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Basic Science Investigations |
1 Department of Nuclear Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Japan
2 Department of Tracer Kinetics, Graduate School of Medicine, Hokkaido University, Sapporo, Japan
3 Department of Radiology, Nikko Memorial Hospital, Muroran, Japan
4 Department of Pathology, Hokkaido Gastroenterology Hospital, Sapporo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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(HIF-1) was also immunostained. Results: ARG images showed that intratumoral 18F-FDG distribution was heterogeneous. The accumulation of 18F-FDG in the CT region was the highest, which was 1.6 and 2.3 times higher than those in the PT and NA regions, respectively (P < 0.001). The expression levels of Glut-1, Glut-3, and HK-II were markedly higher in the CT region (P < 0.001) compared with those in the PT region. The intratumoral distribution of 18F-FDG significantly correlated with the expression levels of Glut-1, Glut-3, and HK-II (r = 0.923, P < 0.001 for Glut-1; r = 0.829, P < 0.001 for Glut-3; and r = 0.764, P < 0.01 for HK-II). The positive staining of HIF-1 was observed in the CT region. Conclusion: These results demonstrate that intratumoral 18F-FDG distribution corresponds well to the expression levels of Glut-1, Glut-3, and HK-II. The elevated expression levels of Glut-1, Glut-3, and HK-II, induced by hypoxia (HIF-1), may be contributing factors to the higher 18F-FDG accumulation in the CT region.
Key Words: 18F-FDG • glucose transporters • hexokinase • heterogeneity • tumor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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5 structurally related proteins (constituting a family of glucose transporters, Glut-1 to Glut-5) (7,8). Significantly elevated expression levels of Glut-1 and Glut-3 are considered to be a factor contributing to the accumulation of 18F-FDG in malignant tumors (9–13). It has also been suggested that the activity level of hexokinase-II (HK-II) contributes to 18F-FDG accumulation in various malignant tumors (14). These studies, however, used PET or tissue-counting techniques and correlated average 18F-FDG accumulation in the tumor with protein expression. It is well known that various components, including nonmalignant components, are involved in most solid tumors (15–17). Tumor tissues also show intratumoral heterogeneity in their various properties, which may originate from the diverse phenotypic properties of tumor cells or may be induced by their metabolic microenvironment (18–20). In this regard, intratumoral heterogeneity in 18F-FDG distribution has been well demonstrated by autoradiography (ARG) (15). However, there have been few reports regarding the biologic mechanisms involved in the intratumoral heterogeneous distribution of 18F-FDG. The relationships between the intratumoral distribution of 18F-FDG and the regional expression of glucose transporters or hexokinases remain to be investigated. Such data should be helpful in understanding the mechanism of 18F-FDG uptake in malignant tumors and should provide the biologic basis for diagnosing, staging, and prognosticating malignant tumors and monitoring therapy response by 18F-FDG PET.
To clarify factors inducing heterogeneous 18F-FDG distribution, we determined in this study the intratumoral distribution of 18F-FDG by ARG and compared it with the regional expression levels of Glut-1, Glut-3, and HK-II in a rat model of malignant tumor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ARG Studies
The frozen samples were cut into 20-µm sections with a CM3050-Cryostat (Leica) at –20°C. The tumor sections were placed in a phosphor image plate cassette, together with a set of calibrated standards (17), and an overnight ARG exposure was used to detect the distribution of 18F-FDG. The tumor sections (10 µm) adjacent to those used for the ARG studies were stained with hematoxylin and eosin (HE) for use as the reference to determine the regions of interest (ROIs) on the autoradiograms.
The ARG images were analyzed using a computerized imaging analysis system (BAS 5000 Bio-Imaging Analyzer; Fuji Photo Film Co., Ltd.). The resolution of ARG of the BAS 5000 is 100 µm. To quantitatively evaluate the distribution of 18F-FDG radioactivity, 32 ROIs (0.36 mm2) were determined on each ARG image. ROIs were placed to cover mainly the central (CT; n = 8) and peripheral (PT; n = 8) regions of the viable tumor tissues, necrotic/apoptotic (NA; n = 8) regions, and the surrounding muscle (SM; n = 8), by referring to the sections stained with HE (Fig. 1); each ROI was selected microscopically by a pathologist and identified by its predominant histologic characteristic as a region of the viable tumor cells and necrotic/apoptotic cells in the CT and PT regions on the HE section. Large necrotic regions occasionally observed in the center of the tumors were excluded from the evaluation. To avoid any bias, these ROIs were determined in a blind manner for ARG images and immunologic staining (Gluts and HK-II). The ROIs placed on HE images were transferred to ARG images by using computer software (MCID-M2 Image Analyzer; Imaging Research Inc.). Briefly, coordinates were set on both HE and ARG images, the coordinates of each ROI on the HE images was determined, and then each ROI was transferred to the same coordinates of the corresponding ARG images. The radioactivity in each ROI was shown by photostimulated luminescence per unit area, PSL/mm2 (PSL = a·D·t: a = constant; D = radioactivity exposed on imaging plate; t = exposed time); then each count of PSL/mm2 from a tumor section was recorded and used to calculate the mean counts per mm2 and converted to the percentage injected dose per gram (%ID/g) of tissue by using activity of the standards, with the assumption that tissue density is 1 g/cm3 (17,24). The mean radioactivities of the 8 ROIs determined for the CT, PT, NA regions, and SM, respectively, were used to evaluate the 18F-FDG accumulation in the tissues.
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(mouse antihypoxia-inducible factor 1 monoclonal IgG 2b, clone H167; Novus Biologicals) using the method of Zhong et al. (26) with slight modification. Briefly, after deparaffinization and rehydration, the slides were initially immersed in a target retrieval solution (10 mmol/L ethylenediaminetetraacetic acid, pH 8.0) and heated in a microwave oven (500 W) for 20 min. After the antigen retrieval, endogenous peroxidase activity was blocked for 5 min in methanol containing 3% hydrogen peroxide. Thereafter, endogenous nonspecific antigens were blocked in 10% hog albumin (Cosmo Bio., Ltd.) for 10 min and then incubated overnight with the primary antibody at 4°C. Finally, the bound antibody was visualized using the ABC procedure with the HISTOFINE MAX-PO (M) kit (Nichirei) and 3,3'-diaminobenzidine tetrahydrochloride. Tumor sections adjacent to those used for the immunostaining were stained with HE.
For immunohistochemical grading, ROIs placed on the HE-stained sections were transferred to immunologically stained sections as described. The intensity of staining and the percentage of positively stained cells in the CT (n = 8) and PT (n = 8) regions of the viable tumor tissues were evaluated microscopically. The intensity of staining (intensity) was graded from 0 to 3 (0 = not stained, 1 = equivocal, 2 = intense, and 3 = very intense) according to the criteria of Higashi et al. (27). Moreover, the percentage of positively stained cells (% positive) was classified from 1 to 5 (1 = 0%–20%, 2 = 20%–40%, 3 = 40%–60%, 4 = 60%–80%, and 5 = 80%–100%). The expression levels of Gluts and HK-II were assessed semiquantitatively using the product of these scores (intensity x % positive) (28). In HIF-1 staining, cells with completely and darkly stained nuclei were regarded as positively stained cells (29).
Statistical Analysis
All values are expressed as mean ± SD. One-way ANOVA and the Bonferroni post hoc test were used to assess the significance of differences due to the intratumoral distribution of 18F-FDG. To evaluate the significance of differences in the expression levels of Glut-1, Glut-3, and HK-II (intensity x % positive) between the CT and PT regions, an unpaired Student t test was performed. Simple regression analysis was used to compare the intratumoral 18F-FDG distribution and the expression levels of Glut-1, Glut-3, and HK-II. A 2-tailed P value < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Immunohistochemical Staining
The typical immunostaining of Glut-1, Glut-3, and HK-II in the CT and PT region is shown in Figure 3. The intensity and extent of staining of Glut-1, Glut-3, and HK-II were markedly higher in the CT region than those in the PT region.
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was clearly observed in the CT region (Fig. 5) but not in the PT region.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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was observed in these regions. Intratumoral 18F-FDG distribution significantly correlated with the expression levels of Glut-1, Glut-3, and HK-II. Consequently, the regional expression levels of Glut-1, Glut-3, and HK-II may increase in a hypoxic environment within tumor tissues and may contribute to heterogeneous 18F-FDG distribution in the tumor tissues. Although intratumoral heterogeneity in 18F-FDG distribution has been demonstrated (15), there have been few reports with regard to the biologic mechanisms involved in the intratumoral heterogeneous distribution of 18F-FDG. The present results showed a significant correlation between the intratumoral distribution of 18F-FDG and the regional expression level of Glut-1, which is consistent with the results of syngeneic rat mammary cancer reported by Brown et al. (30). In addition, our results demonstrated—to our knowledge, for the first time—that intratumoral 18F-FDG distribution significantly correlates with the regional expression levels of Glut-3 and HK-II. The heterogeneity in 18F-FDG distribution may be ascribed to the altered expression levels of Glut-1, Glut-3, and HK-II and may reflect a metabolic microenvironment of tumors.
Aggressive tumors often have insufficient blood supply. Hypoxia occurs in tissue that is >100–200 µm away from a functional blood supply. When the tumors are exposed to a hypoxic environment, HIF-1 is activated to promote the transcription of several genes, including glucose transporters and glycolytic enzymes (31). The increased uptake of 3H-FDG in vitro by hypoxic tumor cells has been well demonstrated (32). Recently, Dearling et al. (33) have extended the previous in vitro work and confirmed the selectivity of 18F-FDG for hypoxic regions over normoxic regions in vivo. In the present study, a relatively higher 18F-FDG accumulation was observed in the CT region, with elevated expression levels of Glut-1, Glut-3, and HK-II. The positive staining of HIF-1 was observed in these regions. Taken altogether, the regional expression levels of Glut-1, Glut-3, and HK-II may be increased by HIF-1 in a hypoxic environment within the tumor tissue and may contribute to the elevated 18F-FDG accumulation.
Several studies have focused on the expression of Gluts and hexokinase activity to define the role of these proteins in the regulation of 18F-FDG accumulation (34,35). Elevated expression levels of Glut-1 and Glut-3 are considered to be factors that contribute to the accumulation of 18F-FDG in malignant tumors (9–13,23,28). It has also been suggested that the activity level of HK-II contributes to 18F-FDG accumulation in various malignant tumors (14). Chromatographic, polyclonal antibody, and amino acid analyses indicated that rat hepatoma hexokinase is most closely related to HK-II and suggests that mitochondrial hexokinase activity determines the rate of accumulation of 18F-FDG in tumors (36). These studies, however, used PET or tissue-counting techniques and correlated average 18F-FDG accumulation in the tumor with protein expression. The biologic mechanisms involved in intratumoral heterogeneity in 18F-FDG distribution have not been fully investigated. The present results demonstrate that the expression levels of Glut-1, Glut-3, and HK-II also contribute to intratumoral heterogeneity in 18F-FDG distribution in our model rats, as determined using an ARG technique. It is crucial to consider the relative contributions of Glut-1, Glut-3, and HK-II to intratumoral 18F-FDG accumulation. Unfortunately, we could not determine the relative contributions of these proteins, mainly due to the limited number of samples used in this study. Further studies are required to clarify the respective contributions of glucose transporters and hexokinase to intratumoral heterogeneity in 18F-FDG accumulation.
Intratumoral 18F-FDG distribution has been described at the cellular level by several investigators (15–20). Kubota et al. showed that 18F-FDG preferentially accumulates in macrophages and young granulation tissues surrounding necrotic foci rather than in tumor cells using a malignant tumor mouse model (15). In contrast, Brown et al. (17) observed relatively less 3H-FDG accumulation in necrotic/inflammatory infiltration compared with that in the tumor cells. Our results in rats confirmed intratumoral heterogeneity in 18F-FDG distribution. The 18F-FDG accumulation in the CT regions was 1.6 and 2.3 times higher than those in the PT and NA regions, respectively. Our results are consistent with the results reported by Brown et al.
In this study, the regions of viable tumor tissues and NA regions in ARG were identified using the HE-stained sections as reference. Apoptosis was most reliably assayed by morphologic counts using HE staining. Since there are some apoptotic cancer cells interwoven in necrotic regions and it was difficult to clearly distinguish apoptotic cells from necrotic cells in the present study, the term NA regions is used. Viable tumor cells and necrotic/apoptotic cells were also interwoven; thus, we used relatively small ROIs to clearly divide the regions between the viable tumor cells and necrotic/apoptotic cells. The larger ROIs appeared to cover both of the viable tumor cells and necrotic/apoptotic cells. Brown et al. (17) also used such ROI analysis to evaluate intratumoral distribution of 18F-FDG. On the other hand, because necrotic cells revealed cell injuries in morphology—such as the cell membrane appearing ruptured, the nuclear chromatin being markedly condensed or pyknotic, vesicular structures filling the cytoplasm, and fusion of organelles (37)—the immunohistochemical staining of Glut-1, Glut-3, and HK-II cannot reflect the antigenicity of cells. Therefore, the expression levels of Glut-1, Glut-3, and HK-II were not investigated by immunostaining and semiquantitative evaluation in the NA regions. The slices immunohistochemically stained for Glut-1, Glut-3, and HK-II were from the formalin-fixed, paraffin-embedded specimens, but those used in ARG imaging to determine regional 18F-FDG distribution were from the frozen samples. Thus, selected areas in the immunohistochemical staining for Glut-1, Glut-3, and HK-II of the CT, PT, and NA regions were not exactly congruent with those for determining regional 18F-FDG distribution on ARG imaging, although both formalin-fixed, paraffin-embedded specimens and frozen samples were adjacent. It is important to note that expression of Glut-1, Glut-3, and hexokinase II (as determined in this study) does not generally imply increased functional activity. Several studies have demonstrated that hexokinase bound to the mitochondrial membrane has a much higher catalytic activity than cytosolic hexokinase. Aloj et al. (14) have also indicated that 18F-FDG uptake correlates better with 18F-FDG phosphorylating activity of mitochondrial preparations than with the level of expression of the Glut-1 or hexokinase I or II genes. The excellent correlation between HK-II expression and 18F-FDG uptake observed in the present study may not be representative for other tumor models.
In our study, ARG imaging was performed 1 h after 18F-FDG injection. The 1-h time point is widely used also in rodent models, and no significant differences in the 18F-FDG uptake were observed in the mouse tumor tissues between 1 and 2 h after 18F-FDG injection (33). However, further studies at more than one time point are needed to give an indication of the time course of 18F-FDG uptake and to compare with the present results in rodent-bearing xenografts.
18F-FDG PET has become increasingly important not only for detecting and staging malignant tumors but also for monitoring therapy response and for differentiating malignant lesions from benign lesions (1–4). However, variable tumor accumulation of 18F-FDG has been indicated to prevent accurate diagnosis by 18F-FDG PET (38,39). Intratumoral heterogeneity in 18F-FDG distribution may also affect such diagnostic accuracy. The present study provides a biologic basis of intratumoral heterogeneity in 18F-FDG distribution, which leads to better understanding of the mechanism of 18F-FDG accumulation in tumors and contributes to the accurate diagnosis of patients with malignant tumors by 18F-FDG PET. On the other hand, recent advances in radiation therapy such as intensity-modulated radiation therapy have exploited new areas for the use of 18F-FDG PET (40). Regions of high 18F-FDG uptake can be treated with a higher radiation dose compared with a hypometabolic portion of the same mass. In addition, glucose transporters (Glut-1 and Glut-3) and glycolytic enzymes (for example, hexokinase) are promoted by activated HIF-1 (31). The present study showed an excellent correlation between regional 18F-FDG uptake and the expression of facilitative glucose transporters (Glut-1 and Glut-3) and HK-II. Furthermore, this study found that 18F-FDG uptake was more intense in tumor regions that express HIF-1. Recent clinical data indicate that hypoxic tumors are known to be more malignant, to be more likely to metastasize, and to have a poor prognosis. HIF-1 is considered to be a key factor for tumor progression by upregulating genes involved in angiogenesis, cell survival, cell invasion, and resistance to drug therapy and radiotherapy. Thus, the findings of this study suggest that tumor areas with high 18F-FDG uptake may represent biologically more aggressive cancer cells. This has important consequences for the use of 18F-FDG PET for treatment planning—particularly, intensity-modulated radiation therapy. 18F-FDG PET could help to identify regions for dose-escalation protocols. We believe that our data also provide useful information in a precision radiation therapy protocol with modern radiotherapy modalities.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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For correspondence or reprints contact: Nagara Tamaki, MD, Department of Nuclear Medicine, Graduate School of Medicine, Hokkaido University, Kita 15 Nishi 7, Kita-ku, Sapporo 060-8638, Japan.
E-mail: natamaki{at}med.hokudai.ac.jp
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| JOURNAL OF NUCLEAR MEDICINE TECHNOLOGY | THE JOURNAL OF NUCLEAR MEDICINE |
, CT region; 
, PT region.