitatively compared. RESULTS: Structural MRI showed that all irradiated tumors still grew in size during the initial days postradiation. The apparent diffusion coefficient (ADC) values of tumors increased significantly postradiation (40 and 20 Gy), except at day 3 postradiation, compared with preradiation. The tumor blood flow decreased significantly postradiation (40 and 20 Gy), but the relative blood flow (tumor vs contralateral) did not show a significant change at most time points postradiation. The amide proton transfer weighted (APTw) signals of the tumor decreased significantly at all time points postradiation (40 Gy), and also at day 9 postradiation (20 Gy). The blood flow and APTw maps demonstrated tumor features that were similar to those seen on gadolinium-enhanced T1-weighted images. CONCLUSIONS: Tumor ADC, blood flow, and APTw were all useful imaging biomarkers by which to predict glioma response to radiotherapy. The APTw signal was most promising for early response assessment in this model.

Micro-computed tomography-based three-dimensional kinematic analysis during lateral bending for spinal fusion assessment in a rat posterolateral lumbar fusion model.

Rat posterolateral lumbar fusion (PLF) models have been used to assess the safety and effectiveness of new bone substitutes and osteoinductive growth factors using palpation, radiography, micro-computed tomography (µCT), and histology as standard methods to evaluate spinal fusion. Despite increased numbers of PLF studies involving alternative bone substitutes and growth factors, the quantitative assessment of treatment efficacy during spinal motion has been limited. The purpose of this study was to evaluate the effect of spinal fusion on lumbar spine segment stability during lateral bending using a µCT-based three-dimensional (3D) kinematic analysis in the rat PLF model. Fourteen athymic male rats underwent PLF surgery at L4/5 and received bone grafts harvested from the ilium and femurs of syngeneic rats (Isograft, n=7) or no graft (Sham, n=7). At 8 weeks after the PLF surgery, spinal fusion was assessed by manual palpation, plain radiography, µCT, and histology. To determine lumbar segmental motions at the operated level during lateral bending, 3D kinematic analysis was performed. The Isograft group, but not the Sham group, showed spinal fusion on manual palpation (6/7), solid fusion mass in radiographs (6/7), as well as bone bridging in µCT and histological images (5/7). Compared to the Sham group, the Isograft group revealed limited 3D lateral bending angular range of motion and lateral translation during lateral bending at the fused segment where disc height narrowing was observed. This µCT-based 3D kinematic analysis can provide a quantitative assessment of spinal fusion in a rat PLF model to complement current gold standard methods used for efficacy assessment of new therapeutic approaches.

Non-invasive in vivo assessment of IDH1 mutational status in glioma.

Gain-of-function mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade gliomas and secondary glioblastoma. They lead to intracellular accumulation of the oncometabolite 2-hydroxyglutarate, represent an early pathogenic event and are considered a therapeutic target. Here we show, in this proof-of-concept study, that [1-(13)C] α-ketoglutarate can serve as a metabolic imaging agent for non-invasive, real-time, in vivo monitoring of mutant IDH1 activity, and can inform on IDH1 status. Using (13)C magnetic resonance spectroscopy in combination with dissolution dynamic nuclear polarization, the metabolic fate of hyperpolarized [1-(13)C] α-ketoglutarate is studied in isogenic glioblastoma cells that differ only in their IDH1 status. In lysates and tumours that express wild-type IDH1, only hyperpolarized [1-(13)C] α-ketoglutarate can be detected. In contrast, in cells that express mutant IDH1, hyperpolarized [1-(13)C] 2-hydroxyglutarate is also observed, both in cell lysates and in vivo in orthotopic tumours.

Comparison of consecutive bolus tracking and flash replenishment measurements for the assessment of tissue hemodynamics using contrast-enhanced ultrasound (CEUS) in an experimental human squamous cell carcinoma model.

PURPOSE: To evaluate "bolus-tracking" (BT) and "flash-replenishment" (FR) for the assessment of tissue hemodynamics by contrast-enhanced ultrasound (CEUS) in an experimental small-animal-squamous-cell-carcinoma-model. Since the underlying tissue is the same, strong correlations between parameter outcomes of both techniques are expected. METHODS AND MATERIALS: Human hypopharynx-carcinoma-cells were subcutaneously injected into the left flank of 18 female athymic-nude-rats. After 10 days of subcutaneous tumour growth, bolus tracking and flash-replenishment measurements were performed consecutively in the same imaging plane in each rat after bolus-injection of SonoVue via the lateral tail vein using a high-end ultrasound system with a 15 MHz probe. Video-sequences were analysed with dedicated software (VueBox®, Bracco-Suisse®). From BT measurements, the parameters peak enhancement (PEBT), wash-in area-under-the-curve (Wi-AUCBT), mean transit time (MTTBT), wash-in-rate (WiRBT) and perfusion-index (Wi-PIBT) were derived; FR yielded estimates of relative-blood-volume (rBVFR), mean transit time MTTFR, relative blood flow rBFFR and wash-in rate Wi-RFR. RESULTS: In all rats, BT and FR measurements could be completed successfully. Highly significant correlations were observed between rBVFR and PEBT, rBVFR and Wi-AUCBT, rBVFR and MTTBT, rBVFR and WiPIBT, MTTFR and MTTBT, rBFFR and PEBT, rBFFR and Wi-AUCBT, rBFFR and WiRBT, rBFFR and WiPIBT, WiRFR and PEBT, WiRFR and Wi-AUCBT, WiRFR and WiRBT and WiRFR and WiPIBT. CONCLUSION: Whereas bolus tracking can be used in a wide range of modalities including CEUS, CT and MR, FR as a technique for the assessment of tissue hemodynamics is unique to CEUS. Although BT and FR yield different parameters, the underlying tissue hemodynamics are equal. In this work, we were able to demonstrate strong correlations between different parameters of both modalities in a small-animal-tumor-model, indicating that flash-replenishment is a valid alternative to the more established bolus-tracking technique. Although the lack of absolute, quantitative parameters hinders a direct comparison of both modalities, FR and BT should both be suitable for a relative comparison, e.g. between baseline and follow-up examinations.

Improved perfusion MR imaging assessment of intracerebral tumor blood volume and antiangiogenic therapy efficacy in a rat model with ferumoxytol.

PURPOSE: To evaluate the consistency of tumor blood volume measurements and antiangiogenic therapy efficacy assessments with a low-molecular-weight gadolinium-based contrast agent (GBCA, gadodiamide) versus an iron oxide nanoparticle (ferumoxytol) in the presence or absence of a loading dose of contrast agent before perfusion magnetic resonance (MR) imaging (preload method). MATERIALS AND METHODS: The protocol was approved by the institutional animal care and use committee. U87MG tumor cells were implanted intracerebrally in 13 rats. All 13 rats underwent 11.75-T MR imaging with gadodiamide (60 µL) 13 days after tumor implantation. The next day, nine rats underwent MR imaging with ferumoxytol (60 µL). Immediately after ferumoxytol imaging, six rats received bevacizumab (45 mg/kg). MR imaging was repeated 48 hours after bevacizumab treatment with gadodiamide and 72 hours after treatment with ferumoxytol. Each study included three consecutive dynamic susceptibility-weighted contrast material-enhanced (DSC) MR acquisitions, which were performed without preload, with single-dose preload, and with double-dose preload. Tumor relative cerebral blood volume (rCBV) was estimated from each DSC MR acquisition. Two-way repeated measures analysis of variance was performed to test for differences between groups with both contrast agents. RESULTS: DSC MR imaging with gadodiamide and without preload showed low rCBV (≤ 1.75) in nine of the 13 tumors; estimated rCBV increased progressively with both single- and double-dose preloads (P < .001). Conversely, rCBVs obtained with ferumoxytol were high (>1.75) and remained constant with all three acquisitions. The magnitude of rCBV decrease after bevacizumab administration was dependent on the dose of gadodiamide preload, whereas the magnitude of rCBV decrease with ferumoxytol was constant regardless of whether contrast agent preload was used. CONCLUSION: With GBCA, tumor rCBV can be underestimated without preload and becomes dose dependent with preload correction. Conversely, ferumoxytol provides consistent assessment of tumor rCBV and antiangiogenic therapy efficacy.

Assessment of multiparametric MRI in a human glioma model to monitor cytotoxic and anti-angiogenic drug effects.

Early imaging or blood biomarkers of tumor response is needed to customize anti-tumor therapy on an individual basis. This study evaluates the sensitivity and relevance of five potential MRI biomarkers. Sixty nude rats were implanted with human glioma cells (U-87 MG) and randomized into three groups: one group received an anti-angiogenic treatment (Sorafenib), a second a cytotoxic drug [1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU (Carmustine)] and a third no treatment. The tumor volume, apparent diffusion coefficient (ADC) of water, blood volume fraction (BVf), microvessel diameter (vessel size index, VSI) and vessel wall integrity (contrast enhancement, CE) were monitored before and during treatment. Sorafenib reduced tumor CE as early as 1 day after treatment onset. By 4 days after treatment onset, tumor BVf was reduced and tumor VSI was increased. By 14 days after treatment onset, ADC was increased and the tumor growth rate was reduced. With BCNU, ADC was increased and the tumor growth rate was reduced 14 days after treatment onset. Thus, the estimated MRI parameters were sensitive to treatment at different times after treatment onset and in a treatment-dependent manner. This study suggests that multiparametric MR monitoring could allow the assessment of new anti-tumor drugs and the optimization of combined therapies.

Establishment of efficacy and safety assessment of human adipose tissue-derived mesenchymal stem cells (hATMSCs) in a nude rat femoral segmental defect model.

Human adipose tissue-derived mesenchymal stem cell (hATMSC) have emerged as a potentially powerful tool for bone repair, but an appropriate evaluation system has not been established. The purpose of this study was to establish a preclinical assessment system to evaluate the efficacy and safety of cell therapies in a nude rat bone defect model. Segmental defects (5 mm) were created in the femoral diaphyses and transplanted with cell media (control), hydroxyapatite/tricalcium phosphate scaffolds (HA/TCP, Group I), hATMSCs (Group II), or three cell-loading density of hATMSC-loaded HA/TCP (Group III-V). Healing response was evaluated by serial radiography, micro-computed tomography and histology at 16 weeks. To address safety-concerns, we conducted a GLP-compliant toxicity study. Scanning electron microscopy studies showed that hATMSCs filled the pores/surfaces of scaffolds in a cell-loading density-dependent manner. We detected significant increases in bone formation in the hATMSC-loaded HA/TCP groups compared with other groups. The amount of new bone formation increased with increases in loaded cell number. In a toxicity study, no significant hATMSC-related changes were found in body weights, clinical signs, hematological/biochemical values, organ weights, or histopathological findings. In conclusion, hATMSCs loaded on HA/TCP enhance the repair of bone defects and was found to be safe under our preclinical efficacy/safety hybrid assessment system.

Dynamic magnetic resonance imaging assessment of vascular targeting agent effects in rat intracerebral tumor models.

We used dynamic MRI to evaluate the effects of monoclonal antibodies targeting brain tumor vasculature. Female athymic rats with intracerebral human tumor xenografts were untreated or treated with intetumumab, targeting α(V)-integrins, or bevacizumab, targeting vascular endothelial growth factor (n = 4-6 per group). Prior to treatment and at 1, 3, and 7 days after treatment, we performed standard MRI to assess tumor volume, dynamic susceptibility-contrast MRI with the blood-pool iron oxide nanoparticle ferumoxytol to evaluate relative cerebral blood volume (rCBV), and dynamic contrast-enhanced MRI to assess tumor vascular permeability. Tumor rCBV increased by 27 ± 13% over 7 days in untreated rats; intetumumab increased tumor rCBV by 65 ± 10%, whereas bevacizumab reduced tumor rCBV by 31 ± 10% at 7 days (P < .001 for group and day). Similarly, intetumumab increased brain tumor vascular permeability compared with controls at 3 and 7 days after treatment, whereas bevacizumab decreased tumor permeability within 24 hours (P = .0004 for group, P = .0081 for day). All tumors grew over the 7-day assessment period, but bevacizumab slowed the increase in tumor volume on MRI. We conclude that the vascular targeting agents intetumumab and bevacizumab had diametrically opposite effects on dynamic MRI of tumor vasculature in rat brain tumor models. Targeting α(V)-integrins increased tumor vascular permeability and blood volume, whereas bevacizumab decreased both measures. These findings have implications for chemotherapy delivery and antitumor efficacy.

Effectiveness of scaffolds with pre-seeded mesenchymal stem cells in bone regeneration--assessment of osteogenic ability of scaffolds implanted under the periosteum of the cranial bone of rats.

To date, there has been no study on the development of novel regimens based on the following tissue engineering principles: seeding and culturing mesenchymal stem cells (MSCs) on a scaffold before surgery or injecting cultured MSCs into a scaffold during surgery. The purpose of this study was to assess the in vivo osteogenic ability of scaffold/MSCs implanted beneath the periosteum of the cranial bone of rats in three different sample groups: one in which MSCs were pre-seeded and cultured on a scaffold to produce the 3-D woven fabric scaffold/MSC composite using osteo-lineage induction medium, one in which cultured MSCs produced by osteo-lineage induction in cell cultivation flasks were injected into a scaffold during surgery and a control group, in which only the 3-D woven fabric scaffold was implanted. The results indicate that pre-seeding MSCs on a scaffold leads to a higher osteogenic ability than injecting cultured MSCs into a scaffold during surgery.

Assessment of acute antivascular effects of vandetanib with high-resolution dynamic contrast-enhanced computed tomographic imaging in a human colon tumor xenograft model in the nude rat.

Tumor size is not a reliable marker for the assessment of early antivascular effects of antiangiogenics. In the present study, we used 200-microm in-plane high-resolution dynamic contrast-enhanced computed tomography (DCE-CT) to noninvasively assess the immediate antivascular effects of vandetanib in a subcutaneous human colon cancer (LoVo) xenograft model in nude rats and to investigate correlation between changes in CT perfusion parameters and tumor volume or immunohistochemical end points. At 3 to 4 weeks after LoVo cell implantation, the animal was gavaged with either vandetanib (50 mg/kg) or vehicle twice (22 hours apart) and scanned with a preclinical DCE-CT scanner before (0 hour) and after treatment (24 hours). Quantitative maps of blood flow (BF) and volume (BV) of the tumor were calculated from the acquired DCE-CT images. The rats were divided into nonhypovascular, hypovascular, and combined (regardless of vascularity) groups. In the nonhypovascular group, significant decreases in both tumor BF and BV were observed in the vandetanib-treated rats compared with increases in the vehicle-treated rats. A significant decrease in BV was detected in the vandetanib-treated rats in the combined group as well. No differences in tumor growth, vascular endothelial growth factor expression, microvessel density, or apoptosis were observed between vandetanib- and vehicle-treated rats in all three groups. These results demonstrate that BF and BV imaging biomarkers from DCE-CT imaging can be used for rapid monitoring of immediate (24 hours after) antimicrovascular effects of vandetanib on tumors, even in the absence of significant changes of tumor volume or clinically relevant immunohistochemical end points.