The purpose of this study was to monitor hypoxia in an orthotopic liver tumor model using a hypoxia-sensitive reporter imaging system and to image enhanced gene expression after clamping the hepatic artery. C6 and RH7777 Morris hepatoma cells were transduced with a triple reporter gene (HSV1-tk/green fluorescent protein/firefly luciferase—triple fusion), placed under the control of a HIF-1–inducible hypoxia responsive element (HRE). The cells showed inducible luciferase activity and green fluorescent protein expression in vitro. Isolated reporter-transduced Morris hepatoma cells were used to produce tumors in livers of nude rats, and the effect of hepatic artery clamping was evaluated. Tumor hypoxia was shown by immunofluorescence microscopy with the hypoxia marker EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl acetamide)] and the fluorescent perfusion marker Hoechst 33342, and by pO2 electrode measurements. For tumor hypoxia imaging with the HRE-responsive reporter, both luciferase bioluminescence and [18F]2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil positron emission tomography was done, and the presence of hypoxia in Morris hepatoma tumors were successfully imaged by both techniques. Transient clamping of the hepatic artery caused cessation of tumor perfusion and severe hypoxia in liver tumors, but not in adjacent liver tissue. These results show that the orthotopic reporter-transduced RH7777 Morris hepatomas are natively hypoxic and poorly perfused in this animal model, and that the magnitude of hypoxia can be monitored using a HRE-responsive reporter system for both bioluminescence and positron emission tomography imaging. However, the severity of tumor ischemia after permanent ligation of the hepatic artery limits our ability to image severe hypoxia in this animal model. [Mol Cancer Ther 2007;6(11):2900–8]

Colorectal cancer is the third most common cancer in both men and women in the United States. Over 150,000 new cases of colorectal cancer are expected to be diagnosed in the United States in 2007 with similar accounts in Europe. Whereas surgical resection may be curative for patients diagnosed with early-stage disease, 61% of the patients are diagnosed with regional or distant metastases (1). Colorectal cancer most commonly metastasizes to the liver. Hepatic metastasis determines the prognosis of patients with colorectal carcinoma. Surgical resection can result in significant prolongation of survival of patients with isolated hepatic metastases. The 5-year survival rate of patients who underwent complete resection of hepatic metastases is ∼35% (2). Unfortunately, <10% of the patients are candidates for surgical resection of their liver lesions and require other regional or systemic treatments (3).

An increasing interest in the tumor microenvironment, particularly neovascularization and hypoxia, has developed in the last decade. Hypoxia has been shown to be a common feature of a wide range of solid tumors (48). Tumor hypoxia has been shown to be an adverse prognostic factor in various cancers, because of the associated increase in potential for tumor progression, metastasis, and resistance to treatment (7, 9). Rather than designing therapeutic strategies to overcome hypoxic resistance, a number of groups have chosen to exploit the hypoxic nature of tumors to gain a therapeutic advantage. Although prolonged hypoxia can induce tumor necrosis, hepatic artery ligation alone does not seem to be useful for colorectal cancer liver metastases (10). Recent studies suggest that hypoxia can be used to target and improve cancer therapy (11, 12). One objective of targeted gene therapy is to minimize gene expression in normal tissue and to maximize gene expression in tumor tissue. In principle, this could be achieved by regulating therapeutic gene expression by tumor-specific promotors. Although hypoxia response elements (HRE) are not tumor specific, especially when one considers the “ischemia” of many organs in the older population with cancer, the severe hypoxia associated with many solid tumors and metastases could be exploited for therapeutic advantage. van Laarhoven et al. (13) recently showed high levels of hypoxia in patients with colorectal liver metastases, based on the postsurgical analysis of tumor specimens stained for the hypoxic cell marker pimonidazole.

With the advent of hypoxia-targeted therapies in combination with hepatic artery ligation (14), the need for noninvasive hypoxia monitoring and therapeutic efficacy will become necessary. Imaging is increasingly being used for noninvasive assessment of tumor physiology and phenotype to enable appropriate patient selection as well as monitor the response of novel therapies. Currently, several noninvasive methods for monitoring tissue hypoxia are under investigation and several of them have the potential for translation to the clinic (15, 16).

The purpose of this study was to develop an orthotopic liver tumor model with a hypoxia-sensitive reporter system to monitor tumor hypoxia by noninvasive imaging. A regulated reporter system was developed, similar to the system that was previously validated (17). Hypoxia imaging of orthotopic liver tumors was done before and after hepatic artery clamping and the results were confirmed by immunohistochemistry and direct oxygen measurements within the liver and liver tumors.

Generation of dxHRE-HSV1-tknes/Green Fluorescent Protein/FLuc-Neo Vector

All DNA manipulations were done by standard procedures using restriction enzymes, T4 DNA ligase, and buffers according to manufacturer's instructions (Life Technologies, Inc., Roche and New England BioLabs). The retroviral vectors SFG-HSV1-tknes/green fluorescent protein (GFP)/FLuc (18), dxFBS-HSV1S-tknes/GFP-Neo,6

6

I. Serganova, V. Ponomarev, and R. Blasberg, unpublished data.

and dxHRE-HSV1-tk/GFP-Neo (17) were used to generate the dxHRE-HSV1-tknes/GFP/FLuc-Neo vector. To develop the construct, several additional vectors were created (Supplementary Data).7
7

Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

First, we used the HpaI-BamHI fragment, containing the GFP/FLuc part from the triple fusion gene (HSV1-tk/GFP/FLuc) in SFG-HSV1-tknes/GFP/FLuc plasmid (18), to replace the GFP part in the vector dxFBS-HSV1-tknes/GFP-Neo (steps 1 and 2, Supplementary Figure).7 The resulting construct dxFBS-HSV1-tknes/GFP/FLuc-Neo was further used to generate dxHRE-tknes/GFP/FLuc-Neo plasmid. The fragment XbaI-BamHI (step 3) was subcloned to the dxHRE-HSV1-tk/GFP-Neo vector to replace the HSV1-tk/GFP gene fusion by the HSV1-tknes/GFP/FLuc. The final plasmid was transfected into the GPG293 cell line for transient retroviral vector production (17). The retrovirus-containing medium was collected for 4 consecutive days and stored at −80°C.

Cell Line Transduction and Cell Culture

C6 and RH7777 rat Morris hepatoma cell lines were obtained from American Type Culture Collection. Cell culture medium was supplemented with 100 units penicillin and 100 μg/mL streptomycin. RH7777 cells were maintained in DMEM supplemented with high glucose (4.5 g/L), 10% FCS, and 5 mmol/L l-glutamine; C6 cells in DMEM medium with only 10% FCS. Cells were transduced with the dxHRE-HSV1-tknes/GFP/FLuc-Neo retrovirus as previously described (19). After transduction, the cells were grown in culture for several days and prepared for subsequent neomycin selection and HRE-activation with the hypoxia mimetic compound CoCl2. GFP-positive cells were sorted by a fluorescence-activated cell sorter (Bioscience) as previously described (17) and visualized by fluorescence microscopy using a Nikon Eclipse T-100.

Flow Cytometry

Transduced C6 and RH7777 cells (1.5 × 105 per well) were plated in six-well tissue culture plates (Becton Dickinson and Company); CoCl2 (100, 200, and 300 μmol/L) as a mimetic hypoxia agent was added at various times (12 and 24 h). Another set of six-well plates were put into a hypoxia incubator (NU 8500, NuAire, Inc.) under hypoxic conditions (0.5% of O2). All cells were collected at the same time. The level of HIF-1 transcriptional activity was assessed using fluorescence-activated cell sorting analysis.

In vitro Bioluminescence Assays

C6 and RH7777 were treated with either CoCl2 or placed under hypoxic conditions as described above. d-Luciferin (Xenogen) at a concentration of 0.5 mmol/L was added immediately before assay. Bioluminescence was measured with an IVIS 100 Imaging system (Xenogen) and analyzed using the LIVINGIMAGE 2.5 software (Xenogen).

In vitro Radiotracer Assays

In vitro radiotracer accumulation studies were done with [14C]2′-fluoro-2′-deoxy-1β-d-arabionofuranosyl-5-iodo-uracil ([14C]FIAU) using the C6 reporter-transduced cells exposed to different concentrations of CoCl2 as previously described (20).

Animal Tumor Model

The experimental animal study protocols were approved by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Cancer Center. Six- to 8-week-old nude rats (200–250 g) were purchased from Charles River Laboratories. All animal procedures were done under anesthesia by inhalation of 2% isoflurane and 100% oxygen at a flow rate of 1.5 L/min. Approximately 5 × 106 RH7777 cells in PBS (50 μL) were injected in the shoulder area to generate s.c. flank tumors. When the tumors reached ∼1 cm in diameter, the animal was sacrificed and the tumors were harvested, cut to small fragments of ∼2 mm3, and kept in PBS on ice. At the same time, other nude rats were anesthetized and opened via midline incision to expose the liver. A total of 21 nude rats were implanted with HRE reporter–transduced RH7777 cells. A 1-cm-long tunnel was punctured into the left liver lobe horizontally just beneath the liver capsule using a scalpel lying flat on the liver surface and a tumor fragment from the flank tumors was inserted into the pouch. Growth of the transplanted liver tumors was monitored with a dedicated small-animal ultrasound system (Vevo 770, VisualSonics). All imaging studies were done 4 to 6 weeks after placement of the hepatic tumor, except for one animal that was imaged later (at 9 weeks) after the development of lung metastases.

During hepatic artery operation, the animal body temperature was maintained between 36°C and 37°C with the use of a heating pad and a heating lamp and monitored with a rectal probe. Following midline laparotomy, the liver hilum was exposed to reveal the common hepatic artery and portal vein. To imitate the clinical procedure of hepatic artery embolization, the hepatic artery was permanently ligated with a silk thread. To test if HRE-driven gene expression could be artificially enhanced, the hepatic artery was transiently clamped (three times for 20 min with 1-min intervals, total of 1 h). None of the animals died during or after operation.

Direct In vivo pO2 Measurements

Direct oxygen measurements were done in the liver tumors and normal liver tissue with the OxyLite system (Oxford Optronix) in two animals. Tissue oxygen measurements were acquired as previously described (21, 22). To explore the potential of hypoxia-triggered reporter expression, we studied the effect of hepatic vessel clamping on the blood flow to the orthotopic liver tumor. Measurements were taken continuously, before, during, and after clamping of the hepatic artery or both the hepatic artery and the portal vein. Animals were sacrificed by CO2 inhalation before coming out of anesthesia.

Hypoxia Imaging at a Microscopic Level

In nine rats, the hypoxia marker EF5 (30 mg/kg; a generous gift from Dr. C. Koch, University of Pennsylvania, Philadelphia, PA) was injected via tail vein under brief anesthesia 24 h before sacrificing the animals (23, 24). In three of those nine animals, we also did hepatic artery ligation before administration of the hypoxia marker. In the remaining six rats, the blood supply to the liver remained unaltered. Two minutes before sacrifice, 60 mg/kg of the perfusion marker Hoechst 33342 (Sigma) was injected via tail vein. All animals were sacrificed by CO2 inhalation and all tumors were harvested with their surrounding liver tissue, placed in OCT cryofixative (Sakura Finetek), and frozen in dry ice and isopentane. Frozen tissue sections (8-μm thick) were prepared from various levels of the specimens. The sections were mounted on poly-l-lysine slides (Fisher Scientific). Hoechst 33342 distribution was assessed using a fluorescent microscope (Axiovert 200M, Carl Zeiss, Inc.) with a DAPI fluorescent filter and ×5 magnification. Up to 330 images were acquired per tumor section and then stitched to a single image using Metamorph 6.2r3 software (Universal Imaging Corporation). The same tissue slides were then fixed with cold acetone, blocked with SuperBlock (ScyTech), and stained with Cy3-conjugated primary mouse anti-EF5 antibody (ELK3-51, provided by Dr. C. Koch). Again, the sections were imaged with the fluorescent microscope, this time with a Cy3 fluorescent filter. Red was assigned to EF5 and the blue was assigned to Hoechst 33342. To compare the distribution of the two tracers, images were overlaid using the Metamorph software.

In vivo Bioluminescence Imaging

For bioluminescence imaging, six new rats with liver tumors were injected with 100 mg/kg of the d-luciferin solution (Xenogen) i.p. and images were acquired for 10 s 20 min after injection. The photon emission (photons/cm2/s/steradian) from rats was measured.

[18F]2′-Fluoro-2′-Deoxyarabinofuranosyl-5-Ethyluracil Production

[18F]2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyluracil (FEAU) was synthesized by coupling the radiolabeled fluoro sugar with the silylated pyrimidine derivative after a procedure previously reported by Serganova and coworkers (17). The product was purified by high-performance liquid chromatography and the radiochemical purity was >98%.

Positron Emission Tomography Imaging

Four of the six rats were also administrated with ∼1,000 μCi (18.5 MBq) [18F]FEAU and imaged in prone position using a dedicated small-animal positron emission tomography (PET) scanner (Focus 120 microPET, Concorde Microsystems). Images were acquired 3 h after tail vein administration of the tracer for 10 min under inhalation anesthesia with 2% isoflurane with transaxial fields of view of 10 cm and axial fields of view of 7.8 cm. An energy window of 350 to 750 keV and a coincidence timing window of 6 ns were used. The resulting list-mode data were sorted into two-dimensional histograms by Fourier rebinning, and transverse images were reconstructed by filtered backprojection into a 128 × 128 × 63 (0.72 × 0.72 × 1.3 mm) matrix. All image data were corrected for nonuniformity of the microPET response, dead-time count losses, and physical decay to the time of injection but there was no attenuation, scatter, or partial-volume averaging correction applied. To convert the counts in the reconstructed images to activity concentration normalized to the administered activity by region of interest analysis in %/ID/g (percentage of injected dose per gram of tissue corrected for radioactive decay to the time of injection) an empirically determined system calibration factor (μCi/mL/cps/voxel) was used. Image analysis was done using the ASIPro software (Concorde Microsystems).

To assess the possibility of enhancing HRE-driven gene expression by transient clamping of the hepatic artery, a second set of four animals with orthotopic reporter-transduced liver tumors was studied. Bioluminescence and PET imaging was done before and 6 h after transient clamping of the hepatic artery for 1 h in the same animals.

Statistics

Mean values and SDs were calculated using the MS Office 2003 Excel 11.0 statistical package (Microsoft).

Generation and Validation of HRE Reporter Cells

The retroviral vector (dxHRE-HSV1-tk/GFP/FLuc-Neo) encoding the HSV1-tk/GFP/FLuc fusion gene was placed under the control of eight repeats of the HRE (ref. 17; Fig. 1A). Two cell lines, C6 rat glioma and RH7777 rat Morris hepatoma, were transduced with this vector. The dxHRE-HSV1-tk/GFP/FLuc-Neo transduced cells were initially selected by neomycin exposure because the neomycin gene was constitutively expressed under the SV40 promoter. Reporter cells were reselected using fluorescence-activated cell sorting based on GFP expression after CoCl2 treatment. Transduced C6 and RH7777 cells were analyzed for the expression of each reporter after exposure to hypoxia and the hypoxia mimetic, CoCl2.

Figure 1.

Structural schema of the retroviral vector (A) and validation of the triple reporter system in transduced C6 cells. Fluorescence-activated cell sorting analysis of GFP expression (B), relative bioluminescence intensity (C), and normalized accumulation of [14C]2′-fluoro-2′-deoxy-1β-d-arabionofuranosyl-5-iodo-uracil (FIAU; D) after the exposure of transduced C6 cells to different concentrations of the hypoxia mimetic CoCl2.

Figure 1.

Structural schema of the retroviral vector (A) and validation of the triple reporter system in transduced C6 cells. Fluorescence-activated cell sorting analysis of GFP expression (B), relative bioluminescence intensity (C), and normalized accumulation of [14C]2′-fluoro-2′-deoxy-1β-d-arabionofuranosyl-5-iodo-uracil (FIAU; D) after the exposure of transduced C6 cells to different concentrations of the hypoxia mimetic CoCl2.

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The responsiveness of the dxHRE-HSV1-tk/GFP/FLuc-Neo reporter system in transduced C6 cells exposed to CoCl2 in culture was evaluated first. Almost 85% of transduced C6 cells became GFP positive (Fig. 1B) and a 4-fold increase in firefly luciferase signal was measured (Fig. 1C). The in vitro radiotracer uptake study showed a 10-fold increase in uptake of [14C]FIAU with 300 μmol/L CoCl2 reflecting HSV1-TK expression (Fig. 1D). Then, we examined the responsiveness of the dxHRE-HSV1-tk/GFP/FLuc-Neo reporter system in transduced RH7777 Morris hepatoma cells by exposure to CoCl2 and to hypoxia (0.5% medium oxygen concentration) for 24 h. The percentage of GFP-positive cells increased from 22 ± 3.6% under normoxia to 49 ± 2.8% under 0.5% oxygen and to 81 ± 3.4% under treatment with 200 μmol/L CoCl2 (Fig. 2A and B). Firefly luciferase expression was measured in transduced cells exposed to 100 and 200 μmol/L CoCl2 for 24 h. A 2.6-fold induction of luciferase activity was observed after treatment with 100 μmol/L CoCl2, and 5.2-fold induction with 200 μmol/L CoCl2 (Fig. 2C). The transduced rat RH7777 Morris hepatoma cell line was chosen for in vivo experiments as an appropriate tumor cell line for an orthotopic liver tumor model in rats.

Figure 2.

Validation of the triple reporter system in transduced Morris hepatoma cells. A, fluorescence-activated cell sorting analysis. B, fluorescence microscopy of transduced Morris hepatoma cells. C, firefly luciferase activity in transduced Morris hepatoma. Baseline, control (normal) conditions.

Figure 2.

Validation of the triple reporter system in transduced Morris hepatoma cells. A, fluorescence-activated cell sorting analysis. B, fluorescence microscopy of transduced Morris hepatoma cells. C, firefly luciferase activity in transduced Morris hepatoma. Baseline, control (normal) conditions.

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Tissue pO2 Measurements

Oxygen tension measurements were obtained in orthotopic, reporter-transduced Morris hepatomas that were 11 and 14 mm in diameter, and in normal liver tissue. Measurements of pO2 in the tumor were highly variable (mean of 30 different probe positions: 1.2 ± 3.5 mm Hg; Fig. 3A), and significantly (P < 0.005) lower than pO2 values in normal liver (41 ± 4.1 mm Hg). To explore the potential for selectively enhancing hypoxia-triggered reporter expression, we investigate the effect of hepatic vessel clamping on hypoxia within the liver and liver tumor. Measurements of tissue pO2 were obtained with and without clamping of the hepatic artery, and after clamping both the hepatic artery and the portal vein. There was only a slight decrease in the pO2 of normal liver after clamping of the hepatic artery and a marked decrease in pO2 (near zero) after clamping of both the hepatic artery and the portal vein (Fig. 3B). The pO2 values returned to baseline when the clamp was released. In contrast, pO2 measurements of the hepatic tumor fell to near zero after clamping the hepatic artery alone, and returned to baseline when the clamp was released (Fig. 3B and C).

Figure 3.

Oxygen tension measurements in hepatic tumors and normal liver tissue. Changes in pO2 in HRE reporter–transduced RH7777 Morris hepatoma after small changes in position of the OxyLite probe (A); in normal liver tissue before, during, and after clamping of the hepatic artery; as well as both the hepatic artery and portal vein (B) and in the same liver tumor (A) in a fourth position with clamping of the hepatic artery alone (C). A to C are from the same animal; measurements were made sequentially.

Figure 3.

Oxygen tension measurements in hepatic tumors and normal liver tissue. Changes in pO2 in HRE reporter–transduced RH7777 Morris hepatoma after small changes in position of the OxyLite probe (A); in normal liver tissue before, during, and after clamping of the hepatic artery; as well as both the hepatic artery and portal vein (B) and in the same liver tumor (A) in a fourth position with clamping of the hepatic artery alone (C). A to C are from the same animal; measurements were made sequentially.

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Hypoxia Imaging at a Microscopic Level

To further assess for the presence of hypoxia in orthotopic HRE-reporter liver tumors, the perfusion pattern (Hoechst 33342 stain) and the distribution of an established hypoxia marker (EF5) were compared. On a macroscopic, nonquantitative level, all tissue regions with intense EF5 staining corresponded to tumor tissue (Fig. 4B) and to areas of low Hoechst 33342 perfusion staining (Fig. 4A). The converse was also observed; liver tissue showed high perfusion as shown by intense Hoechst 33342 staining and low EF5 staining that was at tissue background levels (Fig. 4A–D). The hypoxia and perfusion pattern analysis of tumors after clamping of the hepatic artery showed no perfusion with Hoechst 33342 (Fig. 4E), but also little or no staining with the hypoxia marker (Fig. 4F). The absence of Hoechst 33342 staining indicates the near-complete cessation of tumor perfusion during clamping and probably accounts for the minimal staining with EF5 (Fig. 4E and F).

Figure 4.

Paired perfusion-hypoxia imaging. Matched image sets of the perfusion marker Hoechst 33342 (blue; A) and the hypoxia marker EF5 (red; B). C, overlay of A and B. D, magnified view of the white rectangle outlined in C. Twenty-four hours after permanent ligation of the hepatic artery, cryosections of a tissue block containing two liver tumors were stained for the perfusion marker Hoechst 33342 (E) and the hypoxia marker EF5 (F); the tumor border is outlined by the white line. A to D and E to F were generated from different animals.

Figure 4.

Paired perfusion-hypoxia imaging. Matched image sets of the perfusion marker Hoechst 33342 (blue; A) and the hypoxia marker EF5 (red; B). C, overlay of A and B. D, magnified view of the white rectangle outlined in C. Twenty-four hours after permanent ligation of the hepatic artery, cryosections of a tissue block containing two liver tumors were stained for the perfusion marker Hoechst 33342 (E) and the hypoxia marker EF5 (F); the tumor border is outlined by the white line. A to D and E to F were generated from different animals.

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Hypoxia-Driven Reporter Imaging by Bioluminescence Imaging and PET

The first set of in vivo imaging experiments was done in six nude rats bearing orthotopic HRE reporter–transduced Morris hepatomas. Tumor growth was monitored with a small-animal ultrasound device. When the xenografts were between 5 and 15 mm in diameter, they could be readily visualized by bioluminescence imaging, indicating that the HRE reporter was activated and that the tumors were hypoxic. Bioluminescence intensity of the xenografts was 1.5 ± 0.4 × 106 photons/s/cm2/sr (Fig. 5A). Four of these six animals were selected for [18F]FEAU PET imaging to assess the activation of the HSV1-TK component of the triple-reporter. Radiotracer uptake was 0.18 ± 0.007%ID/g in the liver tumors compared with background radioactivity levels of 0.07 ± 0.002%ID/g, 0.08 ± 0.004%ID/g, and 0.07 ± 0.008%ID/g in lung, liver, and muscle, respectively (Fig. 5B).

Figure 5.

In vivo HRE reporter imaging. Whole-body bioluminescence images (A), and axial and coronal [18F]FEAU microPET images (B) of orthotopic HRE reporter–transduced RH7777 Morris hepatomas in nude rats (arrow, orthotopic liver tumor).

Figure 5.

In vivo HRE reporter imaging. Whole-body bioluminescence images (A), and axial and coronal [18F]FEAU microPET images (B) of orthotopic HRE reporter–transduced RH7777 Morris hepatomas in nude rats (arrow, orthotopic liver tumor).

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To assess the possibility of enhancing HRE-driven gene expression by transient clamping of the hepatic artery, a second set of four animals with orthotopic reporter-transduced liver tumors was studied. Bioluminescence and PET imaging was done before and 6 h after transient clamping of the hepatic artery for 1 h in the same animals. We observed no signal change or a signal decrease in three of the four animals (Fig. 6A). Interestingly, one animal with signal loss in the liver tumor had developed lung metastases that were visualized by both bioluminescence and microPET reporter imaging (Fig. 6A and B) and confirmed at autopsy. These metastases showed no signal change from baseline to postclamping. In one animal, there was a 1.8-fold increase in bioluminescence and a 3-fold increase in radioactivity measured in the tumor after hepatic artery clamping (Fig. 6C and D). No observed difference in the level of background radioactivity (lung, liver, and muscle) was found between preocclusion and postocclusion studies.

Figure 6.

HRE reporter imaging prehepatic and posthepatic artery clamping. Bioluminescence images of HRE reporter–transduced Morris RH7777 hepatomas before and 6 h after transient (1 h) clamping of the hepatic artery. Note the decrease in bioluminescence signal intensity after clamping in this animal and the presence of lung metastasis (T, liver tumor; M, lung metastasis; A). The lung metastases were also visualized by [18F]FEAU microPET imaging (B). Bioluminescence images (C) and [18F]FEAU microPET images (D) of another animal bearing a reporter-transduced liver tumor before and after clamping of the hepatic artery. C and D, images from the same animal; an increase in the tumor signal after occlusion of the hepatic artery is seen in both panels (arrow, orthotopic liver tumor).

Figure 6.

HRE reporter imaging prehepatic and posthepatic artery clamping. Bioluminescence images of HRE reporter–transduced Morris RH7777 hepatomas before and 6 h after transient (1 h) clamping of the hepatic artery. Note the decrease in bioluminescence signal intensity after clamping in this animal and the presence of lung metastasis (T, liver tumor; M, lung metastasis; A). The lung metastases were also visualized by [18F]FEAU microPET imaging (B). Bioluminescence images (C) and [18F]FEAU microPET images (D) of another animal bearing a reporter-transduced liver tumor before and after clamping of the hepatic artery. C and D, images from the same animal; an increase in the tumor signal after occlusion of the hepatic artery is seen in both panels (arrow, orthotopic liver tumor).

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Colorectal cancer has a strong propensity for hepatic metastases. Regional treatment strategies for isolated spread of disease to the liver are an important option for these patients. Surgery is the best therapeutic modality for those who are candidates for partial hepatic resection. Unfortunately, even after complete removal of all liver lesions, ∼50% of patients have a recurrence within 5 years (25). Therefore, novel therapies directed at improved control of liver metastases are required to significantly affect the prognoses of patients with advanced-stage colorectal cancer.

Novel diagnostic and therapeutic approaches under recent investigation exploit the hypoxic microenvironment of tumors to improve the target specificity of gene therapy (26) and oncolytic viral therapy (27). Recently, we showed the possibility of imaging the dose-dependent, temporal dynamics of HIF-1 activity using a HIF-1 responsive reporter system (17). We showed that HIF-1–mediated reporter gene expression can be monitored using PET imaging in a mouse flank tumor model. Here, we expand the application of this reporter system to monitor hypoxia development in an orthotopic tumor model of primary liver cancer and colorectal metastases to the liver. We studied the effect of hepatic artery clamping to simulate hepatic artery embolization, on reporter expression and on tissue oxygen levels.

An orthotopic liver tumor model was developed using reporter cells that contained a triple-fusion reporter gene (HSV1-tk/GFP/firefly luciferase) under the control of a HIF-1–inducible hypoxia-response element. This HRE reporter system is robust and was validated in a previous study (17). In addition, the triple-reporter system provides the opportunity for multimodality imaging of hypoxia both in vitro and in vivo. Multimodality imaging allows for different imaging technologies to be combined for monitoring hypoxia during tumorigenesis and during hypoxia-targeted therapy.

An important observation of this study was the presence of hypoxia in comparatively small orthotopic liver tumors, and this was documented by all imaging modalities and by direct tissue measurements using an oxygen electrode. Interestingly, the immunohistochemical staining for EF5 at a macroscopic level shows a fairly homogenous pattern of hypoxia distribution throughout the entire tumor that extends to the very periphery of the tumor (Fig. 4B). A similar homogenous pattern of tumor hypoxia has been described in a variety of tumor xenografts and also in several types of human tumors, including liver tumors harvested at surgery (13, 28). These observations are in contrast to another common pattern of tumor hypoxia, namely that solid tumors consist of a hypoxic, heterogenous center surrounded by a zone of better-perfused and better-oxygenated cells in the periphery (29). However, at a microscopic level, the pattern of immunohistochemical staining for EF5 and Hoechst 33342 vascular perfusion fluorescence shows a more heterogenous pattern that are inversely correlated (Fig. 4D). This heterogenous pattern of hypoxia in the tumors probably explains the wide range in OxyLite electrode pO2 measurements that were obtained (Fig. 3A and C).

Another interesting observation was the activation of the HRE reporter and positive bioluminescence and microPET imaging in lung metastases that developed in several of these animals. The lung metastases were sizable (size range 2–9 mm3) and could have been hypoxic, but no confirmatory pO2 measurements or EF5 immunohistochemistry was done on the metastases. Alternatively, nonhypoxia-induced up-regulation of HIF-1 transcription factor expression by activation of specific oncogenic signaling pathways (30), rather than physical tumor hypoxia, could also explain the activation of the HRE reporter observed in the lung metastases (Fig. 6A and B; refs. 31, 32).

Hepatic artery embolization is a widely used treatment for unresectable primary or metastatic cancers in the liver. The animal model used in this study provides to emulate hepatic artery embolization by hepatic artery clamping or increase HRE-driven gene expression by transient hepatic artery occlusion, and it provides the opportunity to obtain real-time in vivo measurements of hypoxia in liver tumors before, during, and after hepatic artery ligation. The liver is a unique organ in that there is a dual nutrient and oxygen blood supply. Normal noncancerous liver derives ∼75% of the blood supply via the portal vein. Hepatic tumors, however, derive their nutrient blood source mainly from the hepatic artery. We show that this differential blood supply is also true for our tumor model (Fig. 4), and that we can induce more severe hypoxia in the tumor model compared with normal liver by clamping the hepatic artery (Fig. 3B and C).

To evaluate if we could enhance HRE-driven gene expression, animals were studied before and after transient occlusion of the hepatic artery. In one of these animals, we observed the expected up-regulation of the hypoxia-induced reporter system. What we did not expect was that the opposite effect occurred in three of the four animals. One possible explanation for this is that our orthotopic tumors became severely ischemic during hepatic artery clamping, leading to death of the hypoxia-sensitive tumor cells after 6 h. Another possibility is that in the three animals with signal loss/signal extinction, the hepatic artery clotted during clamping and did not get reperfused anymore, leading to a similar effect as in the ligation experiment shown in Fig. 4E and F.

The research applications of the HRE-triple reporter will remain preclinical, because transduction of human tissue (tumor or specific organs) with reporter systems for diagnostic imaging studies is not likely to occur in the near future. However, for preclinical animal and cell-based studies, reporter systems are increasingly being used. In this context, the bioluminescence readout of the HRE-triple reporter will largely be used in small animals, and along with the fluorescence readout, in high-throughput cell-based drug/compound screening assays. The HSV1-TK component will be most useful for generating microPET tomographic image data sets that can be compared with corresponding coregistered computed tomography and magnetic resonance image data sets. As we assessed the effect of hepatic artery clamping on hepatic tumor hypoxia by bioluminescence and PET imaging in real-time in this study, other similar applications are now available. For example, the effect of antiangiogenesis therapies, cytotoxic or cytostatic chemotherapy, and radiation therapy, as well as the effect of target-specific drugs on tumor hypoxia and HIF-1 transcription factor levels, can now be assessed with this reporter system.

In conclusion, we developed an orthotopic liver tumor model with a hypoxia-sensitive reporter system that enables the monitoring of tumor hypoxia by noninvasive imaging and our results were confirmed by immunohistochemistry and direct oxygen measurements within the liver and liver tumors. Hypoxia imaging of orthotopic liver tumors was successfully done before, but not after, hepatic artery clamping, due to severe ischemia and death of the tumor cells. Our model provides a noninvasive in vivo method for monitoring hypoxia (HIF-1) in a HRE reporter–transduced tumor, and can be used in future studies involving hypoxia-targeted therapies or in combination with hepatic artery infusions of chemotherapeutic drugs.

Grant support: NIH grants R25-CA096945 (R25-CA096945-3: P. Brader, C.C. Riedl, and H. Hricak), P50 CA86438, and R24 CA83084.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: P. Brader and C.C. Riedl contributed equally to this work.

We thank the Molecular Cytology Core Facility staff, especially Eric Suh (Memorial Sloan-Kettering Cancer Center), for their help and support.

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