Combined Injection of ¹⁸F-Fluorodeoxyglucose and 3′-Deoxy-3′-[¹⁸F]fluorothymidine PET Achieves More Complete Identification of Viable Lung Cancer Cells in Mice and Patients than Individual Radiopharmaceutical: A Proof-of-Concept Study^,

Abstract

PURPOSE: The objective is to validate the combination of 3′-deoxy-3′-[¹⁸F]fluorothymidine (¹⁸F-FLT) and ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) as a “novel” positron emission tomography (PET) tracer for better visualization of cancer cell components in solid cancers than individual radiopharmaceutical. METHODS: Nude mice with subcutaneous xenografts of human non-small cell lung cancer A549 and HTB177 cells and patients with lung cancer were included. In ex vivo study, intratumoral radioactivity of ¹⁸F-FDG, ¹⁸F-FLT, and the cocktail of ¹⁸F-FDG and ¹⁸F-FLT detected by autoradiography was compared with hypoxia (by pimonidazole) and proliferation (by bromodeoxyuridine) in tumor section. In in vivo study, first, ¹⁸F-FDG PET and ¹⁸F-FLT PET were conducted in the same subjects (mice and patients) 10 to 14 hours apart. Second, PET scan was also performed 1 hour after one tracer injection; subsequently, the other was administered and followed the second PET scan in the mouse. Finally, ¹⁸F-FDG and ¹⁸F-FLT cocktail PET scan was also performed in the mouse. RESULTS: When injected individually, ¹⁸F-FDG highly accumulated in hypoxic zones and high ¹⁸F-FLT in proliferative cancer cells. In case of cocktail injection, high radioactivity correlated with hypoxic regions and highly proliferative and normoxic regions. PET detected that intratumoral distribution of ¹⁸F-FDG and ¹⁸F-FLT was generally mismatched in both rodents and patients. Combination of ¹⁸F-FLT and ¹⁸F-FDG appeared to map more cancer tissue than single-tracer PET. CONCLUSIONS: Combination of ¹⁸F-FDG and ¹⁸F-FLT PET imaging would give a more accurate representation of total viable tumor tissue than either tracer alone and would be a powerful imaging strategy for cancer management.

Introduction

Positron emission tomography (PET) imaging has emerged as an important clinical tool for cancer detection, staging and monitoring the response to therapy. ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) and 3′-Deoxy-3′-[¹⁸F]fluorothymidine (¹⁸F-FLT) are the most commonly used PET tracers for imaging tumor glucose metabolism and cell proliferation, respectively. Numerous studies in patients and in animal models have been performed to identify which tracer is most optimal in cancer management; however, results were mixed [1–12]. In these studies, although the individual tumor accumulations of each tracer were well studied, investigators failed to fully understand the differences in intratumoral distribution of each tracer.

Solid tumors have complex and highly heterogeneous microenvironment, being composed of viable cancer cells, stroma, and necrotic zones [13–15]. Viable cancer cells are usually either hypoxic or oxic or either minimally or highly proliferative. Cellular proliferation and hypoxia are generally exclusive [16–18]. Although ¹⁸F-FDG is often used as a surrogate for tumor tissue, recent in vivo [9,10,19] and in vitro [8,20,21] studies demonstrated that this tracer preferentially accumulates in hypoxic cancer cells. Conversely, ¹⁸F-fluorothymidine accumulates mostly in proliferative cancer cells, which are usually not hypoxic [19]. Therefore, ¹⁸F-FLT may fail to label or accurately identify minimally proliferative and hypoxic cancer cells that are present in most solid cancers.

Noncancerous intratumoral stroma and necrotic zones accumulate low levels of ¹⁸F-FDG and ¹⁸F-FLT. Therefore, low ¹⁸F-FDG uptake of the oxic cancer cells and low ¹⁸F-fluorothymidine retention of the hypoxic cancer cells are not distinguishable from stroma and necrosis [19]. So, the presence of weak signal from either tracer alone is ambiguous and does not necessarily represent the absence of viable tumor tissue.

There are circumstances where it would be desirable to identify the total tumor tissue burden without identifying the hypoxic and proliferative cancer cell components individually. Obviously, neither ¹⁸F-FDG nor ¹⁸F-FLT has the ability to visualize all of the viable cancer cells present in a tumor. However, their intratumoral complementary distribution patterns may be exploited if the two tracers are given in combination. Recently, Lin and colleagues reported that ¹⁸F-FDG and ¹⁸F-NaF can be combined in a single PET/computed tomography (CT) exam by administering the two radiopharmaceuticals simultaneously or in sequence on the same day. By doing so, the combined PET/CT scan appeared to increase the sensitivity of detection of osseous lesions compared to ¹⁸F-FDG-only PET/CT scans [22], albeit this proposal has been argued [23].

We hypothesized that combined simultaneous ¹⁸F-FDG and ¹⁸F-FLT injection PET scan might encompass both hypoxic (non-proliferative) and oxic (usually proliferative) cancer cells, hence, visualize more cancer cells when compared to scans obtained by each individual radiopharmaceutical injections. In this study, we used human non-small cell lung cancer (NSCLC) HTB177 and A549 cells grown as subcutaneous xenografts in nude mice as well as patients with lung cancer to test our hypothesis. Subjects were intravenously injected with ¹⁸F-FLT and ¹⁸F-FDG in combination in sequence with a 10- to 14-hour interval or simultaneously as “cocktail.” In animal studies, the proliferation marker bromodeoxyuridine (BrdU) and the hypoxia marker pimonidazole were also injected intravenously together. Intratumoral distribution of tracer radioactivity was related to histologic findings of proliferation, hypoxia, perfusion, stroma, and necrosis. Our ex vivo experiments on mice reconfirmed that neither radiotracer alone could identify all of the viable tumor cells but that the combination of both tracers more accurately identified viable cancer cells in the tumors. The combined injection of these tracers was also tested with PET scans in live subjects; this confirmed that the combination of two radiopharmaceuticals visualizes more viable cancer cells. On the basis of this proof-of-concept study, we concluded that, compared to individual injection of either tracer, the simultaneously combined IV injection of ¹⁸F-FDG and ¹⁸F-FLT for PET studies is a novel imaging strategy and may noninvasively achieve more complete and accurate identification of viable lung cancer cells both in mice and humans.

Materials and Methods

Mouse Model of Lung Cancer

In mouse experiments, human NSCLC A549 and HTB177 cell lines were used, which were purchased from American Type Culture Collection (Manassas, VA). A549 and HTB177 cells were maintained in F-12K Medium (American Type Culture Collection) and RPMI 1640 medium (Cellgro, Herndon, VA), respectively. All media were supplemented with 10%FBS (Gemini Bio-Products, West Sacramento, CA), 1% glutamine, and 1% antibiotic mixture (Cellgro). Cells were grown in a humidified incubator at 37°C air atmosphere containing 5% CO₂. Exponentially growing cells were harvested using 0.25%(wt/vol) Trypsin-0.53-mMEDTA solution, and harvested cells were washed and suspended in phosphate-buffered saline. The number of viable cells was counted using a Vi-CELL cell viability analyzer (Beckman Coulter, Miami, FL).

All experiments were performed on 6-week-old female athymic NCr-nu/nu mice purchased from National Cancer Institute (NCI)-Frederick Cancer Research Institute (Bethesda, MD). Nude mice were maintained and used according to the guidelines of University of Louisville Health Center Animal Care and Use Committee. The animal study proposal had been approved by the Committee. Animals were housed five per cage and kept in the institutional small animal facility at a controlled constant temperature and humidity. Food pellets and water were provided ad libitum.

Human Studies

The Institutional Review Board of Inner Mongolia Medical University as well as the local ethics committee approved this investigation. Written consent was obtained from patients. The Institutional Review Board of University of Louisville has approved data transfer and use. Two patients with biopsy-proven pretreated lung adenocarcinoma were approved to include in the proof-of-concept study.

Study Design and Experiment Procedures

In ex vivo mouse studies, to relate the accumulation of ¹⁸F-FDG, ¹⁸F-FLT, and the cocktail ¹⁸F-FDG and ¹⁸F-FLT with tumor microenvironment, both HTB177 and A549 tumor-bearing mice were divided into three groups of three mice in each, and either ¹⁸F-FDG (7.4 MBq) and ¹⁸F-FLT(7.4MBq) or the cocktail of ¹⁸F-FDG (7.4 MBq) and ¹⁸F-FLT (7.4 MBq) was intravenously injected. All animals received pimonidazole (2 mg) and BrdU (4 mg) simultaneously with tracer injection. Animals were killed 1 hour later. Hoechst 33342 (0.5 mg, 0.1 ml; Sigma-Aldrich, St Louis, MO) was given through the tail vein 1 minute before the killing. Individual intratumoral activity distributions of ¹⁸F-FDG and ¹⁸F-fluorothymidine and the activity distribution by simultaneous cocktail injections of both tracers were determined by autoradiography and compared with immunohistochemistry visualization of hypoxia by pimonidazole and proliferation by BrdU from same frozen tumor section.

Noninvasive comparison of intratumoral distribution of ¹⁸F-FDG and ¹⁸F-FLT in same mouse was achieved by acquisitions of 14 hours apart with two subsequent micro-PET scans. Micro-PET scan obtained 1 hour after IV injection of ¹⁸F-FLT (7.4 MBq) and BrdU (4 mg). For the purpose of improving the accuracy of repositioning of a mouse on subsequent studies, before removing the animal from the imaging table of the micro-PET scanner (Concorde MicroSystems, Knoxville, TN), the body contour of the mouse was traced on a piece of paper previously placed under the animal, and also, the midback of the mouse was marked with permanent ink. Approximately 14 hours later, the same mouse was intravenously injected with 7.4MBq of ¹⁸F-FDG together with the hypoxia marker pimonidazole, and ¹⁸F-FDG micro-PET images were obtained 1 hour after the uptake phase. To improve the exact repositioning of the mouse, we used the built-in alignment laser, the body contour that was drawn during the first PET imaging, and the permanent ink cross that was also placed during the initial PET imaging. A total of five mice was studied, each bearing one HTB177 subcutaneous xenograft on the left hind leg and an A549 tumor on the right. After completion of PET scans, animals were killed, and tumors were frozen for histologic procedures.

Intratumoral distribution of ¹⁸F-FDG and ¹⁸F-FLT in same patients was also compared. PET/CT images of the patients were obtained using a GE Discovery ST PET/CT scanner (GE Healthcare, Milwaukee WI), following 6 hours of fasting with blood glucose less than 6 mM. Whole-body ¹⁸F-FDG PET/CT scans were obtained 1 hour after 3.7 MBq/kg IV ¹⁸F-FDG administration. Ten hours later, second PET/CT chest images of each patient were obtained 1 hour after injection of 3.7 MBq/kg ¹⁸F-FLT.

In a separate experiment, animals were intravenously injected with ¹⁸F-FLT (7.4 MBq) and allowed to uptake for 1 hour; animals were then anesthetized and placed and taped on a scanner bed that was covered with a warm pad for PET scan. Immediately after this procedure, ¹⁸F-FDG (7.4 MBq) was given intravenously to the animal; a second micro-PET was performed 1 hour later (e.g., approximately 120–130 minutes after the ¹⁸F-FLT administration). In this study, the animal was anesthetized and typed on the PET scanner bed for the first PET scan and kept the same position throughout the process of second tracer injection, uptake, and the second PET scan. Therefore, paired images from two individual PET scans of same animal were able to accurately select, which avoided any potential misregistration due to animal position changed. A total of three mice were used, each bearing one HTB177 subcutaneous xenograft on the left and A549 tumor on the right hind leg. Similar experiments were conducted in the reversed order of tracer injection in same models of three mice.

Finally, a mixture of ¹⁸F-FDG (7.4 MBq) and ¹⁸F-FLT (7.4 MBq) was coadministrated with pimonidazole (2 mg) and BrdU (4 mg) through the tail vein. Single PET scan was performed 1 hour later to detect whole-body and intratumoral activity distribution of the “cocktail tracers” in three mice. After completion of the imaging and 1 minute before killing the mouse, Hoechst 33342 (0.5 mg) was injected through the tail vein, and harvested tumors were fixed and sectioned for autoradiography and immunohistochemical staining. ¹⁸F-FLT PET was performed 24 hours earlier.

Radiopharmaceuticals

For preclinical animal studies, both ¹⁸F-FDG and ¹⁸F-fluoride were purchased from PETNET Pharmaceuticals Inc facility housed in University of Louisville Hospital (Louisville, KY). ¹⁸F-FLT was purchased from the radiochemical laboratory of Department of Radiology Small Animal Imaging Core Facility (University of Louisville) and synthesized as described previously [19].

For clinical human studies, ¹⁸F-fluoride was generated using an in-house cyclotron; ¹⁸F-FDG and ¹⁸F-FLT were synthesized automatically with FX-FN conventional modules at Inner Mongolia Medical University PET/CT facility (Hohhot, China). Both ¹⁸F-FDG and ¹⁸F-FLT were pyrogen free and qualified for clinical use, with radiochemical purity greater than 98%.

Markers of Hypoxia, Proliferation, and Perfusion for Animal Study

The hypoxia marker pimonidazole hydrochloride (Hypoxyprobe Inc, Burlington, MA) was dissolved in physiological saline at a concentration of 20 mg/ml. The proliferation marker BrdU (Roche Diagnostics, Indianapolis, IN) was first dissolved in dimethylsulfoxide and further diluted in physiological saline to a final concentration of 20 mg/ml. The blood perfusion marker Hoechst 33342 (Sigma-Aldrich) was dissolved in physiological saline at a concentration of 5 mg/ml. In all cases, fresh drug solutions were prepared on the day of animal experiments. These markers were not used for patients in this report.

PET Imaging for Intratumoral Radioactivity Distribution

All animals were imaged in a prone position using a dedicated three-dimensional small-animal R4 micro-PET (Concorde Microsystems) system. The R4 micro-PET scanner has a transaxial field of view of 10 cm and an axial field of view of 7.8 cm. The obtained list-mode data were sorted into two-dimensional histograms by Fourier rebinning, and the images were reconstructed by an iterative reconstruction algorithm into a 128 x 128 x 63 (0.72 x 0.72 x 1.3mm) matrix. In all animal studies, acquisition time was set to 10 minutes for each PET scan. Before each PET study, overnight fasted animals were anesthetized by inhalation of an isoflurane (1.5%)-air mixture. All image sets for each animal were visually examined using a rotating (cine) three-dimensional display. The window and level settings were adjusted for best visual intratumoral tracer distribution. For human studies, a Discovery ST8 PET/CT scanner (GE Healthcare) was used. CT based attenuation correction was applied to all PET studies.

Digital Autoradiography on Frozen Sections

Following animal killing, autoradiography of the harvested tumor sections was obtained by placing the tumor sections against the imaging plate in a film cassette as described previously [10,19,24]. The same plate was used through the experiments; the plate was exposed to tumor sections for approximately 20 hours and read by a Cyclone Plus imaging system (PerkinElmer, Inc, Waltham, MA), which generated digital images with pixel dimensions of 42 x 42 µm.

Visualization of Hypoxia (Pimonidazole), Proliferation (BrdU), and Perfusion (Hoechst 33342)

Pimonidazole, BrdU, and Hoechst 33342 images of the tumor specimens were obtained after completion of autoradiography as described previously [16]. To minimize misalignment of the sections and registration errors, same tumor sections used for digital autoradiography were used to obtain these images. Briefly, slides were air dried, fixed in cold acetone (4°C) for 20 minutes, and incubated with Super-Block (37515; Pierce Biotechnology, Rockford, IL) at room temperature for 30 minutes. All three antibodies were applied in SuperBlock. Sections were then incubated with fluorescein isothiocyanate-conjugated antipimonidazole monoclonal antibody (Hypoxyprobe Inc), diluted 1:25, for 1 hour at room temperature. For BrdU staining, same sections after antipimonidazole antibody and Hoechst 33342 imaging acquired were treated with 2N HCl for 10 minutes at room temperature, followed by 0.1 M Borax for 10 minutes at room temperature. Sections were then exposed to Alexa Fluor 594-conjugated anti-BrdU antibody (1:20 dilution; Molecular Probes, Eugene, OR) for 1 hour at room temperature.

Immunohistochemical staining images were acquired at x40 magnification using a Nikon Eclipse E800 fluorescence microscope (Nikon Americas Inc, Melville, NY) equipped with a motorized stage (Ludl Electronic Products Ltd, Hawthorne, NY). Hoechst 33342 and pimonidazole were imaged using blue and green filters, respectively. BrdU was imaged using a red filter. Hematoxylin and eosin stain of the tumor sections was imaged by light microscopy. Microscopic images from same frozen sections were coregistered using Photoshop 7.0 (Adobe, San Jose, CA).

Results

Ex vivo experiments on HTB177 tumor sections using autoradiography and immunohistochemistry demonstrated excellent spatial colocalization of high levels of ¹⁸F-FDG activity and pimonidazolestained areas of hypoxia. In such areas, there were only few proliferating cancer cells displaying minimal BrdU binding. ¹⁸F-FLT preferentially accumulated in areas of tumors where proliferating cells were present, demonstrating high degree of BrdU binding and low degree of pimonidazole retention indicating oxic cells. Stroma and necrotic zones were also associated with lower ¹⁸F-FDG and ¹⁸F-FLT activity (Figure 1A). However, after injecting ¹⁸F-FLT and ¹⁸F-FDG cocktail, high degree of radioactivity accumulation was noted to associate with hypoxic (pimonidazole-stained positive) and proliferative (BrdU-stained positive) cancer cells; the radiotracer activity in stroma and necrotic zones remained to be low (Figure 1A). In hypoxic zone, ¹⁸F-FLT and ¹⁸F-FDG cocktail uptake ratio relative to that of necrosis was not significantly different from that of ¹⁸F-FDG but was significantly higher than that of ¹⁸F-FLT (Figure 1B). In proliferation zone, the ratio of ¹⁸F-FLT and ¹⁸F-FDG cocktail uptake relative to that of necrosis was not significantly different from that of ¹⁸F-FLT but was significantly higher than ¹⁸F-FDG (Figure 1C). Results were similar in both HTB177 and A549 xenograft tumor tissue sections.

Figure 1.

Intratumoral accumulation of ¹⁸F-FDG, ¹⁸F-FLT, and their cocktail and tumor microenvironment components in HTB177 tumors. Tumor-bearing mice were intravenously injected either with ¹⁸F-FDG (7.4 MBq) or ¹⁸F-FLT (7.4 MBq) or with the mixture of both. All received pimonidazole (2 mg) and BrdU (4 mg) simultaneously. Animals were killed 1 hour later. Hoechst 33342 (0.5 mg, 0.1 ml) was given through the tail vein 1 minute before the killing. Overlay imaging of immunohistochemical stains: hypoxia marker pimonidazole (green), proliferation marker BrdU (red), and perfusion marker Hoechst 33342 (blue). (A) Top panel: ¹⁸F-FDG uptake is high in hypoxic regions (green) as arrow indicated. ¹⁸F-FDG accumulation is absent in delineated proliferating tissue where BrdU (red) was positive and with Hoechst-positive well perfusion regions (arrowhead). Middle panel: ¹⁸F-FLT accumulates in proliferative (red) cancer component as arrowhead indicated, not hypoxic (green) zones (arrow). Bottom panel: Coinjection of ¹⁸F-FDG and ¹⁸F-FLT (cocktail). Both hypoxic (green) and proliferative (red) cancer cells associate with high activity. (B) In hypoxic zone, ¹⁸F-FLT and ¹⁸F-FDG cocktail uptake ratio relative to that of necrosis is not significantly different from that of ¹⁸F-FDG but is significantly higher than that of ¹⁸F-FLT. *P < .01 to all other groups. (C) In proliferation zone, the ratio of ¹⁸F-FLT and ¹⁸F-FDG cocktail uptake relative to that of necrosis is not significantly different from that of ¹⁸F-FLT but is significantly higher than that of ¹⁸F-FDG. *P < .01 to all other groups.

We compared the intratumoral distribution of each tracer in same mouse by PET scans performed 14 hours apart (Figure 2A). In five mice with A549 (right leg) and HTB177 (left leg) NSCLC tumor xenografts, we demonstrated that intratumoral distribution of ¹⁸F-FLT and ¹⁸F-FDG, injected 14 hours apart from each other, displayed different tracer distribution patterns on their PET images. Such differences were better visualized when three-dimensional rotating (cine) maximum intensity projection (MIP) images were displayed side by side. A typical example is shown in Figure 2, B to F, as follows: the distributions of ¹⁸F-FLT and ¹⁸F-FDG did not fully match to each other in either HTB177 (left) or A549 (right)NSCLC tumor xenografts. Pimonidazole and BrdU distribution was also mismatched (Figure 2G). Such mismatch patterns were observed when the agents were injected 14 hours apart or when injected together simultaneously, which indicated that tumor hypoxia and proliferation were relatively stable at least during the 14-hour interval.

Figure 2.

Difference in intratumoral distribution of ¹⁸F-FLT and ¹⁸F-FDG in same mouse during a 14-hour interval. (A) Schematic of experimental design. (B) A coronal slice of ¹⁸F-FLT PET. (C) The coronal slice of ¹⁸F-FDG PET from same level of the mouse. (D) Intratumoral high ¹⁸F-FLT regions in HTB177 slice shown in B were contoured. (E) Intratumoral high ¹⁸F-FDG regions shown in C were contoured. (F) Overlay of the contours of high intratumoral radioactivity-accumulated regions: ¹⁸F-FLT (red) and ¹⁸F-FDG (blue). (G) Pimonidazole (hypoxia, green) and BrdU (proliferation, red) distribution maintains a mutually exclusive pattern when injected 14 hours apart in the frozen tumor section. Hoechst 33342 (blue) indicates functional blood perfusion.

Such mismatched pattern observed in mice was also demonstrated in two patients with lung cancer, when their 10-hours-apart ¹⁸F-FDG PET/CT and ¹⁸F-FLT PET/CT images of lung tumors were compared to each other. The intratumoral distribution of ¹⁸F-FDG and ¹⁸F-FLT appeared mismatched, confirming the findings observed on preclinical mouse studies. A set of representative PET/CT images of a 61-year-old male patient with NSCLC is presented in Figure 3 displaying a significant intratumoral tracer distribution difference and mismatch.

Figure 3.

Difference in intratumoral distribution of ¹⁸F-FLT and ¹⁸F-FDG in a patient. A 61-year-old male with pretreated lung adenocarcinoma underwent ¹⁸F-FLT PET/CT and ¹⁸F-FDG PET/CT scans during 10-hour interval. Apparent difference in intratumoral distribution between the two radiopharmaceuticals is presented as arrow and arrowhead indicated.3

Subsequent PET scans were performed following the flow chart shown in Figure 4A. Coronal PET slices were presented; the initial ¹⁸F-FLT image (scan 1) was compared to a combining ¹⁸F-FDG and ¹⁸F-FLT image (scan 2). There was a clear increase in the fraction of the tumor showing enhanced uptake of radioactivity in combination of ¹⁸F-FDG and ¹⁸F-FLT image than ¹⁸F-FLT-alone imaging (Figure 4, B and C). The same pattern was seen when given radiopharmaceuticals in the reverse order (Figure W1). Results were broadly similar in all the mice we examined.

Figure 4.

¹⁸F-FLT-alone PET versus the combination of ¹⁸F-FLT and ¹⁸F-FDG PET on cancer tissue visualization. (A) Schematic of the experimental design. (B) ¹⁸F-FLT coronal PET slices (scan 1), and combination of ¹⁸F-FLT and ¹⁸F-FDG coronal PET slices (scan 2). (C) Top: Intratumoral distribution of ¹⁸F-FLT in region of interest (ROI) of slice 62 in B (upper panel). Bottom: Intratumoral distribution of the combination of ¹⁸F-FLT and ¹⁸F-FDG in ROI of slice 62 (B, lower panel) maps more higher-radioactivity tissue, indicating better viable cancer tissue visualization.

Intratumoral distributions of the radiotracers were also determined after simultaneous “cocktail” injection of ¹⁸F-FLT and ¹⁸F-FDG PET mixture (Figure 5B) and compared with the ¹⁸F-FLT-only PET images of the same mouse that were obtained 24 hours earlier (Figure 5A). The intratumoral distribution of both radiotracers together clearly showed an increase in the fraction or volume of tumor with increased PET tracers compared to PET images obtained with ¹⁸F-FLT injections alone. Autoradiography of the tumors obtained from the mouse (Figure 5C) revealed that intratumoral radioactivity accumulation was associated with viable cancer cells, which were either hypoxic (pimonidazole-stained positive) or proliferative (BrdU-stained positive) (Figure 5D); stroma and necrotic zones associated with lower radioactivity (Figure 5, C and E).

Figure 5.

The power of ¹⁸F-FLT and ¹⁸F-FDG cocktail PET on viable cancer tissue identification. ¹⁸F-FLT PET (A) was conducted 24 hours before ¹⁸F-FLT and ¹⁸F-FDG cocktail PET (B) in same mouse is presented. Apparent difference in whole-body radioactivity distribution from two scans is noted. Followed the animal killed, tumors were removed, frozen, and sectioned. Histologic findings from same frozen section of A549 tumor on the right thigh were presented. (C) Digital autoradiography of intratumoral distribution of cocktail. Both hypoxic and proliferative cancer cells associate with high radioactivity; “cold” areas on the autoradiograph correspond to either necrotic tissue (N) or stroma (S). (D) Overlay imaging of immunohistochemical stains: hypoxia marker pimonidazole (green), proliferation marker BrdU (red), and perfusion marker Hoechst 33342 (blue). (E) Hematoxylin and eosin stain is provided as reference.

Discussion

Human NSCLC and most solid cancers have a very heterogeneous microenvironment, being composed of viable cancer cells, noncancerous stroma, and necrotic zones. Viable cancer cells are either minimally proliferative and hypoxic cancer cells or oxic and highly proliferative cancer cells, and proliferation and hypoxia are generally exclusive [16–19,24–27]. Therefore, an ideal PET tracer must bind indiscriminately to all viable cancer cells regardless of their status of oxygenation or proliferation.

PET imaging with ¹⁸F-FDG and ¹⁸F-FLT has been widely used in the management of cancer. However, neither of them has fully demonstrated conclusive superiority of one to another [1–12,28–32]. This was presumably due to the highly spatial and temporal heterogeneous nature of the tumor microenvironment [33–35] and the differences in uptake mechanisms of ¹⁸F-FDG and ¹⁸F-FLT. ¹⁸F-FDG is preferentially taken up by hypoxic cancer cells [8–10,19–21]. In contrast, ¹⁸F-FLT accumulates predominantly in proliferative cancer cells and is unlikely taken up by hypoxic cells, as they are minimally proliferative [19]. Therefore, neither ¹⁸F-FDG nor ¹⁸F-FLT alone is perfect for a complete identification of viable cancer cells in solid tumors containing complex tumor microenvironment.

In a previous ex vivo study, we demonstrated that there is a mismatched intratumoral activity distribution pattern of ¹⁸F-FDG and ¹⁸F-FLT (exclusive to each other) [19], which was reconfirmed in this study as well (Figure 1).

Current study noninvasively demonstrated the exclusive pattern distribution mismatch of ¹⁸F-FDG and ¹⁸F-FLT in mouse model of NSCLC when injected into same mice sequentially 10 to 14 hours apart from each other (Figure 2) and in two patients with recent diagnosis of pretreated NSCLC as well (Figure 3). Thus, although preliminary, these findings suggest that the exclusive pattern of intratumoral distribution of ¹⁸F-FDG and ¹⁸F-FLT can be noninvasively imaged in mice and humans with NSCLC.

The 10- to 14-hour time interval, which is slightly over five to seven half-lives decay for F-18, was chosen to minimize the residual radioactivity effect remaining from the first injection into the second imaging (Figures 2 and 3). Because the distribution pattern of BrdU coinjected with ¹⁸F-FLT and pimonidazole coinjected with ¹⁸F-FDG was still mutually exclusive, the tumor microenvironment is probably mostly stable during this period. The possibility of a change in the tumor microenvironment during this period then is not a major concern (Figure 2G). Thus, the demonstrated mismatch pattern of these radiotracers on PET images was valid and reflected the real relationship of ¹⁸F-FDG and ¹⁸F-FLT uptake in vivo and not the consequence of significant spatial change in the intratumoral microenvironment.

Although there is a predominant mismatch in spatial distribution of ¹⁸F-FDG and ¹⁸F-FLT in NSCLC tumors both in mouse (Figure 2) and in human (Figure 3), a detailed evaluation reveals some activity overlaps between ¹⁸F-FDG- and ¹⁸F-FLT-positive areas. One explanation is that this could be due to a possible microenvironmental heterogeneity that is below the resolution of used current PET technology. However, presence of a slight change in tumor microenvironment in some portion of the tumor cannot be totally excluded.

This preliminary study suggests that the simultaneous, combined ¹⁸F-FDG and ¹⁸F-FLT PET imaging identifies more viable cancer tissue than when either tracer is injected individually (Figures 1, 4, and W1). Autoradiography and histologic, immunohistochemical assays have proved that minimally proliferative/hypoxic cancer cells as well as highly proliferative/oxic cells accumulated high levels of radioactivity when ¹⁸F-FDG and ¹⁸F-FLT were administered simultaneously as a cocktail (Figure 1) or in a sequence one after another with a couple of hours apart (data not shown).

The accumulation of ¹⁸F-FDG and ¹⁸F-FLT cocktail might be independent of the oxygenation or proliferation status of cancer cells. Consequently, dual-tracer PET is more capable of better identifying the viable portion of the tumor mass from tumor necrosis and/or stroma; neither ¹⁸F-FDG nor ¹⁸F-FLT alone has the ability to do so.

Tumor microenvironment in terms of proliferation, glucose metabolism, and hypoxia has temporal heterogeneity and is unstable. Ljungkvist et al. reported that hypoxic human head-neck cancer cells growing in mice had a rapid turnover rate [36]. Nehmeh et al. found significant difference in spatial distribution of hypoxia tracer ¹⁸F-misonidazole in the same patients with head and neck cancer when repeat imaging was done over a 3-day interval [37]. Pilot PET studies have indicated that change in intratumoral distribution of ¹⁸F-FLT, ¹⁸F-FDG, and ¹⁸F-misonidazole occurred in as short as ∼48-hour interval in untreated animal model of lung cancer [35]. Furthermore, changes in intratumoral ¹⁸F-FDG distribution in patients with pretreated solid tumors occurring within a few days interval have been reported [34,35]. Accordingly, solid tumors are continuously remodeling, and hypoxic and proliferating cell components are dynamically changing in untreated tumors. Efficient anticancer therapy might also result in a more dramatic change in the microenvironment of a solid tumor. Therefore, ¹⁸F-FLT PET or ¹⁸F-FDG PET in some cases may not accurately identify the precise volume of the viable portion of a tumor to reflect the true effect of the anticancer therapy. However, the combination of ¹⁸F-FDG and ¹⁸F-FLT PET might be more capable of accurately identifying the true extent of the viable cancer cells, which would result in a better-managed anticancer therapy.

In this proof-of-concept study, two individually well-studied PET radiotracers are proposed to be used in a combination, as a cocktail mixture of ¹⁸F-FDG and ¹⁸F-FLT, which might be considered as a novel tracer. Although the drug safety may not be an issue for the use of the combination of ¹⁸F-FDG and ¹⁸F-FLT, burden of the radiation dose issue of the combination may need to be further considered and/or investigated. Recently, Kadrmas and colleagues have reported the technical feasibility of dual-tracer ¹⁸F-FDG and ¹⁸F-FLT on tumor characterization with a single PET scan [38]. We know that, at least in NSCLC experimental models, tumors generally have lower ¹⁸F-FLT uptake than ¹⁸F-FDG; the maximal intratumoral uptake of ¹⁸F-FLT is around 30% of maximal ¹⁸F-FDG uptake [19]. This may be due to the fact that only <∼30% of cancer cells in the nonhypoxic zones was proliferative and accumulated ¹⁸F-FLT, whereas all cancer cells in hypoxic zones were presumably able to accumulate ¹⁸F-FDG. It is also recognized that the cell proliferation rate may differ from one type of cancer to another; therefore, the “cocktail” mixture containing ¹⁸F-FDG and ¹⁸F-FLT probably needs to have a ratio in a range of approximately 1:1 to 1:3 to produce better, accurate PET images, which may be taken as a reference proposal preparing the “cocktail” for future investigations. Thus, before this new imaging strategy is to be accepted for its routine clinical use, in addition to the need for further search of optimization of radiation dose, the mixture ratio of these tracers and the clinical significance (outcome) of their use as a cocktail in the management of cancer therapy needs to be further investigated.

Conclusion

The combination of ¹⁸F-FDG and ¹⁸F-FLT with single PET imaging would give a more accurate representation of viable tumor tissue volume than a PET image obtained with either tracer alone. “Cocktail” PET imaging might be a novel imaging strategy for cancer and produces images that are less dependent on oxygenation and/or proliferative status of cancer cells. The simultaneous combination use of dual tracers as a “cocktail” is a promising PET imaging strategy in cancer management of patients. Its potential clinical significance needs to be further studied.