Name: image-guided resection of glioblastoma and intracranial implantation of therapeutic stem cell-seeded scaffolds
Uploaded: 2018-07-16T12:30:00
Description: Watch this Scientific Journal Video about Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds at JoVE.com

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...

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Laboratory investigation.</a> Journal of Neurosurgery. 108 (2), 320-329 (2008).</li><li>Nakamizo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+marrow-derived+mesenchymal+stem+cells+in+the+treatment+of+gliomas.">Human bone marrow-derived mesenchymal stem cells in the treatment of gliomas.</a> Cancer Research. 65 (8), 3307-3318 (2005).</li><li>Xu, F., Shi, J., Yu, B., Ni, W., Wu, X., Gu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+mediate+mesenchymal+stem+cell+migration+toward+gliomas+in+vitro.">Chemokines mediate mesenchymal stem cell migration toward gliomas in vitro.</a> Oncology Reports. 23 (6), 1561-1567 (2010).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR4-transfected+human+umbilical+cord+blood-derived+mesenchymal+stem+cells+exhibit+enhanced+migratory+capacity+toward+gliomas.">CXCR4-transfected human umbilical cord blood-derived mesenchymal stem cells exhibit enhanced migratory capacity toward gliomas.</a> International Journal of Oncology. 38 (1), 97-103 (2011).</li><li>Ho, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Metalloproteinase+1+Is+Necessary+for+the+Migration+of+Human+Bone+Marrow-Derived+Mesenchymal+Stem+Cells+Toward+Human+Glioma.">Matrix Metalloproteinase 1 Is Necessary for the Migration of Human Bone Marrow-Derived Mesenchymal Stem Cells Toward Human Glioma.</a> STEM CELLS. 27 (6), 1366-1375 (2009).</li><li>Bexell, D., Svensson, A., Bengzon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+therapy+for+malignant+glioma.">Stem cell-based therapy for malignant glioma.</a> Cancer Treatment Reviews. 39 (4), (2012).</li><li>Nouri, F., Wang, X., Hatefi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+theranostic+mesenchymal+stem+cells+for+the+evaluation+of+the+anticancer+efficacy+of+enzyme/prodrug+systems.">Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems.</a> Journal of Controlled Release. 200, 179-187 (2015).</li><li>Kauer, T., Figueiredo, J. -. L., Hingtgen, S., Shah, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulated+therapeutic+stem+cells+implanted+in+the+tumor+resection+cavity+induce+cell+death+in+gliomas.">Encapsulated therapeutic stem cells implanted in the tumor resection cavity induce cell death in gliomas.</a> Nature Neuroscience. 15 (2), 197-204 (2011).</li><li>Bag&#243;, J., Pegna, G., Okolie, O., Hingtgen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+matrices+enhance+the+transplant+and+efficacy+of+cytotoxic+stem+cell+therapy+for+post-surgical+cancer.">Fibrin matrices enhance the transplant and efficacy of cytotoxic stem cell therapy for post-surgical cancer.</a> Biomaterials. 84, 42-53 (2016).</li><li>Bag&#243;, J., Pegna, G., Okolie, O., Mohiti-Asli, M., Loboa, E., Hingtgen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+scaffolds+increase+the+efficacy+of+stem+cell-mediated+therapy+of+surgically+resected+glioblastoma.">Electrospun nanofibrous scaffolds increase the efficacy of stem cell-mediated therapy of surgically resected glioblastoma.</a> Biomaterials. 90, 116-125 (2016).</li><li>Loebinger, M., Eddaoudi, A., Davies, D., Janes, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cell+Delivery+of+TRAIL+Can+Eliminate+Metastatic+Cancer.">Mesenchymal Stem Cell Delivery of TRAIL Can Eliminate Metastatic Cancer.</a> Cancer Research. 69 (10), 4134-4142 (2009).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J. Vis. Exp. (12), e680 (2008).</li><li>Ozawa, T., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Intracranial+Brain+Tumor+Xenografts+With+Subsequent+Analysis+of+Tumor+Growth+and+Response+to+Therapy+using+Bioluminescence+Imaging.">Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging.</a> J. Vis. Exp. (41), e1986 (2010).</li></ol>

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 dur...

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/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&#160;</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).
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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 ...

The overall goal of this procedure is to surgically resect brain cancer in mice and to implant a scaffold seeded with tumor-homing therapeutic stem cells into the postoperative resection cavity. This method can help answer key questions in the cell therapy and neuro-oncology fields, such as how surgical resection affects cell implantation into the surgical cavity, how effective new therapeutic agents are on postsurgical brain cancer, and how scaffold-based delivery systems influence therapeutic outcomes. The main advantage of this technique is that brain cancer interventions can be studied in mice in a way that closely mimics the clinical standard of care in human patients.Though this method can provide insight into stem cell therapy delivered on scaffold, it can also be applied to other interventions, such as nanoparticles, small molecules, oncolytic viruses, or other emerging therapies. Prepare the scaffolds 48 hours prior to implantation in mice by first cutting PLA scaffolds into resection cavity-sized pieces of approximately two by two millimeters. Sterilize the scaffolds by immersing in 70%ethanol for 15 minutes.After 15 minutes, immerse the scaffolds in PBS. Then place scaffolds in Dulbecco's modified Eagle's medium containing 10%fetal bovine serum and 1%penicillin-streptomycin while preparing cells for seeding. To prepare the cells, first lift cultured MSCs using 0.05%trypsin.Incubate at 37 degrees Celsius for five minutes. Following the incubation, ensure that the cells have lifted. Then add DMEM to the flask to inactivate the trypsin.Next, transfer flask contents to a 15-milliliter centrifuge tube. Count the cells using a hemocytometer. Then pellet five times 10 to the fifth MSCs for each scaffold via centrifugation at 100 times g for five minutes.Following the centrifugation, aspirate the supernatant, and resuspend the MSCs in five microliters of DMEM per five times 10 to the fifth cells. Then remove a scaffold from DMEM immersion with forceps, and place it on the lid of the six-well plate. Lift the scaffold off the lid, leaving behind a droplet of excess DMEM.Place the scaffold back on the lid again in a new, dry location. Repeat three to five times. Then place the partially dried scaffold in a new six-well plate for seeding.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 plate below. If the scaffold's too dry, the droplet will not spread over the entire scaffold, resulting in poor initial cell distribution.Using a pipette, gently mix the tube of MSCs to homogenize the suspension, as some cells may have settled to the bottom. Slowly pipette 2.5 microliters of freshly mixed MSC suspension directly on the scaffold, creating a small droplet over the top of the scaffold. Add 300 microliters of DMEM to edges of each well to prevent rapid evaporation of the cell droplet.Incubate at 37 degrees Celsius for 30 minutes, allowing the cells to attach to the scaffold. Following the incubation, use forceps to gently flip the scaffolds over in the well plate. Seed 2.5 microliters of freshly mixed MSC suspension to give a total of five times 10 to the fifth cells seeded per scaffold.After an incubation of 30 minutes to allow the cells to attach to the scaffold, cover the scaffolds in DMEM by adding two milliliters to each well of the six-well plate. Gently lift the scaffolds to allow media to flow underneath them. Incubate at 37 degrees Celsius in this state for 48 hours prior to implantation surgery.Begin by placing the stereotaxic frame on the stage of a fluorescence dissecting stereomicroscope. Then secure the anesthetized mouse in the stereotaxic frame with continuous inhaled anesthesia supply via an isoflurane nose cone adapter. Maintain body temperature with a heating pad.Apply ophthalmic ointment to the eyes to prevent drying of the cornea. Then sterilize the incision site of the scalp with a series of three alcohol and three Betadine wipes. Perform the toe pinch reflex test on each limb, and confirm negative response to ensure proper anesthetization.Using forceps, pinch and gently lift the scalp. After making 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 so that the previously established cranial window is fully visible.Use an 18-gauge needle to gently puncture the dura just interior to the borders of the cranial window. Repeat until the incision fully traces the interior of the window. Remove the dura by peeling it away using fine forceps, revealing the underlying parenchyma and tumor.Next, dim the room lights, and turn the stereomicroscope fluorescence mode on. Locate the U87 mCherry and firefly luciferase-expressing tumor. Load a 200-microliter pipette tip into the end of the tubing of a vacuum pump.Then turn on the vacuum pump. Resect the tumor by gently aspirating the fluorescent tissue until no signal remains. To control bleeding, irrigate with cold PBS, and apply steady pressure with a cotton tip applicator.Use a combination of saline, steady pressure with a cotton tip applicator, and/or a hemostatic agent, such as SURGICEL, to stop bleeding prior to implanting the scaffold. If not proper control it, a potentially dangerous hematoma may form. After resection, turn off the vacuum pump, and discard the pipette tip.Turn the fluorescence off and the room lights back up. 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 one microliter of fibrinogen followed by one microliter of thrombin to secure the scaffold in place. With the dura removed and the bone flap from the cranial window already discarded, seal the wound by closing the skin and applying surgical glue. Remove the animal from inhaled anesthetic, and allow it to recover on a heated surface.These phase-contrast, fluorescent, and combined images show U87 cancer cells engineered with lentiviral constructs to express mCherry and firefly luciferase markers. These phase-contrast, fluorescent, and combined images show corresponding images of stem cells engineered to express diagnostic GFP-Renilla luciferase or therapeutic GFP-TRAIL seeded onto the PLA scaffolding material. This brightfield and fluorescent overlay image shows the location of the tumor.Here, the postsurgical resection cavity with remnant tumor foci is seen. This image shows the implanted PLA scaffold seeded with MSC-TRAIL. Here, postmortem brain tissue is seen with the scaffold overlaid.This fluorescent overlay highlights the GFP-TRAIL cells. Once mastered, this technique can be done in under 30 minutes for each mouse, if it is performed properly. While attempting this procedure, it's important to remember that there will be intrinsic variability in the extent of resection achieved from mouse to mouse, similar to what is seen in clinics.After watching this video, you should have a good understanding of how to perform a surgical resection of a brain tumor and evaluate its impact on therapeutic interventions that include scaffold-based stem cell therapy.

image-guided-resection-glioblastoma-intracranial-implantation

Research

JoVE Journal

Medicine

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 ...

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

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 ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...

Transaxillary First Rib Resection for Treatment of the Thoracic Outlet Syndrome

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol has been used for the decompression of trapped neurovascular structures in the TOS. Among the other surgical procedures, the advantage of the TFRR is that it has the smallest rate of recurrence and better cosmetic outcomes. The disadvantage of TFRR is that it provides a narrow, and deep working corridor that makes obtaining vascular control challenging.

Here, we present a protocol of the transaxillary resection of the first rib for treatment of thoracic outlet syndrome caused by compression of the brachial plexus, subclavian vein and artery.

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol ...

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...