Name: intranasal delivery of therapeutic stem cells to glioblastoma in a mouse model
Uploaded: 2017-06-04T15:00:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model at JoVE.com

This work was supported by NIH R01NS087990 (MSL, IVB).

All animal procedures must be approved by the Institutional Animal Care and Use Committee (IACUC) or equivalent. If there is any uncertainty regarding the specific procedures described herein, do not proceed. Clarify with the institution&#39;s IACUC and a designated veterinary staff member.
1. Ensuring Sterility of Cultured Cells and Follow Principles of Aseptic Techniques
<ol>
	<li>Follow the standard good laboratory practice in all cell culture procedures and IACUC requirements for cell testing and pathogen-free status confirmation prior to administering to mice9,10.</li>
	<li>Follow the NIH requirement for cell authentication to ensure reproducible outcome11.</li>
	<li>Follow established small animal surgery aseptic techniques outlined in this manuscript4.</li>
</ol>
2. Preparation of Glioma Cells for In Vivo Experiment2,4
<ol>
	<li>Culture glioma cells (GBM43, GBM6, and U87MG) in serum-containing (10% fetal bovine serum in Dulbecco&#39;s Modified Eagle&#39;s medium[DMEM]) or serum-free medium (Neurobasal medium with supplements B27, N2, heparin, epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], antibiotics, and L-glutamine12), in a humidified CO2 cell culture incubator.
	<ol>
		<li>Test all cell lines via the mouse clear panel run from commercial service providers.</li>
	</ol>
	</li>
	<li>Collect adherent cells by removing culture medium. Incubate with 0.05% trypsin for 5 min at room temperature (1 mL of trypsin for T25 flask, 2 mL for T75 flask, scale accordingly for culture flasks with larger surface area) and then wash cells with Ca2+ and Mg2+ free phosphate buffered saline (PBS).
	<ol>
		<li>Tap the flask to dislodge cells and immediately neutralize with an excess of serum-containing medium (8 - 9 mL). Then use serological pipettes to aspirate the cell suspension into centrifuge tubes.</li>
		<li>Centrifuge cell suspension at 400 x g for 5 min. Wash the cell at least twice with 10 mL of PBS by repeated centrifugation.</li>
		<li>Collect glioma cells grown as tumor spheres in serum free medium via centrifugation (same speed and duration as above); dissociate the cell pellet by incubation in 1 - 2 mL of cell detachment solution at 37 &#176;C for 5 min before washing twice with 10 mL of PBS by centrifugation (400 x g x 5 min).</li>
		<li>Re-suspend glioma in sterile saline at a concentration of 50,000 - 200,000 cells per 2.5 &#181;L. Cell numbers used for injection into mouse brain depend on the established growth rate for each glioma cell line. Here, use 25,000 and 100,000 for GBM43 and GBM6 patient-derived tumor cells, respectively.</li>
		<li>Prepare double the amount of the necessary cell number for implantation to account for the loss of a fraction during procedure. Transfer the injectable cell suspension to a sterile microcentrifuge tube and place tube on ice. Keep cells on ice for 2 h maximum during surgeries, prepare new batch if extended surgeries are planned.</li>
	</ol>
	</li>
</ol>
3. Intracranial Implantation to Create Xenograft Mouse Models (Figure 1A)2,4,13
<ol>
	<li>Prior to the surgery day, sterilize all surgical tools in an autoclave, and prepare all pre- and post-surgical animal care material per IACUC approved protocol. Sterilize the stereotaxic frame and peripheral equipment to ensure intraoperative aseptic conditions, thus minimizing complications.</li>
	<li>Organize the operation area to minimize clutter and possibility of contamination.</li>
	<li>Dress in appropriate personal protective equipment (PPE) per IACUC requirement.</li>
	<li>Set up appropriate post-operative recovery chamber. Ensure that anesthetics and analgesics are prepared fresh using unexpired reagents. Set up appropriate body heat maintenance instruments per IACUC approved protocol.</li>
	<li>To establish human patient-derived glioma cell xenografts for the testing of intranasal delivery of human NSCs, use immunocompromised mouse species such as nu/nu athymic mouse of any gender at approximately 6 weeks of age.</li>
	<li>Anesthetize the animal based on body weight using the approved reagents, i.e. either via an intraperitoneal (i.p.) injection of a mixture of ketamine (50-100 mg/kg; consult institutional IACUC for approved dose range) and xylazine (5-10 mg/kg; consult institutional IACUC for approved dose range) in 200-250 &#181;L sterile saline, or via 4-5% isoflurane anesthesia using an inhalational device.
	<ol>
		<li>Ensure the animal has reached the surgical anesthesia plane by toe pinching prior to the skin incision. Apply ample sterile ophthalmic gel to ensure the moist state of corneas of the animal.</li>
	</ol>
	</li>
	<li>Sterilize the skull using repeated and alternating circular wipes of betadine and isopropyl alcohol per Pritchett-Corning et al.14.</li>
	<li>Supply the pre-incision analgesia by s.c. injection of Buprenex (0.05 - 2 mg/kg) prior to incision using the surgical blade.</li>
	<li>Use sterilized cotton-tipped applicator to help hemostat while exposing the skull; use 3% hydrogen peroxide to facilitate the clearance of incidental bleeding.</li>
	<li>Use a battery-powered handheld electric ballpoint drill (less than 1 mm in width) to create a burr hole at 2 mm lateral, either left or right, to the midline, and 2 mm behind the Bregma (the position coordinates can be customized based on the tumor model intended for a particular study).</li>
	<li>Mount the animal to the stereotaxic frame to the appropriate head posture, such that the inserted Hamilton micro syringe is perpendicular to the surface of the skull around the burr hole. Take care to ensure that the animal&#39;s anesthetic plane is in good standing by toe pinching. Observe the respiratory rate to make certain that the animal is not under pain stress during the following steps. 
	* We do not emphasize in-ear bar placements because:
	<ol>
		<li>We have predrilled the burr hole to position the needle, and therefore, precise skull coordinates are unnecessary;</li>
		<li>Mouse ear canals are fragile, and the precise in-ear placement of ear bars requires more involved training that is not relevant to our purpose. The risks&#160;of damage to murine ear canals outweigh the benefits of a precise placement;</li>
		<li>Cheek support is much easier to perform and capable of achieving the same levels of mouse head stability and balance during positioning. It is an added benefit for multiple time-sensitive surgeries to ensure timely completion with good cell viability.</li>
	</ol>
	</li>
	<li>Use the stereotaxic frame knobs to position the flat tip micro syringe to the burr hole, then lower it slowly to 3 mm below the dura surface. Retract 0.5 mm to maintain the tip of the needle at 2.5 mm below the dura.</li>
	<li>Inject 2.5 &#181;L of preloaded glioma cells over the period of 1 min. Let the needle maintain its position for another 1-2 min before slowly, over the course of 1 min, withdrawing the needle out of the brain and taking care to minimize cell backflow. Apply sterile bone wax to avoid extracranial tumor outgrowth.</li>
	<li>Close the wound using stainless steel wound clips (7 mm). Deliver a subcutaneous (s.c.) injection of 1 mL of sterile lactated Ringer&#39;s solution (pre-warmed to 37 &#176;C), and place the animal in a recovery chamber which is halfway on a heating pad.</li>
	<li>Return the animals to the ventilated rack once they regain mobility, and follow routine post-op care to ensure smooth recovery.</li>
</ol>
4. In Vivo Bioluminescence Imaging (BLI) of Tumor Growth (Figure 1B)15
NOTE: The patient-derived glioma cells are modified to express firefly luciferase. This allows us to follow tumor progression after intracranial implantation.
<ol>
	<li>Give animals an i.p. injection of the D-luciferin, potassium salt (150 mg/kg)&#160;10 min prior to the imaging session.</li>
	<li>Immediately place animals in an oxygenated isoflurane induction chamber approved by the IACUC.</li>
	<li>Place animals inside the heated (37 &#176;C) imaging chamber to capture the BLI signal using software settings (for details, see manufacturer instruction).</li>
</ol>
5. Whole Brain Irradiation (Figure 1C)2,4,13
NOTE: To enhance the migration of intranasally delivered hNSCs to brain tumors, whole brain irradiation of mice bearing intracranial tumor xenografts can be utilized 4.
<ol>
	<li>Use an i.p. injection of a ketamine and xylazine mixture (50-100 mg/kg and 5-10 mg/kg respectively; consult institutional IACUC for approved dose range) with sub-surgical anesthesia plane to moderately induce anesthesia for mice.</li>
	<li>Utilize a sample tray to position the mice inside the chamber between lead shielding blocks, allowing for head-only exposure to the radiation path (Figure 2).</li>
	<li>Give mice with positive BLI confirmation of tumor growth (normally at day 7-8 after glioma cells implantation) a daily dose of 2 Gy of irradiation for 5 consecutive days. Perform BLI assessment of tumor after the last irradiation dose.</li>
</ol>
6. Genetic Modification of Human Neural Stem Cells (NSCs; Figure 3A)4
<ol>
	<li>Maintain human NSCs (clone HB1.F3.CD)6,7 in DMEM medium with 10% FBS and 1% penicillin-streptomycin in a humidified 37 &#176;C incubator under 95% room air and 5% CO2 (NNSCs).</li>
	<li>Perform genetic overexpression of chemotaxic receptors, such as the C-X-C motif chemokine receptor 4 (CXCR4), in hNSCs by transduction with lentivirus encoding for human CXCR4.
	<ol>
		<li>When NSCs are near 75-80% confluency, add the CXCR4 lentivirus to the culture medium along with polybrene (8 &#181;g/mL), and incubate for 8 h or overnight.</li>
		<li>Replace virus-containing medium with fresh medium containing blasticidin at 4 &#181;g/mL for positive selection of transduced cells.</li>
		<li>Validate the level of CXCR4 expression in modified hNSCs (CXCR4NSCs) via flow cytometry using anti-CXCR4 antibody and matching isotype antibody and via quantitative RT-PCR using a pair of primers amplifying CXCR4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs4.</li>
		<li>Generate vector control NSCs (VCNSCs) using the same strategy, with RFP replacing the CXCR4 gene, and verify via microscopy or flow cytometry due to the presence of RFP fluorescence.</li>
	</ol>
	</li>
</ol>
7. Hypoxic Preconditioning of Human NSCs (Figure 3A)4
<ol>
	<li>Plate cultured NSCs in T75 culture flasks as described above until 90% confluency is reached.</li>
	<li>Place cell culture flasks inside a humidified CO2 chamber/incubator with maintained 1% O2 levels for 24 h, via N2 gas supply controlled by an automated built-in flow meter. These cells are referred to as HNSCs. Control NNSCs are described in step 7.1.</li>
</ol>
8. Loading of Human NSCs with Oncolytic Virus (Figure 3B-3H)5
<ol>
	<li>8.1.Infect NNSCs, HNSCs, VCNSCs, and CXCR4NSCs with 50 conditional replicative adenovirus (CRAd)-S-pk7 viral particles/cell4,16.</li>
	<li>8.2.After 2 h of incubation at room temperature with periodic tapping, wash cells three times with media to remove unbound viral particles and suspend cells in sterile saline for injection. 
	CAUTION: Carry out all virus-related in vitro cell culture procedures in facilities with the appropriate biosafety level designation, such as BSL2. Appropriate personal protective equipment must be worn by researchers handling the oncolytic virus per institutional animal facility guidelines4,16.</li>
</ol>
9. Loading Stem Cells with Micron-size Paramagnetic Iron Oxides (MPIOs) for In Vivo Tracking via Magnetic Resonance Imaging (MRI; Figure 3B-3H)2
<ol>
	<li>To label stem cells, add MPIOs to stem cell cultures at ~80% confluency, in the presence of transfection reagent for overnight incubation. The efficiency of stem cell labeling (NSCs4 or MSCs2) with MPIOs (flash red conjugated) is determined by flow cytometry.</li>
	<li>To visualize stem cell migration and distribution in the brains of glioma-bearing animals, acquire both multi-slice high resolution T2 weighted Rapid Acquisition with Relaxation Enhancement spin echo images and multi-slice T1 weighted Fast Low Angle Shot (FLASH) gradient echo sequences to evaluate the structure via both coronal and axial planes of the mouse brain2.</li>
</ol>
10. Intranasal Delivery of Therapeutic NSCs (Figures 1D and Figure 3D)2,4
<ol>
	<li>Culture and collect NSCs (NNSCs, HNSCs, VCNSCs, and CXCR4NSCs) according the protocol described in section 3. 
	CAUTION: All animal procedures involving OV-loaded NSCs must be carried out in facilities with the appropriate biosafety level designation, such as BL2 or BSL2 facilities.</li>
	<li>Anesthetize mice via i.p. injection of a ketamine and xylazine mixture according to step 3.6.</li>
	<li>Place mice on a clean drape facing up with a heating pad underneath to maintain body temperature. Adjust a padded pillow made of rolled up paper towels with tapes to ensure the upright angle of the nostrils when placed under the head of the mouse. 
	NOTE: Hyaluronidase pre-treatment (total of 100 U as four repeated inoculations at 5-min intervals, 3 &#181;L in each nostril) of the nasal epithelium was described previously2,17.</li>
	<li>Using a micropipette, dispense 2 &#181;L of NSC suspension to each nostril of the mouse. Visually confirm the aspiration of the droplet before moving to the next mouse. The total number of cells delivered can be determined by the user; use 62,500-125,000 cells/&#181;L, such that 0.5-1 million cells can be delivered in 4 x 2 &#181;L doses.</li>
	<li>Use a timer to ensure 5 min intervals between each cell suspension administration, up to 4 times.</li>
	<li>Allow animals to regain mobility in a recovery chamber, with the supine position maintained throughout.</li>
</ol>
11. Survival Analysis (Figure 3E)
<ol>
	<li>Enter the survival duration data of animals in statistical software, and perform statistical comparisons using the log-rank test with the following significance designations: *p &#60;0.05, **p &#60;0.01, ***p &#60;0.001.</li>
</ol>
12. Tissue Collection and Histology/Immunocytochemistry Verification of Intranasal Stem Cell Brain Entry2,4
<ol>
	<li>At the end point of the study, euthanize mice via the IACUC-approved protocols. A standard practice is using a flow rate-controlled CO2 chamber to sacrifice the animals as the primary euthanasia strategy followed by exsanguination by perfusion fixation.</li>
	<li>Immediately after being sacrificed, perform an intracardiac perfusion with PBS on the animal to ensure the elimination of blood residues from blood vessels and the preservation of tissues for subsequent histological analysis.</li>
	<li>Place the CO2-euthanized animal on top of an absorbent pad in the supine position, proceed with a laparotomy to expose the diaphragm muscle, which is dissected along with the rib cage, to expose the heart.</li>
	<li>After making a nick on the right atrium using a pair of dissection scissors, accelerate the exsanguination with a 3-5 mL of PBS injection via the left ventricle at the apex. Inject ice-cold 4% paraformaldehyde (PFA) into circulation using a syringe; peripheral muscle spasms provide visual confirmation that there is no major blockage in the circulation that would prevent proper tissue fixation.</li>
	<li>Surgically remove the head of the carcass and preserve in a 50 mL centrifuge tube in 4% PFA at 4 &#176;C overnight. Change the solution to 30% sucrose the following day prior to brain tissue dissection another 24 h later.</li>
	<li>Embed the brain tissue in OCT compound and flash freeze on isopentane on dry ice. Perform cryosectioning of the resultant tissue block to generate 10-20 &#181;m tissue sections for histology and immunocytochemistry applications.</li>
</ol>

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Authors have no conflict of interest to declare.

Although the intranasal route of drug delivery has been widely explored for small molecules, nanomedicines, and protein compounds alike18, the application of therapeutic stem cells for intranasal brain tumor targeting is very new in the spectrum of brain tumor therapeutics under development2,3,4. There are intrinsic complexities involved regarding the behaviors of stem cells in the nasal cavity, and molec...

Low toxicity, low immunogenicity, and intrinsic brain tumor tropism of human stem cells are attractive traits for the delivery of therapeutics vehicles1. Novel stem cell-based therapeutics for malignant brain tumors are promising innovations developed in recent years, and the intranasal adaptation of this therapeutic strategy represents a leap towards clinical translation, in that non-invasive and repeated administration might dramatically reduce the barrier for patient applications and may be adaptable for out-patient services without general anesthesia or lengthy in-patient service associated with invasive surgical procedures1,2,3,4.
We and others have pioneered the intranasal route of stem cell delivery to brain tumors and have laid the ground work for some of the basic principles of translational research using mouse xenograft models2,3,4, as well as investigated the migration of stem cells in vivo via magnetic resonance imaging (MRI) reagent carriers2. Through these pilot explorations, we have accumulated substantial experience and gained insight on how to best construct a robust pre-clinical evaluation strategy using well-established patient-derived xenograft (PDX) mouse models of malignant glioma, while maintaining the investigative resolution to examine the nuanced mechanistic details of the sophisticated biological phenomena of the intranasal brain entry of therapeutic stem cells delivered to the nasal cavity. Here, we describe the principles of a standardized operating protocol to demonstrate the current state of experimental investigations using a well-established human neural stem cell line HB1.F3.CD5,6,7,8, which is readily modifiable to adapt to specific tumor models or strategies using human stem cells as therapeutic carriers.

<table><tbody><tr><td>Stereotaxic frame</td><td>Kopf Instruments</td><td>Model 900</td><td></td></tr><tr><td>Hypoxic Cell Culture Incubator</td><td>ThermoFisher Scientific</td><td>VIOS 160i</td><td></td></tr><tr><td>Cell culture supplies (Plastics)</td><td>ThermoFisher Scientific</td><td>Varies</td><td>Replaceable with any source</td></tr><tr><td>Legend Micro 21R Refrigerated Microcentrifuge</td><td>ThermoFisher Scientific</td><td>75002490</td><td>Replaceable with any source</td></tr><tr><td>Bench centrifuge Sorvall ST16R&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>75004240</td><td>Replaceable with any source</td></tr><tr><td>Micro syringe 702N 25 &amp;micro;l (22S/2&quot;/2)</td><td>Hamilton Company</td><td>80400</td><td>Flat tip</td></tr><tr><td>Sample Tray for Irradiator</td><td>Best Theratronics</td><td>A13826</td><td>To set up mice protection with lead shield</td></tr><tr><td>Leica DMi8 Microscope</td><td>Leica Microsystem</td><td></td><td>Custom setup</td></tr><tr><td>Leica CM1860 UV cryostat</td><td>Leica Microsystem</td><td></td><td>Custom setup</td></tr><tr><td>Exel International Insulin Syringe</td><td>ThermoFisher Scientific</td><td>14-841-31</td><td></td></tr><tr><td>Corning Phosphate Buffer Saline</td><td>Corning Cellgro/ThermoFisher</td><td>21-031-CV</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium&amp;nbsp;</td><td>Corning Cellgro/ThermoFisher</td><td>11965-084</td><td></td></tr><tr><td>Trypsin 0.05%</td><td>Corning Cellgro/ThermoFisher</td><td>25300054</td><td></td></tr><tr><td>Hyaluronidase from bovine testes</td><td>MilliporeSigma</td><td>H3506</td><td></td></tr></tbody></table>

intranasal delivery of therapeutic stem cells to glioblastoma in a mouse model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Both hypoxic pre-treatment (Figure 4A)4 and CXCR4 overexpression (Figures 4B and 4C)4 significantly upregulate the cell membrane presence of CXCR4 receptors as demonstrated by flow cytometry. The tumor tropism demonstrated by NSCs (blue arrows), is shown in the tumor tissue histology (red circle). The presence of MPIO-labeled (Figure 4D) stem cells ...

Watch this Scientific Journal Video about Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model at JoVE.com

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

intranasal-delivery-therapeutic-stem-cells-to-glioblastoma-mouse

Research

JoVE Journal

Medicine

12.1K Views.  Northwestern University. Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

Video: Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Translational Orthotopic Models of Glioblastoma Multiforme

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain tumors. Unlike xenograft tumors, in GEMs, tumors arise in the native microenvironment in an immunocompetent mouse. However, the use of GBM GEMs in preclinical treatment studies is challenging due to long tumor latencies, heterogeneity in neoplasm frequency, and the timing of advanced grade tumor development. Mice induced via intracranial orthotopic injection are more tractable for preclinical studies, and retain features of the GEM tumors. We generated an orthotopic brain tumor model derived from a GEM model with Rb, Kras, and p53 aberrations (TRP), which develops GBM tumors&#160;displaying linear foci of necrosis by neoplastic cells, and dense vascularization analogous to human GBM. Cells derived from GEM GBM tumors are injected intracranially into wild-type, strain-matched recipient mice and reproduce grade IV tumors, therefore bypassing the long tumor latency period in GEM mice and allowing for the creation of large and reproducible cohorts for preclinical studies. The highly proliferative, invasive, and vascular features of the TRP GEM model for GBM are recapitulated in the orthotopic tumors, and histopathology markers reflect human GBM subgroups. Tumor growth is monitored by serial MRI scans. Due to the invasive nature of the intracranial tumors in immunocompetent models, carefully following the injection procedure outlined here is essential to prevent extracranial tumor growth.

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...

Cancer Research

Common Carotid Artery Repair After Injection in a Mouse Model 

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

Author Spotlight: Advancing Glioblastoma Treatment Through Intraarterial Delivery Strategies for Oncolytic Viruses and MicroRNAs

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

Developing Efficient and Precise Mouse Model for Spinal Cord Glioma

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and comprehensive data on spinal cord gliomas remain limited due to the lack of suitable preclinical animal models. Developing a simple and reproducible animal model has become essential for advancing basic and translational research. A murine model is ideal, as the murine spinal cord shares structural similarities with the human spinal cord. This protocol describes the generation of a reproducible murine model of spinal cord glioma by directly injecting tumor cells into the intervertebral space using the spinous process of the seventh cervical vertebra as a guide. Compared to other methods, this approach is more effective and convenient, involving a smaller incision, reduced invasiveness and blood loss, faster recovery, and more stable tumor formation. This model is expected to advance the understanding of disease mechanisms, optimize surgical strategies, and support the development of therapeutic drugs for spinal cord gliomas.

The present protocol describes the development of a reproducible murine model of spinal cord glioma by injecting tumor cells into the intervertebral space, offering a more effective and less invasive approach for research and therapeutic development.

Spinal cord gliomas are commonly malignant tumors of the spinal cord, leading to a high rate of disability. However, uniform treatment guidelines and ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Immunology and Infection

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...