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Home > A new molecular probe: An NRP-1 targeting probe for the grading diagnosis of glioma in nude mice
A new molecular probe: An NRP-1 targeting probe for the grading diagnosis of glioma in nude mice
Weizhou Wua, Zhejiang Zhong, Yanhua Gongc, Yuheng Shana, Lina Yuan, Li Wangd, Jian Chene, Zhe Zhang
1.Introduction
Glioma is a common central nervous system malignancy, accounting for 75 % of malignant primary brain tumors [1]. According to the histological features, the WHO divides gliomas into grades I-IV [2,3]. Among them, low-grade malignant tumors (LGG, WHO grade I and II) have slow growth and weak invasiveness [4], whereas high-grade malignant tumors (HGG, WHO grade III and IV) have strong invasiveness and poor prognosis [5,6]. At present, glioma is mainly treated with surgery and supplemented with radiotherapy and chemotherapy [7], so the early classification of glioma is crucial for the treatment plan and prognosis [8]. The development of magnetic resonance molecular imaging technique may provide a new opportunity for early grading diagnosis of glioma. A research hotspot of brain tumor research is the use of magnetic resonance imaging of specific biomolecules in vivo to achieve early diagnosis and therapeutic monitoring of diseases.
In this experiment, the novel molecular probe USPIO-PEG-tLyP-1 based on USPIONs with NRP-1 as the target was constructed. Our study aims to inject the probe into tumor-bearing mice through the tail vein, confirm that USPIO-PEG-tLyP-1 can be used for high and low grading diagnosis of glioma with different NRP-1 expression levels by MRI.
2.Materials and methods
2.1.Materials
The human glioma U87 cell line (WHO grade IV) was obtained from the Cell Resource Center, Peking Union Medical College (China). The human glioma CHG-5 cell line (WHO grade II) was purchased from Shenzhen Hao Di Hua Tuo Biological Company (China). The human astrocyte cell line (HA) was purchased from Science USA. Sixteen BALB/c nude mice, female, 4∼6 weeks old, body weight (20 ± 2) g, were purchased from Beijing Hua Fu Kang Company (China), license number SCXK (Jing) 2014-0004.
2.2.USPIO-PEG-tLyP-1 preparation and properties
USPIO-PEG was provided by China Beijing Wande Company. A solution of USPIO-PEG (4 mg Fe, 4.3 mg/mL) was placed into a 2 mL EP tube. Then 1 mg of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), 2.8 mg of sulfur-N-hydroxysuccinimide (NHS), and 100 μL of PBS were combined. The solution was mixed well, and quickly added to the USPIO-PEG solution at room temperature followed of. Reaction in a shaker for 30 min. The solution was then subjected to ultrafiltration to remove excess EDC and NHS from the solution. The polypeptide tLyP-1 was synthesized by the Shanghai Jier Biochemical Company and purified by high-performance liquid chromatography to obtain a polypeptide with a purity of 98 %. The obtained pure peptide was identified by mass spectrometry (Fig. 1 A). Then, 100 μL of 98 % to-1 was added to the above solution, and the coupling reaction was carried out at 37 °C, 2 h, and it was dialyzed overnight with PBS. The resulting solution was concentrated by ultrafiltration to 4 mg/mL and placed in a refrigerator at 4 °C for use. The hydrated particle size and stability of the molecular probe were detected by DLS (Malvern, Britain) and observed by TEM (JEOL).
2.3.The specificity of reactivity of the probe with NRP-1
Synthesized LV-NRP1-RNAi Lentivirus and LV-NRP2-RNAi Lentivirus, and transfected U87 cells separately to obtain U87 cells with low NRP-1 or NRP-2 expression. There are 3 groups in the experiment, including group A: control group (U87 cell), group B: LV-NRP2-RNAi group (U87 with NRP2 low expression), and group C: LV-NRP1-RNAi group (U87 with NRP1 low expression). We cultured these 3 types of cells in a four-well plate, and each cell density was 2.5 × 105/mL. Each well contained 1 mL of medium. These cells were placed in an incubator for 24 h.
The USPIO-PEG-tLyP-1 solution was diluted with DMEM high glucose medium and the final concentration of USPIO-PEG-tLyP-1 solution was 50 μg Fe/ml. A four-well plate was removed from the incubator and the culture medium was discarded. A concentration of 50 μg Fe/ml USPIO-PEG-tLyP-1 (500 μl) was added to each well, which was first washed with PBS. Then, the four-well plate was placed in the incubator for 2 h. Low melting point agarose powder (0.1 mg) was added to 10 mL of PBS to generate a 1 % low melting point agarose gel. The agarose powder solution was heated in a microwave oven to obtain a homogeneous solution. The 1 % low melting point agarose solution was placed in an 80 °C water bath.
After discarding the old culture medium, each well of the four-well plate was washed with PBS 2 times, and add 100 μL of tryptic digestive juice was added, and the plate was placed in an incubator for 5 min. The cells were disaggregated to a single cell suspension and transferred to a 0.6 mL centrifuge tube (1000 rpm for 10 min). The supernatant was discarded, and 200 mL of low melting point agarose solution was added and mixed evenly to suspend the cells. The 24-well plate was moved, and 6 wells were set up for each cell. The R2 value was calculated by the inverse of the relaxation time T2 (R2 = 1/T2) obtained from 3.0 T MRI scan of a 24-well plate.
2.4.Cell culture and NRP-1 protein detection
U87, CHG-5, and HA cells were cultured in DMEM containing 10 % fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Gibco, USA) in a cell culture incubator at 37 °C with 5 % CO2. The medium was changed every other day. When the cells covered 80 % ∼ 90 % of the culture flask, they were digested and passaged with trypsin.
Total protein was extracted by RIPA cell lysis buffer (Dingguo Changsheng Company, China). The protein concentration was detected by a BCA protein assay kit (Biyuntian Company, China). Protein samples (30 μg) were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane, which was blocked for 2 h in 5 % skim milk. Finally, the NRP-1 rabbit anti-human monoclonal antibody (Abcam, USA) (1:2 000) solution was added to the sealed bag containing the membrane, overnight at 4 °C. Next, the membrane was washed with TBST 5 times, incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5 000) (Kangwei Century Company, China), for 1 h at room temperature, washed 5 times with TBST, and developed with an ECL developer. The image was recorded, using β-actin as the internal reference, and greyscale analysis was performed using Image Lab software.
2.5.Establishment and grouping of animal models
All experimental animal procedures were implemented in accordance with the National Institute of Health Animal Protection Guidelines. Sixteen nude mice were randomly divided into two groups: the U87 tumor-bearing mouse group (U87 group) and the CHG-5 tumor-bearing mouse group (CHG-5 group). U87 and CHG-5 cells were inoculated into the nude mice in the right basal ganglia at a concentration of 3.0 × 1010/L. After the nude mice were anesthetized with 0.8.% sodium pentobarbital (20 mL/kg), the head was fixed in the prone position on a stereotaxic instrument (Ruiwosheng, China). The inoculation site was performed as previously described [9], and the white matter region was vertically pierced using a microinjector with 5 μL of tumor cell suspension (U87 or CHG-5). After completion, the mice were raised separately in groups.
After 18 d of modeling, all tumor-bearing mice underwent MRI. All the mice were modeled successfully except for one mouse in group CHG-5. Five tumor-bearing mice with similar tumor sizes were selected from the two groups. The other tumor-bearing mice were sacrificed, and the tumor tissues were taken for pathological examination, which was performed using standard procedures. In addition, the 5 tumor-bearing mice selected from each group were numbered, and USPIO-PEG-type-1 at a dose of 3 mg/kg was injected into each tail vein in order.
2.6.In vivo MRI experiments
The tumor-bearing mice were anesthetized with 2 % isoflurane according to the mouse number. The mice were placed on a 7.0 T animal nuclear magnetic resonance instrument (Siemens, Germany), and an MRI transverse scan of the transplanted tumor was performed at 6 h, 12 h, and 24 h after injection of the probe. The scanning parameters were set to T2WI sequence: TR =3500 ms, TE =72 ms, FOV =400 mm, layer thickness 1.00 mm, layer spacing 0.5 mm, matrix 256 × 256. T2map sequence TR = 1562 ms, TE =9 ms, FOV =400 mm, layer thickness 1.00 mm, layer spacing 0.5 mm, matrix 256 × 256. After scanning, the obtained image was analyzed by the analysis software. The relaxation time (T2 value) of the tumor was measured, and the R2 value (R2 = 1/T2) was calculated.
2.7.Prussian blue staining of the tumor tissue
Tumor-bearing mice were sacrificed by cervical dislocation to isolate the tumor tissue. Partial tumor tissue was taken from each of the two groups. The tumors were fixed in 4 % formaldehyde, embedded in paraffin, and sliced to generate 3 μm thick tissue sections. The samples were stained with Prussian blue (nuclear red fixation method) (Kangwei Century Company, China) and sealed with neutral gum. The distribution of blue-stained iron particles in the tissues was observed under a microscope (Hitachi, Japan), and the particle counts of the two groups were compared. The remaining tumor tissues in both groups were fixed in 2.5.% glutaraldehyde solution, dehydrated with alcohol and embedded in epoxy resin for slicing. Ultrathin sections were stained with uranyl acetate and lead citrate. The incorporation of USPIO particles into U87 and CHG-5 tumor cells was observed by TEM.
2.8.Statistical analysis
Statistical analysis was performed using SPSS version 22 software. The experimental data are presented as the mean ± SD. The R2 values of the transplanted tumours at different time points before and after USPIO-PEG-tLyP-1 treatment were compared between the two groups of tumour-bearing mice, and repeated measures of analysis of variance were used. The R2 values between the two groups were compared by t- test. The differences between the three groups were analysed using the F test. The difference was statistically significant at P < 0.05.
3.Results
3.1.Molecular probe characterization
TEM showed that the USPIO was a round particle with a uniform distribution and size (Fig. 2 A and B). The molecular structure of the probe is shown in Fig. 2 C. The DLS results indicated that the peak value of the hydration diameter of USPIO-PEG at 25 °C was 34.72 nm, and the peak value of the hydration diameter of the molecular probe USPIO- PEG-tLyP-1 was 44.77 nm at 25 °C (Fig. 2 D).
3.2.Molecular probe targeting detection
MRI was performed after the probe was combined with U87 cells. Group A and group B cells showed low signal shadows of the T2- weighted image, but group C cells showed a high signal shadow. There was no significant difference in R2 value between group A (24.09 ± 2.22) and group B (21.38 ± 2.98) (P > 0.05), but there was a significant difference in R2 value between groups A (24.09 ± 2.22) and C (7.64 ± 3.02) (P < 0.01), groups B (21.38 ± 2.98) and C (7.64 ± 3.02) (P < 0.01) (Fig. 1 B).
3.3. Relative expression of NRP-1 in each cell line Western blot analysis showed that the expression of the NRP-1 protein in U87 and CHG-5 cells was higher than that in HA cells (P < 0.01), and the expression level of NRP-1 protein in U87 cells was significantly higher than that in CHG-5 cells (P < 0.01) (Fig. 3).
3.4.In vivo MRI results
After the injection of USPIO-PEG-tLyP-1, no side effects or abnormal behaviors were observed in the tumor-bearing mice. MRI showed that the tumor tissues of the two groups had a high signal before injection (Fig. 4 A and E). After the probe was injected, low signal shadows appeared in the tumor tissues of both groups (Fig. 4 B and F), and the low signal shadow gradually increased with time, reaching a maximum at 12 h (Fig. 4 C and G), after which the low signal shadow began to decrease (Fig. 4 D and H). Moreover, the low signal intensity of the U87 group was significantly higher than that of the CHG-5 group. From Table 1, there is a clear difference in the R2 value of the tumor tissue before and after injection of the molecular probe (F time = 105.520, P = 0.000), there is an interaction between the mole-
color probe and the injection time (F time*group = 6.698, P = 0.002), and the R2 value of the U87 group was higher than that of the CHG-5 group (F group = 43.744, P = 0.000). There was no distinct difference in the R2 values between the two groups before the injection (P > 0.05). But at 6 h, 12 h and 24 h after injection, the R2 values of the tumor tissues of the two groups were significantly different (P < 0.01).
3.5.Pathological findings in animal models
In the U87 group, the tumor cells that were star-shaped or fusiform were densely arrayed and evenly distributed. The nuclei were stained deeply. Pathological mitosis was more common (Fig. 5 A and B). The tumor cells of the CHG-5 group were pleomorphic, with differently sized nuclei, deep nuclear staining, and rare mitotic figures (Fig. 5 C and D).
3.6.Prussian blue staining in tumor tissues
Prussian blue staining of tumor tissue sections showed diffuse iron particle deposition in the U87 group (Fig. 6 A and B), while less deposition was observed in the CHG-5 group (Fig. 6 D and E). The quantity of blue-stained iron particles in the two groups was observed under a microscope. The results showed significantly more blue-stained iron particles in group U87 (851.33 ± 110.23) than those in group CHG-5 (315.50 ± 77.09), and the difference between the two groups was statistically significant (P < 0.01) (Fig. 6 G).
By TEM, more USPIO particles were incorporated into U87 cells than CHG-5 cells (Fig. 6 C and F).
4.Discussion
Because of their strong invasiveness and high recurrence rate, gliomas face great difficulties in diagnosis and treatment. Although treatment is constantly improving, the prognosis of patients is still unsatisfactory. In particular, the median survival of patients with glioblastoma multiforme (GBM) is approximately 14.6 months [10,11]. Therefore, early qualitative and hierarchical diagnosis of glioma directly influences the prognosis of the disease and the life of the patient. Magnetic resonance molecular imaging can reflect the characteristics of specific biomolecules in the body at an early stage and evaluate the microstructure changes of the tissues. Therefore, this may help to achieve an early qualitative and hierarchical diagnosis of the disease.
NRP-1 is a transmembrane glycoprotein that plays a pivotal role in the growth and guidance of axons [12]. Related studies have found that NRP-1, a co-receptor of vascular permeability factor (VEGF), can participate in tumor angiogenesis [13,14]. Blocking the NRP-1 signaling pathway is an effective method for anti-tumor therapy [15]. NRP-1 is expressed at low levels in normal tissue cells [16], but is highly expressed in many tumor cells, such as in breast cancer [17], liver cancer [18], and non-small cell lung cancer [19]. More importantly, the expression of NRP-1 is especially high in glioma, and its expression level is positively correlated with the degree of malignancy of the glioma [20], which may be related to the rich blood vessels and strong infiltration ability of malignant glioma [21]. Three cell lines were selected for this experiment: U87 (HGG) [22], CHG-5 (LGG) [23], and HA (human astrocytes) [24]. The expression of NRP-1 in these three cell lines was verified by Western blot analysis. The results showed that NRP-1 was expressed at low levels in HA cells and was highly expressed in U87 and CHG-5 cells. The expression level of NRP-1 in U87 cells was obviously higher than that in CHG-5 cells, providing theoretical support for the next experiment.
Superparamagnetic iron oxide nanoparticles (SPIONs) are novel MRI contrast agents that can produce a stronger MR signal contrast [25]. The commonly used magnetic nanoparticles for synthetic molecular probes are SPIONs and USPIONs, with USPION having the advantages of a smaller particle size and good compatibility with tumor cells [26]. In 2015, Wang LuNing examined different concentrations of USPION-labelled C6 glioma cells, confirming the feasibility of applying USPION to glioma by MRI [27]. However, a single USPION has poor stability and a short plasma half-life [28], but after modification with certain specific antibodies and ligands, the stability is not only enhanced but the targeted accumulation ability is also improved. In 2014, Shevtsov MA confirmed that the combination of recombinant human epidermal growth factor and SPION-EGF can accumulate in tumors passing through the blood-brain barrier by MRI in the situ model of C6 glioma, and the imaging effect is more significant than that of the unbound SPION [29]. Polyethylene glycol (PEG) is a commonly used surface modifier with high lipid solubility [30]. Modification of US- PIONs with PEG not only enhances the stability of USPIONs, but it also improves the biocompatibility [31] and enhances the blood-brain barrier (BBB) permeability of iron oxide nanoprobes [32]. The type-1 peptide (CGNKRTR) is an easily synthesized linear polypeptide that specifically binds to NRP-1 on the cell surface [33]. type-1 contains a CendR element and can penetrate tissue via the NRP-1 dependent CendR internalization pathway. In 2014, Miao DeYu established the dual targeting molecule Lf-NP-tLyP-1 and verified its good tumor targeting and cell internalization ability [34], making it an ideal ligand for targeting tumor NRP-1 receptors. Therefore, we coupled the specific NRP-1 ligand tLyP-1 with PEG-modified USPION using the carbodiimide method to create USPIO-PEG-tLyP-1 in this experiment. Through the U87 cell experiment, it indicated that this probe could bind to NRP-1 specifically.
To evaluate the imaging effects of this novel probe in the classification diagnosis of glioma in nude mice, U87 and CHG-5 cell lines were used to establish a nude mouse model of high- and low-grade in-situ glioma. Histological images confirmed that the tumor tissues of U87 or CHG-5 tumor-bearing mice were in accordance with the pathological features of high-grade or low-grade glioma, respectively. MRI showed that after injection of the probe, the low signal shadow of the two groups of tumor tissues gradually increased, reaching a maximum at 12 h. Then, the low-signal shadow began to decrease, which may be related to the metabolism of molecular probes in vivo. Moreover, the low signal intensity of the U87 group tumor tissue was obviously higher than that of the CHG-5 group. This indicates that the binding ability of USPIO-PEG-tLyP-1 to U87 tumor tissue is stronger than that of CHG-5 tumor tissue. Since the R2 value is the reciprocal of the T2 relaxation time (unit is ms-1), it has a good correlation with the iron oxide particles and can increase with an increase in the particle concentration [35]. Therefore, the R2 value was used as an analysis index in this experiment. The R2 value of the U87 group tumor tissue was clearly higher than that of the CHG-5 group. Similarly, iron particles were found in the tumor tissue by Prussian blue staining, but the number of blue-stained particles in the U87 group was evidently greater than that in the CHG-5 group.
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