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

Summary

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.

Abstract

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 TNFα-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.

Introduction

Glioblastoma (GBM) is the most common primary brain cancer in adults, with a dismal median survival of just 12-15 months^1,2,3,4,5. 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 adopted^6,7. While this treatment provides patients with a temporary relief of symptoms, standard of care treatment invariably....

Protocol

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 cel....

Results

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).

Discussion

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.......

Materials

List of materials used in this article

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