A Novel Theranostic Strategy for MMP-14-Expressing Glioblastomas Impacts Survival

Abstract

Glioblastoma (GBM) has a dismal prognosis. Evidence from preclinical tumor models and human trials indicates the role of GBM-initiating cells (GIC) in GBM drug resistance. Here, we propose a new treatment option with tumor enzyme-activatable, combined therapeutic and diagnostic (theranostic) nanoparticles, which caused specific toxicity against GBM tumor cells and GICs. The theranostic cross-linked iron oxide nanoparticles (CLIO) were conjugated to a highly potent vascular disrupting agent (ICT) and secured with a matrix-metalloproteinase (MMP-14) cleavable peptide. Treatment with CLIO-ICT disrupted tumor vasculature of MMP-14-expressing GBM, induced GIC apoptosis, and significantly impaired tumor growth. In addition, the iron core of CLIO-ICT enabled in vivo drug tracking with MR imaging. Treatment with CLIO-ICT plus temozolomide achieved tumor remission and significantly increased survival of human GBM-bearing mice by more than 2-fold compared with treatment with temozolomide alone. Thus, we present a novel therapeutic strategy with significant impact on survival and great potential for clinical translation. Mol Cancer Ther; 16(9); 1909-21. ©2017 AACR.

Figure 1. CLIO-ICTs inhibit GBM and GIC survival in vitro

(a) Schematic demonstration of CLIO-ICT activation in presence of tumor enzyme MMP-14, to release the active drug, azademethylcolchicine. Azademethylcolchicine targets tubulin to induce apoptosis in tumor cells. (b) Representative T₂ weighted MR images of CLIO-ICT and ferumoxytol at different dilutions. T₂ MSME sequences were used to generate R₂ relaxivites. R₂ relaxivities of CLIO-ICT (blue line) and ferumoxytol (red line). (c) Graphical representation of MMP-14 expression in different GBM cells lines. MMP-14 expression was measured with q-PCR assay, GAPDH served as endogenous control. (d) Viability analysis of GBMs treated with CLIO-ICT (10nM), ICT (10nM), CLIO (0.01mM) and PBS. GBMs were treated for 96 hrs and viability was assayed using MTS assay. (e) Graph shows fold change for cleaved caspase-3 in control and treated GBM cells. Cleaved caspase-3 fluorescence signals were detected by SensoLyte Homogeneous AMC Caspase-3/7 assay kit and the fold change was represented by the ratio: fluorescence signals in treated/fluorescence signals in control. (f) Annexin-V/PI apoptosis staining in control and treated GBM39 cells (Left panel). Right panel depicts quantification for percentage apoptosis in A172, U87 and pcGBM39 cells after 48 hrs of treatment. (g) Immunofluorescence for α-tubulin in control and treated U87 cells. Upper panel shows confocal images and lower panel depicts quantification of tubulin signals scale bar 10μm. (h) Flow cytometry analysis of GIC markers (CD133, CD15 and CD49F), Annexin V/DAPI staining in control and treated pcGBM39 cells. CLIO-ICT-treated pcGBM39 were subjected to flow cytometric staining for GIC surface markers (upper panel) and apoptosis markers (lower panel). (i) Graph shows percentages of CD133⁺, CD15⁺ and CD49F⁺ GICs (left) and percentage apoptosis (right) in gated CD133⁺, CD15⁺ and CD49F⁺ GICs from control and treated pcGBM39 cells. Results are represented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.005, one-way ANOVA.

Figure 2. CLIO-ICTs retard GBM growth in vivo

(a) Schematic demonstration of CLIO-ICT-mediated anti-GBM effect. In presence of tumor enzyme MMP-14, CLIO-ICT is cleaved to release the active vasculature-disrupting agent, azademethylcolchicine. Activated CLIO-ICT targets tumor vasculature and induces apoptosis in GICs and GBMs, thereby inhibiting GBM growth and improving survival outcomes. (b) Schematic representation of experimental design. Tumors were initiated with orthotopic injections of primary human GBM samples (pcGBM39 cells and pcGBM2 cells that express luciferase and GFP-luciferase construct respectively) into the striatum of NSG mice. Treatment was initiated once the tumors were detected and bioluminescent analyses were performed during and after treatment. (c) Bioluminescent in vivo images of tumors in mice treated with CLIO-ICT (0.5 mmol Fe/kg and 80mg/kg of ICT), ICT (80mg/kg of ICT), CLIO (0.5 mmol Fe/kg) or vehicle. (d) Quantification of the bioluminescent signals. Fold change in total flux represents the ratio: total flux after treatment/total flux before treatment. *P = 0.0002 and *P = 0.0003 for pcGBM39 (n=8) and pcGBM2 (n=6), respectively, one-way ANOVA. (e) Kaplan–Meyer survival curves of control and treated mice demonstrate a significant survival benefit of CLIO-ICT as compared to vehicle, log-rank Mantel–Cox test.

Figure 3. CLIO-ICTs are delivered to GBM tumors and induce tumor apoptosis

Tumors were initiated with orthotopic injections of pcGBM39 tumors and mice were treated with CLIO-ICT (0.5 mmol Fe/kg and 80mg/kg of ICT), ICT (80mg/kg of ICT), CLIO (0.5 mmol Fe/kg) or vehicle. (a) Upper panel: Representative H&E staining for brain coronal sections from control and treated animals. White arrows indicate tumor. Objective 4X. Scale bar represents 100 μm. Middle panel: Prussian blue iron staining for control and treated tumors. Objective 4X. Scale bar represent 100μm. Lower panel: Prussian blue at higher magnification 10× for boxed regions in middle panel. Scale bar represent 40 μm (b) Representative immunofluorescence confocal images for cleaved caspase-3 in control and treated pcGBM39 tumors. Arrows indicate cleaved caspase-3 positive cells. Nuclei counterstained with DAPI. Scale bar represents 75 μm. Graph shows quantification for FITC and cleaved caspase-3 staining in control and treated pcGBM39 tumors. Results are represented as mean ± SD from three independent experiments. *P < 0.05, **P < 0.005, one-way ANOVA. (c) Representative T₂ weighted MR images of mice brain. T₂ FSE sequences were used to capture coronal T₂ weighted images. Nanoparticle and theranostic nanoparticle delivery is demonstrated by T₂ darkening or negative enhancement in CLIO and CLIO-ICT treated animals respectively. On day 14 tumor periphery is marked by dotted yellow line. (d) Quantification of T₂ darkening. T₂ MSME sequences were used to generate T₂ maps, Osirix software was used to calculate T₂ values. CLIO and CLIO-ICT-treated tumors demonstrated shorter T₂ values corresponding to T₂ darkening or negative enhancement. (e) Quantification of tumor volumes before and after treatment. T₂ weighted MR scans were used to calculate tumor volumes using Osirix software. *P < 0.05, **P < 0.005, n=6, one-way ANOVA.

Figure 4. CLIO-ICTs target GBM vasculature and GICs in vivo

Tumors were established with orthotopic injections of pcGBM39 cells. Mice were treated with CLIO-ICT (0.5 mmol Fe/kg and 80mg/kg of ICT), ICT (80mg/kg of ICT), CLIO (0.5 mmol Fe/kg) or vehicle. Representative immunofluorescent confocal images depicting CD31, CD133, CD15 and Nestin staining in pcGBM39 tumors. CD31 is used to outline tumor vasculature. CD133, CD15 and nestin are used to mark GICs. Signal intensity for CD31, CD133, CD15 and nestin in control and treated tumors have been quantified and represented graphically. Scale bar represent 20 μm (CD31) and 10 μm (CD133, CD15 and nestin). Results are representative of three independent experiments. *P < 0.05, **P < 0.005, one-way ANOVA.

Figure 5. CLIO-ICTs induce GIC apoptosis in vivo

Tumors were initiated with orthotopic injections of pcGBM2 cells. Mice were treated with CLIO-ICT (0.5 mmol Fe/kg and 80mg/kg of ICT), ICT (80mg/kg of ICT), CLIO (0.5 mmol Fe/kg) or vehicle. Flow cytometric analysis of GIC markers (CD15), Annexin V and DAPI in (a) vehicle and (b) CLIO-ICT-treated pcGBM2 tumors. GFP positive pcGBM2 tumors were gated and analyzed for percentage apoptosis in both (c) pcGBM2 cells and (d) GICs from pcGBM2. P < 0.05, **P < 0.005, n=6, one-way ANOVA.

Figure 6. CLIO-ICTs increase the anti-tumor efficacy of temozolomide (TMZ) in vivo

(a) In vitro analysis of TMZ chemosensitivity by cell viability assay. Panel of GBM cells were exposed to increasing doses of TMZ (0-500 μM) for 72hrs and cell viability was calculated by MTS assay. For in vivo experiments, tumors were initiated with orthotopic injections of pcGBM39 cells (b-f) and pcGBM2 cells (g-i). Mice were treated with TMZ alone (200mg/kg) or in combination with CLIO-ICT (0.5 mmol Fe/kg and 80mg/kg of ICT) and ICT (80mg/kg of ICT), CLIO (0.5 mmol Fe/kg) or vehicle. (b) Representative T₂ weighted MR images of mice brain. T₂ FSE sequences were used to capture coronal T₂ weighted images. Yellow dotted line represents tumor periphery. (c) Quantification of tumor volumes before and after treatment. T₂ weighted MR scans were used to calculate tumor volumes using Osirix software. Bioluminescent in vivo images of tumors in control and treated mice in pcGBM39 (d) and pcGBM2 (g) tumor models. (e and h) Quantification of the bioluminescent signals. Fold change in total flux represents the ratio: total flux after treatment/total flux before treatment. *P < 0.05, respectively, n = 6 (pcGBM39) and n = 6 (pcGBM2), one-way ANOVA. (f and i) Kaplan–Meyer survival curves of control and treated mice (n=6, pcGBM39; n=6, pcGBM2) demonstrate a significant survival benefit of CLIO-ICT and ICT in combination with TMZ as compared to vehicle, log-rank Mantel–Cox test. *P < 0.05, **P < 0.005, n=6, one-way ANOVA.