Research ArticleExperimental Studies

Pre-clinical Validation of Orthotopically-implanted Pulmonary Tumor by Imaging with 18F-Fluorothymidine-Positron Emission Tomography/Computed Tomography

HIROSHI FUSHIKI, SOSUKE MIYOSHI, AKIHIRO NODA, YOSHIHIRO MURAKAMI, HIROSHI SASAKI, MAKOTO JITSUOKA, KEISUKE MITSUOKA, ICHIRO MATSUNARI and SHINTARO NISHIMURA
Anticancer Research November 2013, 33 (11) 4741-4749;

Abstract

The development of positron-emission tomography (PET) and X-ray computed tomography (CT) imaging has improved the detection of tumor burden and, in turn, pre-clinical drug development and clinical treatment. In pre-clinical drug development, clinically-relevant murine cancer models, such as orthotopic models of lung cancer, have provided an accurate representation of tumor burden in humans. However, evidence demonstrating the capability of imaging-guided evaluation of these clinically-relevant models is limited. Here, we combined 18F-fluorothymidine (FLT)-PET/CT imaging and a murine model of human non-small cell lung cancer (NSCLC) to improve the accuracy of anticancer drug evaluation in pre-clinical studies. We found that FLT-PET/CT imaging enabled the progression of pulmonary tumors to be longitudinally monitored rather than FDG-PET/CT. Furthermore, in an efficacy study of a standard treatment of docetaxel in a murine lung cancer model, FLT-PET imaging detected the anticancer response earlier than volumetric analysis by CT imaging. We, thus, observed a relationship between the alteration of FLT signals and Ki-67 index in the pulmonary tumor during the period of chemotherapy. These results indicate that the combination of FLT-PET/CT imaging and an orthotopic NSCLC model is an effective strategy for evaluating clinical efficacy and potential of an anticancer agent during pre-clinical development.

Lung cancer is a global health problem, representing the leading cause of cancer-related deaths among both men and women (1). The 5-year survival rates of patients with lung cancer is extremely low (<15%) compared to rates for those with prostate (99%), breast (89%), and colon (64%) cancer (2). Despite improvements in diagnosis, a large number of patients with non-small cell lung cancer (NSCLC) are initially diagnosed with advanced stage III or IV disease and therefore cannot be treated via surgical resection with curative intent (2, 3). Although many types of novel anticancer drugs have been approved in the past decade, the overall survival rate of patients receiving chemotherapy in stage III and IV remains low (2, 4).

The success of pre-clinical drug development is dependent on the accurate emulation of human tumor pathology and biology in animal cancer models, and the identification of valid end-point indicators of tumor regression and inhibition of metastasis. Models of solid tumors in humans that comprise of tumor xenografts in rodents have been considered inaccurate due to unnatural tumor vascularization and interaction with the host microenvironment. To improve for clinical relevance of studies examining the pathophysiology of lung cancer, several lung cancer models, including orthotopically-implanted and spontaneous tumorigenic models, have been developed (5-8). Orthotopic models of lung cancer in mice are typically established by the injection of human NSCLC cell line suspensions via the trachea or nose, or directly into the lungs (7-10). These models exhibit a similar clinical pathology with regard to tumor formation and micrometastasis in mediastinal lymph node tissues. Due to the complexity of evaluating tumor progression in murine orthotopic lung cancer models, many researchers evaluating novel anticancer drug candidates have utilized subcutaneous xenograft models, which are a less accurate representation of human lung cancer than orthotopic models. However, with recent advances in small-animal imaging technologies, the evaluation of tumor burden in orthotopic lung cancer models has become more feasible (7, 11-13).

Positron emission tomography (PET) with 18F-fluorodeoxyglucose (18F-FDG) is a standard tool for the diagnosis and staging of cancer (14-19). FDG-PET reflects a function of glucose metabolism in tumors, and is to be a sensitive biomarker for the efficacy of anticancer drugs in the clinical and pre-clinical setting. On the other hand, the utility of 3-deoxy-3-18F-fluorothymidine (18F-FLT), a PET tracer specific for cell proliferation based on thymidine kinase I activity, has been widely recognized as being potentially superior to FDG (20-25). Improvement in the accuracy of measurement of tumor proliferation using FLT-PET may enhance the specificity of detecting neoplasms as well as the specificity and accuracy of the evaluation of response toanticancer therapy (26-30).

We previously demonstrated the successful application of computed tomography (CT) and bioluminescent imaging (BLI) in the evaluation of tumor progression and drug efficacy in an orthotopic model of lung cancer (13). Here, to improve the clinical relevance of preclinical efficacy studies in lung cancer, we conducted an PET/CT imaging study and correlative immunohistochemistry analysis in a orthotopic model of lung cancer in athymic nude mice.

Materials and Methods

Ethics statement. All animal experiments were performed with approval from the Animal Ethics Committee of The Medical and Pharmacological Research Center Foundation (Approval No. 2009-3, Hakui, Japan).

Animals and Cells. Calu-6 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were routinely cultured and maintained in minimal essential medium (MEM) plus GlutaMAX medium (Invitrogen, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 17.9 mM sodium bicarbonate. Cells were passaged no more than six times. Male athymic mice (NAnN.Cg-Foxn1nu/CrlCrlj) were obtained from Charles River Laboratories Japan, Inc. (Yokohama, Japan). All mice were maintained and handled in accordance with the recommendations of the National Institute of Health (Bethesda, MD, USA).

Intra-pulmonary tumor cell implantation. Calu-6 cells grown to sub-confluence were harvested with TrypLE Express (Invitrogen), washed once with phosphate-buffered saline (PBS), and resuspended (1×108 cells/ml) in PBS containing 500 μg/ml Matrigel® (BD Biosciences, San Jose, CA, USA). Intra-pulmonary implantation was performed as previously described (13). Briefly, nude mice (six weeks old) were anesthetized with isoflurane (Forane; Abbott Japan, Tokyo, Japan), and a small skin incision was then made on the left chest wall. While monitoring the motion of the left lung, 20 μl of the cell suspension was injected directly into the lung using a 0.3-ml syringe equipped with a 29-gauge needle (Beckton & Dickinson, Tokyo, Japan). The skin incision was then closed with a surgical skin clip.anticancer drug treatment was initiated three weeks after the implantation of tumor cells (day 0). Four mice in each group received an intravenous (i.v.) injection of 15 mg/kg docetaxel (Taxotel; Sanofi Aventis K.K., Tokyo, Japan) for treatment group, or vehicle on days 0, 4, and 7 for control group.

PET tracers. 18F-FDG and FLT were synthesized in-house via nucleophilic fluorination of 3-N-Boc-5’-O-dimethoxytrityl-3’-O-nosyl-thymidine, a commercially available precursor. 18F was produced via an 18O(p,n)18F nuclear reaction by proton bombardment (12 MeV, 50 μA) of an 18O-water target using a cyclotron-target system (OSCAR-12; JFE Engineering Corp., Tokyo, Japan). 18F-FDG was produced by nucleophilic fluorination of mannose triflate after basic hydrolysis of 2-18F-fluoro-1,3,4,6-tetra-O-acetyl-D-glucose. 18F-FLT was synthesized via nucleophilic fluorination of 3–N–Boc–5’-O-dimethoxytrityl-3’-O–nosyl–thymidine.

X-ray CT and PET. X-ray CT and PET scans were conducted using an Inveon Multimodality system (Siemens, Knoxville, TN, USA). Calu-6 tumor-bearing mice received an i.v. injection of 15 MBq of 18F-FDG or FLT. After 50 min, PET scanning was conducted for 10 min followed by CT scanning for 5 min under anesthesia induced by 2.5% isoflurane. For the comparison study of FDG-PET/CT and FLT-PET/CT, imaging acquisitions were performed at five weeks after cell implantation. For longitudinal monitoring of pulmonary tumor, FLT-PET/CT acquisitions were performed at two, three and five weeks after cell implantation. For treatment experiment, CT acquisitions in order to randomize of tumor-bearing mice were performed at two weeks after cell implantation, and FLT-PET/CT acquisitions were performed at day 0, 3, 7 and 14. Data from PET and CT acquisition were reconstructed and analyzed using ASIproVM software (Siemens). Volumes of interests (VOIs) were manually drawn for pulmonary tumors on PET and CT images. With CT images, tumor volume was determined by analyzing VOI, and tumor diameter was determined from the sagittal sections of CT images. To compare tumor volume by volumetric VOI analysis (3D) and by estimation from tumor diameter (1D), 1D-tumor volume was calculated as a sphere (4/3π×diameter3) (31). To compare of the accumulation of 18F-FDG or FLT, the %ID value was calculated using the following formula: %ID= CPET×100/ID, where CPET is the measured radio activity/cc in the VOI, the ID is the input dose of tracer (MBq). In the chemotherapy study, 18F-FLT accumulation data in tumors were normalized to the uptake in normal right lung tissue, and the 18F-FLT accumulation ratio of tumor to right lung tissue is presented as the normalized uptake value (NUV).

Histological analysis. Following the sacrifice of mice in satellite cohort, tracheas were exposed and lungs were filled with 10% neutral buffered formalin (NBF). Lung tissue samples were fixed in NBF overnight at room temperature and then placed into 70% ethanol before embedding in paraffin blocks. Lung tissue samples were sectioned at 3 μm and stained with either hematoxylin-eosin (HE) or anti-human Ki-67 for evaluation of tumor formation and cell proliferation, respectively. For Ki-67 scoring, sectioned lung samples were deparaffinized and labeled using MIB-1 monoclonal antibody (1:50; Dako Japan Inc., Tokyo, Japan) following autoclaving antigen retrieval in citrate buffer (0.01 M, pH 6.0). Antibody binding was detected using the EnVision Dual Link System HRP (Dako Japan) and a hematoxylin counterstain. The labeled tumor cell count was visually assessed over a 4× microscopic field per 1000 cells in pulmonary tumor. The pathologists were blinded to treatment or non-treatment, and the outcome of FLT-PET assessment.

Statistical analysis. Data are presented as the mean±S.E.M. Comparison of the drug treatment group to the control group for each time point was conducted via Student's t-test. Values of p<0.05 were considered statistically significant. All statistical analyses were performed using Prism 4.0 software for Windows (GraphPad Software, Inc., San Diego, CA, USA).

Results

PET/CT imaging in an orthotopic model of lung cancer in mice. To determine the applicability of PET imaging to pulmonary tumor in an orthotopic lung cancer model in mice, PET/CT imaging with 18F-FDG and FLT were performed five weeks after inoculums with Calu-6 human NSCLC cells into the left lung lobe in nude mice. As shown in Figure 1 (red allow), the implanted pulmonary tumor burden was detected in the left lung lobe by CT, with the benefit of obtaining morphological and spatial information. Regarding the image of FDG-PET, FDG was specifically accumulated in tumor and heart in the thoracic cavity. On imaging by FLT-PET, FLT was accumulated in lung tumor and the tumor burden was clearly visualized with low background. By fusing CT and PET images, we were able to determine the position and condition of lung tumor. As many previous reports have shown, the accumulation of FDG in tumor is higher than FLT (%ID/g: 5.17 and 1.77, in FDG and FLT, respectively). However, there were some high background signals with FDG-PET in the chest, including the heart and skeletal muscle around the lung, indicating that it was sometimes hard to recognize the tumor. In contrast, we easily observed the pulmonary tumor in FLT-PET images, with the advantage of a low background in the thorax. These results suggest that FLT-PET/CT imaging enabled us to detect the pulmonary tumor nodule better than with FDG-PET/CT in this model.

Longitudinal monitoring of pulmonary tumor burden by FLT-PET/CT imaging. To assess the application of longitudinal FLT-PET imaging to pulmonary tumor burden in a murine orthotopic model of lung cancer, FLT-PET/CT imaging was performed two, three, and five weeks after the inoculation of Calu-6 human NSCLC cells into the left lung lobe of nude mice. As shown in Figure 2 (red arrows), the implanted pulmonary tumor burden was detected in the left lung lobe by CT, which was able to reveal morphological and spatial information during the examination period (left panel). Regarding the FLT-PET image, FLT accumulated in the lung tumor, and the tumor burden was clearly visualized, with a low background, at three and five weeks after inoculation, but not at two weeks. These results indicate that in addition to CT imaging, FLT-PET can also detect for pulmonary tumor nodule in a murine model of orthotopic lung cancer.

Histological assessment of tumor regression by docetaxel. To monitor the cellular morphological and proliferation profile in lung tumors during drug treatment, serial histological evaluations of orthotopically-implanted pulmonary tumors were conducted. As shown in Figure 3, three weeks after orthotopic transplantation of Calu-6 human NSCLC tumor cells to mice, docetaxel was administered (15 mg/kg, i.v.) at day 0, 3 and 7. HE staining of lung tissue from tumor-bearing mice revealed that Calu-6 tumors were located in lung parenchyma and progressed continuously (Figure 4, control). While a marked reduction in the size of pulmonary tumors after docetaxel treatment was observed by day 14 (p<0.01), no significant changes were detected by days 3 or 7 (Figure 4, docetaxel panel and lower graph). In contrast, Ki-67 staining of lung tissue bearing Calu-6 tumors showed a significant reduction in proliferative cell numbers following docetaxel treatment on days 3 (p<0.05) and 7 (p<0.001) (Figure 5). These results clearly indicate that tumor regression in response to docetaxel treatment was first detected by the measurement of cell proliferation on day 3 and tumor size on day 14.

Detection of tumor regression due to docetaxel using FLT-PET/CT. Prior to chemotherapy, analysis via Response Evaluation Criteria in Solid Tumors (RECIST) (31) with CT imaging two weeks after tumor implantation showed no difference in tumor size between the control and treatment groups. Sequential FLT-PET/CT acquisition and docetaxel administration (15 mg/kg, i.v.) were initiated three weeks after tumor inoculation (day 0), day3 and day7. Prior to treatment with docetaxel, we confirmed that a single tumor nodule was clearly detected in the lungs of all mice by both CT and FLT-PET (Figure 6A, day 0). Analysis of 18F-FLT accumulation using PET imaging and volumetric analysis of tumors from CT imaging demonstrated a clear difference in tumor progression following docetaxel treatment (Figure 6B and C). Although the accumulation of 18F-FLT in pulmonary tumors increased in a time-dependent manner in the control group, docetaxel administration resulted in reduced FLT accumulation on day 3 (Figure 6B, 57.5% reduction, p=0.149). A significant decline in the accumulation of 18F-FLT was also observed on day 7 (Figure 6, 82.7% reduction, p<0.05) and 14 (Figure 6B, 96.6% reduction, p<0.01). Based on volumetric analysis from CT data, no changes in tumor volume were detected on day 3; however, the tumor volume was slightly reduced in the docetaxel-treated group on day 7, (Figure 6C, 34.8% reduction, p=0.337), and significant regression of the tumor burden was observed on day 14 (Figure 6C, 94.2% reduction, p<0.001).

RECIST in pre-clinical studies. Although we have described here the utility of FLT-PET for tumor imaging in a murine models of orthotopic lung cancer, CT is still the most commonly used technique for detecting pulmonary tumors in the clinical setting. In addition, RECIST has been widely used for clinically evaluating tumor response, however, there is little evidence to support the use of RECIST in pre-clinical studies. Here, we performed a quantitative analysis of the efficacy of docetaxel in a murine orthotopic lung cancer model via 3D (volumetric) and 1D (unidimensional) analysis of pulmonary tumors based on RECIST (Figure 7). Using both measurement parameters, tumor regression induced by docetaxel was detected by day 14 (p<0.01). We also performed a linear regression model analysis to examine the co-linearity between 1D and 3D analysis of tumors. Following the conversion of tumor size as determined from 1D analysis to 3D units (see Materials and Methods), we observed a strong correlation between the 1D and 3D analysis of tumor size (r2=0.7190, p<0.0001).

Figure 1.
Figure 1.

PET/CT imaging of FDG and FLT in an orthotopic lung cancer model. Mice received an intra-pulmonary injection of Calu-6 (human NSCLC) cells (2×106 cells in 20 μl). Five weeks after transplantation, CT (upper) and PET (middle) acquisition with FDG and FLT were performed, and fusion images from CT and PET images were made by commercially available software (bottom). Red arrows and H indicate pulmonary tumor and heart, respectively.

Figure 2.
Figure 2.

Longitudinal monitoring of pulmonary tumor burden by FLT-PET/ CT imaging. Mice received an intra-pulmonary injection of Calu-6 cells (2×106 cells in 20 μl). CT (A) and PET (B) acquisition with 18F-FLT were performed two, three, and five weeks after transplantation. Red arrows and H indicate pulmonary tumor and heart, respectively.

Figure 3.
Figure 3.

Animal study design. CT imaging and grouping of tumor-bearing mice revealed tumor growth two weeks after orthotopic lung tumor implantation. Immediately prior to docetaxel treatment, a baseline pre-therapy FLT-PET/CT image was acquired. Longitudinal FLT-PET/CT imaging was performed 3, 7, and 14 days after the initiation of therapy. At each time point of imaging acquisition, histological samples were obtained from satellite groups of tumor-bearing mice.

Figure 4.
Figure 4.

Histopathological evaluation of orthotopically-implanted Calu-6 human NSCLC pulmonary tumors treated with docetaxel. Mice received an intra-pulmonary implantation of a Calu-6 (human NSCLC) cells (2×106 cells in 20 μl). Docetaxel (15 mg/kg) was administered three weeks after transplantation. After the initiation of docetaxel treatment (15 mg/kg, i.v., administered on days 0, 3, and 7), Calu-6 pulmonary tumors were collected from the docetaxel-treatment group mice on days 3, 7, and 14, and from control group mice on days 0, 3, 7, and 14 (n=4-6). All tumor samples were stained with hematoxylin-eosin (H/E). Representative images of H/E staining of tumors (A), and maximum diameter of pulmonary tumors (B) are shown. Data represent the mean±SEM. *p<0.05, **p<0.01, compared with control by Student's t-test.

Discussion

The application of PET, which is already utilized for the diagnosis of pulmonary tumors, to the evaluation of anticancer drugs might facilitate successful pre-clinical studies and in turn clinical studies. Early assessment of the efficacy of anticancer drugs is important for the estimation of the responsiveness of tumors to treatment and the improvement of patient outcome. For pre-clinical studies, the early assessment of the efficacy of candidate compounds with PET imaging technique may be a critical step in translational research to clinical development. Although several pre-clinical studies have demonstrated that PET with 18F-FDG or 18F-FLT can demonstrate an earlier assessment of anticancer effect in subcutaneous lung cancer xenografts (32-35), PET assessment of subcutaneous xenograft models has potential concerns due to poor blood supply (36, 37). In the present study we demonstrated that FLT-PET imaging enabled earlier assessment of docetaxel efficacy than volumetric CT imaging in a murine model of orthotopic lung cancer. Our findings clearly indicate that the evaluation of tumor progression and drug efficacy using combined PET and CT imaging is an effective approach for the translation of preclinical studies to clinical studies, and may facilitate for prediction of the clinical efficacy of drugs during development.

Murine models of orthotopic lung cancer have unique advantages over subcutaneous models, such as representing an accurate tumor microenvironment and demonstrating anticancer drug efficacy. While application of these models to translational research has been difficult due to complexity of data evaluation, recent advances in in vivo imaging technology have enabled the use of the ‘unevaluable’ murine model of orthotopic lung cancer in the investigation of lung cancer biology and drug discovery. Out of the several approaches and routes of administration for generating orthotopic models of pulmonary tumors in mice, we utilized the intrapulmonary injection of human NSCLC Calu-6 cells directly into the lung, as this method results in the formation of a single tumor nodule in the lung lobe. This method may further bring the advantage of separating the tumor nodule from mediastinal tissue, including the heart, trachea, and bronchi, and enabling longitudinal evaluation of tumor progression or drug responses with tomographic imaging modalities. Despite these advantages, higher uptake of FDG in skeletal muscle and heart make the discrimination of pulmonary tumor difficult. With the demonstration of PET/CT imaging in this model, FLT-PET/CT enabled for visualization of a pulmonary tumor nodule better than FDG-PET/CT did. In addition, contrast-enhanced CT imaging in this model showed that the tumor nodule was surrounded by newly-synthesized blood vessels connected to the pulmonary vein (unpublished data). This observation suggests that the pulmonary tumor in this model enables tumor to obtain fresh supply of oxygen, and PET tracers can then access the tumor burden in this model more easily than in subcutaneous models. Our present findings successfully demonstrate the application of FLT-PET/CT imaging to a murine model of orthotopic lung cancer and may prove clinically relevant in the evaluation of tumor progression and anticancer drug efficacy.

Figure 5.
Figure 5.

Docetaxel treatment results in a decrease in the number of Ki-67-positive cells in orthotopically-implanted Calu-6 human NSCLC pulmonary tumors. Mice received an intrapulmonary implantation of the Calu-6 (human NSCLC) cell line (2×106 cells in 20 μl). Treatment with docetaxel (15 mg/kg) was administered three weeks after transplantation. Calu-6 pulmonary tumors were collected from docetaxel-treatment group mice on days 3, 7, and 14 after initiation of docetaxel treatment (15 mg/kg, i.v., administered on days 0, 3, and 7) and from control group mice on days 0, 3, 7, and 14 (n=4-6). All tumor samples were stained with antibody against human Ki-67. Representative images of Ki-67 immunohistochemical staining from tumors (A), and quantitative measurement of Ki-67-positive cells in pulmonary tumors (B) are shown. Measurements for two out of the four mice in the docetaxel-treatment group on Day 14 were not determined due to insufficient tumor cell numbers. Data represent the mean±SEM. *p<0.05, ***p<0.001, compared with control by Student's t-test.

Figure 6.
Figure 6.

FLT-PET/CT imaging of orthotopically-implanted pulmonary tumors in docetaxel-treated mice. Mice received an intrapulmonary injection of the Calu-6 (human NSCLC) cell line (2×106 cells in 20 μl). Tumor diameters in the lungs were determined by CT acquisition and mice were randomly assigned into study groups four weeks after tumor inoculation. Docetaxel (15 mg/kg) was administered three weeks after transplantation. CT and FLT-PET images were taken on the indicated days after the first treatment. Representative images of the four mice for each group are shown. Red arrows indicate pulmonary tumors (A). Summary of the quantitative analysis of FLT-PET (B) and CT (C) are shown. Data represent the mean±SEM (n=4). Arrows indicate when docetaxel was administered. *p<0.05, **p<0.01, compared with control using Student's t-test.

Figure 7.
Figure 7.

Relationship of RECIST and volumetric analysis as determined by tumor evaluation by CT imaging. Three-dimensional volumetric (A) and 1D diameter (B) quantitative analysis of pulmonary tumors using CT imaging. Data represent the mean±SEM. **p<0.01, n=4 compared with control by Student's t-test. (C) Relationship of tumor volume calculated from tumor diameter (x-axis), and tumor volume determined from CT imaging (y-axis).

PET and CT imaging do have the potential for inaccurate evaluation of imaging data due to the partial volume effect (38). A proportion of our PET and CT data may therefore be inaccurate due to this effect. However, the earlier assessment by FLT-PET is based on data from day 3, in which an equal tumor volume was observed in control and docetaxel treatment groups, in contrast to the decrease in FLT accumulation in docetaxel-treated tumor burden (Figure 6B and C). In addition, we also observed a similar trend in the reduction of FLT accumulation and loss of Ki-67-positive cells in tumors (Figure 5). Furthermore, the period for detecting tumor regression in morphological and histological evaluation was comparable to that of CT and FLT-PET imaging (Figure 6B and C). These observations are consistent with previous reports of FLT-PET enabling the early detection of decreases in tumor burden following anticancer drug therapy (22, 24, 39).

In a clinical setting, the measurement of pulmonary tumors is predominantly performed by CT imaging. Generally, the assessment of lung cancer using CT imaging follows RECIST; however, several limitations to this approach warrant mention, including the potential for tumor misdiagnosis (40). By analyzing tumor progression with CT imaging, we identified a significant correlation between the 3D volumetric and 1D analyses according to RECIST (Figure 7). RECIST based on 1D tumor measurements has been shown to be equivalent to WHO criteria based on bi-dimensional measurements for the purpose of assessing tumor response to therapy (31). Although these criteria only include the evaluation of tumor diameter in response to chemotherapy, findings via our analysis using volumetric data include two sets of clinical criteria based on the evaluation of tumor diameter. Collectively, these results indicate that the widely used clinical methodology for tumor measurement is also applicable to the murine model of orthotopic lung cancer, and that evaluation of overall tumor burden based on RECIST is suitable for evaluation of lung cancer in preclinical and clinical studies (41).

In conclusion, we demonstrated that FLT-PET imaging in a murine model of orthotopic tumor can detect early response of pulmonary tumors to anticancer drugs. To our knowledge, this is the first demonstration of FLT-PET/CT imaging in such a model. We further confirmed that RECIST is a valid approach for the evaluation of tumor burden in a clinically relevant murine model of orthotopic lung cancer. A combined strategy among FLT-PET and RECIST, with advantages regarding earlier assessment and conventional monitoring of drug efficacy in FLT-PET and RECIST, respectively, provides us an accurate evaluation of drug candidates in preclinical lung cancer research. FLT-PET/CT may therefore serve as a useful tool for evaluating the efficacy of anticancer agents with regard to preclinical development and clinical efficacy.

Acknowledgements

The Authors wish to thank Dr. Grierson for his helpful discussion and Taishiro Kimura and Jun Mizusawa for their technical assistance.

Footnotes

  • Conflicts of Interest

    None.

  • Received September 17, 2013.
  • Revision received October 21, 2013.
  • Accepted October 22, 2013.

References

  1. ↵
    1. Mountain CF
    : Revisions in the International System for Staging Lung Cancer. Chest 111: 1710-1717, 1997.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Siegel R,
    2. Naishadham D,
    3. Jemal A
    : Cancer statistics, 2013. CA Cancer J Clin 63: 11-30, 2013.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Herbst RS,
    2. Heymach JV,
    3. Lippman SM
    : Lung cancer. N Engl J Med 359: 1367-1380, 2008.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Janku F,
    2. Stewart DJ,
    3. Kurzrock R
    : Targeted therapy in non-small cell lung cancer – Is it becoming a reality? Nat Rev Clin Oncol 7: 401-414, 2010.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Frese KK,
    2. Tuveson DA
    : Maximizing mouse cancer models. Nat Rev Cancer 7: 645-658, 2007.
    OpenUrlCrossRefPubMed
    1. Sharpless NE,
    2. Depinho RA
    : The mighty mouse: Genetically engineered mouse models in cancer drug development. Nat Rev Drug Discov 5: 741-754, 2006.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Onn A,
    2. Isobe T,
    3. Itasaka S,
    4. Wu W,
    5. O'Reilly MS,
    6. Ki Hong W,
    7. Fidler IJ,
    8. Herbst RS
    : Development of an orthotopic model to study the biology and therapy of primary human lung cancer in nude mice. Clin Cancer Res 9: 5532-5539, 2003.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Doki Y,
    2. Murakami K,
    3. Yamaura T,
    4. Sugiyama S,
    5. Misaki T,
    6. Saiki I
    : Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in mice. Br J Cancer 79: 1121-1126, 1999.
    OpenUrlCrossRefPubMed
    1. Hoffman RM
    : Orthotopic metastatic mouse models foranticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 17: 343-359, 1999.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Wilderman MJ,
    2. Sun J,
    3. Jassar AS,
    4. Kapoor V,
    5. Khan M,
    6. Vachani A,
    7. Suzuki E,
    8. Kinniry PA,
    9. Sterman DH,
    10. Kaiser LR,
    11. Albelda SM
    : Intrapulmonary IFN-β gene therapy using an adenoviral vector is highly effective in a murine orthotopic model of bronchogenic adenocarcinoma of the lung. Cancer Res 65: 8379-8387, 2005.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Olive KP,
    2. Tuveson DA
    : The use of targeted mouse models for preclinical testing of novel cancer therapeutics. Clin Cancer Res 12: 5277-5287, 2006.
    OpenUrlAbstract/FREE Full Text
    1. Hollingshead MG
    : Antitumor efficacy testing in rodents. J Natl Cancer Inst 100: 1500-1510, 2008.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Fushiki H,
    2. Kanoh-Azuma T,
    3. Katoh M,
    4. Kawabata K,
    5. Jiang J,
    6. Tsuchiya N,
    7. Satow A,
    8. Tamai Y,
    9. Hayakawa Y
    : Quantification of mouse pulmonary cancer models by microcomputed tomography imaging. Cancer Sci 100: 1544-1549, 2009.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Adler LP,
    2. Blair HF,
    3. Makley JT,
    4. Williams RP,
    5. Joyce MJ,
    6. Leisure G,
    7. al-Kaisi N,
    8. Miraldi F
    : Noninvasive grading of musculoskeletal tumors using PET. J Nucl Med 32: 1508-1512, 1991.
    OpenUrlAbstract/FREE Full Text
    1. Kalff V,
    2. Hicks RJ,
    3. MacManus MP,
    4. Binns DS,
    5. McKenzie AF,
    6. Ware RE,
    7. Hogg A,
    8. Ball DL
    : Clinical impact of 18F fluorodeoxyglucose positron emission tomography in patients with non-small-cell lung cancer: A prospective study. J Clin Oncol 19: 111-118, 2001.
    OpenUrlAbstract/FREE Full Text
    1. Gambhir SS
    : Molecular imaging of cancer with positron-emission tomography. Nat Rev Cancer 2: 683-693, 2002.
    OpenUrlCrossRefPubMed
    1. Rohren EM,
    2. Turkington TG,
    3. Coleman RE
    : Clinical applications of PET in oncology. Radiology 231: 305-332, 2004.
    OpenUrlCrossRefPubMed
    1. Cullinane C,
    2. Dorow DS,
    3. Kansara M,
    4. Conus N,
    5. Binns D,
    6. Hicks RJ,
    7. Ashman LK,
    8. McArthur GA,
    9. Thomas DM
    : An in vivo tumor model exploiting metabolic response as a biomarker for targeted drug development. Cancer Res 65: 9633-9636, 2005.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Pieterman RM,
    2. van Putten JW,
    3. Meuzelaar JJ,
    4. Mooyaart EL,
    5. Vaalburg W,
    6. Koeter GH,
    7. Fidler V,
    8. Pruim J,
    9. Groen HJ
    : Preoperative staging of non-small cell lung cancer with positron-emission tomography. N Engl J Med 343: 254-261, 2000.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Shields AF,
    2. Grierson JR,
    3. Dohmen BM,
    4. Machulla HJ,
    5. Stayanoff JC,
    6. Lawhorn-Crews JM,
    7. Obradovich JE,
    8. Muzik O,
    9. Mangner TJ
    : Imaging proliferation in vivo with [F-18]FLT and positron emission tomography. Nat Med 4: 1334-1336, 1998.
    OpenUrlCrossRefPubMed
    1. Buck AK,
    2. Schirrmeister H,
    3. Hetzel M,
    4. Von Der Heide M,
    5. Halter G,
    6. Glatting G,
    7. Mattfeldt T,
    8. Liewald F,
    9. Reske SN,
    10. Neumaier B
    : 3-Deoxy-3-[18F]fluorothymidine-positron emission tomography for noninvasive assessment of proliferation in pulmonary nodules. Cancer Res 62: 3331-3334, 2002.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Vesselle H,
    2. Grierson J,
    3. Muzi M,
    4. Pugsley JM,
    5. Schmidt RA,
    6. Rabinowitz P,
    7. Peterson LM,
    8. Vallieres E,
    9. Wood DE
    : In vivo validation of 3’deoxy-3’-[18F]fluorothymidine ([18F]FLT) as a proliferation imaging tracer in humans: Correlation of [18F]FLT uptake by positron-emission tomography with Ki-67 immunohistochemistry and flow cytometry in human lung tumors. Clin Cancer Res 8: 3315-3323, 2002.
    OpenUrlAbstract/FREE Full Text
    1. Muzi M,
    2. Vesselle H,
    3. Grierson JR,
    4. Mankoff DA,
    5. Schmidt RA,
    6. Peterson L,
    7. Wells JM,
    8. Krohn KA
    : Kinetic analysis of 3’-deoxy-3’-fluorothymidine PET studies: Validation studies in patients with lung cancer. J Nucl Med 46: 274-282, 2005.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Kenny LM,
    2. Vigushin DM,
    3. Al-Nahhas A,
    4. Osman S,
    5. Luthra SK,
    6. Shousha S,
    7. Coombes RC,
    8. Aboagye EO
    : Quantification of cellular proliferation in tumor and normal tissues of patients with breast cancer by [18F]fluorothymidine-positron emission tomography imaging: evaluation of analytical methods. Cancer Res 65: 10104-10112, 2005.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. de Langen AJ,
    2. Klabbers B,
    3. Lubberink M,
    4. Boellaard R,
    5. Spreeuwenberg MD,
    6. Slotman BJ,
    7. de Bree R,
    8. Smit EF,
    9. Hoekstra OS,
    10. Lammertsma AA
    : Reproducibility of quantitative 18F-3’-deoxy-3’-fluorothymidine measurements using positron emission tomography. Eur J Nucl Med Mol Imaging 36: 389-395, 2009.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Barthel H,
    2. Cleij MC,
    3. Collingridge DR,
    4. Hutchinson OC,
    5. Osman S,
    6. He Q,
    7. Luthra SK,
    8. Brady F,
    9. Price PM,
    10. Aboagye EO
    : 3’-Deoxy-3’-[18F]fluorothymidine as a new marker for monitoring tumor response to antiproliferative therapy in vivo with positron-emission tomography. Cancer Res 63: 3791-3798, 2003.
    OpenUrlAbstract/FREE Full Text
    1. Leyton J,
    2. Latigo JR,
    3. Perumal M,
    4. Dhaliwal H,
    5. He Q,
    6. Aboagye EO
    : Early detection of tumor response to chemotherapy by 3’-deoxy-3’-[18F]fluorothymidine positron emission tomography: the effect of cisplatin on a fibrosarcoma tumor model in vivo. Cancer Res 65: 4202-4210, 2005.
    OpenUrlAbstract/FREE Full Text
    1. Pio BS,
    2. Park CK,
    3. Pietras R,
    4. Hsueh WA,
    5. Satyamurthy N,
    6. Pegram MD,
    7. Czernin J,
    8. Phelps ME,
    9. Silverman DH
    : Usefulness of 3’-[F-18]fluoro-3’-deoxythymidine with positron-emission tomography in predicting breast cancer response to therapy. Mol Imaging Biol 8: 36-42, 2006.
    OpenUrlCrossRefPubMed
    1. Solit DB,
    2. Santos E,
    3. Pratilas CA,
    4. Lobo J,
    5. Moroz M,
    6. Cai S,
    7. Blasberg R,
    8. Sebolt-Leopold J,
    9. Larson S,
    10. Rosen N
    : 3’-Deoxy-3’-[18F]fluorothymidine positron-emission tomography is a sensitive method for imaging the response of BRAF-dependent tumors to MEK inhibition. Cancer Res 67: 11463-11469, 2007.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Sohn HJ,
    2. Yang YJ,
    3. Ryu JS,
    4. Oh SJ,
    5. Im KC,
    6. Moon DH,
    7. Lee DH,
    8. Suh C,
    9. Lee JS,
    10. Kim SW
    : [18F]Fluorothymidine positron emission tomography before and seven days after gefitinib treatment predicts response in patients with advanced adenocarcinoma of the lung. Clin Cancer Res 14: 7423-7429, 2008.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Therasse P,
    2. Arbuck SG,
    3. Eisenhauer EA,
    4. Wanders J,
    5. Kaplan RS,
    6. Rubinstein L,
    7. Verweij J,
    8. Van Glabbeke M,
    9. van Oosterom AT,
    10. Christian MC,
    11. Gwyther SG
    : New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 92: 205-216, 2000.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Pardo OE,
    2. Latigo J,
    3. Jeffery RE,
    4. Nye E,
    5. Poulsom R,
    6. Spencer-Dene B,
    7. Lemoine NR,
    8. Stamp GW,
    9. Aboagye EO,
    10. Seckl MJ
    : The fibroblast growth factor receptor inhibitor PD173074 blocks small cell lung cancer growth in vitro and in vivo. Cancer Res 69: 8645-8651, 2009.
    OpenUrlAbstract/FREE Full Text
    1. Ullrich RT,
    2. Zander T,
    3. Neumaier B,
    4. Koker M,
    5. Shimamura T,
    6. Waerzeggers Y,
    7. Borgman CL,
    8. Tawadros S,
    9. Li H,
    10. Sos ML,
    11. Backes H,
    12. Shapiro GI,
    13. Wolf J,
    14. Jacobs AH,
    15. Thomas RK,
    16. Winkeler A
    : Early detection of erlotinib treatment response in NSCLC by 3’-deoxy-3’-[F]-fluoro-L-thymidine ([F]FLT) positron-emission tomography (PET). PLoS One 3: e3908, 2008.
    OpenUrlCrossRefPubMed
    1. Takeuchi S,
    2. Zhao S,
    3. Kuge Y,
    4. Zhao Y,
    5. Nishijima K,
    6. Hatano T,
    7. Shimizu Y,
    8. Kinoshita I,
    9. Tamaki N,
    10. Dosaka-Akita H
    : 18F-Fluorothymidine PET/CT as an early predictor of tumor response to treatment with cetuximab in human lung cancer xenografts. Oncol Rep 26: 725-730, 2011.
    OpenUrlPubMed
  35. ↵
    1. Lee SJ,
    2. Kim EJ,
    3. Lee HJ,
    4. Kim SY,
    5. Oh SJ,
    6. Ryu JS,
    7. Moon DH,
    8. Ahn JH,
    9. Kim SW
    : A pilot study for the early assessment of the effects of BMS-754807 plus gefitinib in an H292 tumor model by [18F]fluorothymidine positron-emission tomography. Invest New Drugs 31: 506-515, 2013.
    OpenUrlPubMed
  36. ↵
    1. Ebenhan T,
    2. Honer M,
    3. Ametamey SM,
    4. Schubiger PA,
    5. Becquet M,
    6. Ferretti S,
    7. Cannet C,
    8. Rausch M,
    9. McSheehy PM
    : Comparison of [18F]-tracers in various experimental tumor models by PET imaging and identification of an early response biomarker for the novel microtubule stabilizer patupilone. Mol Imaging Biol 11: 308-321, 2009.
    OpenUrlCrossRefPubMed
  37. ↵
    1. O'Reilly T,
    2. Wartmann M,
    3. Brueggen J,
    4. Allegrini PR,
    5. Floersheimer A,
    6. Maira M,
    7. McSheehy PM
    : Pharmacokinetic profile of the microtubule stabilizer patupilone in tumor-bearing rodents and comparison of anticancer activity with other MTS in vitro and in vivo. Cancer Chemother Pharmacol 62: 1045-1054, 2008.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Soret M,
    2. Bacharach SL,
    3. Buvat I
    : Partial-volume effect in PET tumor imaging. J Nucl Med 48: 932-945, 2007.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Zander T,
    2. Scheffler M,
    3. Nogova L,
    4. Kobe C,
    5. Engel-Riedel W,
    6. Hellmich M,
    7. Papachristou I,
    8. Toepelt K,
    9. Draube A,
    10. Heukamp L,
    11. Buettner R,
    12. Ko YD,
    13. Ullrich RT,
    14. Smit E,
    15. Boellaard R,
    16. Lammertsma AA,
    17. Hallek M,
    18. Jacobs AH,
    19. Schlesinger A,
    20. Schulte K,
    21. Querings S,
    22. Stoelben E,
    23. Neumaier B,
    24. Thomas RK,
    25. Dietlein M,
    26. Wolf J
    : Early prediction of nonprogression in advanced non-small cell lung cancer treated with erlotinib by using [18F]fluorodeoxyglucose and [18F]fluorothymidine positron emission tomography. J Clin Oncol 29: 1701-1708, 2011.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Wahl RL,
    2. Jacene H,
    3. Kasamon Y,
    4. Lodge MA
    : From RECIST to PERCIST: Evolving Considerations for PET response criteria in solid tumors. J Nucl Med 50(Suppl 1): 122S-150S, 2009.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Namati E,
    2. Thiesse J,
    3. Sieren JC,
    4. Ross A,
    5. Hoffman EA,
    6. McLennan G
    : Longitudinal assessment of lung cancer progression in the mouse using in vivo micro-CT imaging. Med Phys 37: 4793-4805, 2010.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Pre-clinical Validation of Orthotopically-implanted Pulmonary Tumor by Imaging with 18F-Fluorothymidine-Positron Emission Tomography/Computed Tomography
HIROSHI FUSHIKI, SOSUKE MIYOSHI, AKIHIRO NODA, YOSHIHIRO MURAKAMI, HIROSHI SASAKI, MAKOTO JITSUOKA, KEISUKE MITSUOKA, ICHIRO MATSUNARI, SHINTARO NISHIMURA
Anticancer Research Nov 2013, 33 (11) 4741-4749;
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords