Introduction
Gliomas are the most common central nervous system neoplasms in adults 1 2 3 4,5
PET, which uses radiolabeled tracers, is a promising noninvasive imaging method for a more accurate evaluation of initial staging, regional and distant metastasis, treatment response, and recurrence 6–9 18 F-FDG), a commonly used tracer for neoplasm detection, and also a marker of glycolytic metabolism, is widely used in the clinical field. Malignant cells demonstrate increased uptake of 18 F-FDG because of increased levels of aerobic glycolysis and increased expression of glucose transporter (GLUT) proteins, which is the foundation of quantitative PET analysis 10 18 F-FDG uptake may be associated with variations in the tumor microenvironment 11 18 F-FDG PET/computed tomography (CT) in the assessment of the effect of radiosensitizers remains unclear.
Oleanolic acid (OA), a triterpenoid saponin, widely distributed in nature 12 in vitro 13 18 F-FDG PET/CT might be a good way of assessing the clinical effect of OA. Therefore, the aim of this study was to evaluate the radiosensitization effect of OA on a C6 rat glioma model using 18 F-FDG PET/CT and Ki67 and GLUT-1 immunohistochemistry (IHC) and evaluate the utility of 18 F-FDG PET/CT in assessing early changes after radiotherapy.
Materials and methods
Cell and tissue culture
C6 rat glioma cells were obtained from the Teaching and Research Section of Nuclear Medicine of Anhui Medical University and were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Invitrogen) at 37°C in 5% CO2 and 95% air. Cell cultures were split two to three times per week, and experiments were performed when cell confluence reached 80%.
Animals and experiment design
All of the experimental protocols in this study were approved by the Ethics Review Committee for Animal Experimentations of Anhui Medical University. Thirty-two male Sprague–Dawley rats, weighing 190–250 g, were purchased from the Experimental Animal Center of Anhui Medical University. Cells were digested with pancreatin, washed twice with PBS, and then suspended in serum-free Dulbecco’s modified Eagle’s medium. C6 rat glioma cells (1.0Ă—107 /ml, 0.3 ml for each rat) were implanted subcutaneously in the right thigh region of the rats. At that time, the C6 rat glioma tumors had grown to ∼1 cm in diameter, which was considered a mark of success of the vaccination. Thereafter, all of the tumor-bearing rats were observed for one more week and were used at day 21 of tumor growth. OA was prepared by suspension in dimethyl sulfoxide solution (0.1% dimethyl sulfoxide, 1.8% NaCl in distilled water), with a final solution concentration of 20 mg/ml.
These tumor-bearing rats were randomly divided into four groups of eight rats each: the control group (group A), the OA group (group B), the radiotherapy group (group C), and the radiotherapy/OA combined group (group D). Group A rats were given saline through gavage (2 ml daily for 7 days), group B rats were given OA solution through gavage (2 ml daily for 7 days), group C rats were given radiotherapy only, and group D rats were given OA solution (as per group B); at day 2 after intragastric administration, group D rats also received radiotherapy during the intragastric administration period. Groups C and D rats were anesthetized with chloral hydrate (mass fraction 10%, 0.3 ml/100 g) by means of intraperitoneal injection before radiotherapy. These two groups were irradiated once every 2 days for 6 days with a fractionated dose of 4 Gy using a Varian 23EX medical linear accelerator (Varian Medical Systems Inc., Palo Alto, California, USA).
18 F-FDG PET/CT imaging18 F-FDG was provided by NanJing Jiangyuan Andike Positron Research and Development Co. Ltd (Wuxi, Jiangsu, China), and its radiochemical purity was higher than 95%. For the imaging experiment, all rats were laid in the prone position after being kept in a fasting state for at least 6 h before 18 F-FDG PET/CT imaging. Each of the rats received 37 MBq 18 F-FDG through tail vein injection. PET/CT images were acquired 1 h after 18 F-FDG administration using a Syngo MMWP system (Siemens Biograph 64 Truepoint PET/CT; Siemens, Munich, Germany). The parameters used were as follows: 120 kV, 80 mA, slice thickness of 1.5 mm, and 5 min per bed position.
18 F-FDG PET/CT imaging for groups C and D was carried out before radiotherapy, 1 day after treatment, and 7 days after treatment. Imaging for groups A and B was carried out at the same time.
The image plane with the largest tumor appearance on the PET/CT fusion image was selected for data collection. An irregular region of interest (ROI) covering the entire tumor was drawn manually. At this time, ROIs were drawn on the contralateral paraspinal muscles. The uptake value of tracer in tumor and muscle tissue was determined in the attenuation-corrected transaxial tomographic slices by calculating the standard uptake value (SUV) and was measured by means of ROI. The maximal SUVs for 18 F-FDG were obtained from the selected ROI and compared with the SUVs of the contralateral paraspinal to calculate the tumor/muscle (T/M) ratio. Two experienced PET/CT clinicians analyzed the images independently, and any controversies were resolved by consultation. 18 F-FDG PET/CT images were compared with HIF-1α and GLUT-1 IHC of paraffin sections. During the study, the PET/CT clinicians and pathologist were blinded to the PET/CT and IHC images, respectively.
Histology and IHC
Two tumors were prepared from each group before treatment, 1 day after treatment, and 7 days after treatment for IHC or hematoxylin and eosin examination. Primary tumors were collected, fixed in 10% para-formaldehyde for 1 week, embedded in paraffin, and then sectioned at a thickness of 4 μm using a microtome. We used hematoxylin and eosin for histological staining, GLUT-1 as a marker for metabolism, and Ki67 as a marker for proliferation. Immunostaining for Ki67 (Santa Cruz Biotechnology, Santa Cruz, California, USA) and GLUT-1 (Bioss, Beijing, China) was carried out using rabbit polyclonal antibodies at dilutions of 1 : 300. Tumor cell density was counted using a Ă—200 objective. Ki-67 images were observed under a Ă—400 high-power field, and nuclear staining was counted per 200 tumor cells using an Olympus microscope (Olympus, Tokyo, Japan). The proliferation activity of the tumor cells was defined as the percentage of positively stained cells 14 15
Statistical analysis
All values were presented as mean±SE and analyzed using the SPSS statistic 13.0 software package (SPSS Inc., Chicago, Illinois, USA). Univariate repeated-measures analysis of variance was carried out for the T/M ratio and tumor volume. The density of tumor cells and expression of Ki-67 and GLUT-1 were analyzed by means of one-way analysis of variance. The associations between T/M, tumor cell density, and expression of Ki67 and GLUT-1 were tested by means of bivariate correlation analysis. A P -value of 0.05 or less was considered significant.
Results
C6 rat glioma model and 18 F-FDG PET/CT imaging
C6 rat glioma tumors (>1 cm in diameter) were observed within 2 weeks of tumor transplantation in 32 Sprague–Dawley rats. No obvious tumor metastases were found among these tumor-bearing rats.
There were no significant differences in T/M values between any of the four groups – groups A, B, C, or D (F =0.147, P =0.931), which were 10.086±1.983, 9.750±2.197, 9.388±2.097, and 9.786±2.158, respectively. However, there were differences between the T/M values of the four groups 1 day after treatment (F =2.891, P =0.05), with T/M values of 10.408±1.958, 9.946±2.198, 8.085±1.989, and 8.065±2.019, respectively. The T/M values of groups C and D were lower when compared with those of group A (t CA =2.354, t DA =2.356, P <0.05), but there were no significant differences between groups C and D (t CD =0.020, P =0.984) or groups A and B (t AB =0.444, P =0.664). Significant differences were observed 7 days after treatment (F =33.726, P <0.01), with T/M values of 11.865±2.056, 11.411±1.923, 6.398±1.814, and 4.558±1.159, respectively. The T/M values of group D were lower than those of the other three groups (t DC =2.416, P =0.03; t DA =8.133, t DB =7.656, P <0.01), and the T/M values of group C were lower than those of group A and group B (t CA =5.637, t CB =4.632, P <0.01), whereas there were no significant differences between groups A and B (t AB =0.456, P =0.656). A decreasing trend was observed in the three different time points of group D (F =16.164, P <0.01). 18 F-FDG PET/CT images of the three different time points of group D are shown in Fig. 1 .
Fig. 1: 18 F-FDG PET/CT images from the combined oleanolic acid /radiotherapy group. From left to right: CT (a), PET (b), PET/CT (c); from top to bottom: before treatment, 1 day after treatment, and 7 days after treatment. CT, computed tomography; 18 F-FDG, fluorine-18-deoxyglucose.
The tumor volumes of the four groups (3.685±0.833, 3.735±0.825, 3.481±0.878, and 3.501±0.904 cm3 , respectively) showed no significant differences before treatment (F =0.177, P =0.911), nor were there significant differences 1 day after treatment (3.707±0.852, 3.780±0.828, 3.495±0.309, and 3.468±0.901 cm3 ) (F =0.255, P =0.857). However, there were significant differences between tumor volumes in the four groups 7 days after treatment (F =7.681, P <0.01). Tumor volumes of groups A and B had increased slightly (F =2.493 and 2.344, P >0.05) and those of groups C and D had decreased slightly (F =0.381 and 2.392, P >0.05), with a more significant decrease seen in group D than in group C (t =2.351, P =0.02). The T/M ratios and tumor volume changes of the four groups are shown in Table 1 .
Table 1: Changes in tumor/muscle ratios and tumor volumes for the four groups A, B, C, and D
Histopathological observations
All of the tumor-bearing rats were killed under anesthesia after the final imaging. The tumors were clearly delineated from the surrounding normal tissue. Fresh tumor tissue was bright red, with partially necrotic gray areas seen within the tumors. The tumor cells showed a disordered arrangement. Obviously abnormal and hyperchromatic nuclei without much necrosis were seen microscopically in groups A and B. Large areas of patchy necrosis, nuclear pyknosis, and karyorrhexis were seen in groups C and D. More areas of necrosis were seen in group D than in group C (Fig. 2I ). There were significant differences between tumor cell densities in the four groups (F =16.667, P <0.001), which were 790.250±127.731, 780.500±179.255, 536.625±120.759, and 397.000±90.473, respectively. The tumor cell densities of group D were lower than those of group C (t =2.617, P <0.05).
Fig. 2: I. Hematoxylin and eosin staining of the four groups (Ă—200): (a), control group; (b), oleanolic acid group; (c), radiotherapy group; (d), combined oleanolic acid /radiotherapy group. II. Ki67 immunohistochemical staining of the four groups (Ă—200): (a), control group; (b), oleanolic acid group; (c), radiotherapy group; (d), combined oleanolic acid /radiotherapy group. III. GLUT-1 immunohistochemical staining of the four groups (Ă—200): (a), control group; (b), oleanolic acid group; (c), radiotherapy group; (d), combined oleanolic acid /radiotherapy group. GLUT-1, glucose transporter-1.
Immunohistological examinations
Ki67 staining was mainly located in the cell nuclei of the glioma tumor cells, although some cytoplasmic staining was seen. Significant differences were observed between the four groups (F =22.082, P <0.001), of which the percentages of positively stained cells were 71.6±11.7, 66.7±15.8, 40.0±16.5, and 23.8±8.7%, respectively. The percentages of positive cells were much lower in group D than in group C (t =2.438, P =0.029; Fig. 2II ).
GLUT-1 staining was mainly membranous, with some cytoplasmic staining. There were significant differences in GLUT-1 expression between the four groups (F =39.555, P <0.001). The staining grades of the four groups were 9.500±1.851, 9.875±1.457, 4.250±1.281, and 4.125±0.991, respectively (Fig. 2III ).
Correlation between T/M ratio, Ki67/GLUT-1 expression, and tumor cell densities
In the C6 rat glioma tumor model, the T/M ratio had a significant positive relationship with the expression of Ki67 (r =0.523, P =0.002) and GLUT-1 (r =0.676, P <0.001) and with tumor cell densities (r =0.628, P <0.001). The correlation between the T/M ratio and Ki67 expression, GLUT-1 expression, and tumor cell densities is shown in Fig. 3a–c .
Fig. 3: (a–f) Correlation between T/M ratio, expression of Ki67, expression of GLUT-1, and tumor cell densities. GLUT-1, glucose transporter-1; T/M, tumor/muscle.
Correlation between tumor cell densities and Ki67/GLUT-1 expression
In the C6 rat glioma tumor model, the tumor cell densities had a significant positive relationship with the expression of Ki67 (r =0.750, P <0.001) and also with the expression of GLUT-1 (r =0.734, P <0.001; Fig. 3d and e ).
Correlation between Ki67 and GLUT-1 expression
A significant relationship between Ki67 expression and GLUT-1 expression was found (r =0.584, P <0.001; Fig. 3f ).
Discussion
Radiotherapy, combined with surgery and chemotherapy, is a keystone in glioma treatment 16 17–19 20 18 F-FDG PET/CT, which is based on glucose metabolism, is a well-established tool in diagnostics, tumor staging and restaging, and assessment of response to treatment 21
In the present study, to our knowledge, we reported for the first time a radiosensitization effect of OA on 18 F-FDG uptake in a C6 rat gliomas model using PET/CT imaging. Herein, we demonstrated that the addition of OA to radiation significantly inhibited the glucose uptake and tumor growth, suggesting that OA enhances the effect of radiotherapy in C6 rat glioma models. Furthermore, we compared the PET/CT results with the pathology results, finding high consistency, which would suggest that 18 F-FDG PET/CT would provide more internal information for the tumors.
In our study, the T/M ratios of the four groups showed significant differences on day 1 after treatment, whereas the tumor volumes did not. Changes in functionality that occur before morphological changes would account for this phenomenon 22,23 22,24 in vivo . Interestingly, we found that the tumor volumes of group C had no significant decrease. More swollen areas and inflammatory cells were found in group C, as compared with group D. We believe that the radiation necrosis induced by the treatment may account for this situation 25
Recent studies 26–29 18 F-FDG uptake values have a strong relationship with the Ki67 and GLUT-1 IHC results, and our data are consistent with their findings. GLUT-1 expression related to the radioresistance of tumor at a clinically relevant level has been reported in several studies 30,31
There are a number of limitations in this study. First, the number of experimental animals used is small because of time constraints and experimental conditions, which would explain the observed discrepancy between our study and other studies. Second, we used a rat model rather than a human tumor xenograft model, and thus the result of this study cannot be extrapolated to a clinical study directly. More preclinical experiments are needed to confirm the suitability of OA as a radiosensitizer.
Conclusion
The current study indicates that OA can increase the therapeutic impact of radiotherapy efficiently, which provides more evidence to extend the usage of OA. 18 F-FDG PET/CT, which can detect the therapy-induced reduction of glucose uptake before morphologic imaging, is a potential, sensitive imaging modality for evaluating the effect of radiosensitizers.
Acknowledgements
This study was supported by the Natural Science Foundation of China (NSFC: 81371587). The authors thank Professors Tang Hong and Xiao Liang for their excellent assistance.
Conflicts of interest
There are no conflicts of interest.
References
1. Shah U, Morrison T. A review of the symptomatic management of malignant gliomas in adults. J Natl Compr Canc Netw 2013; 11:424–429.
2. Cuddapah VA, Robel S, Watkins S, Sontheimer H. A neurocentric perspective on glioma invasion. Nat Rev Neurosci 2014; 15:455–465.
3. Omuro A, DeAngelis LM. Glioblastoma and other malignant gliomas: a clinical review. JAMA 2013; 310:1842–1850.
4. Cohen-Jonathan Moyal E. From bench to bedside: experience of the glioblastoma model for the optimization of radiosensitization. Cancer Radiother 2012; 16:25–28.
5. Verheij M, Vens C, van Triest B. Novel therapeutics in combination with radiotherapy to improve cancer treatment: rationale, mechanisms of action and clinical perspective. Drug Resist Updat 2010; 13:29–43.
6. Evangelista L, Cervino AR, Chondrogiannis S, Marzola MC, Maffione AM, Colletti PM, et al.. Comparison between anatomical cross-sectional imaging and
18 F-FDG PET/CT in the staging, restaging, treatment response, and long-term surveillance of squamous cell head and neck cancer: a systematic literature overview. Nucl Med Commun 2014; 35:123–134.
7. Eugene T, Corradini N, Carlier T, Dupas B, Leux C, Bodet-Milin C.
18 F-FDG-PET/CT in initial staging and assessment of early response to chemotherapy of pediatric rhabdomyosarcomas. Nucl Med Commun 2012; 33:1089–1095.
8. Kruse M, Sherry SJ, Paidpally V, Mercier G, Subramaniam RM. FDG PET/CT in the management of primary pleural tumors and pleural metastases. Am J Roentgenol 2013; 201:W215–W226.
9. Histed SN, Lindenberg ML, Mena E, Turkbey B, Choyke PL, Kurdziel KA. Review of functional/anatomical imaging in oncology. Nucl Med Commun 2012; 33:349–361.
10. Schillaci O. Use of dual-point fluorodeoxyglucose imaging to enhance sensitivity and specificity. Semin Nucl Med 2012; 42:267–280.
11. Huang T, Civelek AC, Li J, Jiang H, Ng CK, Postel GC, et al.. Tumor microenvironment-dependent
18 F-FDG,
18 F-fluorothymidine, and
18 F-misonidazole uptake: a pilot study in mouse models of human non-small cell lung cancer. J Nucl Med 2012; 53:1262–1268.
12. Zeng YB, Hsiao HM, Chan SH, Wang YH, Lin YY, Kuo YH, et al.. Synthesis and anti-cancer activity of a glycosyl library of
N -acetylglucosamine-bearing
oleanolic acid . Mol Divers 2014; 18:13–23.
13. Wang J, Yu M, Xiao L, Xu S, Yi Q, Jin W. Radiosensitizing effect of
oleanolic acid on tumor cells through the inhibition of GSH synthesis in vitro. Oncol Rep 2013; 30:917–924.
14. Kramer E, Herman O, Frand J, Leibou L, Schreiber L, Vaknine H. Ki67 as a biologic marker of basal cell carcinoma: a retrospective study. Isr Med Assoc J 2014; 16:229–232.
15. Huang XQ, Chen X, Xie XX, Zhou Q, Li K, Li S, et al.. Co-expression of CD147 and GLUT-1 indicates radiation resistance and poor prognosis in cervical squamous cell carcinoma. Int J Clin Exp Pathol 2014; 7:1651–1666.
16. Juratli TA, Schackert G, Krex D. Current status of local therapy in malignant gliomas: a clinical review of three selected approaches. Pharmacol Ther 2013; 139:341–358.
17. Jayakumar S, Kunwar A, Sandur SK, Pandey BN, Chaubey RC. Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: role of reactive oxygen species, GSH and Nrf2 in radiosensitivity. Biochim Biophys Acta 2014; 1840:485–494.
18. El-Hadaad HA, Wahba HA, Gamal AM, Dawod T. Adjuvant pelvic radiotherapy vs. sequential chemoradiotherapy for high-risk stage I–II endometrial carcinoma. Cancer Biol Med 2012; 9:168–171.
19. Cante D, Franco P, Sciacero P, Girelli G, Marra AM, Pasquino M, et al.. Hypofractionation and concomitant boost to deliver adjuvant whole-breast radiation in ductal carcinoma in situ (DCIS): a subgroup analysis of a prospective case series. Med Oncol 2014; 31:838.
20. Ahmed R, Oborski MJ, Hwang M, Lieberman FS, Mountz JM. Malignant gliomas: current perspectives in diagnosis, treatment, and early response assessment using advanced quantitative imaging methods. Cancer Manag Res 2014; 6:149–170.
21. Sharma P, Khangembam BC, Suman KC, Singh H, Rastogi S, Khan SA, et al.. Diagnostic accuracy of
18 F-FDG PET/CT for detecting recurrence in patients with primary skeletal Ewing sarcoma. Eur J Nucl Med Mol Imaging 2013; 40:1036–1043.
22. Nakatani K, Nakamoto Y, Togashi K. FDG-PET/CT assessment of misty mesentery: feasibility for distinguishing viable mesenteric malignancy from stable conditions. Eur J Radiol 2013; 82:e380–e385.
23. Bhatnagar P, Subesinghe M, Patel C, Prestwich R, Scarsbrook AF. Functional imaging for radiation treatment planning, response assessment, and adaptive therapy in head and neck cancer. Radiographics 2013; 33:1909–1929.
24. Michielsen K, Vergote I, Op de Beeck K, Amant F, Leunen K, Moerman P, et al.. Whole-body MRI with diffusion-weighted sequence for staging of patients with suspected ovarian cancer: a clinical feasibility study in comparison to CT and FDG-PET/CT. Eur Radiol 2014; 24:889–901.
25. Siu A, Wind JJ, Iorgulescu JB, Chan TA, Yamada Y, Sherman JH. Radiation necrosis following treatment of high grade glioma: a review of the literature and current understanding. Acta Neurochir (Wien) 2012; 154:191–201.
26. Sauter AW, Winterstein S, Spira D, Hetzel J, Schulze M, Mueller M, et al.. Multifunctional profiling of non-small cell lung cancer using
18 F-FDG PET/CT and volume perfusion CT. J Nucl Med 2012; 53:521–529.
27. Campanile C, Arlt MJ, Krämer SD, Honer M, Gvozdenovic A, Brennecke P, et al.. Characterization of different osteosarcoma phenotypes by PET imaging in preclinical animal models. J Nucl Med 2013; 54:1362–1368.
28. Begent J, Sebire NJ, Levitt G, Brock P, Jones KP, Ell P, et al.. Pilot study of F(18)-fluorodeoxyglucose positron emission tomography/computerised tomography in Wilms’ tumour: correlation with conventional imaging, pathology and immunohistochemistry. Eur J Cancer 2011; 47:389–396.
29. Ekmekcioglu O, Aliyev A, Yilmaz S, Arslan E, Kaya R, Kocael P, et al.. Correlation of
18 F-fluorodeoxyglucose uptake with histopathological prognostic factors in breast carcinoma. Nucl Med Commun 2013; 34:1055–1067.
30. Yan SX, Luo XM, Zhou SH, Bao YY, Fan J, Lu ZJ, et al.. Effect of antisense oligodeoxynucleotides glucose transporter-1 on enhancement of radiosensitivity of laryngeal carcinoma. Int J Med Sci 2013; 10:1375–1386.
31. Kunkel M, Moergel M, Stockinger M, Jeong JH, Fritz G, Lehr HA, Whiteside TL. Overexpression of GLUT-1 is associated with resistance to radiotherapy and adverse prognosis in squamous cell carcinoma of the oral cavity. Oral Oncol 2007; 43:796–803.
