Claudio E. Tatsui, Frederick F. Lang, Joy Gumin, Dima Suki, Naoki Shinojima and Laurence D. Rhines
There is currently no reproducible animal model of human spinal metastasis that allows for laboratory study of the human disease. Consequently, the authors sought to develop an orthotopic model of spinal metastasis by using a human lung cancer cell line, and to correlate neurological decline with tumor growth.
To establish a model of spinal metastasis, the authors used a transperitoneal surgical approach to implant PC-14 lung tumors into the L-3 vertebral body of nude mice via a drill hole. In 24 animals, motor function was scored daily by using the validated semiquantitative Basso-Beattie-Bresnahan (BBB) scale. A second group of 26 animals (6 or 7 per time point) were sacrificed at specific times, and the spines were removed, sectioned, and stained. Canal compromise was analyzed quantitatively by determining the ratio of the area of the neural elements to the area of the spinal canal on histological sections (neural/canal ratio). Correlations between BBB score and histological evaluation of tumor growth were assessed.
Lung cancer xenografts grew in all animals undergoing functional evaluation (24 mice) according to a reliable and reproducible time course, with paraplegia occurring at a median interval of 30 days following tumor implantation (95% CI 28.1–31.9 days). Importantly, the analysis defined 4 key milestones based on components of the BBB score; these were observed in all animals, were consistent, and correlated with histological progression of tumor. From Days 1 to 14, the mean BBB score declined from 21 to 19. The animals progressed from normal walking with the tail up to walking with the tail constantly touching the ground (milestone 1). The median time to tail dragging was 12 days (95% CI 10.8–13.2). Histological studies on Day 14 demonstrated that tumor had progressed from partial to complete VB infiltration, with initial compression of the neural elements and epidural tumor extension to adjacent levels (mean neural/canal ratio 0.32 ± 0.05, 7 mice). From Days 15 to 20/21 (left/right leg), the mean BBB score declined from 19 to 14. Animals showed gait deterioration, with the development of dorsal stepping (milestone 2). The median time to dorsal stepping was 21 days (95% CI 19.4–22.6) in the left hindlimb and 23 days (95% CI 20.6–25.4) in the right hindlimb. Histological studies on Day 21 demonstrated an increase in the severity of the neural element compression, with tumor extending to adjacent epidural and osseous levels (mean neural/canal ratio 0.19 ± 0.05, 6 mice). From Days 22 to 26/27 (left/right leg), the mean BBB score declined from 14 to 8. Animals had progressive difficulty ambulating, to the point where they showed only sweeping movements of the hindlimb (milestone 3). The median time to hindlimb sweeping was 26 days (95% CI 23.6–28.4) and 28 days (95% CI 27.1–28.9) in the left and right hindlimbs, respectively. Histological studies on Day 28 revealed progressive obliteration of the spinal canal (mean neural/canal ratio 0.09 ± 0.01, 7 mice). From Days 29 to 36, the animals progressed to paralysis (milestone 4). The median time to paralysis was 29 days (95% CI 27.6–30.4) and 30 days (95% CI 28.1–31.9) in the left and right hindlimbs, respectively.
The authors have developed an orthotopic murine model of human spinal metastasis in which neurological decline reproducibly correlates with severity of tumor progression. Although developed for lung cancer, this model can be expanded to study other types of metastatic or primary spinal tumors. Ultimately, this will allow testing of targeted therapies against specific tumor types.
Daniel K. Fahim, Claudio E. Tatsui, Dima Suki, Joy Gumin, Frederick F. Lang and Laurence D. Rhines
There is currently no reproducible animal model of human primary malignant bone tumors in the spine to permit laboratory investigation of the human disease. Therefore, the authors sought to adapt their previously developed orthotopic model of spinal metastasis to a model for primary malignant bone tumors of the spine.
A transperitoneal surgical approach was used to implant osteosarcoma (Krib-1) into the L-3 vertebral body of nude mice via a drill hole. Motor function was evaluated daily using the previously validated qualitative key milestones of tail dragging, dorsal stepping, hindlimb sweeping, and paralysis. A subset of these animals was euthanized upon reaching the various milestones, and the spines were removed, sectioned, and stained. The degree of spinal cord compression was correlated with the occurrence of milestones and assessed by a ratio between the neural elements divided by the area of the spinal canal. Another subset of animals received stably transfected Krib-1 cells with the luciferase gene, and bioluminescence was measured at 10, 20, and 30 days postimplantation.
Osteosarcoma xenografts grew in all animals according to a reliable and reproducible time course; the mean time for development of behavioral milestones was noted in relation to the day of implantation (Day 1). Tail dragging (Milestone 1) occurred on Day 19.06 (95% CI 16.11–22.01), dorsal stepping (Milestone 2) occurred on Day 28.78 (95% CI 26.79–30.77), hindlimb sweeping (Milestone 3) occurred on Day 35.61 (95% CI 32.9–38.32), and paralysis of the hindlimb (Milestone 4) occurred on Day 41.78 (95% CI 39.31–44.25). These clinically observed milestones correlated with increasing compression of the spinal cord on histological sections. The authors observed a progressive increase in the local bioluminescence (in photons/cm2/sec) of the implanted level over time with a mean of 2.17 (range 0.0–8.61) at Day 10, mean 4.68 (range 1.17–8.52) at Day 20, and mean 5.54 (range 1.22–9.99) at Day 30.
The authors have developed the first orthotopic murine model of a primary malignant bone tumor in the spine, in which neurological decline reproducibly correlates with tumor progression as evidenced by pathological confirmation and noninvasive bioluminescence measurements. Although developed for osteosarcoma, this model can be expanded to study other types of primary malignant bone tumors in the spine. This model will potentially allow animal testing of targeted therapies against specific primary malignant tumor types.
Jonathan G. Thomas, Brittany C. Parker Kerrigan, Anwar Hossain, Joy Gumin, Naoki Shinojima, Felix Nwajei, Ravesanker Ezhilarasan, Patrice Love, Erik P. Sulman and Frederick F. Lang
Mesenchymal stem cells (MSCs) have been shown to localize to gliomas after intravascular delivery. Because these cells home to areas of tissue injury, the authors hypothesized that the administration of ionizing radiation (IR) to tumor would enhance the tropism of MSCs to gliomas. Additionally, they sought to identify which radiation-induced factors might attract MSCs.
To assess the effect of IR on MSC migration in vitro, transwell assays using conditioned medium (CM) from an irradiated commercially available glioma cell line (U87) and from irradiated patient-derived glioma stem-like cells (GSCs; GSC7-2 and GSC11) were employed. For in vivo testing, green fluorescent protein (GFP)-labeled MSCs were injected into the carotid artery of nude mice harboring orthotopic U87, GSC7-2, or GSC17 xenografts that were treated with either 0 or 10 Gy of IR, and brain sections were quantitatively analyzed by immunofluorescence for GFP-positive cells. These GSCs were used because GSC7-2 is a weak attractor of MSCs at baseline, whereas GSC17 is a strong attractor. To determine the factors implicated in IR-induced tropism, CM from irradiated GSC7-2 and from GSC11 was assayed with a cytokine array and quantitative ELISA.
Transwell migration assays revealed statistically significant enhanced MSC migration to CM from irradiated U87, GSC7-2, and GSC11 compared with nonirradiated controls and in a dose-dependent manner. After their intravascular delivery into nude mice harboring orthotopic gliomas, MSCs engrafted more successfully in irradiated U87 (p = 0.036), compared with nonirradiated controls. IR also significantly increased the tropism of MSCs to GSC7-2 xenografts (p = 0.043), which are known to attract MSCs only poorly at baseline (weak-attractor GSCs). Ionizing radiation also increased the engraftment of MSCs in strong-attractor GSC17 xenografts, but these increases did not reach statistical significance. The chemokine CCL2 was released by GSC7-2 and GSC11 after irradiation in a dose-dependent manner and mediated in vitro transwell migration of MSCs. Immunohistochemistry revealed increased CCL2 in irradiated GSC7-2 gliomas near the site of MSC engraftment.
Administering IR to gliomas enhances MSC localization, particularly in GSCs that attract MSCs poorly at baseline. The chemokine CCL2 appears to play a crucial role in the IR-induced tropism of MSCs to gliomas.
Visish M. Srinivasan, Joy Gumin, Kevin M. Camstra, Stephen R. Chen, Jeremiah N. Johnson, Yuzaburo Shimizu, Brittany C. Parker Kerrigan, Elizabeth J. Shpall, Frederick F. Lang and Peter Kan
Bone marrow–derived human mesenchymal stem cells (BM-hMSCs) have been used in clinical trials for the treatment of several neurological disorders. MSCs have been explored as a delivery modality for targeted viral therapeutic agents in the treatment of intracranial pathologies. Delta-24-RGD, a tumor-selective oncolytic adenovirus designed to target malignant glioma cells, has been shown to be effective in animal models and in a recent clinical trial. However, the most efficient strategy for delivering oncolytic therapies remains unclear. BM-hMSCs have been shown to home toward glioma xenografts after intracarotid delivery. The feasibility of selective intraarterial infusion of BM-hMSCs loaded with Delta-24-RGD (BM-hMSC-Delta-24) to deliver the virus to the tumor is being investigated. To evaluate the feasibility of endovascular intraarterial delivery, the authors tested in vitro the compatibility of BM-hMSC-Delta-24 with a variety of commercially available, clinically common microcatheters.
BM-hMSCs were cultured, transfected with Delta-24-RGD, and resuspended in 1% human serum albumin. The solution was then injected via 4 common neuroendovascular microcatheters of different inner diameters (Marathon, Echelon-14, Marksman, and SL-10). Cell count and viability after injection through the microcatheters were assessed, including tests of injection velocity and catheter configuration. Transwell assays were performed with the injected cells to test the efficacy of BM-hMSC-Delta-24 activity against U87 glioma cells. BM-hMSC-Delta-24 compatibility was also tested with common neuroendovascular medications: Omnipaque, verapamil, and heparin.
The preinfusion BM-hMSC-Delta-24 cell count was 1.2 × 105 cells/ml, with 98.7% viability. There was no significant difference in postinfusion cell count or viability for any of the catheters. Increasing the injection velocity from 1.0 ml/min to 73.2 ml/min, or modifying the catheter shape from straight to tortuous, did not significantly reduce cell count or viability. Cell count and viability remained stable for up to 5 hours when the cell solution was stored on ice. Mixing BM-hMSC-Delta-24 with clinical concentrations of Omnipaque, verapamil, and heparin prior to infusion did not alter cell count or viability. Transwell experiments demonstrated that the antiglioma activity of BM-hMSC-Delta-24 was maintained after infusion.
BM-hMSC-Delta-24 is compatible with a wide variety of microcatheters and medications commonly used in neuroendovascular therapy. Stem cell viability and viral agent activity do not appear to be affected by catheter configuration or injection velocity. Commercially available microcatheters can be used to deliver stem cell neurotherapeutics via intraarterial routes.
Markus M. Luedi, Sanjay K. Singh, Jennifer C. Mosley, Islam S. A. Hassan, Masumeh Hatami, Joy Gumin, Lukas Andereggen, Erik P. Sulman, Frederick F. Lang, Frank Stueber, Gregory N. Fuller, Rivka R. Colen and Pascal O. Zinn
Dexamethasone, a known regulator of mesenchymal programming in glioblastoma (GBM), is routinely used to manage edema in GBM patients. Dexamethasone also activates the expression of genes, such as CEBPB, in GBM stem cells (GSCs). However, the drug’s impact on invasion, proliferation, and angiogenesis in GBM remains unclear. To determine whether dexamethasone induces invasion, proliferation, and angiogenesis in GBM, the authors investigated the drug’s impact in vitro, in vivo, and in clinical information derived from The Cancer Genome Atlas (TCGA) cohort.
Expression profiles of patients from the TCGA cohort with mesenchymal GBM (n = 155) were compared with patients with proneural GBM by comparative marker selection. To obtain robust data, GSCs with IDH1 wild-type (GSC3) and with IDH1 mutant (GSC6) status were exposed to dexamethasone in vitro and in vivo and analyzed for invasion (Boyden chamber, human-specific nucleolin), proliferation (Ki-67), and angiogenesis (CD31). Ex vivo tumor cells from dexamethasone-treated and control mice were isolated by fluorescence activated cell sorting and profiled using Affymetrix chips for mRNA (HTA 2.0) and microRNAs (miRNA 4.0). A pathway analysis was performed to identify a dexamethasone-regulated gene signature, and its relationship with overall survival (OS) was assessed using Kaplan-Meier analysis in the entire GBM TCGA cohort (n = 520).
The mesenchymal subgroup, when compared with the proneural subgroup, had significant upregulation of a dexamethasone-regulated gene network, as well as canonical pathways of proliferation, invasion, and angiogenesis. Dexamethasone-treated GSC3 demonstrated a significant increase in invasion, both in vitro and in vivo, whereas GSC6 demonstrated a modest increase. Furthermore, dexamethasone treatment of both GSC3 and GSC6 lines resulted in significantly elevated cell proliferation and angiogenesis in vivo. Patients with mesenchymal GBM had significant upregulation of dexamethasone-regulated pathways when compared with patients with proneural GBM. A prognostic (p = 0.0007) 33-gene signature was derived from the ex vivo expression profile analyses and used to dichotomize the entire TCGA cohort by high (median OS 12.65 months) or low (median OS 14.91 months) dexamethasone signature.
The authors present evidence that furthers the understanding of the complex effects of dexamethasone on biological characteristics of GBM. The results suggest that the drug increases invasion, proliferation, and angiogenesis in human GSC-derived orthotopic tumors, potentially worsening GBM patients’ prognoses. The authors believe that careful investigation is needed to determine how to minimize these deleterious dexamethasone-associated side effects in GBM.