Detecting functional changes with [18F]FAZA in a renal cell carcinoma mouse model following sunitinib therapy
© Chapman et al.; licensee Springer. 2014
Received: 13 February 2014
Accepted: 5 May 2014
Published: 10 September 2014
The multitargeting tyrosine kinase inhibitor (TKI) sunitinib is currently the first-line drug therapy for metastasizing renal cell carcinoma (RCC). TKIs have profound effects on tumor angiogenesis, leading to modifications of the tumor microenvironment. The goal of this study was to determine whether these treatment-induced changes can be detected with [18F]FAZA.
The present study utilized positron emission tomography (PET) to analyze tumor oxygenation status during and after sunitinib therapy in the murine Caki-1 RCC tumor model. Dynamic and static scans were performed, as well as ex vivo biodistributions at 3 h post injection (p.i.). Immunohistochemical analysis of tumor tissue was carried out for the quantification of pimonidazole binding and the hypoxia-associated factors CD-31, Ki-67, and Von Willebrand factor (VWF). In addition, in vitro cellular uptake studies were done to analyze the direct effects of sunitinib on the Caki-1 cells.
During therapy with sunitinib (40 mg/kg/day), uptake of [18F]FAZA into Caki-1 mice decreased by 46±5% (n = 4; 5 days) at 3 h post injection (p.i.) during the first study and 22±5% (n = 8; 9 days) during the long-term study, indicating a decrease in the tumor's hypoxia level. However, when drug therapy was stopped, this effect was reversed completely, and the tumor [18F]FAZA uptake increased to 126±6% (n = 6) of the control tumor uptake, indicative of an even higher level of tumor hypoxia compared to the therapy starting point. Sunitinib had no direct effect on [18F]FAZA uptake into Caki-1 cells in vitro.
[18F]FAZA PET could be used to monitor drug response during sunitinib therapy in RCC and may guide combination therapies based on the tumor's hypoxia status.
Renal cell carcinoma (RCC) is the third most diagnosed urological malignancy, with 75% of cases being the clear cell RCC histological subtype . RCC arises in the proximal tubule of the nephron within the kidney; its symptoms present late, often preventing diagnosis early enough to lead to a favorable prognosis . The American Cancer Society estimated 65,150 new cases and 13,680 deaths in the USA in 2013, a significant increase compared to the 2005 prediction statistics of 36,160 new cases and 12,660 deaths ,. Between 2005 and 2009, the overall incidence of kidney cancer in the USA increased 3.1% per year , which is comparable to the observed 2% increase over the past two decades worldwide and within Europe . Barring early intervention, many patients will experience metastatic disease and require systemic therapy. Metastatic RCC (mRCC) rarely shows a complete and durable response to immuno- or radiation therapy . Significant improvements in patient management have been achieved, however, with the development of targeted small molecules including tyrosine kinase inhibitors (TKIs) such as sunitinib, sorafenib, pazopanib, and axitinib -. Two types of TKIs are known to date. Termed type I and type II, they differ in their mode of action at the ATP binding site of the tyrosine kinase . Sunitinib represents a TKI of type I, which targets mainly the vascular endothelial growth factor receptor 2 (VEGFR 2) and the platelet-derived growth factor receptors (PDGFR) α and β, the stem cell growth factor c-KIT (CD117), the RET proto-oncogene, and the Fms-like tyrosine kinase 3 (FLT-3). In addition, sunitinib has also been shown to inhibit 72 other kinases ,, justifying its classification as a multitargeting receptor kinase inhibitor ,. Sunitinib is currently approved for the treatment of gastrointestinal stromal tumors (GIST), pancreatic neuroendocrine tumors, and as first-line therapy for mRCC ,.
Sunitinib not only acts directly on tumors cells (GIST and RCC) but also indirectly by disrupting the development of blood vessels, leading to increased microenvironmental stress on the tumor tissue . A number of receptor tyrosine kinases are controlled by multikinase inhibitors such as sunitinib and sorafenib. They modulate hypoxia-inducible factor 1α (HIF-1α), indicating that antiangiogenic effects might be achieved not only by direct action on vascular endothelial cells but also by blocking compensatory hypoxia- and ligand-induced changes in HIF-1α and HIF-2α expression in the target tumor cells . HIF-1α levels are controlled by the oxygen tension within a cell. Under hypoxic conditions, HIF-1α is activated and, in turn, triggers the production of proteins such as VEGF, PDGF, TGF, EGF, GLUT1 and CA IX, which are all essential for angiogenesis ,.
Positron emission tomography (PET), a non-invasive imaging method, is a tool well suited for in vivo detection and characterization of hypoxia in a tumor's microenvironment ,. As a clinical tool, PET can be helpful in the diagnosis, outcome prediction, and therapy monitoring of RCC cancer patients . Its broader clinical utilization in this context is, however, subject to discussion . Several PET radiotracers for the imaging of tumor hypoxia have been developed, such as the 2-nitroimidazoles [18F]FMISO, [18F]FAZA, [18F]EF5, and the copper-64-based thiosemicarbazone [64Cu]ATSM ,. Among them, [18F]FMISO is the most extensively studied and widely used PET radiotracer for quantitative assessment of tumor hypoxia in both preclinical and clinical settings . [18F]FAZA and [64Cu]ATSM may also be useful, depending on the patient cohort. [18F]FAZA, because of its lower lipophylicity and therefore faster blood and non-target tissue clearance, exhibits superior image contrast . [18F]FAZA and [18F]FMISO yield tumor-to-kidney ratios that are similar to each other and larger when compared to [64Cu]ATSM -. At present, it is unknown which hypoxia PET radiotracer would provide the greatest tumor-to-background ratio in RCC patients.
PET radiotracers were used in several clinical and numerous preclinical studies to detect tumor hypoxia and predict and analyze tumor response to chemotherapy. These tracers may play a role in guiding hypoxia-specific treatment in cancer patients . Most commonly, [18F]FMISO and [18F]FDG were used to detect effects of a therapy with TKIs such as sunitinib or sorafenib -. To date, only one preclinical study was performed using [18F]FAZA . Here, the effects of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, were monitored. The current study utilizes [18F]FAZA to detect functional changes in the microenvironment of a renal cell carcinoma mouse model during sunitinib therapy and subsequent withdrawal. It is known that a TKI therapy regimen, which is typically split into therapeutic fractions, induces a withdrawal flare phenomenon in RCC patients ,. The goal of this study was to determine whether these treatment-induced changes in tumor hypoxia can be detected and quantified using a PET radiotracer for hypoxia in an animal model. This ability could help optimize the timing of combination therapy in metastatic RCC patients. [18F]FAZA has been chosen because of its lower lipophylicity and elevated clearance.
All animal experiments were carried out in compliance with the guidelines of the Canadian Council on Animal Care and with approval from the local Animal Care Committee of the Cross Cancer Institute under the protocol number AC 11183. Caki-1 is a human epithelial kidney clear cell carcinoma cell line which expresses the wild-type von Hippel-Lindau tumor suppressor protein and was derived from a metastatic site. Donor tumors in mice were generated by injecting approximately 100,000 Caki-1 tumor cells subcutaneously into a BALB/c nude mouse (Charles River Laboratories, Quebec, Canada). Once the tumors reached a suitable size of 400 to 500 mm3, they were fragmented into roughly equal size of 2×2-mm tissue chunks and implanted into the left and right flanks of the experimental BALB/c nude mice. The tumors became palpable after 5 days, and when they reached an appropriate size of 5 to 10 mm in diameter, they were randomly assigned to the experimental groups: one receiving sunitinib therapy, the other a control group receiving vehicle injections only. Three experimental setups were performed: (A) short-term effects of sunitinib (5 days of therapy) on tumor oxygenation, (B) long-term sunitinib therapy (14 days of therapy; 5 days on, 2 days off), and (C) treatment withdrawal (another 12 days after finishing therapy). After 4, 9, and 14 days of treatment, as well as the 12 days after treatment withdrawal, selected mice from both groups were analyzed with PET. Sunitinib malate (Sutent™) was a gift from Pfizer Canada (Kirkland, Quebec, Canada). The mice in the treatment groups received intraperitoneal injection of 40 mg/kg/day of sunitinib malate in saline/1%DMSO; the control groups received vehicle injections (saline/1%DMSO).
Positron emission tomography
PET detection of tumor hypoxia was performed on BALB/c nude mice bearing subcutaneous Caki-1 tumors following the injection of 1-(5-deoxy-5-[18F]fluoro-α-d-arabinofuranosyl)-2-nitroimidazole ([18F]FAZA). Radiosynthesis of [18F]FAZA was performed in automated synthesis units according to a standard literature procedure . The precursor for the [18F]FAZA synthesis, 1-(2,3-diacetyl-5-tosyl-(α-d-arabinofuranosyl)-2-nitroimidazole, was purchased from ABX GmbH (Radeberg, Germany). For PET experiments, a catheter was placed into the tail vein of the sedated mouse. Under inhalation anesthesia, the mice were placed in prone position with their medial axis parallel to the axis of the microPET® R4 scanner (Siemens Preclinical Solutions, Knoxville, TN, USA) and their thorax, abdomen, and hind legs centered in the field of view. A transmission scan for attenuation correction was not acquired. [18F]FAZA (4 to 5 MBq in 100- to 150-µL solution - H2O/max. 8% EtOH and diluted with normal saline (NS)) was injected intravenously through the catheter into the tail vein. For the different experimental protocols, an emission scan of either 180-min (dynamic experiments in setup A) or 10-min (static scans in setup B) PET acquisitions was subsequently performed. The dynamic list mode data were sorted into sinograms with 65 time frames (10 × 2, 8 × 5, 6 × 10, 6 × 20, 8 × 60, 10 × 120, 5 × 300, and 12 × 600 s). The frames were reconstructed using ordered subset expectation maximization (OSEM) or maximum a posteriori (MAP) reconstruction modes. The pixel size was 0.085 × 0.085 × 0.12 cm, and the resolution in the center field of view was 1.8 mm. Correction for partial volume effects was not performed. The image files were further processed using the ROVER v2.0.51 software (ABX GmbH). Masks defining 3D regions of interest (ROI) were set, and the ROIs were defined by thresholding. ROIs covered all visible tumor mass of the subcutaneous tumors, and the thresholds were defined by 50% of the maximum radioactivity uptake level of each Caki-1 tumor in each animal. Mean standardized uptake values (SUVmean = (activity/mL tissue)/(injected activity/body weight), mL/g) were calculated for each ROI. For comparison, SUVmax was also analyzed for the static PET experiments. Time-activity curves (TAC) were generated for the dynamic scans only. All semi-quantified PET data are presented as means ± SEM.
After each PET scan, the mice were immediately sacrificed by cervical dislocation, and the tumors and muscle tissue were collected in scintillation vials, weighed, and counted using a Wizard-3 1480 automatic gamma counter (Perkin-Elmer Life Sciences, Woodbridge, ON, Canada). Biodistribution experimental results were analyzed as percentage of injected dose per gram of tissue (%ID/g) or as SUV. All biodistribution values were obtained at 3 h post injection (p.i.) of the radiotracer. Data are presented as means ± SEM.
Based on investigations by Verwer et al. , a reversible two-tissue compartment model with blood volume parameter was applied to the data sets. For this purpose, an ROI defining the heart content was generated by thresholding the averaged early time frames which visualized the blood flow through the heart. The plasma input TAC generated from this ROI was convolved with the compartmental model and numerically fitted to the tumor TACs of the same animal. This procedure was implemented in Matlab software (version R2014a), utilizing a Nelder-Mead simplex direct search. The fit was governed by the minimization of the sum over all time points of square differences between the measured values and the model prediction.
Following euthanization, the tumor tissue samples were collected for preparation of formalin-fixed paraffin-embedded tissue slides. One hour (short-term experiment with 5-day sunitinib treatment only) or 3 h (long-term experiment with 14 days of sunitinib therapy plus therapy withdrawal) prior to sacrificing the animal, pimonidazole HCl (Hypoxyprobe, Inc., Burlington, MA, USA) was injected intraperitoneally at a dose of 60 mg/kg. Additionally, immunostaining for platelet endothelial cell adhesion molecule (PECAM) CD-31 (rabbit polyclonal anti-mouse CD-31, Santa Cruz Biotechnology Inc., Dallas, TX, USA), Von Willebrand factor (rabbit polyclonal von Willebrand factor antibody), and Ki67 (rabbit monoclonal anti-mouse Ki67, Abcam Inc., Toronto, ON, Canada) were performed to illustrate changes in neoangiogenesis and vascular density. Briefly, following rehydration and antigen retrieval, endogenous peroxidase activity was blocked using methanol containing 0.3% hydrogen peroxide. After that, the sections were incubated for 1 h with the subsequent primary monoclonal antibody. The bound antibodies were visualized using DakoCyomation Envision-horseradish peroxidase and chromagen using the EnVision™ FLEX kit (Dako Canada, Burlington, Ontario, Canada). The slides were counterstained with hemotoxylin and analyzed under a microscope (Axiocam HR, Zeiss, Oberkochen, Germany). The images were edited with Adobe® Photoshop (Adobe Systems Inc., San Jose, CA, USA) to remove connective and non-tumor tissue surrounding the tumor. Then, using MetaMorph® Microscopy Automation & Image Analysis Software (Molecular Devices, LLC, Sunnyvale, CA, USA), color thresholds were defined for the entire tumor area and the specific antibody bound tissue only. Percent of binding was calculated from the resulting number of pixels for each of the investigated factors.
In vitro cell uptake
The human renal cell carcinoma cell line, Caki-1 (American Tissue Cell Culture (ATCC)), and for comparison, the human mammary carcinoma cell line MCF-7 (a gift from Dr. David Murray, Cross Cancer Institute, University of Alberta) were used. The cell lines were cultured at 37°C and 5% CO2 (standard conditions) in F-12/DMEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% penicillin/streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, and 1% non-essential amino acids. Using glass cell culture dishes with a diameter of 55 mm, the cells were plated at a density of approximately 500,000 cells per plate and incubated for 48 h under normal conditions at 37°C and 5% CO2. Following that time, half of the plates were taken for culturing the cells under hypoxic conditions over the next 24 h; the others were kept under normal cell culture conditions. Hypoxic cell culture conditions were achieved using a special sealed vacuum chamber device (Division of Experimental Oncology, Department of Oncology, University of Alberta) in which O2 was almost entirely replaced with N2 (O2 < 0.1%) . For the radiotracer incubation, the medium was replaced with Krebs-Ringer buffer (115 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCl2, 25 mM NaHCO3, and 5 mM glucose at a pH of 7.4) at room temperature. Around 2.5 to 3.5 MBq of [18F]FAZA, containing buffer was added per plate and incubated for 4 h under either normal or hypoxic conditions. Sunitinib maleate 90 µL of 10‒4‒M (in DMSO) was added to selected plates to analyze the effect of sunitinib on cellular [18F]FAZA binding. After incubation, the cells were rinsed three times with ice-cold Krebs-Ringer buffer to stop further cell uptake and then immediately lysed using 1 mL 5% trichloroacetic acid (TCA) for 1 h. One plate of each condition was used for protein quantification using the BCA protein assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's recommendations, and bovine serum albumin was used as the protein standard. Following the 1-h lysis with TCA, the supernatants were collected and placed into scintillation vials to be counted using the Wizard gamma counter (Wallac 1480 Wizard-3, Perkin-Elmer) in order to determine radioactivity uptake levels in the cells. Radiotracer cell uptake levels were normalized to percent of the total added radioactivity per milligram protein (% radioactivity/mg protein). Data are presented as means ± SEM.
Effect of sunitinib on Caki-1 tumor growth
Characterization of the Caki-1 tumor model with [18F]FAZA
Effect of sunitinib on [18F]FAZA uptake and trapping in vivo
Uptake and retention of [18F]FAZA in Caki-1 tumors was further analyzed by kinetic modeling using a two-compartment model . All four kinetic parameters k1 to k4 were calculated for control as well as for the sunitinib-treated tumors (Figure 3, bottom right). In the presence of sunitinib, all constants were found to be increased, significantly even for k2 and k4 with a p < 0.05. Once diffused into the cell, [18F]FAZA either diffuses back out of the cell or is reduced for further intracellular binding. The first process is governed by the rate constant k2 and is elevated under sunitinib therapy, as is the latter, which is governed by k3. The ratio k3/(k2 + k3), however, remains unchanged, indicating that once it has crossed the cell membrane, a similar fraction of [18F]FAZA will be further reduced in the presence or absence of sunitinib. Once reduced, re-oxygenation of [18F]FAZA is governed by the rate constant k4. It is amplified in the presence of sunitinib, resulting in the observed reduction in uptake under treatment (Figure 3). Analysis of the fractional blood volume (fbv) revealed similar values in the absence and presence of sunitinib: 0.160 ± 0.032 and 0.148 ± 0.036 (n = 4), respectively, indicating similar overall perfusion of the tumor volume (yet not necessarily the microenvironment, which can display hypoxia).
Monitoring therapy effects and withdrawal of sunitinib with [18F]FAZA
Following the investigation of short-term effects of sunitinib on tumor oxygenation, a second experimental series was performed in order to analyze the longitudinal effects of anti-angiogenic therapy on Caki-1 tumor oxygenation. Figure 5 summarizes the PET results with [18F]FAZA for the effect of sunitinib during the therapy and after therapy withdrawal. Treated Caki-1 tumors clearly showed higher radioactivity uptake levels than the control tumors, confirming that in the presence of sunitinib, the amount of tumor hypoxia is decreased in the tumor microenvironment. After 9 days of sunitinib therapy (Figure 5, left), the SUVmean,3h of control tumors was found to be 0.24 ± 0.02, while the SUVmean,3h of sunitinib-treated tumors was 0.19 ± 0.01 (n = 8; p < 0.05). Similar results were obtained when analyzing SUVmax values, which are more relevant for clinical use: 0.38 ± 0.03 (control) versus 0.29 ± 0.02 (9 days of sunitinib; n = 8, p < 0.05). Pimonidazole staining from tissue slices confirmed a reduction in hypoxia of the treated tumors. Twelve days after sunitinib therapy was withdrawn, [18F]FAZA uptake into the treated tumors increased again, leading now to even higher uptake levels than the control tumors (Figure 5, right): SUVmean, 3 h of 0.18 ± 0.01 (control) versus 0.23 ± 0.01 (sunitinib-treated; n = 6, p < 0.05). Analysis of SUVmax revealed the following values: 0.30 ± 0.02 (control) versus 0.36 ± 0.02 (after therapy withdrawal; n = 6, p < 0.05). This was indicative of higher levels of hypoxia and corresponded to a flare effect after withdrawal of sunitinib. The pimonidazole staining confirmed this phenomenon with similar levels of binding in both the control and the treated groups.
In vitro cell uptake
Four main findings of the current study may be summarized as follows: (i) xenograft Caki-1 RCC tumors show uptake of [18F]FAZA, revealing a base level of hypoxia; (ii) sunitinib therapy reduces [18F]FAZA tumor uptake, indicating reduced tumor hypoxia; (iii) TKI withdrawal flare phenomenon was observed after sunitinib was discontinued, resulting in a once more increased tumor hypoxia; and (iv) sunitinib did not influence the direct cellular uptake and retention of [18F]FAZA in the Caki-1 and MCF-7 cells under normoxic or hypoxic conditions in vitro.
Currently, treatment of metastatic RCC with the TKI sunitinib is recommended as one of the first therapy options ,,. Sunitinib causes reduced tumor size or growth rate. In the current study, smaller Caki-1 tumor sizes were observed in the treated mice. Meta-analysis of metastatic RCC studies has shown that higher exposure to sunitinib is correlated with improved treatment outcomes. Patients with the highest exposure to sunitinib had longer times to progression, and the probability of decreases in tumor size or of halting tumor growth was higher in patients with the highest exposure to sunitinib ,. However, after therapy, Caki-1 tumors reached similar sizes again, indicating that the tumors regained their molecular characteristics which are essential for tumor development and growth. Changes occurring during therapy with sunitinib seem to be reversed after treatment disruption, leading to further tumor progression. The goal in patients would be to have a maximum exposure time to sunitinib which is associated with longer time to tumor progression, longer overall survival, and greater decrease in tumor size .
Therapy of metastatic RCC with TKIs can be monitored with non-invasive imaging methods to analyze therapy success as well as predict patient outcomes ,. Several preclinical and clinical studies have been carried out to analyze therapeutic effects of a TKI such as sunitinib or sorafenib utilizing PET -. Besides monitoring tumor metabolism in RCC with [18F]FDG, [18F]FMISO was used to analyze effects on tumor oxygenation status and [18F]FLT for determination of proliferation. During the present study, [18F]FAZA was used. Although [18F]FAZA in comparison to [18F]FMISO may exhibit a better clearance pattern from non-targeting muscle tissue based on its lower lipophylicity, there is still a debate about the radiotracer of choice for imaging tumor hypoxia -. In Caki-1 tumors, [18F]FAZA uptake amounted to 1.5% to 2.2% ID/g after 3 h p.i. As analyzed from the dynamic PET studies, initial uptake reached a maximum level after approximately 40 to 60 min which did not further increase. Image contrast with [18F]FAZA is achieved based on the non-target tissue clearance over time. In Caki-1 tumors, a tumor-to-muscle ratio of approximately 1.5 was reached after 3 h p.i.. The current findings are in line with observations in other murine and human xenograft models of different origin, where 1.3% to 3% ID/g or even lower levels were measured at the same time p.i. ,,. Similar to the Caki-1 model, low uptake of [18F]FMISO was found in A498, another human RCC xenografted tumor model .
PET is an ideal non-invasive imaging tool to use for monitoring response to therapy. However, there are still ongoing discussions of how patients would benefit from monitoring tumor hypoxia status and how hypoxia-directed therapy approaches could be used clinically ,. During the present study, sunitinib therapy led to a reduction of tumor hypoxia levels by 22% to 46% as determined with [18F]FAZA. Although sunitinib mainly targets tumor vasculature and function, it can be concluded that it also leads to a reduction of tumor hypoxia. As Verwer et al.  have proposed, a reversible two-tissue compartment model best describes uptake through passive diffusion and initial retention based on a chemical reduction of a nitroimidazole such as [18F]FAZA. Both steps are reversible: higher oxygen levels can re-oxygenate the reduced radiotracer, and [18F]FAZA may leave cells again via diffusion into the plasma. Kinetic analysis of the present dynamic data revealed that k1 to k4 were all elevated in the presence of sunitinib, k2 and k4 even significantly. Increased k1 shows that sunitinib-treated tumors allow even faster cellular uptake of [18F]FAZA. Delivery of the radiotracer through the tumor vasculature is rather improved and not impaired. On the other hand, elevated k2 indicates a higher rate of back diffusion into the plasma for treated mice. One can conclude that in this case, more re-oxygenated [18F]FAZA is present in the intracellular compartment. The intracellular presence or absence of [18F]FAZA is determined in this work to be due chiefly to the difference in k4, i.e., the re-oxygenation of the reduced [18F]FAZA, which is significantly elevated under sunitinib therapy. The perfused percentage of each tumor, as determined by the fbv, was found to be equal (within error margins) in each case, meaning that delivery of the radiotracer through the blood vessels is not impaired under sunitinib therapy. In summary, sunitinib treatment leads to decreased uptake in tumor cells due to increased re-oxygenation and back diffusion of [18F]FAZA caused by overall lower hypoxia levels.
Congruent with the current findings, sunitinib also reduces [18F]FMISO uptake by 22% to 35% in metastatic RCC patients . The EGFR-blocker gefitinib showed even higher inhibition of [18F]FAZA uptake (60% to 70%) in xenograft A431 human squamous cell carcinoma . Similar observations with [18F]FAZA and [18F]FMISO were described in A431 tumor-bearing mice under therapy with CI-1033, a pan-Erb-B inhibitor . Studies with [18F]FDG analyzing glucose metabolism revealed 17% to 75% reduction in uptake during a sunitinib therapy -, although with high variability for basic [18F]FDG levels in RCC patients ,, reducing its utility in the clinics. In addition, the proliferation marker [18F]FLT also showed a 16% reduced uptake in RCC patients in the presence of sunitinib . Taken together, this supports the conclusion that systemic therapy with the TKI sunitinib achieves therapeutic efficacy by decreasing tumor hypoxia, metabolism, and proliferation. Monitoring of tumor oxygen levels is feasible in lesions with a well-defined population of hypoxic cells. This allows detection of reduced hypoxia during sunitinib therapy, while non-hypoxic metastases would not be expected to change .
Immunostaining with pimonidazole did only show a tendency for decreased binding to hypoxic cells in the short-term study. This may be explained by the short incubation time of only 1 h compared to 3 h in the long-term study. As the dynamic PET study reveals, effects of sunitinib on [18F]FAZA are likewise not detectable at 1 h p.i. In theory, pimonidazole should behave similarly to [18F]FAZA since both are 2-nitroimidazoles. Pimonidazole binding was reduced after 3 h p.i. by 30% which is similar to the 21% reduction of [18F]FAZA. Considering the small numbers of animals, n, and thus relatively large SEM, this difference was insignificant. Immunohistochemistry with CD-31 and Ki67 resulted in a reduction in endothelial cells (and therefore mean vessel density and cell proliferation), pointing to a decrease in oxygen consumption and demand. This may have contributed to improved oxygenation of the sunitinib-treated Caki-1 tumors which correlates well with the fact that [18F]FAZA delivery to the tumor cells was not impaired under sunitinib. From a clinical perspective, improved oxygenation has been shown to improve response to conventional chemotherapy, and radiotherapy, as well as reduce metastatic potential .
While sunitinib therapy leads to a pronounced reduction in tumor hypoxia, termination of therapy resulted in an increased hypoxia with even higher levels of [18F]FAZA uptake compared to the control tumors. Following the release of TK inhibition, the tumor cell activity rebounds and experiences accelerated proliferation. This withdrawal flare phenomenon has not been thoroughly investigated subsequent to discontinuation of anti-angiogenic therapy. Similar rebound effects were observed in human renal cell cancer, where the SUV of [18F]FLT increased by 15% after sunitinib withdrawal . Currently, patients receive sunitinib in an on-and-off regimen . Following treatment discontinuation, patients sometimes experience recurrent pain in metastatic sites, as a result of flare re-growth. It has been proposed that during this period of withdrawal, tumor cells are stuck in the `S' phase and therefore are more vulnerable to combination cytotoxic chemotherapy . This may be a useful strategy for therapy monitoring of RCC with PET, e.g., determining the hypoxia status.
Since direct effects of sunitinib on [18F]FAZA uptake into the Caki-1 cells can be excluded from the in vitro studies, the observed reduction in tumor hypoxia in vivo must be related to other effects of sunitinib. Sunitinib was originally designed to specifically inhibit VEGFR, PDGFR, and C-kit; however, it has been found to inhibit other types of kinases as well, including non-receptor tyrosine kinases, receptor tyrosine kinases, tyrosine kinase-like kinases, cyclin-dependant kinases, and mitogen-activated protein kinase . For this reason, it is reasonable to assume that sunitinib, as a type I TKI, has a greater number of off-target effects compared to some of the more specific TKIs categorized as type II, such as sorafenib and imatinib. This may help explain some contrasting effects observed in different cell lines following TKI therapy ,,–.
The present results show that [18F]FAZA PET could be used to detect functional changes in the hypoxia status of renal cell carcinoma. It was found that during therapy with the TKI sunitinib, [18F]FAZA uptake and trapping in Caki-1 tumors was reduced, indicating improved tumor oxygenation. Upon withdrawal of sunitinib therapy, [18F]FAZA uptake into the tumor cells again increased, demonstrating a rebound in tumor hypoxia to even higher levels. This work demonstrates that [18F]FAZA PET could be used to monitor TKI therapy clinically and may guide combination therapies dependent on tumor hypoxia status.
DC, JM, LW, MW, and RM contributed to the concept and study design. DC, IM, and MW collected the data. DC and MW performed the data analysis. DC, JM, LW, MW, and RM were involved in the interpretation of the data. HSJ performed the kinetic analysis. All authors were involved in the writing process and all approved the manuscript before submission.
The authors would like to thank Dr. John Wilson, David Clendening, and Blake Lazurko from the Edmonton PET Center for providing 18F produced on a biomedical cyclotron. In addition, we would like to thank Ali Akbari for the synthesis of [18F]FAZA on an automated synthesis unit, Monica Wang for the help with the in vitro cell experiments, Kevin McCloy from the Technical Management Group at the Cross Cancer Institute (CCI) for maintenance of the small animal PET, and also Gail Hipperson and Dan McGinn from the Vivarium of the CCI for helping in the animal handling. This work was financially supported by the Edmonton PET Center, the Mr. Lube Foundation, and Alberta Innovates Health Solution (AIHS).
- Linehan WM, Walter MM, Zbar B: The genetic base of cancer of the kidney. J Urol 2003, 170: 163–172.Google Scholar
- Tan PH, Cheng L, Rioux-Leclercq N, Merino MJ, Netto G, Reuter VE, Shen SS, Grignon DJ, Montironi R, Egevad L, Srigley JR, Delahunt B, Moch H: Renal tumors: diagnostic and prognostic biomarkers. Am J Surg Pathol 2013, 37: 1518–1531. 10.1097/PAS.0b013e318299f12eView ArticlePubMedGoogle Scholar
- Cancer Facts and Figures. American Cancer Society, Atlanta ACS; 2013.Google Scholar
- Cohen HT, McGovern FJ: Renal-cell carcinoma. N Engl J Med 2005, 353: 2477–2490. 10.1056/NEJMra043172View ArticlePubMedGoogle Scholar
- Ljungberg B, Bensalah K, Bex A, Canfield S, Dabestani S, Hofmann F, Hora M, Kuczyk MA, Lam T, Marconi L, Merseburger AS, Mulders PFA, Staehler M, Volpe A: Guidelines on Renal Cell Carcinoma. European Association of Urology (EAU), Arnhem; 2013.Google Scholar
- Basu B, Eisen T: Perspectives in drug development for metastatic renal cell cancer. Target Oncol 2010, 5: 139–156. 10.1007/s11523-010-0149-2View ArticlePubMedGoogle Scholar
- Dutcher JP: Recent developments in the treatment of renal cell carcinoma. Ther Adv Urol 2013, 5: 338–353. 10.1177/1756287213505672PubMed CentralView ArticlePubMedGoogle Scholar
- Schrader AJ, Hofmann R: Metastatic renal cell carcinoma: recent advances and current therapeutic options. Anticancer Drugs 2008, 19: 235–245. 10.1097/CAD.0b013e3282f444deView ArticlePubMedGoogle Scholar
- Gross-Goupil M, Massard C, Ravaud A: Targeted therapies in metastatic renal cell carcinoma: overview of the past year. Curr Urol Rep 2012, 13: 16–23. 10.1007/s11934-011-0232-yView ArticlePubMedGoogle Scholar
- Zhang J, Yang PL, Gray NS: Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 2009, 9: 28–39. 10.1038/nrc2559View ArticlePubMedGoogle Scholar
- Mendel DB, Laird AD, Xin X, Louie SG, Christensen JG, Li G, Schreck RE, Abrams TJ, Ngai TJ, Lee LB, Murray LJ, Carver J, Chan E, Moss KG, Haznedar JO, Sukbuntherng J, Blake RA, Sun L, Tang C, Miller T, Shirazian S, McMahon G, Cherrington JM: In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res 2003, 9: 327–337.PubMedGoogle Scholar
- Adams VR, Leggas M: Sunitinib malate for the treatment of metastatic renal cell carcinoma and gastrointestinal stromal tumors. Clin Ther 2007, 29: 1338–1353. 10.1016/j.clinthera.2007.07.022View ArticlePubMedGoogle Scholar
- Petrelli A, Giordano S: From single- to mutli-target drugs in cancer therapy: when aspecificity becomes an advantage. Curr Med Chem 2008, 15: 422–432. 10.2174/092986708783503212View ArticlePubMedGoogle Scholar
- Wood L: Sunitinib malate for the treatment of renal cell carcinoma. Expert Opin Pharmacother 2012, 13: 1323–1336. 10.1517/14656566.2012.689130View ArticlePubMedGoogle Scholar
- Escudier B, Albiges L, Sonpavde G: Optimal management of metastatic renal cell carcinoma: current status. Drugs 2013, 73: 427–438. 10.1007/s40265-013-0043-1View ArticlePubMedGoogle Scholar
- Castellano D, Ravaud A, Schmidinger M, De Velasco G, Vazquez F: Therapy management with sunitinib in patients with metastatic renal cell carcinoma: key concepts and the impact of clinical biomarkers. Cancer Treat Rev 2013, 39: 230–240. 10.1016/j.ctrv.2012.04.009View ArticlePubMedGoogle Scholar
- Nilsson MB, Zage PE, Zeng L, Xu L, Cascone T, Wu HK, Saigal B, Zweidler-McKay PA, Heymach JV: Multiple receptor tyrosine kinases regulate HIF-1alpha and HIF-2alpha in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors. Oncogene 2010, 29: 2938–2949. 10.1038/onc.2010.60View ArticlePubMedGoogle Scholar
- Marín-Hernández A, Gallardo-Pérez JC, Ralph SJ, Rodríguez-Enríquez S, Moreno-Sánchez R: HIF-1alpha modulates energy metabolism in cancer cells by inducing over-expression of specific glycolytic isoforms. Mini Rev Med Chem 2009, 9:1084–1101. 10.2174/138955709788922610View ArticlePubMedGoogle Scholar
- Cassavaugh J, Lounsbury KM: Hypoxia-mediated biological control. J Cell Biochem 2011, 112: 735–744. 10.1002/jcb.22956View ArticlePubMedGoogle Scholar
- Chapman JD, Schneider RF, Urbain JL, Hanks GE: Single-photon emission computed tomography and positron-emission tomography assays for tissue oxygenation. Semin Radiat Oncol 2001, 11: 47–57. 10.1053/srao.2001.18103View ArticlePubMedGoogle Scholar
- Krohn KA, Link JM, Mason RP: Molecular imaging of hypoxia. J Nucl Med 2008, 49(Suppl 2):129S-148S. 10.2967/jnumed.107.045914View ArticlePubMedGoogle Scholar
- Sacco E, Pinto F, Totaro A, D'Addessi A, Racioppi M, Gulino G, Volpe A, Marangi F, D'Agostino D, Bassi P: Imaging of renal cell carcinoma: state of the art and recent advances. Urol Int 2011, 86: 125–139. 10.1159/000322724View ArticlePubMedGoogle Scholar
- Krajewski KM, Giardino AA, Zukotynski K, Van den Abbeele AD, Pedrosa I: Imaging in renal cell carcinoma. Hematol Oncol Clin North Am 2011, 25: 687–715. 10.1016/j.hoc.2011.04.005View ArticlePubMedGoogle Scholar
- Carlin S, Humm JL: PET of hypoxia: current and future perspectives. J Nucl Med 2012, 53: 1171–1174. 10.2967/jnumed.111.099770View ArticlePubMedGoogle Scholar
- Wuest M, Wuest F: Positron emission tomography radiotracers for imaging hypoxia. J Labelled Comp Radiopharm 2013, 56: 244–250. 10.1002/jlcr.2997View ArticlePubMedGoogle Scholar
- Sorger D, Patt M, Kumar P, Wiebe LI, Barthel H, Seese A, Dannenberg C, Tannapfel A, Kluge R, Sabri O: [18F]Fluoroazomycinarabinofuranoside (18FAZA) and [18F]Fluoromisonidazole (18FMISO): a comparative study of their selective uptake in hypoxic cells and PET imaging in experimental rat tumors. Nucl Med Biol 2003, 30: 317–326. 10.1016/S0969-8051(02)00442-0View ArticlePubMedGoogle Scholar
- Piert M, Machulla HJ, Picchio M, Reischl G, Ziegler S, Kumar P, Wester HJ, Beck R, McEwan AJ, Wiebe LI, Schwaiger M: Hypoxia-specific tumor imaging with 18F-fluoroazomycin arabinoside. J Nucl Med 2005, 46: 106–113.PubMedGoogle Scholar
- Reischl G, Dorow DS, Cullinane C, Katsifis A, Roselt P, Binns D, Hicks RJ: Imaging of tumor hypoxia with [124I]IAZA in comparison with [18F]FMISO and [18F]FAZA–first small animal PET results. J Pharm Pharm Sci 2007, 10: 203–211.PubMedGoogle Scholar
- Lewis JS, McCarthy DW, McCarthy TJ, Fujibayashi Y, Welch MJ: Evaluation of 64Cu-ATSM in vitro and in vivo in a hypoxic tumor model. J Nucl Med 1999, 40: 177–183.PubMedGoogle Scholar
- Dunphy MP, Lewis JS: Radiopharmaceuticals in preclinical and clinical development for monitoring of therapy with PET. J Nucl Med 2009, Suppl 1: 106S-121S. 10.2967/jnumed.108.057281View ArticleGoogle Scholar
- Valable S, Petit E, Roussel S, Marteau L, Toutain J, Divoux D, Sobrio F, Delamare J, Barré L, Bernaudin M: Complementary information from magnetic resonance imaging and (18)F-fluoromisonidazole positron emission tomography in the assessment of the response to an antiangiogenic treatment in a rat brain tumor model. Nucl Med Biol 2011, 38: 781–793.PubMedGoogle Scholar
- Hugonnet F, Fournier L, Medioni J, Smadja C, Hindié E, Huchet V, Itti E, Cuenod CA, Chatellier G, Oudard S, Faraggi M: Metastatic renal cell carcinoma: relationship between initial metastasis hypoxia, change after 1 month's sunitinib, and therapeutic response: an 18F-fluoromisonidazole PET/CT study. J Nucl Med 2011, 52: 1048–1055. 10.2967/jnumed.110.084517View ArticlePubMedGoogle Scholar
- Murakami M, Zhao S, Zhao Y, Chowdhury NF, Yu W, Nishijima K, Takiguchi M, Tamaki N, Kuge Y: Evaluation of changes in the tumor microenvironment after sorafenib therapy by sequential histology and 18F-fluoromisonidazole hypoxia imaging in renal cell carcinoma. Int J Oncol 2012, 41: 1593–1600.PubMed CentralPubMedGoogle Scholar
- Revheim ME, Winge-Main AK, Hagen G, Fjeld JG, Fosså SD, Lilleby W: Combined positron emission tomography/computed tomography in sunitinib therapy assessment of patients with metastatic renal cell carcinoma. Clin Oncol (R Coll Radiol) 2011, 23: 339–343. 10.1016/j.clon.2010.11.006View ArticleGoogle Scholar
- Vercellino L, Bousquet G, Baillet G, Barré E, Mathieu O, Just PA, Desgrandchamps F, Misset JL, Hindié E, Moretti JL: 18F-FDG PET/CT imaging for an early assessment of response to sunitinib in metastatic renal carcinoma: preliminary study. Cancer Biother Radiopharm 2009, 24: 137–144. 10.1089/cbr.2008.0527View ArticlePubMedGoogle Scholar
- Lyrdal D, Boijsen M, Suurküla M, Lundstam S, Stierner U: Evaluation of sorafenib treatment in metastatic renal cell carcinoma with 2-fluoro-2-deoxyglucose positron emission tomography and computed tomography. Nucl Med Commun 2009, 30: 519–524. 10.1097/MNM.0b013e32832cc220View ArticlePubMedGoogle Scholar
- Solomon B, Binns D, Roselt P, Weibe LI, McArthur GA, Cullinane C, Hicks RJ: Modulation of intratumoral hypoxia by the epidermal growth factor receptor inhibitor gefitinib detected using small animal PET imaging. Mol Cancer Ther 2005, 4: 1417–1422. 10.1158/1535-7163.MCT-05-0066View ArticlePubMedGoogle Scholar
- Liu G, Jeraj R, Vanderhoek M, Perlman S, Kolesar J, Harrison M, Simoncic U, Eickhoff J, Carmichael L, Chao B, Marnocha R, Ivy P, Wilding G: Pharmacodynamic study using FLT PET/CT in patients with renal cell cancer and other solid malignancies treated with sunitinib malate. Clin Cancer Res 2011, 15(17):7634–7644. 10.1158/1078-0432.CCR-11-1677View ArticleGoogle Scholar
- Bruce JY, Kolesar JM, Hammers H, Stein MN, Carmichael L, Eickhoff J, Johnston SA, Binger KA, Heideman JL, Perlman SB, Jeraj R, Liu G: A phase I pharmacodynamic trial of sequential sunitinib with bevacizumab in patients with renal cell carcinoma and other advanced solid malignancies. Cancer Chemother Pharmacol. 2014.Google Scholar
- Reischl G, Ehrlichmann W, Bieg C, Solbach C, Kumar P, Wiebe LI, Machulla H-J: Preparation of the hypoxia imaging PET tracer [18F]FAZA: reaction parameters and automation. Appl Radiat Isot 2005, 62: 897–901. 10.1016/j.apradiso.2004.12.004View ArticlePubMedGoogle Scholar
- Verwer EE, van Velden FHP, Bahce I, Yaqub M, Schuit RC, Windhorst AD, Raijmakers P, Lammertsma AA, Smit EF, Boellaard R: Pharmacokinetic analysis of [18F]FAZA in non-small cell lung cancer patients. Eur J Nucl Med Mol Imaging 2013, 40: 1523–1531. 10.1007/s00259-013-2462-3View ArticlePubMedGoogle Scholar
- Koch CJ, Stobbe CC, Baer KA: Metabolism induced binding of 14C-misonidazole to hypoxic cells: kinetic dependence on oxygen concentration and misonidazole concentration. Int J Radiat Oncol Biol Phys 1984, 10: 1327–1331. 10.1016/0360-3016(84)90342-0View ArticlePubMedGoogle Scholar
- Houk BE, Bello CL, Poland B, Rosen LS, Demetri GD, Motzer RJ: Relationship between exposure to sunitinib and efficacy and tolerability endpoints in patients with cancer: results of a pharmacokinetic/pharmacodynamic metaanalysis. Cancer Chemother Pharmacol 2010, 66: 357–371. 10.1007/s00280-009-1170-yView ArticlePubMedGoogle Scholar
- Maier FC, Kneilling M, Reischl G, Cay F, Bukala D, Schmid A, Judenhofer MS, Röcken M, Machulla HJ, Pichler BJ: Significant impact of different oxygen breathing conditions on noninvasive in vivo tumor-hypoxia imaging using [18F]-fluoro-azomycinarabino-furanoside ([18F]FAZA). Radiat Oncol 2011, 6: 165–175. 10.1186/1748-717X-6-165PubMed CentralView ArticlePubMedGoogle Scholar
- Rischin D, Hicks RJ, Fisher R, Binns D, Corry J, Porceddu S, Peters LJ: Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumor hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of Trans-Tasman Radiation Oncology Group Study 98.02. J Clin Oncol 2006, 24: 2098–2104. 10.1200/JCO.2005.05.2878View ArticlePubMedGoogle Scholar
- Dorow DS, Cullinane C, Conus N, Roselt P, Binns D, McCarthy TJ, McArthur GA, Hicks RJ: Multi-tracer small animal PET imaging of the tumour response to the novel pan-Erb-B inhibitor CI-1033. Eur J Nucl Med Mol Imaging 2006, 33: 441–452. 10.1007/s00259-005-0039-5View ArticlePubMedGoogle Scholar
- Majhail NS, Urbain JL, Albani JM, Kanvinde MH, Rice TW, Novick AC, Mekhail TM, Olencki TE, Elson P, Bukowski RM: F-18 fluorodeoxyglucose positron emission tomography in the evaluation of distant metastases from renal cell carcinoma. J Clin Oncol 2003, 21: 3995–4000. 10.1200/JCO.2003.04.073View ArticlePubMedGoogle Scholar
- Dilhuydy MS, Durieux A, Pariente A, de Clermont H, Pasticier G, Monteil J, Ravaud A: PET scans for decision-making in metastatic renal cell carcinoma: a single-institution evaluation. Oncology 2006, 70: 339–344. 10.1159/000097946View ArticlePubMedGoogle Scholar
- Arabi M, Piert M: Hypoxia PET/CT imaging: implications for radiation oncology. Q J Nucl Med Mol Imaging 2010, 54: 500–509.PubMedGoogle Scholar
- Atkinson BJ, Kalra S, Wang X, Bathala T, Corn P, Jonasch E: Clinical outcomes in patients with metastatic renal cell carcinoma treated with alternative sunitinib schedules. J Urol: Tannir NM. 2013.Google Scholar
- Murakami M, Zhao S, Zhao Y, Yu W, Fatema CN, Nishijima KI, Yamasaki M, Takiguchi M, Tamaki N, Kuge Y: Increased intratumoral fluorothymidine uptake levels following multikinase inhibitor sorafenib treatment in a human renal cell carcinoma xenograft model. Oncol Lett 2013, 6: 667–672.PubMed CentralPubMedGoogle Scholar
- Gaustad JV, Simonsen TG, Leinaas MN, Rofstad EK: Sunitinib treatment does not improve blood supply but induces hypoxia in human melanoma xenografts. BMC Cancer 2012, 12: 388–403. 10.1186/1471-2407-12-388PubMed CentralView ArticlePubMedGoogle Scholar
- Gaustad JV, Simonsen TG, Roa AM, Rofstad EK: Tumors exposed to acute cyclic hypoxia show increased vessel density and delayed blood supply. Microvasc Res 2013, 85: 10–15. 10.1016/j.mvr.2012.11.002View ArticlePubMedGoogle Scholar