Increased Antiangiogenic Effect by Blocking CCL2-dependent Macrophages in a Rodent Glioblastoma Model: Correlation Study with Dynamic Susceptibility Contrast Perfusion MRI

When glioblastoma multiforme (GBM) is treated with anti-vascular endothelial growth factor (VEGF) agents, it commonly exhibits tumor progression due to the development of resistance, which results in a dismal survival rate. GBM tumors contain a large number of monocytes/macrophages, which have been shown to be resistant to the effects of bevacizumab. It has been reported that tumor-associated macrophages (TAMs) promote resistance to bevacizumab treatment. Therefore, it is important to target TAMs in the GBM microenvironment. TAMs, which depend on chemokine ligand 2 (CCL2) for differentiation and survival, induce the expression of proangiogenic factors such as VEGF. Dynamic susceptibility contrast (DSC)-MR imaging is an advanced technique that provides information on tumor blood volume and can potentially predict the response to several treatments, including anti-angiogenic agents such as bevacizumab, in human GBM. In this study, we used a CCL2 inhibitor, mNOX-E36, to suppress the recruitment of TAMs in a CCL2-expressing rat GBM model and investigated the effect of combination therapy with bevacizumab using DSC-MR imaging. We demonstrated that the inhibition of CCL2 blocked macrophage recruitment and angiogenesis, which resulted in decreased tumor volume and blood volume in CCL2-expressing GBM in a rat model. Our results provide direct evidence that CCL2 expression can increase the resistance to bevacizumab, which can be assessed noninvasively with the DSC-MR imaging technique. This study shows that the suppression of CCL2 can play an important role in increasing the efficacy of anti-angiogenic treatment in GBM by inhibiting the recruitment of CCL2-dependent macrophages.


Cell culture
The human GBM cell lines U87 MG, LN18 and the mouse GBM cell line GL261 were chosen for this study. Both human GBM cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in RPMI with 10% FBS at 37 °C . In case of GL261, the cells were purchased from DSMZ (Leibniz Institute DSMZ-German Collection of Microorganism and Cell Cultures) and cultured in 90% DMEM + 10% FBS + 4mM Glutamine. Rat primary bone marrow macrophages and rat primary brain microvascular endothelial cells were purchased from Cell Applications, Inc. (San Diego, CA, USA) and cultured according to the manufacturer's protocols.

Proliferation assay
To assess cell viability in two different human GBM cell lines, U87 MG and LN 18 were seeded on 96-well plates at an initial density of 5 × 10 4 cells/well treated mNOX-E36 or DMSO as vehicle, and proliferation was measured 24, 48, and 72 hours. To assess the toxicity, various concentration (0 -400 µg/mL) of mNOX-E36 was treated 24 hours after cell seeding and measured by cell counting kit-8 (CCK-8, Dojindo). Briefly, 10 μl of 5 mg/mL CCK-8 agent was added and incubated for 2 hours. Then, the amount of formazan formed by viable cancer cells was determined by absorbance at 450 nm on a microplate reader. The number of viable cells was expressed as a percentage of control cells. All treatment conditions were performed in sextuplicate and final concentration for in vitro experiments in this study was determined by proliferation assay.

Western blot analysis
rCCL2 protein levels were evaluated by western blot analysis. Cells were lysed in ice-cold lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin and protease inhibitor cocktail (Sigma)], and the concentration of lysate protein was evaluated using the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL, USA). Approximately 30 μg of protein was loaded in each lane of a polyacrylamide denaturing gel for electrophoresis. After electrophoresis, the protein was transferred to nitrocellulose membranes for blotting. We used a rabbit polyclonal antibody to rat CCL2 (MyBioSource) and a rabbit polyclonal antibody to β-actin (Abcam). Primary antibodies were detected by horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology).

Immunocytochemistry
Cells were seeded on glass coverslips. The next day, the cells were fixed in 4% formaldehyde, rinsed three times in PBS and permeabilized in PBS containing 0.1% Triton X-100. After rinsing with PBS, cells were incubated in 3% BSA for 30 minutes at room temperature for blocking. The samples were incubated for 1 hour with the primary antibody (rabbit antibody to rat CCL2, MyBioSource) and for 1 hour at room temperature with the secondary antibody (Alexa Fluor ® 594-conjugated goat rabbit-specific antibody, Santa Cruz) after mounting with DAPI. For fluorescence imaging, images were taken using a confocal microscope (Zeiss LSM 510 META).

Cytokine array
Cytokine arrays (Merck Millipore) for rat CCL2 detection were conducted according to the manufacturers' protocols using cell lysate and 48-hour cell-conditioned media that were controlled for protein concentration and media volume. To analyze the effect of mNOX-E36 on CCL2 release in cells, CCL2 expressing cells were grown in media supplemented with 400 μM mNOX-E36 or PBS as vehicle. After 2 weeks, whole-cell lysates and cell conditioned were collected and measured.

Macrophage migration assay
Cell suspensions (2 x 10 5 cells/100 μl) were added to the upper chamber of 8-μm pore Transwell inserts (Corning) for 2 hours at 37 °C to attach to the membrane. Transwells were then moved to 24-well plates containing 600 μl of cell-conditioned media and incubated at 37 °C for 24 hours. Non-migratory cells were removed from the upper surface of the membrane by scraping with cotton swabs. Migrated cells were fixed in 4% paraformaldehyde, stained with 0.2% crystal violet and analyzed at ⅹ 20 magnification in five randomly chosen microscope fields per filter.

Matrigel in vitro tube formation assay for angiogenesis
An experimental design for the angiogenesis is shown in Figure 3A. First, 10 µl of growth factor-reduced Matrigel (BD Bioscience) was placed in the wells of µ -slides (Ibidi, Germany) and incubated at 37 °C for 30 minutes to allow polymerization. Then, 50 μl of rat brain microvascular endothelial cells (1 x 10 4 / well) was seeded subconfluently onto each Matrigel-coated well and allowed to adhere for 4 hours at 37 °C. Conditioned medium from co-cultured tumor cells with macrophages or without macrophages were prepared, of which 50 μl was added to the 50 μl cell suspension already in the wells, resulting in a final volume of 100 μl. After an 8-hour incubation at 37 °C, tube formation was assessed. The number of tubes formed was quantitated as previously described. Briefly, a connecting branch between 2 discreet endothelial cells was counted as one "tube."

MR Imaging protocol in a rat GBM model
MRI studies were performed on a 9.4T MR scanner (Agilent Technologies) fitted with a rapid 72 volume transmit coil and a rapid 72 rat brain receive coil. The rats and mice were anesthetized with 1.5-2% isoflurane/oxygen (v/v), and oxygen saturation and heart rate were

Immunohistochemistry
Immunohistochemistry was performed using formalin-fixed paraffin-embedded tumor blocks. Briefly, 4-µm-thick tissue sections were deparaffinized in xylene and hydrated by immersing in a series of graded ethanol. Antigen retrieval was performed in a microwave by placing the sections in epitope retrieval solution (0.01 M citrate buffer, pH 6.0) for 20 minutes; endogenous peroxidase was inhibited by immersing the sections in 0.3% hydrogen peroxide for 10 minutes. Sections were then incubated with primary rabbit polyclonal antibody to rat CCL2/ MCP-1 (MBS691393) or mouse monoclonal antibody to rat CD68 (UM800047, ORIGENE), goat polyclonal antibody to rat CD34 (AF4117), mouse monoclonal antibody to human KI-67 (UM800033, ORIGENE) in Dako REAL antibody diluent (Dako). To investigate the necrosis, rabbit polyclonal antibody to human LDH (bs-3827R, Bioss) and HMGB1 (OABF00245, Aviva Systems Biology) were used as primary antibodies. Staining for the detection of bound antibody was evaluated by DAB and all imaging were analyzed by Image J. Moreover, the blood vessel numbers which were evaluated by CD34 were expressed based on previous study 1 .

Mouse CCL2 expressing GBM cell line
To prepare mCCL2 expressing C57BL/6J mouse GBM model, we established mCCL2- To express mCCL2 transgenes in cells, GL261 was transfected with lentivirus and analyzed by fluorescence microscopy using green filters (Leica, Wetzlar, Germany).

RNA isolation and real-time PCR for human study
Total RNA of each human sample was isolated by using the Qiaquick RNeasy Mini kit Real-time PCR was performed in a Rotor-Genes Q cycler machine (Qiagen) using Rotor-Genes SYBR Green PCR kit (Qiagen) according to the manufacturer's instructions in a total volume of 20 µl. Cycling conditions for CCL2 and CD68, GAPDH genes were 10 minutes at 95 °C , 40 cycles of 20 seconds at 95 °C , 20 seconds at optimal Tm, 20 seconds at 72 °C . The sequences of primers were as follows: CCL2 5'-agcaagtgtcccaaagaagc-3' and 5'-tggaatcctgaa cccacttc-3', CD68 5'-cccacacaggggtctttg-3' and 5'-gatcaggccgatgatgagag-3', GAPDH 5'-ggc attgctctcaatgacaa-3' and 5'-atgtaggccatgaggtccac-3'. To correlate the threshold (Ct) values from the amplification plots to copy number, a standard curve was generated, and a nontemplate control was run with every assay. All samples were run in duplicate, and the average value was used.

MR imaging protocol in GBM patients
For human MR study, twenty-six patients underwent conventional MR imaging and DSC perfusion MR imaging using a 3T-scanner (Verio; Siemens Healthcare Sector) with a 32channel head coil. The conventional MR imaging included T1-weighted imaging (T1WI), such as transverse spin-echo imaging, before and after contrast enhancement or multi-planar reconstructed transverse, coronal imaging with a sagittal three-dimensional magnetization

Statistical analyses
All statistical analyses were performed using a commercial software program (MedCalc version 13.1.0.0, MedCalc Software). A p value < 0.05 was considered statistically significant. Kolmogorov-Smirnov's test was used to determine whether the non-categorical variables were normally distributed. Non-parametric data are presented as the median and interquartile range (IQR, range from the 25th to the 75th percentile), and parametric data are shown as the mean ± standard deviation. According to the results of the Kolmogorov-Smirnov's test, a paired or unpaired Student's t-test, Wilcoxon test or Mann-Whitney U-test was performed, as appropriate, to compare the values between two groups. Bonferroni correction was applied for the multiple comparisons of the values (e.g. a p value < 0.017 was considered statistically significant in three groups comparison). Survival data were analyzed by using a Kaplan-Meier survival analysis with a log rank method of statistics in a mouse GBM model. Pearson's correlation analysis was performed to measure the significance of the association between the nCBV parameters and RNA expression level in GBM patients.