Vazgen Khankaldyyan

Effect of the angiogenesis inhibitor Cilengitide (EMD 121974) on glioblastoma growth in nude mice.

OBJECTIVETo determine the effect of the angiogenesis inhibitor Cilengitide (EMD 121974) on glioblastoma growth and associated angiogenesis in the brains of nude mice. METHODSHuman glioblastoma cells (105 U87MG cells) in 1 μl of medium were stereotactically injected during a 20-minute period into the caudate/putamen of nude mice. The mice were intraperitoneally treated daily with Cilengitide or solvent (control) beginning 5 days after tumor injection. The mice were sacrificed from 1 hour to 63 days after tumor implantation and examined for tumor size, vascularity, apoptosis, and cell replication. RESULTSThis injection technique resulted in a highly reproducible, localized, spherical tumor cell placement in the parenchyma without reflux into the subarachnoid space or penetration into the ventricle. Serial brain sections showed the tumor size remained unchanged at 1 to 2 mm3 for approximately 30 to 40 days. Thereafter, the control tumors showed exponential growth to a volume of 120 mm3, with death of the mice at approximately 8 to 9 weeks. Serial staining for Ki-67, a marker for cell replication, and CD31, an indicator for angiogenesis, demonstrated an increase in proportion to the growth of the tumor. In contrast, the tumor volume in Cilengitide-treated mice stayed unchanged at 1 to 2 mm3 during the entire length of the experiment, with staining for Ki-67 and CD31 remaining low. CONCLUSIONThis standardized brain tumor model is highly reproducible and useful for testing new treatment regimens. Cilengitide is highly effective in suppressing blood vessel growth, thereby controlling orthotopic growth of this glioblastoma cell line.

Magnetic Resonance Imaging Tracking of Ferumoxytol-Labeled Human Neural Stem Cells: Studies Leading to Clinical Use

Numerous stem cell‐based therapies are currently under clinical investigation, including the use of neural stem cells (NSCs) as delivery vehicles to target therapeutic agents to invasive brain tumors. The ability to monitor the time course, migration, and distribution of stem cells following transplantation into patients would provide critical information for optimizing treatment regimens. No effective cell‐tracking methodology has yet garnered clinical acceptance. A highly promising noninvasive method for monitoring NSCs and potentially other cell types in vivo involves preloading them with ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) to enable cell tracking using magnetic resonance imaging (MRI). We report here the preclinical studies that led to U.S. Food and Drug Administration approval for first‐in‐human investigational use of ferumoxytol to label NSCs prior to transplantation into brain tumor patients, followed by surveillance serial MRI. A combination of heparin, protamine sulfate, and ferumoxytol (HPF) was used to label the NSCs. HPF labeling did not affect cell viability, growth kinetics, or tumor tropism in vitro, and it enabled MRI visualization of NSC distribution within orthotopic glioma xenografts. MRI revealed dynamic in vivo NSC distribution at multiple time points following intracerebral or intravenous injection into glioma‐bearing mice that correlated with histological analysis. Preclinical safety/toxicity studies of intracerebrally administered HPF‐labeled NSCs in mice were also performed, and they showed no significant clinical or behavioral changes, no neuronal or systemic toxicities, and no abnormal accumulation of iron in the liver or spleen. These studies support the clinical use of ferumoxytol labeling of cells for post‐transplant MRI visualization and tracking.

Improved Oral Delivery of N-(4-Hydroxyphenyl)Retinamide with a Novel LYM-X-SORB Organized Lipid Complex

Purpose: Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is a cytotoxic retinoid that suffers from a wide interpatient variation in bioavailability when delivered orally in a corn oil capsule. The poor bioavailability of the capsule formulation may have limited responses in clinical trials, and the large capsules are not suitable for young children. To support the hypothesis that a novel organized lipid matrix, LYM-X-SORB, can increase the oral bioavailability of fenretinide, fenretinide in LYM-X-SORB matrix and in a powderized LYM-X-SORB formulation was delivered to mice. Experimental Design: Fenretinide was delivered orally to mice as the contents of the corn oil capsule, in LYM-X-SORB matrix (4-HPR/LYM-X-SORB matrix) or in a LYM-X-SORB matrix powderized with sugar and flour (4-HPR/LYM-X-SORB oral powder). Levels of 4-HPR, and its principal metabolite, N-(4-methoxyphenyl)retinamide, were assayed in plasma and tissues. Results: In a dose-responsive manner, from 120 to 360 mg/kg/d, delivery to mice of 4-HPR in LYM-X-SORB matrix, or as 4-HPR/LYM-X-SORB oral powder, increased 4-HPR plasma levels up to 4-fold (P < 0.01) and increased tissue levels up to 7-fold (P < 0.01) compared with similar doses of 4-HPR delivered using capsule contents. Metabolite [N-(4-methoxyphenyl)retinamide] levels mirrored 4-HPR levels. Two human neuroblastoma murine xenograft models showed increased survival (P < 0.03), when treated with 4-HPR/LYM-X-SORB oral powder, confirming the bioactivity of the formulation. Conclusions: 4-HPR/LYM-X-SORB oral powder is a novel, oral drug delivery formulation, suitable for pediatric use, which warrants further development for the delivery of fenretinide in the treatment of cancer. A phase I clinical trial in pediatric neuroblastoma is in progress.

In Vivo Testing of Renilla Luciferase Substrate Analogs in an Orthotopic Murine Model of Human Glioblastoma

In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma; the relatively high absorbance by tissue of the light emitted by the enzyme–substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit. A number of analogs of coelenterazine are being investigated that may accomplish one or both of these goals. In this study, we report on the testing of two new substrate analogs, EnduRen™ and ViviRen™, manufactured by Promega Corporation, in an orthotopic murine model of human glioblastoma expressing Renilla luciferase. We have tested these analogs in this cell line, both in vitro and in vivo, and find that the substrate ViviRen results in significantly greater light output than the natural substrate or the other analog EnduRen. This new substrate could be valuable for studies where greater sensitivity is important.

Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration

Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations. Cell lines established in 20% oxygen exhibited lower proliferation, survival, expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo.

An allograft mouse model for the study of hearing loss secondary to vestibular schwannoma growth.

Vestibular schwannoma is a benign neoplasm arising from the Schwann cell sheath of the auditory-vestibular nerve. It most commonly affects both sides in the genetic condition Neurofibromatosis type 2, causing progressive high frequency sensorineural hearing loss. Here, we describe a microsurgical technique and stereotactic coordinates for schwannoma cell grafting in the vestibular nerve region that recapitulates local tumor growth in the cerebellopontine angle and inner auditory canal with resulting hearing loss. Tumor growth was monitored by bioluminescence and MRI in vivo imaging, and hearing assessed by auditory brainstem responses. These techniques, by potentially enabling orthotopic grafting of a variety of cell lines will allow studies on the pathogenesis of tumor-related hearing loss and preclinical drug evaluation, including hearing endpoints, for NF2-related and sporadic schwannomas.

A noninvasive, multimodality approach based on MRS and MRI techniques for monitoring intracranial brain tumor angiogenesis

The understanding of tumor oxygenation at the microvascular level in an orthotopic model may provide useful insight into tumor physiology, therapeutic response and development of protocols to study tumor behavior. In this paper the vascular status and the patho-physiological changes occurring during angiogenes is are studied in an orthotopic brain tumor model using a noninvasive multimodality approach based on near infrared (NIR) diffuse optical spectroscopy (DOS) along with magnetic resonance imaging (MRI) We report a direct correlation between tumor size and intratumoral microvessel density MVD, and tumor oxygenation. The relative decrease in the oxygen saturation value with tumor growth indicates that though blood vessels infiltrate and proliferate the tumor region, a hypoxic trend is clearly present

20. Carboxylesterase-Secreting Neural Stem Cells Increase Efficacy of Irinotecan in Orthotopic Glioma Models: Translation Toward the Clinic

Human neural stem cells (NSCs) are serving as vehicles for targeted delivery of cytosine deaminase/5-flucytosine enzyme/prodrug therapy to recurrent glioma patients in first in human Phase 1 clinical trials (IND 14041). The initial safety/feasibility clinical study demonstrated safety, non-tumorigenicity, non-immunogenicity with 1 round of treatment, and proof of concept for localized conversion of 5-Flucytosine to the active chemotherapeutic agent, 5-Fluorouracil. We have now developed a second generation NSC brain tumor therapy, engineering NSCs to secrete a modified human carboxylesterase (hCE1m6) that converts irinotecan (IRN) to the potent topoisomerase inhibitor SN-38. Biodistribution, efficacy, and safety/toxicity studies have been completed and an Investigational New Drug application has been filed with the Food and Drug Administration (IND 16265).Early studies on the CE-expressing human NSCs was described in Metz, et al., Stem Cells Transl Med. 2013 Dec;2(12):983), demonstrating tumor tropism in orthotopic human glioma models and tumor localized conversion of IRN to SN-38 in a time-dependent manner. The current preclinical studies show in vivo biodistribution of varying doses of intracranially delivered CE-NSCs and optimization of a clinically relevant adult IRN treatment regimen based on pharmacokinetic and long term survival studies. These studies demonstrated a statistically significant increase in long-term survival distributions with the addition of CE-NSCs + IRN as compared to IRN alone (see figure). These data, together with our preclinical safety/toxicity data, support clinical translation of this schema to phase I clinical trials for patients with recurrent glioma. View Large Image | Download PowerPoint Slide

Low Magnification Confocal Microscopy of Tumor Angiogenesis

Blood vessels are critical to normal mammalian development, tissue repair, and growth and treatment of cancer. Mouse research models enable mechanistic studies of blood vessels. We detail how to perfuse mice with fluorescent tomato lectin or the lipophilic fluorophore DiI. We provide details on how to image fluorescently labeled blood vessels.