Irisin, an exercise myokine, potently suppresses tumor proliferation, invasion, and growth in glioma.

Glioblastoma multiforme is the most common and aggressive glial tumor with poor prognosis. Importantly, effective treatment options for glioblastoma are unmet needs. Obesity and low physical activity have been linked with a high risk of cancer, and exercise is related to delayed cancer development and progression. Epidemiological studies have revealed a correlation between exercise and the survival rate of patients with glioblastoma. Nevertheless, the mechanisms by which exercise exerts its anticancer effects in glioblastoma remain unclear. Here, we found that irisin, an exercise-induced myokine, induced G2 /M cell cycle arrest and increased p21 levels in glioblastoma cells, leading to the inhibition of cell proliferation. In addition, irisin inhibited glioblastoma cell invasion by upregulating TFPI-2 and even reversed the aggressive tumor phenotype promoted by co-cultivation with cancer-associated adipocytes. Furthermore, irisin retarded xenograft glioblastoma tumor growth, and radiolabeled irisin demonstrated specific tumor-targeting capability in vivo. Therefore, this study identified one potential molecular mechanism by which exercise prevents cancer progression via irisin. Intriguingly, irisin has the potential to be developed as a molecular imaging and therapeutic anticancer agent.

Integrin α2ß1-targeting ferritin nanocarrier traverses the blood-brain barrier for effective glioma chemotherapy.

BACKGROUND: Ferritin, the natural iron storage protein complex, self-assembles into a uniform cage-like structure. Human H-ferritin (HFn) has been shown to transverse the blood-brain barrier (BBB) by binding to transferrin receptor 1 (TfR1), which is abundant in endothelial cells and overexpressed in tumors, and enters cells via endocytosis. Ferritin is easily genetically modified with various functional molecules, justifying that it possesses great potential for development into a nanocarrier drug delivery system. RESULTS: In this study, a unique integrin α2ß1-targeting H-ferritin (2D-HFn)-based drug delivery system was developed that highlights the feasibility of receptor-mediated transcytosis (RMT) for glioma tumor treatment. The integrin targeting α2ß1 specificity was validated by biolayer interferometry in real time monitoring and followed by cell binding, chemo-drug encapsulation stability studies. Compared with naïve HFn, 2D-HFn dramatically elevated not only doxorubicin (DOX) drug loading capacity (up to 458 drug molecules/protein cage) but also tumor targeting capability after crossing BBB in an in vitro transcytosis assay (twofold) and an in vivo orthotopic glioma model. Most importantly, DOX-loaded 2D-HFn significantly suppressed subcutaneous and orthotopic U-87MG tumor progression; in particular, orthotopic glioma mice survived for more than 80 days. CONCLUSIONS: We believe that this versatile nanoparticle has established a proof-of-concept platform to enable more accurate brain tumor targeting and precision treatment arrangements. Additionally, this unique RMT based ferritin drug delivery technique would accelerate the clinical development of an innovative drug delivery strategy for central nervous system diseases with limited side effects in translational medicine.

Focused Ultrasound Enhances Central Nervous System Delivery of Bevacizumab for Malignant Glioma Treatment.

Purpose To demonstrate that magnetic resonance (MR) imaging-monitored transcranial focused ultrasound can enhance the delivery of the antiangiogenic monoclonal antibody bevacizumab into the central nervous system (CNS) for glioblastoma multiforme (GBM) treatment. Materials and Methods All animal experiments were approved by the animal committee and adhered to experimental animal care guidelines. Transcranial focused ultrasound exposure in the presence of microbubbles was used to open the blood-brain barrier (BBB) to enhance bevacizumab penetration into the CNS in healthy and glioma-bearing mice. Bevacizumab concentration was quantitated with high-performance liquid chromatography, and Western blot testing was performed to confirm the specific biologic form in the CNS. Penetration of bevacizumab into brain tissue was estimated in vivo by means of contrast material-enhanced MR imaging and quantitative gallium 68 ((68)Ga)-bevacizumab micro-positron emission tomography, and glioma progression was longitudinally followed with T2-weighted MR imaging. Hematoxylin-eosin staining and cluster of differentiation 31 immunostaining were used to assess morphologic changes and vascular inhibition at histologic examination. The two-tailed Student t test and the Mantel-Cox log-rank test were used for statistical analyses, with a significance level of .05. Results Focused ultrasound significantly enhanced bevacizumab penetration into the CNS by 5.7- to 56.7-fold compared with that in nonexposed brain (both P < .0001). Contrast-enhanced MR imaging indexes correlated with bevacizumab concentration (r = 0.748-0.857) in vivo. Focused ultrasound-enhanced bevacizumab delivery significantly retarded glioma progression, with a significantly increased median survival (median increase in survival time = 135% in the group treated with bevacizumab and focused ultrasound, P < .0001; as compared with 48% in the group treated with bevacizumab alone, P = .0002). Conclusion Focused ultrasound-enhanced bevacizumab delivery can provide an antivascularization normalization effect to suppress glioma. (©) RSNA, 2016 Online supplemental material is available for this article.

Human ATP-Binding Cassette Transporter ABCG2 Confers Resistance to CUDC-907, a Dual Inhibitor of Histone Deacetylase and Phosphatidylinositol 3-Kinase.

CUDC-907 is a novel, dual-acting small molecule compound designed to simultaneously inhibit the activity of histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K). Treatment with CUDC-907 led to sustained inhibition of HDAC and PI3K activity, inhibition of RAF-MEK-MAPK signaling pathway, and inhibition of cancer cell growth. CUDC-907 is currently under evaluation in phase I clinical trials in patients with lymphoma or multiple myeloma, and in patients with advanced solid tumors. However, the risk of developing acquired resistance to CUDC-907 can present a significant therapeutic challenge to clinicians in the future and should be investigated. The overexpression of ATP-binding cassette (ABC) drug transporter ABCB1, ABCC1, or ABCG2 is one of the most common mechanisms of developing multidrug resistance (MDR) in cancers and a major obstacle in chemotherapy. In this study, we reveal that ABCG2 reduces the intracellular accumulation of CUDC-907 and confers significant resistance to CUDC-907, which leads to reduced activity of CUDC-907 to inhibit HDAC and PI3K in human cancer cells. Moreover, although CUDC-907 affects the transport function of ABCG2, it was not potent enough to reverse drug resistance mediated by ABCG2 or affect the expression level of ABCG2 in human cancer cells. Taken together, our findings indicate that ABCG2-mediated CUDC-907 resistance can have serious clinical implications and should be further investigated. More importantly, we demonstrate that the activity of CUDC-907 in ABCG2-overexpressing cancer cells can be restored by inhibiting the function of ABCG2, which provides support for the rationale of combining CUDC-907 with modulators of ABCG2 to improve the pharmacokinetics and efficacy of CUDC-907 in future treatment trials.

Human ATP-Binding Cassette Transporter ABCB1 Confers Resistance to Volasertib (BI 6727), a Selective Inhibitor of Polo-like Kinase 1.

The overexpression of the serine/threonine specific polo-like kinase 1 (Plk1) is associated with poor prognosis in many types of cancer. Consequently, Plk1 has emerged as a valid therapeutic target for anticancer drug design. Volasertib is a potent inhibitor of Plk1 that inhibits the proliferation of multiple human cancer cell lines by promoting cell cycle arrest at nanomolar concentrations. However, the risk of developing drug resistance, which is often associated with the overexpression of the ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein), can present a therapeutic challenge for volasertib and many other therapeutic drugs. Although volasertib is highly effective against the proliferation of numerous cancer cell lines, we found that the overexpression of ABCB1 in cancer cells leads to cellular resistance to volasertib and reduces the level of volasertib-stimulated G2/M cell cycle arrest and subsequent onset of apoptosis. Furthermore, we demonstrate that volasertib competitively inhibits the function of ABCB1 and stimulates the basal ATPase activity of ABCB1 in a concentration-dependent manner, which is consistent with substrate transport by ABCB1. More importantly, we discovered that the coadministration of an inhibitor or drug substrate of ABCB1 restored the anticancer activity of volasertib in ABCB1-overexpressing cancer cells. In conclusion, the results of our study reveal that ABCB1 negatively affects the efficacy of volasertib and supports its combination with a modulator of ABCB1 to improve clinical responses.

Evaluation of prognostic integrin α2ß1 PET tracer and concurrent targeting delivery using focused ultrasound for brain glioma detection.

The ability to early detect and assess the treatment response of recurrent and/or disseminated metastatic glioblastoma is critical for the effective management of this group of patients. Accumulating experimental evidence indicates that integrin α2ß1 might be a prognostic biomarker for advanced phenotype of cancers. In this study, a novel (68)Ga-labeled integrin α2ß1-targeted PET tracer (68)Ga-NOTA-PEG4-cyclo (GDGEAyK) ((68)Ga-A2B1) was designed and evaluated for the potential prognostic imaging of glioblastoma tumor in preclinical model. To prospectively verify the prognostic value of integrin α2ß1, the in vitro Western blot and flow cytometry studies were performed to validate the integrin expression level of human glioblastoma (U87MG) cells. Extremely high expression level of integrin α2ß1 justifies its role as a potential targeting marker. Thus, (68)Ga-A2B1 positron emission tomography was performed in subcutaneous U87MG tumor bearing athymic mice at 15 min postinjection after injection of 7-8MBq tracers. The receptor targeting specificity was confirmed in a competition blocking experiment. The tumor uptake of (68)Ga-A2B1 in the control and blockage groups was 1.57 ± 0.13 %ID/g (n = 3) and 0.96 ± 0.23 %ID/g** (n = 3), respectively. However, because of the quick renal washout rate and labile nature of peptide tracers in circulation conditions, the focus ultrasound (FUS) mediated delivery method was adopted to enhance tumor uptake and retention of tracers. To test the FUS delivery efficacy in vivo, three experimental arms were designed as follows: tumor bearing mice were administrated with (68)Ga-A2B1 only or microbubbles (MBs) with FUS treatment ((68)Ga-A2B1 + FUS + MBs) or embedded (68)Ga-A2B1-microbubbles ((68)Ga-A2B1-MBs + FUS) followed with FUS sonication. The average radioactivity accumulation within a tumor was quantified from the multiple region of interest volumes using the %ID/g value and was analyzed in accordance with the ex vivo autoradiographic and pathologic data. The significant tumor uptake in (68)Ga-A2B1 + FUS +MBs group (n = 6) and (68)Ga-A2B1-MBs + FUS group (n = 4) following FUS treatment were calculated as 2.25 ± 0.50 %ID/g* and 2.6 ± 0.49 %ID/g**, comparing with (68)Ga-A2B1 only group 1.48 ± 0.42 %ID/g (n = 10). These results suggest that there is significant difference in (68)Ga-A2B1 tumor uptake by FUS treatment either with or without tracer integration with microbubbles, which demonstrate a promising delivery strategy and critical multimodal setting for phenotyping imaging of aggressive glioma tumor. In conclusion, (68)Ga labeled (68)Ga-A2B1 allows noninvasive imaging of tumor-associated α2ß1 expression and can be embedded in MB lipid shell for enhanced delivery and controlled release by sonoporation.

Radioactive smart probe for potential corrected matrix metalloproteinase imaging.

Although various activatable optical probes have been developed to visualize metalloproteinase (MMP) activities in vivo, precise quantification of the enzyme activity is limited due to the inherent scattering and attenuation (limited depth penetration) properties of optical imaging. In this investigation, a novel activatable peptide probe (64)Cu-BBQ650-PLGVR-K(Cy5.5)-E-K(DOTA)-OH was constructed to detect tumor MMP activity in vivo. This agent is optically quenched in its native form, but releases strong fluorescence upon cleavage by selected enzymes. MMP specificity was confirmed both in vitro and in vivo by fluorescent imaging studies. The use of a single modality to image biomarkers/processes may lead to erroneous interpretation of imaging data. The introduction of a quantitative imaging modality, such as PET, would make it feasible to correct the enzyme activity determined from optical imaging. In this proof of principle report, we demonstrated the feasibility of correcting the activatable optical imaging data through the PET signal. This approach provides an attractive new strategy for accurate imaging of MMP activity, which may also be applied for other protease imaging.

Novel α(2)ß(1) integrin-targeted peptide probes for prostate cancer imaging.

Accumulating experimental evidence indicates that overexpression of α(2)ß(1) integrin may correlate with progression in human prostate cancer. The objective of this study was to design a novel imaging probe based on the Asp-Gly-Glu-Ala (DGEA) peptide for near-infrared-fluorescent (NIRF) imaging of α(2)ß(1) integrin expression in prostate cancer. The peptides were conjugated with appropriate fluorescent dyes, and the binding affinity of these probes was evaluated by flow cytometry in three human prostate cell lines (PC-3, CWR-22, and LNCaP). In vivo NIRF imaging of the α(2)ß(1)-positive PC-3 xenograft model was performed to evaluate the α(2)ß(1) targeted probe. In vitro immunofluorescence staining was carried out to confirm the α(2)ß(1) integrin expression level. Flow cytometry analysis showed that PC-3 had the highest probe uptake, followed by CWR-22 and LNCaP tumor cells. In the subcutaneous PC-3 model, the tumor demonstrated prominent uptake with good tumor to background contrast. Immunohistochemistry staining also supported the in vivo optical imaging results. DGEA-based optical agents have been developed for specific imaging of α(2)ß(1) integrin expression. In vitro and in vivo localization demonstrated the potential of this agent to identify tumor subtypes amenable to anti-α(2)ß(1) integrin treatment and potentially provide prognostic information regarding tumor progression.

Biological stability evaluation of the α2ß1 receptor imaging agents: diamsar and DOTA conjugated DGEA peptide.

Robust chelating stability under biological condi-tions is critical for the design of copper-based radiopharmaceuticals. In this study, the stabilities of (64)Cu-DOTA and diamsar (two bifunctional Cu-64 chelators (BFCs)) conjugated DGEA peptides were evaluated. The in vitro stabilities of (64)Cu-DOTA-DGEA, (64)Cu-DOTA-Ahx-DGEA, and (64)Cu-Z-E(diamsar)-Ahx-DGEA were evaluated in PBS. A carboxyl-protected DOTA-DGEA was also synthesized to study the potential inter- and intramolecular interactions between DOTA and the carboxylate groups of DGEA peptide. microPET imaging of (64)Cu-DOTA-DGEA and (64)Cu-Z-E(diamsar)-Ahx-DGEA were performed in PC-3 prostate tumor model to further investigate the in vivo behavior of the tracers. DOTA-DGEA, DOTA-Ahx-DGEA, Z-E(diamsar)-Ahx-DGEA, and protected DOTA-DGEA peptides were readily obtained, and their identities were confirmed by MS. (64)Cu(2+) labeling was performed with high radiochemical yields (>98%) for all tracers after 1 h incubation. Stability experiments revealed that (64)Cu-DOTA-DGEA had unexpectedly high (64)Cu(2+) dissociation when incubated in PBS (>55% free (64)Cu(2+) was observed at 48 h time point). The (64)Cu(2+) dissociation was significantly reduced in the carboxyl-protected (64)Cu-DOTA-DGEA complex but not in the (64)Cu-DOTA-Ahx-DGEA complex, which suggests the presence of competitive binding for (64)Cu(2+) between DOTA and the carboxyl groups of the DGEA peptide. In contrast, no significant (64)Cu(2+) dissociation was observed for (64)Cu-Z-E(diamsar)-Ahx-DGEA in PBS. For microPET imaging, the PC-3 tumors were clearly visualized with both (64)Cu-DOTA-DGEA and (64)Cu-Z-E(diamsar)-Ahx-DGEA tracers. However, (64)Cu-DOTA-DGEA demonstrated 5× higher liver uptake than (64)Cu-Z-E(diamsar)-Ahx-DGEA. This biodistribution variance could be attributed to the chelating stability difference between these two tracers, which correlated well with the PBS stability experiments. In summary, the in vitro and in vivo evaluations of (64)Cu-Z-E(diamsar)-Ahx-DGEA and (64)Cu-DOTA-DGEA have demonstrated the significantly superior Cu-chelation stability for the diamsar derivative compared with the established DOTA chelator. The results also suggest that diamsar may be preferred for Cu chelation especially when multiple carboxylic acid groups are present. Free carboxyl groups may naturally compete with DOTA for (64)Cu(2+) binding and therefore reduce the complex stability.

Design, synthesis and validation of integrin α2ß1-targeted probe for microPET imaging of prostate cancer.

PURPOSE: The ability of PET to aid in the diagnosis and management of recurrent and/or disseminated metastatic prostate cancer may be enhanced by the development of novel prognostic imaging probes. Accumulating experimental evidence indicates that overexpression of integrin α(2)ß(1) may correlate with progression in human prostate cancer. In this study, (64)Cu-labeled integrin α(2)ß(1)-targeted PET probes were designed and evaluated for the imaging of prostate cancer. METHODS: DGEA peptides conjugated with a bifunctional chelator (BFC) were developed to image integrin α(2)ß(1) expression with PET in a subcutaneous PC-3 xenograft model. The microPET images were reconstructed by a two-dimensional ordered subsets expectation maximum algorithm. The average radioactivity accumulation within a tumor or an organ was quantified from the multiple region of interest volumes. RESULTS: The PET tracer demonstrated prominent tumor uptake in the PC-3 xenograft (integrin α(2)ß(1)-positive). The receptor specificity was confirmed in a blocking experiment. Moreover, the low tracer uptake in a CWR-22 tumor model (negative control) further confirmed the receptor specificity. CONCLUSION: The sarcophagine-conjugated DGEA peptide allows noninvasive imaging of tumor-associated α(2)ß(1) expression, which may be a useful PET probe for evaluating the metastatic potential of prostate cancer.

Tetrazine-trans-cyclooctene ligation for the rapid construction of integrin αvß3 targeted PET tracer based on a cyclic RGD peptide.

Labeling biomolecules with (18)F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating (18)F labeled probes based on the tetrazine-trans-cyclooctene ligation. Starting with only 30 µg (78 µM) of a tetrazine-RGD conjugate and 2 mCi (5 µM) of (18)F-trans-cyclooctene, the (18)F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The (18)F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction.

Rational Design, Pharmacomodulation, and Synthesis of [68Ga]Ga-Alb-FAPtp-01, a Selective Tumor-Associated Fibroblast Activation Protein Tracer for PET Imaging of Glioma.

Dynamic changes in the tumor-associated fibroblast activation protein (FAP) expression in tumors of different stages may be helpful for prognostic evaluation and treatment response monitoring, making this protein a promising surveillance biomarker for timely diagnosis of malignant tumors and effective planning of patient care. To prospectively verify the diagnostic efficacy value of the developed FAP tracers, [68Ga]Ga-FAPtp and [68Ga]Ga-Alb-FAPtp-01, dynamic/static positron emission tomography (PET)/computed tomography scans were acquired for tumor-targeting studies in vivo and in comparison with the well-established clinically used tracer [68Ga]Ga-FAPI-04. The optimized rationally designed FAP-targeting PET tracer, [68Ga]Ga-Alb-FAPtp-01, with albumin-binding capability demonstrated prominent tumor uptake over time. The mean standard uptake value (SUV) and the tumor/muscle (T/M) ratio were as high as 1.775 ± 0.179 SUV and T/M = 5.9, 1.533 ± 0.222 SUV and T/M = 6.7, and 1.425 ± 0.204 SUV and T/M = 9.5, respectively, at 1, 2, and 3 h. Its improved tumor uptake and pharmacokinetics suggest that the [68Ga]Ga-Alb-FAPtp-01 tracer can noninvasively detect FAP activation in vivo, permitting a precise definition of its roles in tumors of different stages and yielding insights regarding FAP-targeted radiotherapeutic strategies at the molecular level.

Evaluation of copper-64 labeled AmBaSar conjugated cyclic RGD peptide for improved microPET imaging of integrin alphavbeta3 expression.

Recently, we have developed a new cage-like bifunctional chelator 4-((8-amino-3,6,10,13,16,19-hexaazabicyclo [6.6.6] icosane-1-ylamino) methyl) benzoic acid (AmBaSar) for copper-64 labeling and synthesized the positron emission tomography (PET) tracer (64)Cu-AmBaSar-RGD. In this study, we further evaluate the biological property of this new AmBaSar chelator by using (64)Cu-AmBaSar-RGD as the model compound. In vitro and in vivo stability, lipophilicity, cell binding and uptake, microPET imaging, receptor blocking experiments, and biodistribution studies of (64)Cu-AmBaSar-RGD were investigated, and the results were directly compared with the established radiotracer (64)Cu-DOTA-RGD. The (64)Cu-AmBaSar-RGD was obtained with high radiochemical yield (> or =95%) and purity (> or =99%) under mild conditions (pH 5.0-5.5 and 23-37 degrees C) in less than 30 min. For in vitro studies, the radiochemical purity of (64)Cu-AmBaSar-RGD was more than 97% in PBS or FBS and 95% in mouse serum after 24 h of incubation. The log P value of (64)Cu-AmBaSar-RGD was -2.44 +/- 0.12. For in vivo studies, (64)Cu-AmBaSar-RGD and (64)Cu-DOTA-RGD have demonstrated comparable tumor uptake at selected time points on the basis of microPET imaging. The integrin alpha(v)beta(3) receptor specificity was confirmed by blocking experiments for both tracers. Compared with (64)Cu-DOTA-RGD, (64)Cu-AmBaSar-RGD demonstrated much lower liver accumulation in both microPET imaging and biodistribution studies. Metabolic studies also directly supported the observation that (64)Cu-AmBaSar-RGD was more stable in vivo than (64)Cu-DOTA-RGD. In summary, the in vitro and in vivo evaluations of the (64)Cu-AmBaSar-RGD have demonstrated its improved Cu-chelation stability compared with that of the established tracer (64)Cu-DOTA-RGD. The AmBaSar chelator will also have general applications for (64)Cu labeling of various bioactive molecules in high radiochemical yield and high in vivo stability.

The effect of caffeine on cerebral metabolism during alpha-chloralose anesthesia differs from isoflurane anesthesia in the rat brain.

RATIONALE: Caffeine is a widely studied psychostimulant, even though its exact effect on brain activity remains to be elucidated. Positron emission tomography (PET) allows studying mechanisms underlying cerebral metabolic responses to caffeine in caffeine-naïve rats. Rodent studies are typically performed under anesthesia. However, the anesthesia may affect neurotransmitter systems targeted by tested drugs. OBJECTIVES: The scope of the present study was to address the impairing or enhancing effect of two common anesthetics, alpha-chloralose and isoflurane, on the kinetics of caffeine. METHODS: The first group of rats (n = 15) were anesthetized under 1.5% isoflurane anesthesia. The second group of rats (n = 15) were anesthetized under alpha-chloralose (80 mg/kg). These rats received an intravenous injection of saline (n = 5) or of 2.5 mg/kg (n = 5) or 40 mg/kg (n = 5) caffeine for both groups. RESULTS: With 2.5 mg/kg or 40 mg/kg caffeine, whole-brain cerebral metabolism was significantly reduced by 17.2% and 17% (both P < 0.01), respectively, under alpha-chloralose anesthesia. However, the lower dose of caffeine (2.5 mg/kg) had a limited effect on brain metabolism, whereas its higher dose (40 mg/kg) produced enhancements in brain metabolism in the striatum, hippocampus, and thalamus (all P < 0.05) under isoflurane anesthesia. CONCLUSION: These findings demonstrate significant differences in brain responses to caffeine on the basic of the anesthesia regimen used, which highlights the importance of attention to the anesthetic used when interpreting findings from animal pharmacological studies because of possible interactions between the anesthetic and the drug under study.

Effects of Hemodynamic Response Function Selection on Rat fMRI Statistical Analyses.

The selection of the appropriate hemodynamic response function (HRF) for signal modeling in functional magnetic resonance imaging (fMRI) is important. Although the use of the boxcar-shaped hemodynamic response function (BHRF) and canonical hemodynamic response (CHRF) has gained increasing popularity in rodent fMRI studies, whether the selected HRF affects the results of rodent fMRI has not been fully elucidated. Here we investigated the signal change and t-statistic sensitivities of BHRF, CHRF, and impulse response function (IRF). The effect of HRF selection on different tasks was analyzed by using data collected from two groups of rats receiving either 3 mA whisker pad or 3 mA forepaw electrical stimulations (n = 10 for each group). Under whisker pad stimulation with large blood-oxygen-level dependent (BOLD) signal change (4.31 ± 0.42%), BHRF significantly underestimated signal changes (P < 0.001) and t-statistics (P < 0.001) compared with CHRF or IRF. CHRF and IRF did not provide significantly different t-statistics (P > 0.05). Under forepaw stimulation with small BOLD signal change (1.71 ± 0.34%), different HRFs provided insignificantly different t-statistics (P > 0.05). Therefore, the selected HRF can influence data analysis in rodent fMRI experiments with large BOLD responses but not in those with small BOLD responses.

Convection-Enhanced Delivery of a Virus-Like Nanotherapeutic Agent with Dual-Modal Imaging for Besiegement and Eradication of Brain Tumors.

Convection-enhanced delivery (CED) is a promising technique for infusing a therapeutic agent directly into the brain, bypassing the blood-brain barrier (BBB) with a pressure gradient to increase drug concentration specifically around the brain tumor, thereby enhancing tumor inhibition and limiting the systemic toxicity of chemotherapeutic agents. Herein, we developed a dual-imaging monitored virus-like nanotherapeutic agent as an ideal CED infusate, which can be delivered to specifically besiege and eradicate brain tumors. Methods: We report one-pot fabrication of green-fluorescence virus-like particles (gVLPs) in Escherichia coli (E. coli) for epirubicin (EPI) loading, cell-penetrating peptide (CPP) modification, and 68Ga-DOTA labeling to form a positron emission tomography (PET)-fluorescence dual-imaging monitored virus-like nanotherapeutic agent (68Ga-DOTA labeled EPI@CPP-gVLPs) combined with CED for brain tumor therapy and image tracking. The drug delivery, cytotoxicity, cell uptake, biodistribution, PET-fluorescence imaging and anti-tumor efficacy of the 68Ga-DOTA labeled EPI@CPP-gVLPs were investigated in vitro and in vivo by using U87-MG glioma cell line and U87-MG tumor model. Results: The 68Ga-DOTA-labeled EPI@CPP-gVLPs showed excellent serum stability as an ideal CED infusate (30-40 nm in size), and can be disassembled through proteolytic degradation of the coat protein shell to enable drug release and clearance to minimize long-term accumulation. The present results indicated that 68Ga-DOTA-labeled EPI@CPP-gVLPs can provide a sufficiently high drug payload (39.2 wt% for EPI) and excellent detectability through fluorescence and PET imaging to accurately represent drug distribution during CED infusion. In vivo delivery of the 68Ga-DOTA-labeled EPI@CPP-gVLPs through CED demonstrated that the median survival was prolonged to over 50 days when the mice received two administrations (once per week) compared with the control group (median survival: 26 days). Conclusion: The results clearly indicated that a combination of 68Ga-DOTA-labeled EPI@CPP-gVLPs and CED can serve as a flexible and powerful synergistic treatment in brain tumors without evidence of systemic toxicity.

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery.

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

Optimized analysis of blood flow and wall shear stress in the common carotid artery of rat model by phase-contrast MRI.

The present study systemically investigated the influence of gated/non-gated sequences, velocity encoding (VENC), and spatial resolution on blood flow, wall shear stress (WSS), and artery area evaluations when scanning the common carotid artery (CCA) in rats using phase-contrast magnetic resonance imaging (PC-MRI). We first tested whether or not non-gated PC-MRI was appropriate for evaluating blood flow and WSS in rats. For both gated and non-gated techniques, VENC values in the range of 60-120 cm/s with an interval of 10 cm/s were also tested. Second, we optimized the in-plane resolution of PC-MRI for blood flow and WSS measurements. Results showed the usage of a gated instrument can provide more reproducible assessments, whereas VENC had an insignificant influence on all hemodynamic measurements (all P > 0.05). Lower resolutions, such as 0.63 mm, led to significant overestimations in blood flow and artery area quantifications and to an underestimation in WSS measurements (all P < 0.05). However, a higher resolution of 0.16 mm slightly increased measurement variation. As a tradeoff between accuracy and scan time, we propose a gated PC-MRI sequence with a VENC of 120 cm/s and a resolution of 0.21 mm to be used to extract hemodynamic information about rat CCA.

The Use of PET Imaging for Prognostic Integrin α2ß1 Phenotyping to Detect Non-Small Cell Lung Cancer and Monitor Drug Resistance Responses.

PURPOSE: Growing evidence has demonstrated that aberrant expression of integrin α2ß1 might contribute to the invasion, metastasis and drug resistance of non-small cell lung cancer (NSCLC). Thus, the integrin α2ß1 targeting 68Ga-DOTA-A2B1 tracer was validated in NSCLC in contrast to accumulation of the clinically used 18F-FDG PET tracer to see if 68Ga-DOTA-A2B1-PET imaging can offer a valuable and critical diagnostic imaging criterion for the identification of phenotypes of aggressive lung cancer. METHODS: To verify the prognostic value of integrin α2ß1, several quantitative and functional in vitro assays were validated in different NSCLC cell lines (CL1-0, CL1-5, A549 and selected A549++ cells). Positron emission tomography (PET) imaging studies using both standard 18F-FDG and a newly developed 68Ga-labeled integrin α2ß1 (68Ga-DOTA-A2B1) tracer were sequentially performed on mice with lung tumor xenografts in different anatomic locations (subcutaneous, orthotopic and osseous) to validate the targeting capability of the 68Ga-DOTA-A2B1 tracers. Treatment responses were monitored by injecting animals with metastatic bone tumors with 5 mg/kg doxorubicin. All in vivo treatment responses in each treatment subgroup were monitored with a PET imaging system to evaluate the up-regulation of integrin expression at the earliest stage of treatment (6 h). RESULTS: The PET and computed tomography (CT) images from NSCLC xenograft animals unambiguously demonstrated accumulation of the integrin tracer 68Ga-DOTA-A2B1 in the tumor lesions at all locations. The average tumor uptake and tumor-to-normal (T/N) ratio were 2.51 ± 0.56 %ID/g and T/N = 2.82, 3.40 ± 0.42 %ID/g and T/N = 1.52, and 1.58 ± 0.108 %ID/g and T/N = 2.31 in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). The xenograft tumors were all clearly visible. In contrast, the accumulation of 18F-FDG reached 3.6 ± 0.76 %ID/g, 1.39 ± 0.075 %ID/g and 3.78 ± 0.73 %ID/g in subcutaneous, orthotopic and osseous tumors, respectively (n = 5; p < 0.05). However, due to the high background uptake by normal tissue, the T/N values were less than or close to 1, making the tumors almost indistinguishable in the PET imaging analysis. Furthermore, 68Ga-DOTA-A2B1-PET imaging of the treated osseous tumor model demonstrated more than 19% tracer uptake in A549 lesions (1.72 ± 0.95 %ID/g vs. pretreatment 1.44 ± 0.12 %ID/g,p = 0. 015) 6 h post-treatment with doxorubicin. The elevated intensity of tracer uptake was in accordance with the results of in vitroWestern blot and ex vivo integrin staining, demonstrating elevated integrin α2ß1 expression. CONCLUSION: In this study, integrin α2ß1 was identified as a biomarker of aggressive malignant NSCLC. Thus, efforts should be devoted to validating integrin α2ß1 as a potential target for non-invasive diagnosis and as a predictive marker for monitoring treatment responses using a preclinical PET imaging system.