Despite many refinements in current therapeutic strategies, the overall prognosis for a patient with glioblastoma is dismal. Neural precursor cells (NPCs) are capable of tracking glioma tumors and thus could be used to deliver therapeutic molecules. We have engineered mouse NPCs to deliver a secreted form of tumor necrosis factor–related apoptosis–inducing ligand (S-TRAIL); S-TRAIL is optimized to selectively kill neoplastic cells. Furthermore, we have developed means to simultaneously monitor both the migration of NSCs toward gliomas and the changes in glioma burden in real time. Using a highly malignant human glioma model expressing Renilla luciferase (Rluc), intracranially implanted NPC-FL-sTRAIL expressing both firefly luciferase (Fluc) and S-TRAIL was shown to migrate into the tumors and have profound antitumor effects. These studies demonstrate the potential of NPCs as therapeutically effective delivery vehicles for the treatment of gliomas and also provide important tools to evaluate the migration of NPCs and changes in glioma burden in vivo. Ann Neurol 2005;57:34–41
Glioblastomas are the most common primary malignant brain tumors. Over the past 20 years, the incidence of gliomas has increased considerably and the median survival for patients with glioblastomas is less than 2 years.1 Gliomas are diffuse and infiltrating with no clear border between normal brain and tumor. Surgical resection is almost always followed by regrowth of tumor cells residing in adjacent regions of normal brain. New therapies are needed that specifically target tumor cells, especially those cells that have escaped the main tumor mass. Although oncolytic viral therapy appears promising,2 it is limited by relatively short survival of viral vectors caused by immune rejection and by their difficulty in reaching glioblastoma cells infiltrating the brain parenchyma.
Neural precursor cells (NPCs) have the ability to migrate widely throughout diseased and aged brain3, 4 and to differentiate into neurons, glia, and oligodendroglia.5 NPCs also can be used to deliver therapeutic proteins, particularly those with selective antineoplastic effects. One such protein that has been used in this context is tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)6 which selectively induces apoptosis in transformed cells7–9 and compared with other proapoptotic proteins, such as FasL, has little toxic side effects on normal cells.10 The presence of death domain containing TRAIL receptors in transformed cells such as gliomas makes them susceptible to TRAIL-mediated apoptosis.11, 12 In contrast, the absence of such receptors in normal cells such as NPCs makes them resistant to TRAIL-mediated apoptosis, thus allowing them to be used as therapeutic vehicles to deliver TRAIL.
We have recently engineered a secretable form of TRAIL (S-TRAIL) and shown that it has an enhanced apoptosis-inducing and bystander effect for tumor cells.13 To extend the release time of S-TRAIL and deliver it directly to invasive tumor cells, we evaluated NPCs expressing S-TRAIL as a unique delivery modality for brain tumor therapy. Quantitative evaluation was facilitated by engineering cell lines with luminescent and fluorescent transgenes and by the use of dual bioluminescence imaging to track growth of highly malignant gliomas, Gli36ΔEGFR, and migration of NPCs. Results demonstrate that intracranially implanted S-TRAIL–secreting NPCs have significant antitumor effects.
Materials and Methods
Generation of Tumor Necrosis Factor–Related Apoptosis–Inducing Ligand Amplicons
The cDNA sequence encoding amino acids 114 to 281 TRAIL fused to a cDNA fragment encoding a leucine zipper and the extracellular domain of the hFlt3 ligand were cloned into the multiple cloning site (MCS) of the pKSR2–Herpes simplex virus type 1 amplicon resulting in the S-TRAIL construct.13 Similarly the cDNA fragment encoding Renilla luciferase (Rluc) was cloned into the MCS of the pHGCX amplicon resulting in the Rluc construct.6
Cell Lines and Cell Culture
NPC-FL is a vmyc immortalized mouse neuroprogenitor cell line (NPC line) derived from the C17.2 cell line14 which stably expresses β-galactosidase (β-gal3) and firefly luciferase (Fluc).15 NPC-FLs were cotransfected with the S-TRAIL amplicon plasmid and pcDNA3.1 Hyg (−) (Invitrogen, Carlsbad, CA). Resulting stable S-TRAIL–positive clones were selected using 200μg/ml hygromycin in the growth medium, isolated, and checked for S-TRAIL secretion in the medium by ELISA (described below). To create a glioma cell line expressing Rluc, we stably cotransfected highly malignant human primary glioma cells, Gli36ΔEGFR,16, 17 with the Rluc amplicon6 and pcDNA3.1 Hyg (−) (Invitrogen) as described above. The clone showing the highest Rluc activity (see luciferase activity section below), Gli36-RL cell line (also green fluorescent protein (GFP)-positive), was used for further studies.
Cell Viability Assay and ELISA
Gli36ΔEGFR cells and NPC-FLs were plated in 96-well Primaria plates (Falcon, Bedford, MA) at a concentration of 1.5 × 104 cells/well and 3 × 103 cells/well, respectively. Twenty-four hours later, cells were incubated in a medium containing 0 to 240ng/ml of recombinant TRAIL (rTRAIL; PeproTech, Rocky Hill, NJ). Forty-eight hours after TRAIL addition, cell viability was assessed using 10μl WST (Tetrazolium salt; Roche, Indianapolis, IN) in a final volume of 100μl growth medium. After 4 hours of incubation, plates were read at 450nm using a Vmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA). TRAIL concentration in the conditioned culture medium from NPC-FLs or NPC-FL-sTRAILs was measured by ELISA with the TRAIL Immunoassay Kit (Biosource International, Camarillo, CA) according to manufacturer's protocol using recombinant human (h) TRAIL expressed in Escherichia coli as a standard.
To assess transgene expression and cell type, we fixed NPC-FL-sTRAILs and permeabilized and incubated them with: (1) a rabbit anti–firefly luciferase antibody (1:100, Molecular Probes, Eugene, OR); (2) a rabbit anti–β-gal antibody (1;100; Research Products International, Mount Prospect, IL); or (3) a antinestin monoclonal antibody (MAb) (1:200, Chemicon International, Temecula, CA) for 1 hour at 37°C. Cells then were washed and incubated with either goat anti–rabbit Alexa dye 488nm (1, 2) or goat anti–mouse Alexa dye 496nm (3) conjugated secondary antibodies (Molecular Probes, Eugene, OR) for 1 hour, then washed, mounted, and examined microscopically.
To assess apoptotic effects of S-TRAIL secreted from NPCs on glioma cells, we grew either 1 × 106 NPC-FLs or NPC-FL-sTRAILs in a total volume of 1ml, and 72 hours later 400μl of either of the growth medium was added to the cultures of 2 × 105 Gli36ΔEGFR cells. After 48 hours, cells were fixed and immunostained using a rabbit anti–caspase-3 primary antibody (Cell Signaling, Beverly, MA) and a goat anti–rabbit Alexa dye 488nm-conjugated secondary antibody, as described above.
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Western blotting
Gli36ΔEGFR, NPC-FL, or NPC-FL-sTRAIL cells were lysed, and proteins were denatured, resolved by SDS-PAGE, transferred to membranes, and blocked, as described previously13 using rabbit anti–TRAIL-R1 (1:500; AXXORA, LLC, San Diego, CA) or goat anti–TRAIL-R2 (1:500; Alexis Biochemicals, San Diego, CA) antibodies.
Athymic nude mice (nu/nu; 6–7 weeks old; Charles River Laboratories, Wilmington, MA) were stereotactically implanted with Gli36-RL glioblastoma cells and NPC-FL or NPC-FL-sTRAILs. Two to 3 days after glioma cell implantations, mice were imaged for Rluc activity before their distribution into various experimental groups as described below. All animal protocols were approved by an institutional review board. Three sets of experiments were performed. (1) For studying migration of NPC-FL-sTRAILs toward Gli36-RL gliomas, Gli36-RL cells (0.7 × 106 in 4μl phosphate-buffered saline [PBS]) were implanted stereotactically into the right frontal lobe of nude mice (n =20) and 2 days later, NPC-FL-sTRAIL (n =8) or NPC-FLs (n =8) (1 ×106 in 4μl PBS) or saline (n =4) were injected into the left frontal lobe of tumor-bearing mice. NPC-FL-sTRAILs were also implanted into the left frontal lobe of non–tumor-bearing mice (n =4). (2) For studying the effect of S-TRAIL secreted by NPCs within the tumors 0.25 × 106 Gli36-RL cells (in 4μl PBS; n =5) or a mix of 0.25 × 106 Gli36-RL cells with 0.75 ×106 NPC-FL-sTRAILs (in 5μl PBS; n =5) or NPC-FLs (in 5μl PBS; n =5) were implanted stereotactically into the right frontal lobe of nude mice. (3) To determine the influence of S-TRAIL secreted by NPC-FL-sTRAILs implanted in the close vicinity of the gliomas, we implanted Gli36-RL cells (0.25 ×106 in 4μl PBS) into the right frontal lobe of nude mice (n =20), and 3 days later NPC-FL-sTRAILs or NPC-FLs (0.75 × 106 in 4μl PBS) were stereotactically implanted into the right frontal lobe.
In Vivo Photonflux Imaging
Mice were imaged for Rluc activity by injecting coelenterazine (100μg/animal in 150μl saline) intravenously via the tail vein, and 5 minutes later photon counts were recorded for 5 minutes using a cryogenically cooled high efficiency charge-coupled device camera system (Roper Scientific, Trenton, NJ). For Fluc imaging, mice were given intraperitoneal injection of D-luciferin (4.5mg/animal in 150μl saline), and photon counts were recorded 10 minutes after D-luciferin administration. Postprocessing and visualization were performed as described previously.6 Mice were imaged every 5 to 7 days for Fluc and or Rluc activity.
Immediately after the last imaging session, mice were sacrificed, and brains were immersed in Tissue Freezing Medium (Triangle Biomedical Sciences, Durham, NC) on dry ice and 30μm coronal brain sections were cut. For X-gal staining, brain sections on slides were incubated overnight in X-gal solution. After washing slides were counterstained with eosin, dried, and mounted for microscopy. Parallel sections also were visualized for GFP fluorescence on a fluorescence microscope (Axiovert, Carl Zeiss, Ag, Germany). Brain sections from NPC-Fl-sTRAIL cell implanted mice were also stained with caspase-3 antibody (Cell Signaling Technology, Beverly, MA) according to manufacturer's protocol and counterstained with eosin.
To verify whether NPC-FLs could serve as cellular vehicles to produce a secretable form of TRAIL, we first evaluated the expression of death domain–containing TRAIL binding receptors, TRAIL-R1 and TRAIL-R2 by western blotting of both NPC-FL and Gli36ΔEGFR cell lysates. Immunoreactive proteins of expected sizes were present in the glioma cells and absent in the NPC-FLs (Fig 1A). To establish that S-TRAIL was lethal to tumor cells and had no effect on NPC viability, we conducted dose–response curves using rTRAIL. As predicted, there was no effect of TRAIL on NPC-FL viability, whereas Gli36ΔEGFR cell viability was reduced to 50% at 40ng/ml and to 20% at 240ng/ml rTRAIL (see Fig 1B). These results confirmed that these NPCs do not express death domain–bearing TRAIL receptors and therefore can be engineered to stably express S-TRAIL. Furthermore, Gli36ΔEGFR glioma cells express these receptors and are indeed susceptible to TRAIL.
To convert NPC-FLs to therapeutic vehicles, we stably transfected them with an expression cassette for S-TRAIL incorporated into an Herpes simplex virus amplicon. Quantitation of TRAIL in the cell supernatant confirmed secretion of 140ng/106 cells/24 hours by NPC-FL-sTRAILs with no significant amounts produced by control cells, NPC-FLs, or Gli36ΔEGFR cells (Fig 2A). To verify that NPC-FL-sTRAILs retained the characteristics of NPC-FLs, we immunostained NPC-FL-sTRAILs with antibodies against nestin, β-gal, and Fluc (Fig 2B). These experiments confirmed the retention of the NPC phenotype and exogenous genes expression of its parental cell line. Furthermore, Gli36ΔEGFR cells exposed to conditioned medium from NPC-FL-sTRAILs showed activation of caspase-3 (see Fig 2D), whereas no such activation occurred with the medium from NPC-FLs (see Fig 2C). These results show that TRAIL secreted by NPCs is active and can induce apoptosis in glioma cells in culture.
We created a glioma cell line (derived from Gli36ΔEGFR), stably expressing GFP and Rluc, termed Gli36-RL. Both Gli36-RL cells and NPC-FL-sTRAILs were implanted in the brain parenchyma, and animals were administered coelenterazine or D-luciferin, respectively. The bioluminescence signal generated by both cell types correlated linearly with cell number within the ranges tested (Fig 3A, B), thus allowing quantification of glioma cells, as well as NPC-FL-sTRAIL in mice brains. To determine if dual reporter luciferase imaging could be performed to monitor glioma growth and NPC migration, we performed dual substrate experiments. Gli36-RL cells were implanted into the right frontal lobe, and 3 days later NPC-FL-sTRAILs into the left frontal lobe of the same mice (Fig 4A). The glioma growth in mice was monitored every 5 to 7 days for up to 3 weeks (see Fig 4B–D), and the migration of NPC-FL-sTRAILs toward these glioma was monitored in parallel in the same mice by imaging Fluc activity (see Fig 4E–G). Tumor volumes increased exponentially and NPCs migrated across the corpus collosum into the tumor by day 21 (see Fig 4G), as correlated by GFP fluorescence of tumor cells (see Fig 4H) and X-gal staining of NPCs (see Fig 4I). To check the behavior of NPC-FL-sTRAILs within the brains of non–tumor-bearing mice, we implanted NPC-FL-sTRAILs into the left hemisphere of mice and imaged for Fluc activity over 4 weeks. NPC-FL-sTRAILs did not migrate but proliferated somewhat within 1 month after their implantation (data not shown; see also Tang and colleagues15). Immunohistochemistry with caspase-3 antibodies in these mice showed no apoptotic cells in the brain parenchyma in the region of these S-TRAIL–releasing cells 3 weeks after implantation (see Fig 4J). These observations indicate that TRAIL-secreting NPCs migrate selectively toward gliomas and that both tumor volumes and NPC migration can be monitored in the brain of the same living animal by dual luciferase/substrate imaging. Furthermore, S-TRAIL does not appear to have any harmful effect on normal brain tissues.
Two approaches to NPC brain tumor therapy were addressed. In the first approach, NPC-FL-sTRAILs were implanted in close vicinity to established gliomas (Fig 5A), and, in the second approach, NPC-FL-sTRAILs were mixed with glioma cells and implanted into mouse brains. In the former approach, glioma growth was reduced after a period of 3 weeks (Fig 5B, G), as compared with the control NPC-FL–implanted animals (Fig 5C, G), where the tumor grew more rapidly. X-gal staining on the tissue sections confirmed the presence of NPC-FL-sTRAILs in the tumors (see Fig 5F). These results show that S-TRAIL–secreting NPCs when implanted in the close vicinity of an established glioma migrate into the glioma and reduce the rate of tumor growth.
In the second approach, Gli36-RL cells alone, or a mix of Gli36-RL cells and NPC-FL-sTRAILs or NPC-FLs, were implanted into the left frontal lobe of mice. Nine and 16 days after cell implantation, tumor growth was reduced considerably in Gli36-RL/NPC-FL-sTRAIL–implanted animals (Fig 6A, B, G) as compared with the control Gli36-RL/NPC-FL–implanted mice (see Fig 6C, D, G). Fluc imaging and X-gal staining confirmed the presence of NPC-FL-sTRAILs (see Fig 6E, H) and NPC-FLs (see Fig 6F) in the tumors. Quantitative analysis showed a significant reduction (p < 0.05) in glioma-bearing animals 2 weeks after implantation of S-TRAIL–producing NPCs as compared with the controls (see Fig 6G). These results show that S-TRAIL–secreting NPC are effective in significantly reducing glioma growth in the brains of experimental animals.
The ability of TRAIL to selectively induce apoptosis of transformed cells makes it an attractive therapeutic agent for cancer.18 In this study, we have engineered NPCs to express S-TRAIL and have shown that these cells migrate into gliomas in mouse brain and result in considerable reduction in growth of highly malignant gliomas. Furthermore, we have applied real-time imaging techniques that allow us to precisely monitor the magnitude and timing of migration of NPCs and subsequent changes in glioma volumes over time.
In this study, NPCs and normal brain were found to be immune to the toxic effects of TRAIL, whereas glioblastoma cells were highly sensitive. There are at least two possible mechanisms by which normal cells and not the neoplastic cells are resistant to TRAIL-mediated apoptosis.19 TRAIL-resistant cells do not express TRAIL-R1 or TRAIL-R2 death receptors but rather express either defective or decoy TRAIL (truncated death domain) receptors that fail to trigger an intracellular apoptotic cascade. In contrast, the presence of death domain-containing TRAIL receptors in neoplastic cells makes them susceptible to TRAIL-mediated apoptosis.11 Resistance in normal cells also can be mediated by inhibition of cytoplasmic caspase-8 cleavage by the apoptosis-inhibitory proteins, cFLIP, and PED/PEA-15, mechanisms that are frequently lacking in TRAIL-sensitive neoplastic cells.20
A critical issue in treating brain tumors is delivery of therapeutic molecules in a way that they can access the tumors, which for drugs means crossing the brain–blood barrier and reaching invasive tumor cells. Gene therapy offers the possibility of direct delivery of therapeutic agents or prodrug activating enzymes within the brain.21 This can be done using replication defective and oncolytic vectors for on-site delivery with a broad range of virus vectors. However, none of these vectors target invasive tumor cells. The sensitivity of gliomas to TRAIL has been demonstrated by intratumoral delivery of TRAIL using viruses.22–25 Virus vectors allow extended delivery of TRAIL on site and at a sufficiently high concentration to be therapeutic.8 Although the tumoricidal effect of TRAIL is evident from these studies, they are limited, in part, by their ineffectiveness against tumor microsatellites because of the limited range of delivery of TRAIL in the brain. Recent studies have used secretable forms of TRAIL and migratory cells to extend the range of delivery.13, 26, 27
It has been shown that NPCs injected contralateral to an established glioma migrate across the brain and become interspersed within the tumor tissue, as well as being present at the tumor periphery and in association with microsatellite foci occurring some distance from the main tumor mass.15, 26 Thus, NPCs are attractive candidates for delivering therapeutic proteins and can be used to target both the primary tumor mass and invasive tumor foci in experimental animal models. In this study, when implanted contralaterally, NPC-FL-sTRAILs expressing Fluc and S-TRAIL migrated across the brain into an established glioma but were not very effective in reducing glioma volumes. This may be attributed to the fact that the glioma volumes had increased substantially over the week after implantation, and, by the time the NPC-FL-sTRAILs reached the gliomas, the tumor volumes were too large to be influenced by TRAIL. The Gli36ΔEGFR glioma cell line used for these studies is highly malignant and grows much faster as compared with the other glioma lines, such as U87. When the same number of NPC-FL-sTRAILs were implanted into or close to tumors of smaller size, even the highly malignant Gli36ΔEGFR glioma volumes were reduced considerably as compared with the untreated or NPC-FL–treated tumors.
The ability to image both the migration of NPCs and the changes in tumor volumes in vivo is critical in assessing the efficacy of gene delivery and in quantitating therapeutic effects. Migration properties of NPCs producing therapeutic proteins have been established by immunostaining for β-gal,26 but this involved the use of sequential animal death to follow the cell migration and to evaluate tumor growth and invasiveness. In the in vivo results reported in this study, the bioluminescence imaging allowed the real-time imaging of these events in individual mice over successive time intervals. Fluc imaging allowed tracking of NPC migration and their presence in the tumors, whereas the influence of S-TRAIL on glioma growth was followed by Rluc imaging.
In conclusion, we have engineered an NPC line such that it expresses both Fluc and a form of S-TRAIL with an enhanced apoptosis-inducing ability and created a system that allows real-time bioluminescence imaging of both the migration of NPCs and changes in glioma volumes in living animals. Using this study as a template, advances can be made in the way therapeutic agents, such as TRAIL, and delivery vehicles, such as NPCs are used for brain tumor therapy. Although the model NPC line used here is of mouse origin with consequent problems28 and not of clinical grade, in a clinical situation, one would envision neurosurgical removal of the main tumor mass with infusion of S-TRAIL–releasing human NPCs to migrate to invasive tumor foci.
This work was supported by the Goldhirsh Medical Foundation (K.S., X.O.B.), American Brain Tumor Association (K.S.), the NIH (National Cancer Institute, NCI CA69246, X.O.B., NCI CA86355 R.W., X.O.B., NCICA92782, X.O.B.) and the Center for Molecular Imaging Research (CMIR, NCIP50, NCIR24).
We thank Dr E. Snyder for providing us with the mouse C17.2 NPCs and S. Mcdavitt for skilled editorial assistance.