Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells.

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site-specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell-based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.

Development of CD133 Targeting Multi-Drug Polymer Micellar Nanoparticles for Glioblastoma - In Vitro Evaluation in Glioblastoma Stem Cells.

PURPOSE: Glioblastoma (GBM) is a malignant brain tumor with a poor long-term prognosis due to recurrence from highly resistant GBM cancer stem cells (CSCs), for which the current standard of treatment with temozolomide (TMZ) alone will unlikely produce a viable cure. In addition, CSCs regenerate rapidly and overexpress methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to resistance. The objective of this research was to apply the concepts of nanotechnology to develop a multi-drug therapy, TMZ and idasanutlin (RG7388, a potent mouse double minute 2 (MDM2) antagonist), loaded in functionalized nanoparticles (NPs) that target the GBM CSC subpopulation, reduce the cell viability and provide possibility of in vivo preclinical imaging. METHODS: Polymer-micellar NPs composed of poly(styrene-b-ethylene oxide) (PS-b-PEO) and poly(lactic-co-glycolic) acid (PLGA) were developed by a double emulsion technique loading TMZ and/or RG7388. The NPs were covalently bound to a 15-nucleotide base-pair CD133 aptamer to target the CD133 antigen expressed on the surfaces of GBM CSCs. For diagnostic functionality, the NPs were labelled with radiotracer Zirconium-89 (89Zr). RESULTS: NPs maintained size range less than 100 nm, a low negative charge and exhibited the ability to target and kill the CSC subpopulation when TMZ and RG7388 were used in combination. The targeting function of CD133 aptamer promoted killing in GBM CSCs providing impetus for further development of targeted nanosystems for localized therapy in future in vivo models. CONCLUSIONS: This work has provided a potential clinical application for targeting GBM CSCs with simultaneous diagnostic imaging.

Advanced imaging techniques for neuro-oncologic tumor diagnosis, with an emphasis on PET-MRI imaging of malignant brain tumors.

PURPOSE OF REVIEW: This review will explore the latest in advanced imaging techniques, with a focus on the complementary nature of multiparametric, multimodality imaging using magnetic resonance imaging (MRI) and positron emission tomography (PET). RECENT FINDINGS: Advanced MRI techniques including perfusion-weighted imaging (PWI), MR spectroscopy (MRS), diffusion-weighted imaging (DWI), and MR chemical exchange saturation transfer (CEST) offer significant advantages over conventional MR imaging when evaluating tumor extent, predicting grade, and assessing treatment response. PET performed in addition to advanced MRI provides complementary information regarding tumor metabolic properties, particularly when performed simultaneously. 18F-fluoroethyltyrosine (FET) PET improves the specificity of tumor diagnosis and evaluation of post-treatment changes. Incorporation of radiogenomics and machine learning methods further improve advanced imaging. The complementary nature of combining advanced imaging techniques across modalities for brain tumor imaging and incorporating technologies such as radiogenomics has the potential to reshape the landscape in neuro-oncology.

Challenges, limitations, and pitfalls of PET and advanced MRI in patients with brain tumors: A report of the PET/RANO group.

Brain tumor diagnostics have significantly evolved with the use of positron emission tomography (PET) and advanced magnetic resonance imaging (MRI) techniques. In addition to anatomical MRI, these modalities may provide valuable information for several clinical applications such as differential diagnosis, delineation of tumor extent, prognostication, differentiation between tumor relapse and treatment-related changes, and the evaluation of response to anticancer therapy. In particular, joint recommendations of the Response Assessment in Neuro-Oncology (RANO) Group, the European Association of Neuro-oncology, and major European and American Nuclear Medicine societies highlighted that the additional clinical value of radiolabeled amino acids compared to anatomical MRI alone is outstanding and that its widespread clinical use should be supported. For advanced MRI and its steadily increasing use in clinical practice, the Standardization Subcommittee of the Jumpstarting Brain Tumor Drug Development Coalition provided more recently an updated acquisition protocol for the widely used dynamic susceptibility contrast perfusion MRI. Besides amino acid PET and perfusion MRI, other PET tracers and advanced MRI techniques (e.g. MR spectroscopy) are of considerable clinical interest and are increasingly integrated into everyday clinical practice. Nevertheless, these modalities have shortcomings which should be considered in clinical routine. This comprehensive review provides an overview of potential challenges, limitations, and pitfalls associated with PET imaging and advanced MRI techniques in patients with gliomas or brain metastases. Despite these issues, PET imaging and advanced MRI techniques continue to play an indispensable role in brain tumor management. Acknowledging and mitigating these challenges through interdisciplinary collaboration, standardized protocols, and continuous innovation will further enhance the utility of these modalities in guiding optimal patient care.

Hybrid 18F-Fluoroethyltyrosine PET and MRI with Perfusion to Distinguish Disease Progression from Treatment-Related Change in Malignant Brain Tumors: The Quest to Beat the Toughest Cases.

Conventional MRI has important limitations when assessing for progression of disease (POD) versus treatment-related changes (TRC) in patients with malignant brain tumors. We describe the observed impact and pitfalls of implementing 18F-fluoroethyltyrosine (18F-FET) perfusion PET/MRI into routine clinical practice. Methods: Through expanded-access investigational new drug use of 18F-FET, hybrid 18F-FET perfusion PET/MRI was performed during clinical management of 80 patients with World Health Organization central nervous system grade 3 or 4 gliomas or brain metastases of 6 tissue origins for which the prior brain MRI results were ambiguous. The diagnostic performance with 18F-FET PET/MRI was dually evaluated within routine clinical service and for retrospective parametric evaluation. Various 18F-FET perfusion PET/MRI parameters were assessed, and patients were monitored for at least 6 mo to confirm the diagnosis using pathology, imaging, and clinical progress. Results: Hybrid 18F-FET perfusion PET/MRI had high overall accuracy (86%), sensitivity (86%), and specificity (87%) for difficult diagnostic cases for which conventional MRI accuracy was poor (66%). 18F-FET tumor-to-brain ratio static metrics were highly reliable for distinguishing POD from TRC (area under the curve, 0.90). Dynamic tumor-to-brain intercept was more accurate (85%) than SUV slope (73%) or time to peak (73%). Concordant PET/MRI findings were 89% accurate. When PET and MRI conflicted, 18F-FET PET was correct in 12 of 15 cases (80%), whereas MRI was correct in 3 of 15 cases (20%). Clinical management changed after 88% (36/41) of POD diagnoses, whereas management was maintained after 87% (34/39) of TRC diagnoses. Conclusion: Hybrid 18F-FET PET/MRI positively impacted the routine clinical care of challenging malignant brain tumor cases at a U.S. institution. The results add to a growing body of literature that 18F-FET PET complements MRI, even rescuing MRI when it fails.

The tauopathies: Neuroimaging characteristics and emerging experimental therapies.

The tauopathies are a heterogeneous group of neurodegenerative disorders in which the prevailing underlying disease process is intracellular deposition of abnormal misfolded tau protein. Diseases often categorized as tauopathies include progressive supranuclear palsy, chronic traumatic encephalopathy, corticobasal degeneration, and frontotemporal lobar degeneration. Tauopathies can be classified through clinical assessment, imaging findings, histologic validation, or molecular biomarkers tied to the underlying disease mechanism. Many tauopathies vary in their clinical presentation and overlap substantially in presentation, making clinical diagnosis of a specific primary tauopathy difficult. Anatomic imaging findings are also rarely specific to a single tauopathy, and when present may not manifest until well after the point at which therapy may be most impactful. Molecular biomarkers hold the most promise for patient care and form a platform upon which emerging diagnostic and therapeutic applications could be developed. One of the most exciting developments utilizing these molecular biomarkers for assessment of tau deposition within the brain is tau-PET imaging utilizing novel ligands that specifically target tau protein. This review will discuss the background, significance, and clinical presentation of each tauopathy with additional attention to the pathologic mechanisms at the protein level. The imaging characteristics will be outlined with select examples of emerging imaging techniques. Finally, current treatment options and emerging therapies will be discussed. This is by no means a comprehensive review of the literature but is instead intended for the practicing radiologist as an overview of a rapidly evolving topic.

Novel therapeutics and drug-delivery approaches in the modulation of glioblastoma stem cell resistance.

Glioblastoma (GBM) is a deadly malignancy with a poor prognosis. An important factor contributing to GBM recurrence is high resistance of GBM cancer stem cells (GSCs). While temozolomide (TMZ), has been shown to consistently extend survival, GSCs grow resistant to TMZ through upregulation of DNA damage repair mechanisms and avoidance of apoptosis. Since a single-drug approach has failed to significantly alter prognosis in the past 15 years, unique approaches such as multidrug combination therapy together with distinctive targeted drug-delivery approaches against cancer stem cells are needed. In this review, a rationale for multidrug therapy using a targeted nanotechnology approach that preferentially target GSCs is proposed with discussion and examples of drugs, nanomedicine delivery systems, and targeting moieties.

Use of multimodality imaging, histology, and treatment feasibility to characterize a transgenic Rag2-null rat model of glioblastoma.

Many drugs that show potential in animal models of glioblastoma (GBM) fail to translate to the clinic, contributing to a paucity of new therapeutic options. In addition, animal model development often includes histologic assessment, but multiparametric/multimodality imaging is rarely included despite increasing utilization in patient cancer management. This study developed an intracranial recurrent, drug-resistant, human-derived glioblastoma tumor in Sprague-Dawley Rag2-Rag2 tm1Hera knockout rat and was characterized both histologically and using multiparametric/multimodality neuroimaging. Hybrid 18F-fluoroethyltyrosine positron emission tomography and magnetic resonance imaging, including chemical exchange saturation transfer (18F-FET PET/CEST MRI), was performed for full tumor viability determination and characterization. Histological analysis demonstrated human-like GBM features of the intracranially implanted tumor, with rapid tumor cell proliferation (Ki67 positivity: 30.5 ± 7.8%) and neovascular heterogeneity (von Willebrand factor VIII:1.8 to 5.0% positivity). Early serial MRI followed by simultaneous 18F-FET PET/CEST MRI demonstrated consistent, predictable tumor growth, with exponential tumor growth most evident between days 35 and 49 post-implantation. In a second, larger cohort of rats, 18F-FET PET/CEST MRI was performed in mature tumors (day 49 post-implantation) for biomarker determination, followed by evaluation of single and combination therapy as part of the model development and validation. The mean percentage of the injected dose per mL of 18F-FET PET correlated with the mean %CEST (r = 0.67, P < 0.05), but there was also a qualitative difference in hot spot location within the tumor, indicating complementary information regarding the tumor cell demand for amino acids and tumor intracellular mobile phase protein levels. Finally, the use of this glioblastoma animal model for therapy assessment was validated by its increased overall survival after treatment with combination therapy (temozolomide and idasanutlin) (P < 0.001). Our findings hold promise for a more accurate tumor viability determination and novel therapy assessment in vivo in a recently developed, reproducible, intracranial, PDX GBM.

Aerosolized In Vivo 3D Localization of Nose-to-Brain Nanocarrier Delivery Using Multimodality Neuroimaging in a Rat Model-Protocol Development.

The fate of intranasal aerosolized radiolabeled polymeric micellar nanoparticles (LPNPs) was tracked with positron emission tomography/computer tomography (PET/CT) imaging in a rat model to measure nose-to-brain delivery. A quantitative temporal and spatial testing protocol for new radio-nanotheranostic agents was sought in vivo. LPNPs labeled with a zirconium 89 (89Zr) PET tracer were administered via intranasal or intravenous delivery, followed by serial PET/CT imaging. After 2 h of continuous imaging, the animals were sacrificed, and the brain substructures (olfactory bulb, forebrain, and brainstem) were isolated. The activity in each brain region was measured for comparison with the corresponding PET/CT region of interest via activity measurements. Serial imaging of the LPNPs (100 nm PLA-PEG-DSPE+89Zr) delivered intranasally via nasal tubing demonstrated increased activity in the brain after 1 and 2 h following intranasal drug delivery (INDD) compared to intravenous administration, which correlated with ex vivo gamma counting and autoradiography. Although assessment of delivery from nose to brain is a promising approach, the technology has several limitations that require further development. An experimental protocol for aerosolized intranasal delivery is presented herein, which may provide a platform for better targeting the olfactory epithelium.

Imaging of intranasal drug delivery to the brain.

Intranasal (IN) delivery is a rapidly developing area for therapies with great potential for the treatment of central nervous system (CNS) diseases. Moreover, in vivo imaging is becoming an important part of therapy assessment, both clinically in humans and translationally in animals. IN drug delivery is an alternative to systemic administration that uses the direct anatomic pathway between the olfactory/trigeminal neuroepithelium of the nasal mucosa and the brain. Several drugs have already been approved for IN application, while others are undergoing development and testing. To better understand which imaging modalities are being used to assess IN delivery of therapeutics, we performed a literature search with the key words "Intranasal delivery" and "Imaging" and summarized these findings in the current review. While this review does not attempt to be fully comprehensive, we intend for the examples provided to allow a well-rounded picture of the imaging tools available to assess IN delivery, with an emphasis on the nose-to-brain delivery route. Examples of in vivo imaging, for both humans and animals, include magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), gamma scintigraphy and computed tomography (CT). Additionally, some in vivo optical imaging modalities, including bioluminescence and fluorescence, have been used more in experimental testing in animals. In this review, we introduce each imaging modality, how it is being utilized and outline its strengths and weaknesses, specifically in the context of IN delivery of therapeutics to the brain.

An analog of thyrotropin-releasing hormone (TRH) is neuroprotective against glutamate-induced toxicity in fetal rat hippocampal neurons in vitro.

TRH has been found to be efficacious in treating certain neurodegenerative disorders such as epilepsy, Alzheimer's disease, neurotrauma and depression, however, its mechanism of action is poorly understood. Since glutamate (Glu) toxicity has been implicated in these disorders, we utilized primary enriched cultures of rat fetal (E 17) hippocampal neurons to test the hypothesis that an analog of TRH, 3-Methyl-Histidine TRH (3Me-H TRH), given concurrently with Glu would protect such neurons against cell damage and cell death. Cell viability was assessed via Trypan Blue exclusion cell counts, and neuronal damage was determined by assaying lactic acid dehydrogenase (LDH) released in the conditioned media. Fetal hippocampal neurons were cultured in neurobasal media for 7 days. On day 7, neurons (10(6)/well) were treated with: control media, 10 microM 3Me-H TRH, 500 microM Glu or 500 microM Glu with either 10, 1, 0.1, 0.01 or 0.001 microM 3Me-H TRH. Both media and neurons were harvested 16 h after treatment. Prolonged exposure to 10 microM 3Me-H TRH was not toxic to the cells, whereas neurons exposed to 500 microM Glu resulted in maximal cell death. Notably, 10, 1 and 0.1 microM 3Me-H TRH, when co-treated with 500 microM Glu, protected fetal neurons against cell death in a concentration-dependent manner. These results provide support for an important neuroprotective effect of TRH/analogs against glutamate toxicity in primary hippocampal neuronal culture and implicate a potentially beneficial role of TRH/analogs in neurodegenerative diseases.

Perspective: the potential of student organizations for developing leadership: one school's experience.

Leadership development is vital to the future of medicine. Some leadership development may take place through the formal curriculum of the medical school, yet extracurricular activities, such as student government and affiliated student organizations, can provide additional, highly valuable leadership development opportunities. These organizations and their missions can serve as catalysts for students to work with one another, with the faculty and administration of the medical school, with the community, and with local, regional, and national organizations. The authors have organized this discussion of the leadership development potential of student organizations around six important principles of leadership: ownership, experience, efficacy, sense of community, service learning, and peer-to-peer mentoring. They provide practical examples of these leadership principles from one institution. They do not presume that the school is unique, but they do believe their practical examples help to illuminate the potential of extracurricular programs for enhancing the leadership capabilities of future physicians. In addition, the authors use their examples to demonstrate how the medical school, its surrounding community, and the profession of medicine can benefit from promoting leadership through student organizations.

Intranasal delivery of neuropeptides.

A major barrier to entry of neuropeptides into the brain is low bioavailability and presence of the blood-brain barrier. Intranasal delivery of neuropeptides provides a potentially promising alternative to other routes of administration, since a direct pathway exists between the olfactory neuroepithelium and the brain. Use of the rat as an animal model in nose to brain delivery of neuropeptides allows for several advantages, including a large surface area within the nasal cavity dedicated to olfactory epithelium and robust neuronal pathways extending to and from most areas of the brain from the nose via the olfactory cortex. A major disadvantage to using rats for nose to brain delivery is the difficulty in selectively targeting the posterior olfactory epithelium (which facilitates delivery to the brain) over the more anterior respiratory epithelium (which facilitates delivery to the lungs and secondarily to the peripheral blood) in the nasal cavity. We have developed a novel delivery system that consists of surgically implanting stainless-steel cannulas in the dorsal aspect of the nasal cavity overlying the olfactory neuroepithelium, thereby allowing neuropeptide compounds to bypass the respiratory epithelium.

Attenuation of kindled seizures by intranasal delivery of neuropeptide-loaded nanoparticles.

Thyrotropin-releasing hormone (TRH; Protirelin), an endogenous neuropeptide, is known to have anticonvulsant effects in animal seizure models and certain intractable epileptic patients. Its duration of action, however, is limited by rapid tissue metabolism and the blood-brain barrier. Direct nose-to-brain delivery of neuropeptides in sustained-release biodegradable nanoparticles (NPs) is a promising mode of therapy for enhancing CNS neuropeptide bioavailability. To provide proof of principle for this delivery approach, we used the kindling model of temporal lobe epilepsy to show that 1) TRH-loaded copolymer microdisks implanted in a seizure focus can attenuate kindling development in terms of behavioral stage, afterdischarge duration (ADD), and clonus duration; 2) intranasal administration of an unprotected TRH analog can acutely suppress fully kindled seizures in a concentration-dependent manner in terms of ADD and seizure stage; and 3) intranasal administration of polylactide nanoparticles (PLA-NPs) containing TRH (TRH-NPs) can impede kindling development in terms of behavioral stage, ADD, and clonus duration. Additionally, we used intranasal delivery of fluorescent dye-loaded PLA-NPs in rats and application of dye-loaded or dye-attached NPs to cortical neurons in culture to demonstrate NP uptake and distribution over time in vivo and in vitro respectively. Also, a nanoparticle immunostaining method was developed as a procedure for directly visualizing the tissue level and distribution of neuropeptide-loaded nanoparticles. Collectively, the data provide proof of concept for intranasal delivery of TRH-NPs as a viable means to 1) suppress seizures and perhaps epileptogenesis and 2) become the lead compound for intranasal anticonvulsant nanoparticle therapeutics.

Thyrotropin-releasing hormone d,l polylactide nanoparticles (TRH-NPs) protect against glutamate toxicity in vitro and kindling development in vivo.

Thyrotropin-releasing hormone (TRH) is reported to have anticonvulsant effects in animal seizure models and certain intractable epileptic patients. However, its duration of action is limited by rapid tissue metabolism and the blood brain barrier. Direct nose-brain delivery of neuropeptides in sustained-release biodegradable nanoparticles (NPs) is a promising mode of therapy for enhancing CNS bioavailability. Bioactivity/neuroprotection of d,l polylactide nanoparticles containing TRH was assessed against glutamate toxicity in cultured rat fetal hippocampal neurons. Subsequently, we utilized the kindling model of temporal lobe epilepsy to determine if intranasal administration of nanoparticles containing TRH (TRH-NPs) could inhibit kindling development. Animals received daily treatments of either blank (control) or TRH-NPs for 7 days before initiation of kindling. On day 8 and each day thereafter until either fully kindled or until day 20, the animals received daily treatments before receiving a kindling stimulus 3 h later. Afterdischarge duration (ADD) was assessed via electroencephalographs recorded from electrodes in the basolateral amygdalae and behavioral seizure stereotypy was simultaneously recorded digitally. Intranasal application of TRH-NPs resulted in a significant reduction in seizure ADD as kindling progressed, while the number of stimulations required to reach stage V seizures and to become permanently kindled was significantly greater in TRH-NP-treated subjects. Additionally, delay to clonus was significantly prolonged while clonus duration was reduced indicating a less severe seizure in TRH-NP-treated subjects. Our results provide proof of principle that intranasal delivery of sustained-release TRH-NPs may be neuroprotective and can be utilized to suppress seizures and perhaps epileptogenesis.

Isoflurane exacerbates electrically evoked seizures in amygdala-kindled rats during recovery.

Neuroexcitatory effects of isoflurane during or following anesthesia are controversial, particularly in epileptic patients. In contrast, halothane is generally considered to be highly anticonvulsant. Kindling is an animal model of epilepsy suitable for studying the effects of anesthetic agents on the epileptic brain. Fully kindled, Sprague-Dawley rats were either untreated or received a 5 min exposure to isoflurane or halothane 30 min prior to a seizure and compared to seizures in the absence of prior anesthesia. Afterdischarge duration was assessed via electroencephalographs recorded from electrodes implanted in the basolateral amygdala and behavioral seizure stereotypy (stages I-V) was simultaneously recorded and analyzed using digital video for all seizures. Total seizure duration and clonus duration were significantly (P<0.05) increased 30 min after isoflurane but not halothane exposure relative to pre-treatment control. These results are the first to demonstrate that isoflurane exacerbates electrically evoked secondarily generalized seizures in fully kindled animals during recovery. These results also show that the kindling paradigm is useful for evaluating the mechanism of anesthetic agents that may be proconvulsant in epileptic subjects.

Intranasal delivery of a thyrotropin-releasing hormone analog attenuates seizures in the amygdala-kindled rat.

PURPOSE: Thyrotropin-releasing hormone (TRH) is known to have anticonvulsant effects in several animal seizure models and is efficacious in treating patients with certain intractable epilepsies. However, the duration of TRH's action is limited due to low bioavailability and difficulty penetrating the blood-brain barrier (BBB). Since direct nose to brain delivery of therapeutic compounds may provide a means for overcoming these barriers, we utilized the kindling model of temporal lobe epilepsy to determine if intranasal administration of a TRH analog, 3-methyl-histidine TRH (3Me-H TRH), could significantly inhibit various seizure parameters. METHODS: Kindling was accomplished using a 1s train of 60 Hz biphasic square wave (200 microA peak to peak) administered daily to the basolateral amygdala until the animal was fully kindled. Afterdischarge duration (ADD) was assessed via electroencephalographs (EEGs) recorded bilaterally from bipolar electrodes in the basolateral amygdala and behavioral seizure severity (stage I-V) was simultaneously recorded digitally. Kindled subjects received 3Me-H TRH (10(-9), 10(-8), 10(-7) M) intranasally 60 and 30 min prior to amygdala stimulation. The ADD and seizure stage was compared to control kindled animals receiving physiological saline intranasally. RESULTS: Intranasal application of 3Me-H TRH resulted in a concentration-dependent reduction in total seizure ADD. Additionally, the analog had significant concentration-dependent effects on behavioral stages I through IV (partial) and stage V (generalized) seizures. However, 3Me-H TRH significantly reduced clonus duration only at the highest concentration. DISCUSSION: The results indicate that intranasal delivery of TRH/analogs may be a viable means to suppress temporal lobe seizures and perhaps other seizure disorders.