The authors acknowledge editorial contributions from Dr. Kathryn Pietrosimone. PLA scaffolds were manufactured by Dr. Elizabeth Loboa&#39;s lab at North Carolina State University. This work was supported by the UNC Lineberger Comprehensive Cancer Center&#39;s University Cancer Research Fund and the UNC Translational and Clinical Sciences Institute (KL2TR001109, UL1TR001111).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of The University of North Carolina at Chapel Hill.
NOTE: This procedure is performed on mice with an open craniotomy, as a modified version of the protocol by Mostany and Portera-Cailliau19 wherein the bone tissue that is surgically removed is discarded, leaving the brain accessible for establishment and eventual resection of GBM tumors. Following craniotomy, tumors are established via stereotactic implantation as described in Ozawa and James20. We implant 1 x 105 U87 mCh-Fl cells at the following stereotactic coordinates (in mm from bregma): (2.5, 0, -0.5).
1. Cell Culture and Scaffold Preparation
NOTE: Scaffolds should be prepared 48 h prior to implantation in mice. The following volumes are provided on a per-scaffold basis. Multiply quantities as needed for additional scaffolds.
<ol>
	<li>Cut PLA scaffolds into resection cavity-sized (approximately 2 mm x 2 mm) pieces. Scaffolds can be cut by hand using scissors or using a hole punch for repeatability.</li>
	<li>Sterilize by immersing in 70% ethanol for 15 min followed by immersing in PBS. Place scaffold in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin while preparing cells for seeding.</li>
	<li>Lift cultured MSCs using 0.05% trypsin (3-5 mL for a T75 flask). Incubate at 37 &#176;C for 5 min. Ensure that the cells have lifted, then add 7-10 mL DMEM to the flask to inactivate trypsin.</li>
	<li>Transfer flask contents to a 15-mL centrifuge tube. Count cells using a hemocytometer. Pellet 5 x 105 MSCs for each scaffold via centrifugation at 100 x g for 5 min.</li>
	<li>Aspirate off supernatant and resuspend MSCs in 5 &#181;L DMEM per 5 x 105 cells.</li>
	<li>Prepare the scaffolds for seeding by patting them dry.
	<ol>
		<li>Remove a scaffold from DMEM immersion with forceps and temporarily place it on the lid of the 6-well plate. Lift the scaffold off the lid, leaving behind a droplet of excess DMEM.</li>
		<li>Place the scaffold back on the lid again in a new, dry location. Repeat 3-5 times, then place the partially dried scaffold in a new 6-well plate for seeding. Repeat for each scaffold. 
		NOTE: A partially dried scaffold provides optimal cell seeding results. If the scaffold is too wet, cells will slide off the scaffold and adhere to the well plate below. If the scaffold is too dry, the droplet will not spread over the entire scaffold, resulting in poor initial cell distribution.</li>
	</ol>
	</li>
	<li>Using a pipette, gently mix the vial of stem cells to homogenize the suspension as some cells may have settled to the bottom.</li>
	<li>Slowly pipette 2.5 &#181;L freshly mixed MSC suspension directly on the scaffold, creating a small droplet over the top of the scaffold.</li>
	<li>Add 300 &#181;L DMEM to edges of each well. This will prevent rapid evaporation of the cell droplet. Incubate at 37 &#176;C for 30 min, allowing the cells to attach to the scaffold.</li>
	<li>With forceps, gently flip the scaffolds over in the well plate. Seed 2.5 &#181;L freshly mixed MSC suspension (this results in a total of 5 x 105 cells seeded per scaffold). Incubate at 37 &#176;C for 30 min, allowing the cells to attach to the scaffold.</li>
	<li>Cover the scaffolds in DMEM by adding 2 mL to each well of the 6-well plate. Gently lift the scaffolds to allow media to flow underneath them. Incubate at 37 &#176;C in this state for 48 h prior to implantation surgery.</li>
	<li>Check the scaffolds after 24 h and add/change media if excess evaporation or discolored media due to pH imbalance is observed.</li>
</ol>
2. Fluorescence-guided Resection and Scaffold Implantation
NOTE: Sterilize all tools prior to initializing surgery. Administer prophylactic analgesia as outlined in your&#160;institutional IACUC protocol.
<ol>
	<li>Place the stereotaxic frame on the stage of a fluorescence dissecting stereomicroscope.</li>
	<li>Anesthetize the mouse under inhaled isoflurane in an induction chamber. Once anesthetized, secure the mouse in the stereotaxic frame with continuous inhaled anesthesia supply via an isoflurane nose cone adapter. Maintain body temperature with a heating pad and/or probe. 
	NOTE: 4-5% isoflurane is suitable for induction and 2-3% is suitable for maintenance, but this must be adapted and monitored for each mouse.</li>
	<li>Apply ophthalmic ointment to the eyes to prevent drying of the cornea. Sterilize the incision site of the scalp with a series of three alcohol and three betadine wipes.</li>
	<li>Perform toe pinch reflex test on each limb and confirm negative response to ensure proper anesthetization. Ensure a steady respiratory rate at approximately 55-65 breaths/min.</li>
	<li>Using forceps, pinch and gently lift the scalp. Make a midline linear rostral-caudal incision with surgical scissors. Irrigate the incision site with PBS and remove subdermal fat with a cotton tip applicator in a circular brushing motion. Arrange the skin such that the previously-established cranial window is fully visible.</li>
	<li>Gently puncture the dura just interior to the borders of the cranial window using an 18-G needle. Repeat until the incision fully traces the interior of the window.</li>
	<li>Remove the dura by peeling it away using fine forceps, revealing the underlying parenchyma and tumor.</li>
	<li>Locate the U87 mCh-Fl tumor by turning room lights off and turning the stereomicroscope fluorescence-mode on. Prepare for resection by loading a 1-200 &#181;L pipette tip into the end of the tubing of a vacuum pump.</li>
	<li>Turn the vacuum pump on. Resect the tumor by gently aspirating fluorescent tissue until no signal remains. Turn the vacuum pump off and discard the pipette tip. Turn the fluorescence off and the room lights back on. 
	NOTE: To control bleeding, irrigate with cold PBS and apply steady pressure with a cotton tip applicator. In more severe cases, the resection cavity can also be temporarily packed with a hemostatic agent&#160;as long as it is removed after the bleeding has stopped followed by another PBS irrigation.</li>
	<li>Immediately prior to implantation, slowly dip an MSC-seeded PLA scaffold in PBS to remove unwanted media and associated components. Implant the scaffold into the resection cavity. If needed, add 1 &#181;L fibrinogen (67-106 mg/mL) followed by 1 &#181;L thrombin (400-625 U/mL) to secure the scaffold in place.</li>
	<li>With the dura removed and the bone flap from the cranial window already discarded, seal the wound simply by closing the skin and applying surgical glue. Remove the animal from inhaled anesthetic and allow to recover on a heated surface.</li>
	<li>Once ambulatory, return mouse to cage. Administer analgesic on IACUC-approved schedule. Repeat procedure for each mouse.</li>
</ol>
3. Post-Operative Imaging
<ol>
	<li>Using the 28-G insulin syringe, inject mice with D-luciferin (150 mg/kg intraperitoneal).</li>
	<li>Wait 10 min. This will allow the luciferin to circulate throughout the body and react with the luciferase-expressing cancer cells in the brain to obtain peak expression.</li>
	<li>Under isoflurane anesthesia (as mentioned previously), image mice in a bioluminescence imaging (BLI) system to visualize the tumors. Vary exposure times as needed (seconds to minutes) depending on tumor size.</li>
	<li>Repeat imaging procedure as needed to determine tumor growth kinetics. Continue to monitor animal health. 
	NOTE: The therapeutic MSCs will constitutively express TRAIL, killing cancer cells and suppressing overall tumor growth. Imaging every 2-5 days provides sufficient frequency for this purpose.</li>
	<li>When predetermined endpoints outlined in the IACUC protocol have been met, sacrifice mice by transcardial perfusion and collect tissues for analysis.</li>
</ol>

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Drs. Sheets, Bag&#243;, and Hingtgen have equity in Falcon Therapeutics who has licensed aspects of the stem cell and scaffold technology from UNC Chapel Hill.

Surgery can typically be completed within 30 min per mouse, given that the following points are taken into consideration to maximize precision and avoid time-consuming pitfalls. First, ensure the mouse is properly positioned in the stereotaxic instrument prior to starting the procedure. Unwanted head movements will limit surgical accuracy of the craniotomy, location of the tumor implantation, and degree of tumor resection. Prior to resection, fully remove the portion of the dura covering the tumor. The tough, fibrous dura hardens as it dehydrates during aspiration. If incompletely removed, the hardened dura may limit aspirator tip mobility and prevent complete tumor resection. During resection, blood vessels are often inadvertently resected along with the tumor, causing significant bleeding. Excess blood diminishes the intensity of the fluorescent signal and obscures the tumor. Maintaining tumor visibility during resection ensures proper extent of resection, and limits the removal of healthy tissue and excess bleeding. For smaller bleeds, irrigate the damaged vessel with cold saline&#160;and apply gentle pressure with a cotton tip applicator. If bleeding continues, pack a hemostatic agent into the cavity for 2-3 min and then remove with forceps. Irrigate with PBS afterwards to restore physiological pH prior to implanting the cell-seeded scaffold. When resection is completed&#160;and bleeding has stopped, the final pitfall is inadequate skin wound closure. This is most often caused by excess moisture (PBS or blood) that prevents the surgical glue from bonding the incised skin. Avoid this by gently drying the skin with a cotton tip applicator prior to applying the glue. It is also possible to accidentally glue the skin directly to the skull if the wound gap is not first held in a closed position with forceps prior to gluing.
With these considerations in mind, the above protocol produces consistent and reliable GBM surgical resections that&#160;mimic the standard of care for accurate in vivo testing of emerging cell and small molecule therapies. Still, several components can be modified to serve particular experimental needs. For instance, tumor properties such as size, location, and extent of invasion can be adjusted by changing the initial number and depth of injected cells, time between tumor establishment and resection, or by switching the tumor cell line itself. Furthermore, this study is performed in athymic nude mice whose compromised immune system tolerates human cells. Immune competent&#160;mice may be more appropriate for studies using mouse cells.
Compared to a closed system (i.e., the bone or a coverslip is put back into place), the open craniotomy performed in this protocol reduces intracranial pressure (ICP). Since increased ICP is responsible for the onset of symptoms including seizures,&#160;mice will experience an artificial prolongation of survival in this model. While the impact is minimized due to the delay applying to both control and treatment groups, future models could re-close the bone flap to restore ICP and determine the role that it plays in cell therapy for GBM.

Glioblastoma (GBM) is the most common primary brain cancer in adults, with a dismal median survival of just 12-15 months1,2,3,4,5. Survival has not significantly improved since 2005 when the current clinical standard of maximal surgical resection followed by radiation and concomitant and adjuvant temozolomide chemotherapy was adopted6,7. While this treatment provides patients with a temporary relief of symptoms, standard of care treatment invariably results in recurrence as invasive cancer foci evade resection and are protected from systemic therapies by the blood-brain barrier (BBB). Strategies which target invasive tumor foci while circumventing the BBB are urgently needed to gain traction against this aggressive and debilitating disease.
Human mesenchymal stem cells (MSCs) show promise as drug delivery vehicles for GBM due to their native tumor tropism8,9. MSCs possess receptors for and migrate towards soluble factors that tumors secrete, including stromal cell-derived factor 1&#945; (SDF-1&#945;), matrix metalloproteinase-1 (MMP-1), and monocyte chemoattractant protein-1 (MCP-1) among others10,11,12,13. Engineering MSCs to express and secrete cytotoxic drugs allows them to be harnessed as tumor-homing drug delivery vehicles. Engineered MSCs move toward invasive tumor foci and deliver therapeutic proteins. This approach has demonstrated feasibility in a variety of preclinical GBM models9,14. However, the vast majority of these models do not include surgical resection despite the clinical relevance of this component. Emerging studies using new models of resection revealed that surgical tumor removal reduces the persistence of stem cells that are directly injected into the surgical cavity15. Loss of viability resulted in reduced efficacy, likely due to decreases in the dose and duration of drug delivered to the invasive tumor foci.
To increase stem cell viability and drug delivery, MSCs can be seeded onto scaffolds prior to implantation. In this protocol, biocompatible and resorbable electrospun nanofibrous poly(lactic acid) (PLA) is used as scaffolding for the MSCs. PLA flexes and conforms to the shape of the resection cavity upon implant, which maximizes therapeutic coverage and minimizes the distance MSCs must travel to reach tumor cells. MSCs remain on the scaffold during implantation and then migrate off the scaffold toward tumor cells after implantation16,17. MSCs and the cytotoxic drugs they carry then accumulate at the tumor foci. Delivery of cytotoxic drug to the tumor requires MSC viability and persistence, both of which are aided by implantation on scaffolds.
In this procedure, lentiviral vectors are used to induce stable expression of fluorescent (in vitro tracking) and bioluminescent (in vivo tracking) markers in both cancer and stem cell lines. The human GBM line U87 is infected with mCherry and firefly luciferase (U87 mCh-Fl), and the non-therapeutic MSCs with GFP and renilla luciferase (MSC GFP-Rluc). The therapeutic variant of MSCs express TNF&#945;-related apoptosis inducing ligand (MSC-TRAIL). TRAIL, a constitutively-secreted protein, binds to nearby death receptors on cancer cells and initiates caspase-mediated apoptosis18.
Here, we provide a protocol for preclinical image-guided GBM surgical resection and implantation of MSC-seeded scaffolds. In brief, nude mice are given a craniotomy followed three days later by stereotactic orthotopic injection of U87 mCh-Fl to establish the primary tumor. The engrafted tumor grows for a period of approximately one week. PLA scaffolds are seeded with MSCs 48 h prior to resection surgery. The tumor is then resected under fluorescent guidance, and the MSC-loaded scaffold is implanted into the resection cavity. Tumor burden and mouse survival are then tracked post-operatively with bioluminescence imaging (BLI). A timeline of these procedures is provided below (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57452/57452fig1.jpg" /> 
Figure 1: Timeline of procedures. Mice initially receive a cranial window (Day 0). After a recovery period of three days, tumors are implanted (Day 3) and grow for approximately one week. Scaffolds are seeded with MSCs (Day 8) two days in advance of the tumor resection and implantation procedure (Day 10). Tumor progression and therapeutic efficacy are evaluated via post-operative imaging thereafter (Day 10+). <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57452/57452fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Just for mouse stereotaxic instrument</td><td>Stoelting</td><td>51730</td><td>Maintains steady head positioning during surgery</td></tr><tr><td>Fluorescence dissecting stereomicroscope</td><td>Leica</td><td>M165 FC&amp;nbsp;</td><td>Allows real-time imaging of tumor during resection</td></tr><tr><td>Motorized integrated stereotaxic injector (ISI) system</td><td>Stoelting</td><td>53315</td><td>Allows precise tumor cell injection volume and rate</td></tr><tr><td>Vetbond tissue adhesive</td><td>3M</td><td>1469</td><td>Sugical glue to close skin wound</td></tr><tr><td>Artificial tears</td><td>Akorn</td><td>664268</td><td>Prevents eyes from drying during surgery</td></tr><tr><td>Webcol alcohol preps</td><td>Covidien</td><td>6818</td><td>Sterilize incision site</td></tr><tr><td>Betadine surgical scrub</td><td>Purdue Fredick Company</td><td>6761815117</td><td>Sterilize incision site</td></tr><tr><td>Cotton-tipped applicators</td><td>Fisherbrand</td><td>23-400-115</td><td>Surgery tool</td></tr><tr><td>E-vac aspirating system</td><td>Argos</td><td>EV310</td><td>Vacuum pump used to resect tumor</td></tr><tr><td>Fibrinogen and thrombin extracted from as-received TISSEEL</td><td>Baxter</td><td></td><td>To temporarily secure the scaffold in the resection cavity</td></tr><tr><td>IVIS Kinetic in vivo optical imaging system</td><td>Caliper Life Science</td><td></td><td>Bioluminescent Imager</td></tr><tr><td>D-Luciferin potassium salt</td><td>PerkinElmer</td><td>122799</td><td>In vivo imaging agent</td></tr></tbody></table>

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.

U87 tumor cells were engineered to express mCherry and firefly luciferase (U87 mCh-Fl) prior to injection, and to allow for image-guided resection and bioluminescence tracking (Figure 2A-C). Stem cells were similarly engineered with diagnostic GFP-Rluc (MSC-GFP) or therapeutic GFP-TRIAL (MSC-TRAIL) and seeded onto the PLA scaffolds to confirm attachment and proliferation (Figure 2D-F).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57452/57452fig2.jpg" /> 
Figure 2: Representative images of fluorescent cancer cells and MSCs seeded on PLA. A-C) Phase, fluorescent, and combined images, respectively, show U87 cancer cells engineered with lentiviral constructs to express mCh-Fl markers. D-F) Corresponding images of stem cells engineered to express GFP-Rl or therapeutic GFP-TRAIL seeded onto the PLA scaffolding material. Scale bars = 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57452/57452fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Intraoperative images are provided below to highlight portions of the surgical procedure for tumor resection and scaffold implantation (Figure 3). The animal is first anesthetized and aligned in the stereotaxic instrument (Figure 3A). The skin is opened, and the dura is peeled back. The tumor is resected (Figure 3B), and when examined under white light appears to be fully removed. However, fluorescent images of the tumor before (Figure 3C) and after (Figure 3D) resection indicate while the majority of the tumor was removed, a residual amount remains. This phenomenon resembles clinical cases wherein surgeons are unable to remove tumor cells that quickly migrate away from the primary mass and into inoperable areas in the brain. Migratory therapeutic MSCs move toward residual tumor foci that remain following surgical resection. To deliver MSCs into the resection cavity, the MSCs are first seeded onto PLA, and then the scaffold construct is placed in the resection cavity. Presence of the MSCs on the scaffold are confirmed post-implant by fluorescently imaging GFP signal (Figure 3E). Ex vivo whole brain images show scaffold dimensions relative to the resection cavity (Figure 3F-H). The scaffold appears significantly larger when laid flat, but can be molded into the shape of the resection cavity during implantation. By coating the entire cavity, the distance therapeutic MSCs have to migrate to reach tumor foci is minimized. After surgery, BLI is used to track tumor growth over time and determine scaffold-delivered MSC-TRAIL efficacy.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57452/57452fig3.jpg" /> 
Figure 3: Representative intraoperative and postoperative images. A) Intraoperative series showing mouse before incision. B) Incised mouse with cranial window revealing tumor mass. C) Bright field and fluorescent overlay image showing location of tumor. D) Post-surgical resection cavity with remnant tumor foci. E) Implanted PLA scaffold seeded with MSC-TRAIL. F) Post-mortem brain tissue with scaffold overlaid. G) Fluorescent overlay highlighting GFP-TRAIL cells. H) Magnified version of G. Scale bars = 5 mm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57452/57452fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds at JoVE.com

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

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 ...

image-guided-resection-glioblastoma-intracranial-implantation

Nanyang Technological University

Research

JoVE Journal

Medicine

8.2K Views.  University of North Carolina at Chapel Hill. Tumor-zoekende therapeutische mesenchymale stamcellen (MSCs) tonen belofte als een behandeling voor invasieve glioblastoma. Optimale transplantatie gaat om levering van MSCs in de holte van de resectie tumor op steigers. Hier, preklinische technieken om te studeren MSC behandeling van glioblastoma zijn geleverd inclusief: afbeelding-geleide tumor resectie; implantatie van MSC-geplaatste steigers; en postoperatieve therapie volgen.

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

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

Flexible Organic Electronic Devices for Pulsed Electric Field Therapy of Glioblastoma

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed electric fields (PEFs) are promising for the improvement of treatment efficiency. However, they rely on rigid electrodes that cause acute and chronic damage, especially in soft tissues such as the brain. In this work, flexible electronics were used to deliver PEFs to tumors and the biological response was evaluated with fluorescent microscopy. Interdigitated gold electrodes on a thin, transparent parylene-C substrate were coated with the conducting polymer PEDOT:PSS, resulting in a conformable and biocompatible device. The effects of PEFs on tumors and their microenvironment were examined using various biological models. First, monolayers of glioblastoma cells were cultured on top of the electrodes to investigate phenomena in vitro. As an intermediate step, an in ovo model was developed where engineered tumor spheroids were grafted in the embryonic membrane of a quail. Due to the absence of an immune system, this led to highly vascularized tumors. At this early stage of development, embryos have no immune system, and tumors are not recognized as foreign bodies. Thus, they can develop fast while developing their own vessels from the existing embryo vascular system, which represents a valuable 3D cancer model. Finally, flexible electrode delivery of PEFs was evaluated in a complete organism with a functional immune system, using a syngenic, orthograft (intracranial) mouse model. Tumor spheroids were grafted into the brain of transgenic multi-fluorescent mice prior to the implantation of flexible organic electrode devices. A sealed cranial window enabled multiphoton imaging of the tumor and its microenvironment during treatment with PEFs over a period of several weeks.

This work describes the development of flexible interdigitated electrodes for implementation in 3D brain tumor models, namely, in vitro culture, in ovo model, and in vivo murine model. The proposed method can be used to evaluate the effects of pulsed electric fields on tumors at different levels of complexity.

Glioblastoma is difficult to eradicate with standard oncology therapies due to its high degree of invasiveness. Bioelectric treatments based on pulsed ...

Cancer Research

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

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

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.

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 ...

Fluorescence-guided Tumor Resection and MSC-seeded Scaffold Implantation in Murine Model: A Surgical Procedure to Deliver Therapeutic Mesenchymal Stem Cells into Brain Tumor Resection Cavity in Mouse Model

Source: Sheets, K. T. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57452" target="_blank">Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds.</a>&#160;J. Vis. Exp.&#160;(2018)
This video presents the surgical procedure for image-guided tumor resection and implantation of a bio-scaffold seeded with genetically engineered mesenchymal stem cells or MSCs expressing tumor necrosis factor-related apoptosis-inducing ligand or TRAIL. These therapeutic MSCs have the potential in treatment of brain tumors.

Source: Sheets, K. T. et al. Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds.&#160;J. Vis. ...

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

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