Background

Renal cell carcinoma (RCC) is the third most diagnosed urological malignancy, with 75% of cases being the clear cell RCC histological subtype [1]. 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 [2]. 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 [3],[4]. Between 2005 and 2009, the overall incidence of kidney cancer in the USA increased 3.1% per year [3], which is comparable to the observed 2% increase over the past two decades worldwide and within Europe [5]. 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 [6]. 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 [7]-[9]. 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 [10]. 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 [3). As dynamic PET experiments revealed, lower levels of radiotracer were detected and these decreased over the 3-h time frame. In treated animals, [18F]FAZA uptake into the Caki-1 tumors reached its maximum at 30 min p.i. (SUVmean of 0.38 ± 0.02 (n = 4/2)) and decreased to a final SUVmean,3h of 0.23 ± 0.02 (n = 4/2; p < 0.05). In control tumors, a SUVmean of 0.44 ± 0.05 was measured after 60 min and 0.42 ± 0.05 (n = 4 tumors/two mice) after 3 h p.i. This observation showed clearance of [18F]FAZA and therefore less trap** of the radiotracer, indicative for a lower level of hypoxia in the treated versus the non-treated tumors. Biodistribution analysis of tumor tissue after the PET experiments confirmed these findings: 2.20 ± 0.30% ID/g in the controls versus 1.38 ± 0.13% ID/g (n = 8/4; p < 0.05) in the sunitinib-treated tumors.

Figure 3
figure 3

[18F]FAZA images in Caki-1 tumor mice in the absence (left) and presence (right) of sunitinib. The insets show the respective transaxial slides of both mice. Time-activity curves for the radioactivity uptake into Caki-1 tumor in the absence and presence of sunitinib in vivo (bottom left). Image data are shown as maximum intensity projection (MIP) at 3 h post injection and TAC data as SUV and mean ± SEM from four tumors grown in two mice. Kinetic constants k1 to k4 were calculated based on a two-compartment model (bottom right). Changes were significant for k2 and k4 (*p < 0.05).

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Uptake and retention of [18F]FAZA in Caki-1 tumors was further analyzed by kinetic modeling using a two-compartment model [41]. 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).

Following PET imaging and biodistribution experiments, tumor tissue slices were analyzed immunohistochemically. Figure 4 summarizes the results for pimonidazole, CD-31, VWF, and Ki67 staining. Treatment of Caki-1 tumor-bearing mice with sunitinib led to reduced staining of Ki67, CD-31, and an increase in VWF. Although no significant difference in the quantification of the pimonidazole staining was detectable, a qualitative trend of decreased staining density was observed in the long-term study (see Figure 5). A potential reason for this observation could be that animals were sacrificed at 1 h p.i. of pimonidazole for the short-term experiments. Since pimonidazole, like FAZA, is a 2-nitromidazole, no effects of sunitinib were detected after 1 h p.i. in accordance with the PET results (Figure 3). A 1.83 ± 0.10% CD-31 binding was determined in the tumor tissue from the control mice, whereas the samples from sunitinib-treated animals showed reduced binding of 0.58 ± 0.05% CD-31 (n = 8/4 - tumors from four mice; p < 0.001). Additionally, in the controls, 0.53 ± 0.05% VWF positive cells were found versus 0.31 ± 0.08% in sunitinib-treated samples (n = 8/4; p < 0.001). Ki67 immunohistochemistry showed a decreased binding from 75 ± 2% Ki67 positive cells in the controls to 54 ± 6% Ki67 positive cells in the treated samples (n = 8/4; p < 0.001).

Figure 4
figure 4

Immunohistochemical staining with pimonidazole HCl, CD-31, Ki-67, and VWF in Caki-1 tumor tissue slices. Slices are shown from control animals and mice treated with sunitinib (40 mg/kg/day) for 5 days (top). Quantified staining data for the percent positive stained tissue. Data as mean ± SEM with **p < 0.01; ***p < 0.001 and n.s. for not significant.

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Figure 5
figure 5

Static [18F]FAZA images in BALB/c nude mice bearing two Caki-1 tumors on each upper flank. Image data are presented as maximum intensity projection (MIP) at 3 h post injection. Top left: control mouse versus sunitinib-treated mouse (9 days 40 mg/kg). Top right: control mouse versus a sunitinib-treated mouse which had received 13 days of drug therapy followed by 12 days after therapy was finished. Bottom: semi-quantified PET data as SUV in comparison to percent pimonidazole binding in tumor slices. Data as mean ± SEM from n analyzed tumors grown in x mice. *p < 0.05.

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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

In order to assess the overall functional effects of a sunitinib treatment in vivo, uptake of [18F]FAZA into the Caki-1 cells was analyzed directly in the absence and presence of sunitinib in vitro. Hypoxia selective uptake and retention (= trap**) of [18F]FAZA occurs only under hypoxic conditions. After a 4-h incubation time, [18F]FAZA uptake was significantly higher in the Caki-1 cells (Figure 6): 2.42 ± 0.09% (hypoxia) versus 0.24 ± 0.02% radioactivity/mg protein (n = 9; p < 0.001; normoxia). For comparison and proof of the experimental setup, MCF-7 cells, known to have a higher uptake of the hypoxia PET tracer under hypoxic conditions, were also analyzed: 2.63 ± 0.44% (hypoxia) versus 0.37 ± 0.06% radioactivity/mg protein (n = 9; p < 0.001; normoxia). Cellular trap** of [18F]FAZA into the Caki-1 and MCF-7 cells was increased approximately eightfold to tenfold under hypoxic conditions, confirming increased cellular retention of [18F]FAZA under hypoxic conditions. The presence of sunitinib (3 µM) did not change the uptake properties of [18F]FAZA into the cell lines (Figure 6), indicating no direct effect occurring from this compound on the cellular uptake of a hypoxic radiotracer.

Figure 6
figure 6

Uptake of [18F]FAZA into Caki-1 (renal cell carcinoma) and MCF-7 cells (human breast cancer cell line). Cell uptake was normalized to percent radioactivity per milligram protein, and data are shown as mean ± SEM from n analyzed cell culture dishes from x number of experiments. Cell uptake was measured under normoxic and hypoxic conditions and in the presence and absence of 3 µM sunitinib.

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Discussion

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 [7],[15],[16]. 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 [16],[43]. 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 [43].

Therapy of metastatic RCC with TKIs can be monitored with non-invasive imaging methods to analyze therapy success as well as predict patient outcomes [22],[23]. Several preclinical and clinical studies have been carried out to analyze therapeutic effects of a TKI such as sunitinib or sorafenib utilizing PET [31]-[38]. 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 [26]-[28]. 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. [26],[27],[44]. Similar to the Caki-1 model, low uptake of [18F]FMISO was found in A498, another human RCC xenografted tumor model [33].

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 [30],[45]. 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. [41] 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 [32]. The EGFR-blocker gefitinib showed even higher inhibition of [18F]FAZA uptake (60% to 70%) in xenograft A431 human squamous cell carcinoma [37]. 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 [46]. Studies with [18F]FDG analyzing glucose metabolism revealed 17% to 75% reduction in uptake during a sunitinib therapy [34]-[36], although with high variability for basic [18F]FDG levels in RCC patients [47],[48], 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 [38]. 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 [32].

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 [49].

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 [38]. Currently, patients receive sunitinib in an on-and-off regimen [50]. 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 [38]. 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 [

Authors' contributions

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.