Prediction of the anti-glioma therapeutic effects of temozolomide through in vivo molecular imaging of MMP expression

Currently, there is no effective way to assess the therapeutic response of temozolomide (TMZ) for the glioma. In this study, the human U87MG-fLuc glioma animal models were set up and the antitumor efficacy of TMZ was evaluated using bioluminescence imaging (BLI) and MRI. Then, bioluminescence tomography (BLT) was reconstructed using an adaptive sparsity matching pursuit (ASMP) algorithm. Second, the expression level of the MMP-750 probe was examined with or without TMZ treatment using FMI. Third, the expression of MMP2 and MMP3 was specifically examined after treatment. The results showed that TMZ effectively inhibited glioma growth. The targeted imaging of MMP-750 was decreased during the treatment of glioma with TMZ. Moreover, the MMP2 and MMP3 expression was found to correlate with the inhibition effect of TMZ. Our study indicated that the therapeutic effects of TMZ can be effectively evaluated at an early stage using molecular imaging, and MMP targeting the fluorescence probe could be utilized for the prediction and assessment of the therapeutic effects of TMZ. © 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement OCIS codes: (170.3880) Medical and biological imaging; (110.6955) Tomographic imaging; (170.6280) Spectroscopy, fluorescence and luminescence; (170.4580) Optical diagnostics for medicine. References and links 1. R. Stupp, W. P. Mason, M. J. van den Bent, M. Weller, B. Fisher, M. J. Taphoorn, K. Belanger, A. A. Brandes, C. Marosi, U. Bogdahn, J. Curschmann, R. C. Janzer, S. K. Ludwin, T. Gorlia, A. Allgeier, D. Lacombe, J. G. Cairncross, E. Eisenhauer, and R. O. Mirimanoff, “Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma,” N. Engl. J. Med. 352(10), 987–996 (2005). 2. E. G. Van Meir, C. G. Hadjipanayis, A. D. 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Introduction
Malignant gliomas are one of the most intractable and fatal cancers due to their location, aggressive biological behavior and invasive growth [1,2].Although neuroimaging and chemotherapy have made rapid progress in modern times, it is difficult for cancer patients to recover completely [3].Glioma shows certain resistance to many chemotherapy agents (e.g., nitrosourea or the combination regimen procarbazine, lomustine and vincristine) [4].Even if the blood-brain barrier is broken down in glioma patients, most chemotherapeutic drugs do not effectively infiltrate the brain [5].
Temozolomide (TMZ) is a novel imidazotetrazinone methylating agent.TMZ is a small lipophilic molecule and it can effectively cross the blood-brain barrier [6,7].TMZ has shown a schedule-dependent antitumor activity in malignant gliomas [8][9][10][11][12].TMZ is commonly used to treat malignant brain tumors; currently there is no effective way for monitoring and evaluating TMZ treatment effects at the early stage.Medical imaging technologies, as computed x-ray tomography (CT), ultrasound (US) and magnetic resonance imaging (MRI), play an indispensable role in clinical practice.However, these imaging methods usually detect cancer in areas that are a centimeter or larger in diameter, at which point patients are difficult to cure.There are tremendous incentives to develop novel imaging technologies for the early detection of cancer.In comparison with traditional anatomical imaging methods, molecular imaging is unique in specificity and sensitivity.Molecular imaging has important clinical value in predicting the anti-tumor effect of drugs.It becomes an important imaging tool to diagnose diseases, evaluate therapeutic efficacies, and facilitate drug development and other biomedical applications as well [13][14][15][16][17][18].However, such twodimensional bioluminescence imaging is incapable to provide 3-dimensional information of internal features of interest, and does not reveal in-depth information.Bioluminescence tomography (BLT) reconstructs an internal bioluminescent source distribution from external optical measures which can be localized and quantified in 3D [19,20], and BLT allows the integration of molecular and physiological information with anatomical information.
Proteinases endow tumor cells with the ability to invade and metastasize to different tissue sites in vivo [21].Matrix metalloproteinases (MMPs), a family of calcium-dependent endopeptidases [22], have been identified as one of the major proteinase systems responsible for extracellular proteolysis.MMPs enable tumor cells to break through basement membranes and invade [23].MMPs promote tumor cell invasion by degrading extracellularmatrix proteins, and activating signal-transduction pathways [24].MMP activity in the tumor environment was dynamically assessed by using an MMPSense 750 imaging probe.MMPSense 750 was quenched with near-infrared fluorochromes positioned on a nonimmunogenic backbone previously designed as to sense proteinase activities directly in vivo in intact tumor environments [21].This probe is a metalloprotease substrate, optically silent in its inactive state and highly fluorescent following MMP-mediated activation [21,25].Our previous work indicates that MMP-750 can specifically target gliomas and fluorescence tomography (FMT) of MMP-750 provides 3D information for the spatial localization of gliomas in situ, and hence works as an ideal fluorescence probe for gliomas.
The expression level of some MMPs in cancer tissues is higher than that in normal tissues and the extent of expression is related to the tumor stage [26], invasiveness [27,28], metastasis [29] and angiogenesis [30].Moreover, it is reported that the expression of MMP family members are highly expressed in gliomas, and the expression levels are related to TMZ treatment [31][32][33][34].Hence, it is interesting to study whether targeted imaging of MMP-750 probes can predict antitumor activities of TMZ therapy.Moreover, it is known that MMPSense 750 can be activated by a series of MMPs, but which specific MMP members are functional for the regulation of TMZ treatment is still not known.
The aims of this work are to investigate the treatment effects of TMZ using BLT on both xenograft and orthotopic glioma mouse models; second, the FMI of gliomas using an MMP-750 probe was studied during TMZ treatment.Finally, the expression of MMPs specifically correlated with the treatment efficacy of TMZ was studied using western blot and immunohistochemistry (IHC) experiments.

Materials and reagents
A human U87MG-fLuc glioma cell line was obtained from the American type culture collection (ATCC).Culturing medium and fetal bovine serum (FBS) were purchased from HyClone (Thermo Scientific, USA).D-Luciferin was bought from Biotium (CA, Fremont, USA).Temozolomide (TMZ) was obtained from Schering-Plough Corporation (Kenilworth, NJ, USA).The TMZ solution (0.5 mg/µl) was prepared in dimethyl sulfoxide (DMSO).Before the injection, the stock drug solution was thawed, diluted in sterile saline to a final concentration of 0.005 mg/µl.MMP-750 was purchased from PerkinElmer (Waltham, MA, USA).The emission spectra peak was 775 nm, and excitation spectra peak was 749 nm.

Cell culture
U87MG-fLuc is a human GBM cell line.Cells were grown in Dulbecco's modified eagle medium and supplemented with 10% fetal bovine serum (FBS), and were maintained under a humidified atmosphere of 5% CO 2 at 37°C.

Mouse glioma models
The experiments were carried out in male BALB/c athymic nude mice, 4-5 weeks (Vital River Laboratory Animal Technology Corporation, Beijing, China).The mice were maintained under specific-pathogen-free conditions in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Peking University (Permit No: 2011-0039).The experiments were carried out in accordance with the approved guidelines.The U87MG xenograft animal models were inoculated with 7  10 U87MG cells.Only singlecell suspensions with >90% viability were used for injections.The mice were randomly divided into two groups (n = 10 for control group and n = 10 for TMZ group).For TMZ group, animals received six doses of TMZ (50 mg/kg/day) on consecutive days, starting 6 days after tumor inoculation.At the same time, however, the control group was given an equal amount of 0.9% saline i.p.The tumor volume was measured with calipers as follows: (1) where v is the tumor size, x is the large diameter of the tumor, and y is the small diameter of the tumor.At the same time, the weight and survival of mice were recorded during the experiment.
The orthotopic tumor bearing mice (N = 20) were anesthetized by i.p. injection of sodium pentobarbital.After disinfection and incision of the skin, a small hole was made in the skull through the skin overlying the cranium.The mice were stationed in a stereotactic frame with an ear bar.About 6  10 glioma cells in a volume of 4 µl PBS were injected slowly into the brain, and then the scalp was closed with sutures.A Hamilton syringe (Anting Co. Shanghai, China) attached to the stereotaxic system was used to implant all of the tumor cells.

TMZ treatment
Ten days after tumor inoculation, the mice were randomly divided into two groups (n = 10 for the control group and n = 10 for the TMZ group).The TMZ and control groups were administered TMZ i.p. (50 mg/kg/day) and an equal amount of 0.9% saline for 6 days, respectively.The mouse body weight and tumor volume were recorded every five days.To obtain the data, the survival rates were monitored daily and mice were euthanized at the appearance of severe neurological damage from tumor growth.

Magnetic resonance imaging (MRI)
Mice were required to be anesthetized for the image acquisition.Mice were lightly anesthetized with 2% isoflurane mixed with oxygen using a vaporizer (Lumic International).To assess the tumor size, mice underwent MRI scanning on the 10th day after completion of drug treatment.T1-weighted MRI images were obtained with a compact high-performance MRI system (1.5T,M3TM, Aspect Imaging, Israel) after injection of 0.1 ml contrast medium (Gadodiamide, Omniscan, Amersham) into the tail vein.In vivo multi-slice images of mouse brains were acquired in the axial plane (TR 6000 ms, TE 50 ms, slice thickness 0.8 mm and slice spacing 0.2 mm).

In vivo bioluminescent imaging (BLI)
For the sake of evaluating the antitumor efficacy of TMZ, BLI of the glioma-bearing mice was acquired every 5 days after treatment began.Before BLI was acquired, the mice were anesthetized and injected with D-luciferin i.p. (150 mg/kg) for 8 minutes.The data were collected with an IVIS Spectrum Imaging System (PerkinElmer, Germany).

Fluorescence imaging (FMI) of MMP-750
The MMP-750 probe was reconstituted with 1.2 mL of 1 × phosphate-buffered saline (PBS) before intravenous injection of animals at a dose of 2 nmol (100μL) per mouse.The in vivo bio-distribution of the MMP-750 probe was dynamically monitored using the IVIS Spectrum Imaging System (PerkinElmer, USA) at different time points after intravenous injection.The procedure of the proposed algorithm was as follows:

Micro-CT
Algorithm 1: Adaptive Sparsity Matching Pursuit (ASMP) Input: A M N − × matrix, y -measurements vector, max N -the number of maximum iterations allowed.
Initialization:  0 x = , the residual r y = Outer iteration: compute the signal proxy x x = Inner iteration: identify supports prune the estimate ( ) , then

Histology and immunohistochemistry
Mice were sacrificed after the imaging experiment, and the brain and tumor tissues were excised, fixed, and they were processed for paraffin embedding by a standard procedure.All sections which included glioma cells were used for histology and immunohistochemistry. Continuous sections (4 µm) were obtained, and preceded for hematoxylin and eosin (H&E) staining.For immunohistochemistry, tissue sections (thickness of 8 µm) were immunostained using anti-mouse MMP2 (Abcam, ab86607) and anti-rabbit MMP3 (CST, #14351) antibodies.

Statistical analysis
The experimental data were performed using Prism 5.0 (San Diego, CA, USA).Statistical analysis was calculated using the Student's t-test (two-tailed).The significance of differences in groups was compared using Tukey-Kramer's Multiple Comparison test.Data are represented as the mean ± SEM.In all tests, statistical significance was assumed to be p<0.05.

Monitoring the anti-tumor efficacy of TMZ on the in vivo gioma tumor model
The anti-glioma therapeutic effects of TMZ were analyzed on subcutaneous and orthotopic glioma mouse models by measuring the BLI and tumor volume for 20 continuous days during treatment.In the glioma xenograft tumor model, BLI imaging in Fig. 2(a) showed that the BLI light signal increased rapidly in the control group, but the BLI light signal decreased during TMZ treatment suggesting that TMZ can effectively inhibit tumor growth during the observation period.As shown in Fig. 2(b), the tumor volume was also measured and the data were consistent with the in vivo BLI observation confirming the treatment efficacy of TMZ.The mouse body weight was measured and we did not observe significant weight loss suggesting that the dosing regimen of TMZ was well tolerated for the tumor bearing mice.Moreover, mouse survival was monitored, and the prolongation of mouse survival was found in the TMZ treatment group compared to the control group.

Evaluatio
For the orthot imaging can o the antitumor reconstruction volume chang treatment gro and 4(e) from slice (Fig. 4(c found that the group.The B was greatly in could be relia

Discussion
In this work, we found that it is feasible to effectively evaluate the therapeutic effects of TMZ at the early stage using bioluminescence tomography (BLT).At the same time, the smart MMP-750 probe can be used to predict the antitumor activities of TMZ therapy.Finally, the expression of MMP2 and MMP3 may be involved in the regulation of therapeutic effects of TMZ.BLI is an ideal in vivo imaging method to observe the biological behavior of the tumor and assess the drug therapeutic efficacy at cellular levels.Edinger et al. revealed lymphoma growth and the efficacy of immune cell therapies using in vivo BLI [38].The experiments of Folaron et al. showed the safety and efficacy of the vascular disrupting agent in experimental glioma models using BLI and MRI [39].Galiger et al. assessed the efficacy of antifungals against Aspergillus fumigatus using BLI [40].In this study, we evaluated the antitumor efficacy of TMZ treatment using BLI.It showed that TMZ effectively inhibited glioma growth in both subcutaneous and orthotopic human U87MG-fLuc glioma animal models.MRI and HE histology confirmed the in vivo BLI observation.
Second, a multi-modality imaging method was utilized to make a more comprehensive assessment of TMZ treatment on gliomas.BLT utilizes in vivo optical imaging technology and mathematical modeling to produce three dimensional (3D) bioluminescent images.In combination with the bioluminescent signal of the mouse surface, we propose the reconstruction process of light sources using the ASMP method, which could provide 3D tumor volume information.Hence, we can evaluate drug treatment efficacy of TMZ more accurately and comprehensively with BLT.The results demonstrated that TMZ can effectively inhibit tumor growth and the feasibility of our methodology for localization and quantification of the optical imaging in vivo.It can contribute to the early diagnosis and better treatment of cancer, and thereby substantially improving health-related quality of life.
Third, the expression level of the MMP-750 probe was examined on both subcutaneous and orthotopic human U87MG gliomas with TMZ treatment using FMI.MMPs are key proteolytic enzymes of tumor invasion and metastasis [41,42].Based on their extracellular matrix degradation capacity in the extracellular mediators of various tissues, MMPs can regulate tumor invasion and metastasis [43][44][45][46][47].So we hypothesize that the expression of smart MMP-750 probes in the glioma can help predict antitumor activities of TMZ therapy.Our experimental results showed that the targeted imaging of MMP-750 was decreased in the glioma after treatment with TMZ compared with the control group.Our experimental results proved the above statement.
Finally, MMP members were screened after treatment with TMZ.The results showed that MMP2 and MMP3 expression was relatively decreased in the TMZ-treated group compared to the control group suggesting that the chemotherapeutic effects of TMZ are possible through down-regulation of the expression of MMP2 and MMP3, which has a marked impact in tumor invasion and metastasis in both physiological and pathological processes of brain cancer.
Our future work will focus on combining multiple imaging modalities, such as fluorescence molecular tomography (FMT), magnetic resonance imaging (MRI) and positron emission tomography (PET), to comprehensively assess the anti-tumor efficacy of TMZ or other drugs for glioma treatment to provide early prediction and assessment of therapeutic effects.Moreover, we will try to utilize the intelligent MMP fluorescence probe for targeted glioma imaging in clinical trials.At the same time, more work should focus on the in-depth molecular mechanism study of MMPs to provide a deeper understanding of the roles of MMPs in glioblastoma.The smart MMP-750 probe we used in this study is currently for research only.More experiments and safety evaluation will be made for the MMP-targeted fluorescence probes to pass FDA requirements for future clinical application.
Fig. 2 (a) BL intens and co Based on TMZ treatmen decreased in t by the measu treatment gro observation, M the TMZ treat histology stain

3 .
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Fig. 7 vitro e compared to the general subspace pursuit algorithms, which enhances the reconstruction accuracy and robustness.
Fi 2.9 In vivo B ΔΔCt was utilized for the calculation of relative gene expression.