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>

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 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

8.2K Views.  University of North Carolina at Chapel Hill. 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 therapeut...