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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nucl Med Commun. Author manuscript; available in PMC 2010 September 27.
Published in final edited form as:
PMCID: PMC2946081
NIHMSID: NIHMS235177

99mTc glucarate high-resolution imaging of drug sensitive and drug resistant human breast cancer xenografts in SCID mice

Abstract

Background and aim

Previous studies have showed that 99mTc labelled glucarate (GLA) might be an agent for non-invasive detection of breast tumours. In xenografted BT20 breast tumours, GLA was found to have higher uptake than 99mTc sestamibi (MIBI). It is unclear whether GLA can localize in all cell line breast cancer xenografts, as well as breast tumours with multidrug resistance (MDR). The present study aimed to investigate the properties of GLA in detecting drug sensitive and drug resistant MCF7 breast cancer xenografts in mice by using dynamic single photon emission computed tomography (SPECT) imaging.

Methods

MCF7/S cells are drug sensitive breast carcinoma cells. MCF7/D40 cells are 40-fold more resistant to doxorubicin compared to MCF7/S. Subcutaneous tumours were grown in SCID mice for 10–14 days after injection of 1 × 106 cells into the right thigh. Anaesthetized mice with MCF7/S (MIBI, n= 9; GLA, n= 8) and MCF7/D40 (MIBI, n= 6; GLA, n= 5) tumours were imaged using a high-resolution SPECT system called FASTSPECT. Dynamic images were acquired for 2 h after intravenous injection of GLA or MIBI. Expression of MDR P-glycoprotein (Pgp) in the tumours was demonstrated in the MCF7/D40 tumours by western blotting, not in the MCF7/S tumours.

Results

The xenografted tumours were visualized unequivocally within 10–30 min in GLA images and remained detectable for at least 2 h after injection. Drug resistant tumours, from which MIBI was rapidly expelled, retained GLA as readily as did drug sensitive tumours. The biodistribution data of GLA demonstrated significantly higher accumulation (%ID/g) compared to MIBI.

Conclusion

MCF7 tumour xenografts can be detected by 99mTc glucarate imaging. More importantly, 99mT glucarate can potentially localize drug resistant breast tumours.

Keywords: 99mTc glucarate, 99mTc sestamibi, breast cancer, multidrug resistant, SCID mice, high-resolution SPECT

Introduction

Scintimammography (SMM) is an adjunct to conventional mammography in identifying patients with breast cancer using several radiopharmaceuticals either currently available or still under investigation [18]. 99mTc sestamibi (MIBI) scintimammography has been shown to have clinical utility in identifying breast tumours that are difficult to study with conventional mammography [13]. It has been approved by the U.S. Food and Drug Administration as an adjunct to mammography in the detection of breast cancer. Compared to conventional mammography, 99mTc sestamibi imaging has superior utility in patients with large amounts of glandular tissue and dense breasts [911].

99mTc sestamibi is a lipophilic monocationic radiotracer that accumulates predominantly within the mitochondria and cytoplasm of cells on the basis electrical potentials generated across membrane bilayers. Overall institutional sensitivity and specificity in 99mTc sestamibi imaging are 75.4% and 82.7% for breast cancer detection [3]. The positive predictive value is 74.5% and the negative predictive value is 83.4%. Perhaps because of either poor gamma camera resolution or lower 99mTc sestamibi uptake in the tumours, the sensitivity of 99mTc sestamibi SMM for small lesions is not as good as it is in larger lesions. For example, the sensitivity for tumours under 1 cm in size was only 48.2% [3]. Larger lesions may also be undetected by 99mTc sestamibi SMM due to low cellular proliferation or overexpression of a multidrug resistance (MDR) gene, MDR1, which encodes for a membrane glycoprotein, P-glycoprotein (Pgp) [12]. Pgp acts as an energy dependent drug-efflux pump and allows transport of a wide range of structurally and functionally unrelated cytotoxic drugs out of tumour cells [13,14]. In addition to the drugs, many surrogate markers of Pgp function in vivo such as 99mTc sestamibi can also be pumped out [15,16]. Faster clearance of 99mTc sestamibi has been observed in tumours that express Pgp compared with tumours that do not [17]. Consequently, there is a need for an alternative marker that is not as readily eliminated by Pgp.

D-glucaric acid is a six-carbon dicarboxylic acid sugar and a natural catabolite of glucuronic acid metabolism in mammals [18,19]. All mammals excrete glucaric acid (glucarate) as a physiological end product. Glucaric acid can be readily radiolabelled with sodium pertechnetate, resulting in 99mTc glucarate (GLA), which was originally designed for detecting early necrosis of the heart and brain [1921]. It was reported that 99mTc glucarate could also concentrate in malignant breast tumours [7,8]. Biodistribution studies indicated that primary breast tumours in patients were visualized at early imaging time with 99mTc glucarate [22]. In SCID mice with xenografted BT20 tumours, 99mTc glucarate was found to have significantly higher uptake than 99mTc sestamibi [7]. The mechanisms of 99mTc glucarate uptake and accumulation in the breast tumours are as yet unknown. It is unclear if 99mTc glucarate will localize in all cell line breast cancer xenografts.

The purpose of this study was to determine the properties of 99mTc glucarate in detecting and localizing human MCF-7 breast cancer xenografts including drug sensitive and drug resistant breast tumours. We compared the in vivo kinetics of 99mTc glucarate with 99mTc sestamibi in SCID mice using high-resolution SPECT imaging. In addition to studying the properties of 99mTc glucarate, we wanted to clarify whether a novel high-resolution stationary single photon emission computed tomography (SPECT) system, FASTSPECT, would yield high-resolution images combined with the capability of fast dynamic imaging for radiopharmaceutical kinetic study in a mouse tumour model.

Materials and methods

Tumour cell lines

Drug sensitive cell lines (MCF7/S) were doxorubicin sensitive MCF7 breast carcinoma cells originally obtained from the American Type Culture Collection (ATCC #HTB-22, Rockville, MD).

Drug resistant cell lines (MCF7/D40) were generated in vitro by successively culturing parental MCF7 cells in slowly increasing concentrations of doxorubicin in a multiple step procedure [23]. Fresh drug was added when the medium was changed three times a week. The concentration of doxorubicin was increased from 1 × 10−8 M to 7 × 10−8 M over 19 months. During an additional 12 months, the concentration was increased to 4 × 10−7 M, representing full development of the doxorubicin resistant variant cells. The presence of Pgp in the MCF7/D40 cells was confirmed by immunoblot analysis using the C219 mouse monoclonal antibody (IgG) with 125I labelled rabbit anti-mouse IgG as the secondary antibody. The drug resistant cells were maintained in a drug-free medium for 1 week prior to experiments.

Tumour models

Severe combined immunodeficient (SCID) mice weighing 18–22 g were obtained from the SCID mouse core facility at the University of Arizona Comprehensive Cancer Center. Mice were housed under pathogen-free conditions in microisolator cages with laboratory chow and water available. Subcutaneous breast adenocarcinomas were established by injecting 9 × 106 MCF7 cells into the mouse right thigh. The volume of tumour was monitored and measured daily. After 14 days, tumours reached a size of 200–500mm3 for imaging.

Animals were anaesthetized with 1.0–1.5% isoflurane, and the jugular vein was catheterized with a PE10 catheter via a surgical procedure. The catheter was secured proximal to the opening with a ligature, and further secured distal to the opening with a second ligature.

Experimental groups

MCF7/S tumour model

A total of 17 SCID mice with drug sensitive tumours were studied. FASTSPECT imaging was performed in nine mice with 99mTc sestamibi (MIBI/S group), eight with 99mTc glucarate (GLA/S group).

MCF7/D40 tumour model

There were 11 mice with drug resistant tumours subjected to FASTSPECT imaging. The images were obtained in six mice with 99mTc sestamibi (MIBI/D40 group), five with 99mTc glucarate (GLA/D40 group).

Preparation of the radiopharmaceutical

Glucarate kits were provided by Molecular Targeting Technologies, Inc., (West Chester, PA) as single-vial doses containing 12.5 mg of glucarate. The reagent in the vial was a sterile, non-pyrogenic, lyophilized composition of stannous chloride, glucarate, sodium bicarbonate and hydrochloric acid. One millilitre of 99mTc as sodium pertechnetate (not less than 2.59GBq (70 mCi)) was added to the vial and allowed to react at room temperature for 15 min. The radiochemical purity (RCP) of the 99mTc glucarate was determined by Gelman instant thin-layer silica gel (ITLC-SG) strips developed in saline and acetone. In the ITLC-SG strip developed by saline, 99mTc colloid remained at the origin while 99mTc glucarate and [99mTc]pertechnetate migrated near the solvent front. In the strip developed by acetone, 99mTc glucarate and 99mTc colloid remained at the origin while [99mTc]pertechnetate moved near the solvent front. The RCP of 99mTc GLA was greater than 95% for all experimental injections. 99mTc glucarate was used within 3 h after preparation.

99mTc sestamibi was prepared with a Cardiolite kit (Bristol–Myers Squibb) provided by Syncor Corporation. The RCP was greater than 95%.

Acquisition of high-resolution SPECT images

Dynamic SPECT images were acquired using a high-resolution stationary SPECTsystem, FASTSPECT, which consists of 24 small modular gamma cameras and a cylindrical aperture [24,25]. The system was built at the Radiology Research Laboratory of the University of Arizona. Twenty-four 1mm diameter pinholes were drilled in the aperture such that a point source in the centre of the field of view simultaneously is projected to the centre of each camera. The total magnification is 3.5 in a 3.0 cm × 3.2 cm × 3.2 cm field of view. The spatial resolution of the system in the reconstructed image is about 1.0mm in all directions. Point source sensitivity in air is 359.5 cps/MBq (13.3 cps/μCi).

Anaesthetized animals were placed inside the FASTSPECT aperture using a translation stage. 99mTc sestamibi or 99mTc glucarate (0.15 ml, 111–222 MBq (3–6 mCi)) was injected via the jugular vein catheter followed by a 0.08ml saline flush. Beginning immediately upon injection, dynamic images were acquired every 1 min for the first 10 min, followed by 5 min acquisitions at 15, 20 and 30 min. Then 5 min images were obtained every 15 min from 30 to 120 min after injection. Twenty-four projections, one from each camera, were generated using a look-up-table scheme. In this scheme, each scintillation event within the camera’s NaI crystal was registered as the digitized outputs from the camera’s four photomultiplier tubes. In order to estimate energy and interaction position, the four outputs were then compared to a 20-bit look-up table. This table was precalculated using a calibration procedure that involved moving a collimated source across the camera face.

Image processing

The maximum likelihood expectation maximization (MLEM) reconstruction algorithm was applied to generate 3-dimensional images. All images were reconstructed using 100 iterations. The projection model built into this algorithm was generated using a calibration scheme that involved moving an uncollimated source through the imaging system’s field of view in 1mm steps and recording the system response at each calibration point. Using the software of SlicerDicer (PIXOTEC LLC, Renton, WA), transaxial, coronal and sagittal slices of the tumour were generated with 1 pixel thickness (1.0 mm). A lower threshold value was set around 60 in a colour range from 0 to 255 to display the uptake of 99mTc glucarate or 99mTc sestamibi in the tumour.

In all the images from 1 to 120 min after injection, regions of interest (ROIs) over the tumours were created from one coronal slice with the highest accumulation of 99mTc glucarate or 99mTc sestamibi. An ROI was also created over a non-tumour area to determine the radioactivity of background. After correction for the background and radioactive decay, the radioactivity in the tumour was projected onto the dynamic images for determinations of time–activity curve.

Biodistribution measurements

After images were acquired, the mice were killed with a lethal dose of barbiturate. Blood samples, tumour, skeletal muscle, heart, lung, kidney and liver were harvested. The tissue samples were weighed and measured using a CRC-4 Radioisotope Dose Calibrator (Capintec, Ramsey, NJ). The radioactivity in the tissue samples was expressed as percentage of injected dose per gram tissue (%ID/g). The samples were counted in a well counter adjusted to the 99mTc window if the radioactivity was lower than 74 kBq (2.0 μCi). Standards prepared from an aliquot of the administered 99mTc glucarate source were counted at the same time to convert the counts per minute from the gamma well counter into kilobequerels or microcuries.

Western blot analysis

In order to determine the drug resistant properties in the xenografted tumours, western blot analysis was applied to detect Pgp expression. The tumour samples were frozen in liquid nitrogen for more than 3 days. Randomly selected MCF7/S and MCF7/D40 tumour tissues were homogenized in lysis buffer after 99mTc decayed to background level. Protein concentration was determined using a BioRad DC (BioRad, Hercules, CA) protein assay. A total of 100mg protein was loaded per lane on a 10% SDS–PAGE gel. Completed gel was then transferred to a PVDF membrane and probed with a mouse monoclonal antibody C219 (1:200 dilution) against Pgp (Signet, Dedham, MA). A rabbit anti-mouse HRP secondary antibody was used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and detected with an ECL western blot analysis system (Amersham, Piscataway, NJ).

Data analysis

All results were expressed as mean ± SEM. Comparisons between groups were performed with one-way analysis of variance. Comparisons between two variables within a group were made by means of the two-tailed paired t-test. Probability values less than 0.05 were considered significant.

Individual 99mTc glucarate and 99mTc sestamibi clearance curves from the peak uptake were fit using non-linear regression procedures available in TableCurve 2D software (Systat Software Inc., Richmond, CA).

Ethics

All experiments were performed in accordance with the guidelines for animal research from the National Institutes of Health (NIH publication 85-23, revised 1985) and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Arizona.

Results

Pgp expression

Western blot analysis indicated that human MDR1 Pgp was well expressed in the xenografted MCF7/D40 tumours. A prominent band was observed in tumour cell membrane preparations with C219 antibody, indicating Pgp expression in the MCF7/D40 tumour samples. No immunodetectable MDR1 Pgp was presented in the xenografted MCF7/S tumour samples.

FASTSPECT tumour imaging

All tumours of 99mTc glucarate groups were initially visualized within 5 min by FASTSPECT imaging after injection of 99mTc glucarate, and they were unequivocally localized within 10–30 min. Figure 1 shows representative 99mTc glucarate coronal tomographic images in a mouse with subcutaneous MCF7/S tumour 20 min post injection. Dynamic 99mTc glucarate images demonstrated that the radioactive accumulation in the MCF7 tumours remained for at least 120 min post-injection. Representative dynamic images in MCF7/S and MCF7/D40 are shown in Fig. 2. Relative to the drug sensitive tumours, the drug resistant tumours exhibited the same level uptake of 99mTc glucarate during the entire period for dynamic acquisition. Two hours post-administration, the MCF7/D40 tumour xenograft could be well visualized in all directions of tomographic 99mTc glucarate images (Fig. 3). In comparison with 99mTc sestamibi imaging, 99mTc glucarate imaging demonstrated lower background activity and higher tumour accumulation (Figs 2 and and44).

Fig. 1
99mTc glucarate FASTSPECT coronal images (right panel) in a mouse with MCF7/S tumour xenograft on right thigh (left panel) 20 min after injection. Tumour (arrow) was localized clearly with low radioactive background on 99mTc glucarate images.
Fig. 2
(A and B) 99mTc glucarate dynamic FASTSPECT images (serial transaxial slices) from a mouse with MCF7/S breast tumour (arrow). The tumour (arrow) was visualized within 5 min and stayed well-defined for at least 120 min after 99mTc glucarate injection. ...
Fig. 3
99mTc glucarate FASTSPECT images 120 min after injection in a mouse with MCF7/D40 tumour xenograft on right thigh (A). Tumour (arrow) was well localized in all directions of tomographic images (B: coronal; C: sagittal; D: transaxial slices).
Fig. 4
(A and B) Dynamic FASTSPECT images from a mouse with MCF7/S breast tumour on right thigh (arrow) using 99mTc sestamibi (serial sagittal slices). The tumour was unequivocally visualized after about 10 min and remained detectable until 120 min after injection. ...

The MCF7/S tumours could be detected initially by FASTSPECT imaging 2–10 min after intravenous administration with 99mTc sestamibi. Two hours post-injection, dynamic images demonstrated that tumour activity remained detectable (Figs. 4A and B). In contrast, as shown in Fig. 4(C and D), the MCF7/D40 tumours could be localized only 2–4 min post-injection of 99mTc sestamibi; then the radioactivity in the tumours dropped quickly to background levels.

Kinetics of 99mTc glucarate and 99mTc sestamibi in MCF7 tumours

Figure 5 shows time–activity curves plotted from 1 min to 120 min for 99mTc glucarate and 99mTc sestamibi in the MCF7/S and MCF7/D40 xenografts determined by the computerized ROI analysis of FASTSPECT images that were background subtracted and decay corrected. The time–activity curves of 99mTc sestamibi were significantly lower for the MCF7/D40 tumours than for the MCF7/S tumours at each point in time from 5 min to 120 min. There was no such difference at each point between the 99mTc glucarate time–activity curves of the MCF7/S and MCF7/D40 tumours. There was significantly higher peak uptake and end radioactivity for 99mTc glucarate than for 99mTc sestamibi in each xenografted tumour model (P<0.05; Fig. 6).

Fig. 5
Time–activity curves of 99mTc sestamibi and 99mTc glucarate in the MCF7/S and MCF7/D40 tumours. The radioactivity in both tumour models with 99mTc glucarate was significantly higher at all points than with 99mTc sestamibi. In contrast to 99mTc ...
Fig. 6
The peak uptake and end radioactivity detected by FASTSPECT imaging using 99mTc glucarate were significantly higher than that using 99mTc sestamibi in both of the MCF7/S and MCF7/D40 tumours. The end activity of 99mTc sestamibi in the MCF7/D40 tumours ...

Clearance of 99mTc glucarate plotted as the percentage of peak activity did not differ significantly in the MCF7/D40 tumours compared with MCF7/S. In contrast, the clearance of 99mTc sestamibi was greater in MCF7/D40 tumours (Fig. 7). Both 99mTc glucarate and 99mTc sestamibi exhibited biphasic clearance curves, and biexponential equations were found to provide the best fits to the curves. The early phase showed fast clearance and the late phase showed slow clearance. The half-time values (minutes) of biexponential washout (T1/2, time to reach half of initial activity) from the tumours are shown in Table 1. The T1/2 values were significantly lower for MCF7/D40 than for MCF7/S during both the early and late phases with 99mTc sestamibi (P<0.05). With 99mTc glucarate, however, there was no difference in T1/2 values between MCF7/D40 and MCF7/S. 99mTc glucarate had a significantly shorter T1/2 in the late phase compared to 99mTc sestamibi in MCF7/S. 99mTc sestamibi in the MCF7/D40 tumours exhibited shorter T1/2 than 99mTc glucarate in the early phase (P<0.05).

Fig. 7
99mTc glucarate and 99mTc sestamibi washout curves from the MCF7/S and MCF7/D40 breast tumours. The radioactivity is plotted as a percentage of the peak activity in the tumour and extended to 2 h using the TableCurve curve-fitting calculation. Abbreviations ...
Table 1
Biexponential tumour clearance half-time (min)

The 2 h fractional washout (percent peak activity) of 99mTc sestamibi was significantly greater in MCF7/D40 than that in MCF7/S (71.7±1.7% vs 50.2±5.3%, P<0.05), but there was no difference (71.9±2.9% vs 72.8±3.1%, P>0.05) for 99mTc glucarate.

Biodistribution data

The average tumour weight for the 28 mice was 0.12±0.02 g. No difference was found in tumour weight between the MCF7/S and MCF7/D40 tumours imaged with 99mTc glucarate or 99mTc sestamibi. The biodistribution data are shown in Table 2. 99mTc sestamibi exhibited significantly lower radioactive accumulation (%ID/g) in the MCF7/D40 tumours than that in the MCF7/S tumours, (P<0.05). 99mTc glucarate showed significantly higher accumulation (%ID/g) than 99mTc sestamibi in either the MCF7/S or MCF/D40 tumours. There was no difference in 99mTc glucarate accumulations between the two kinds of xenografted tumour models. 99mTc glucarate demonstrated significantly lower radioactivity in non-tumour soft tissue (muscle) compared to 99mTc sestamibi (P<0.05). As a result, 2 h after injection, the tumour-to-muscle ratios (tumour/muscle) for 99mTc glucarate were 9.4-fold higher than for 99mTc sestamibi in the MCF7/S tumours and 19.2-fold higher than in the MCF7/D40 tumours. The blood activity of 99mTc sestamibi was significantly lower than 99mTc glucarate. Relative to 99mTc sestamibi, 99mTc glucarate showed a significant lower accumulation in liver.

Table 2
Biodistribution data (%ID/g)

Discussion

In this study, the feasibility of 99mTc labelled glucarate for detecting MCF7 breast tumours was assessed and compared to 99mTc sestamibi imaging using the high-resolution SPECT system, FASTSPECT. In the xenografted tumour models, 99mTc glucarate showed higher tumour uptake than 99mTc sestamibi. As described previously, 99mTc sestamibi was demonstrated to be a substrate for MDR in the drug resistant MCF7 tumours. The expression of Pgp is responsible for the high washout rate of 99mTc sestamibi. 99mTc glucarate, in contrast, exhibited the same kinetics in the drug resistant tumours as in the MCF7/S tumours, which are sensitive to doxorubicin. Thus, MDR is not a factor affecting the accumulation of 99mTc glucarate in the MCF7 breast tumours.

99mTc sestamibi is an isonitrile compound and is concentrated primarily in mitochondria. The uptake of cationic 99mTc sestamibi depends on negative transmembrane and mitochondrial potentials. Although the lipophilic sestamibi might diffuse through plasma membranes by a passive mechanism, Na+/K+ ATPase pump activity is required to maintain the membrane potential [2629]. 99mTc sestamibi is fixed intracellularly as long as the cell membrane integrity is intact and nutrient blood flow persists. This active energy dependent mechanism might explain the slower washout of 99mTc sestamibi compared to that of 99mTc glucarate from the tumour xenografts in the present study.

Since glucarate is a six-carbon dicarboxylic acid sugar and behaves as a glucose analogue, the increased uptake of GLA may relate to upregulated glucose transport in the tumour cells. Overexpression of glucose transporters directly involves accelerated metabolic processes to accommodate the energy requirements of rapidly dividing cells and provide the carbon backbone for DNA and RNA synthesis for cell proliferation in growing tumours. The uptake of 99mTc glucarate in certain cells was inhibited by fructose [20]. Insulin administration was found to increase 99mTc glucarate uptakes in the rat hearts, livers and skeletal muscles significantly [30]. These experimental results suggest that 99mTc glucarate may enter certain cells by the sugar transport system.

The exact mechanism of 99mTc glucarate uptake in tumour is currently unclear. In acute infarcted myocardium, 99mTc glucarate was observed to target the nuclei of the necrotic myocardium. High nuclear uptake of 99mTc glucarate was also reported in the BT20 human breast tumour cell line [7]; the mitochondrial and cytosolic fractions also sequestered a substantial amount of 99mTc glucarate. However, the nuclear fraction of 99mTc glucarate distribution in tumour cells was lower than that in infarcted myocytes. When [14C]glucarate was administrated to rats bearing primary mammary tumours, significant binding of glucarate to proteins was found in the cytosolic fraction [31]. However, it is unclear how 99mTc glucarate is transported into the nuclei and mitochondria. Experimental evidence suggests that the activity of glucarate is mediated via signal transduction pathways involving cAMP and protein kinase C [32]. In acute infarcted myocardium, it is believed that 99mTc glucarate is associated with disruption of the myocyte and nuclear membranes, allowing free intracellular diffusion and electrochemical binding of the negatively charged glucarate complex to positively charged histones. However, the uptake of 99mTc glucarate associated with tumour necrosis or membrane breakdown is less likely in the current study, in which we used the xenografted tumours 10–14 days after tumour implantation. The tumour size was limited to about 0.12 g. No significant tumour necrosis was found in histological and electron microscopic examinations. Under the electron microscope, the tumour cells demonstrated prominent nuclei and abundant mitochondria with no significant breakage (unreported data).

The higher tumour uptake of 99mTc glucarate is not simply due to the distribution of the blood pool radioactivity and enhanced tumour vasculature with increased blood supply. Petrov et al. observed the tumour blood pool activity in BT20 breast-tumour-bearing SCID mice and compared 99mTc glucarate and 99mTc-DTPA uptakes [7]. The tumour uptake was significantly greater with 99mTc glucarate than with 99mTc-DTPA. 99mTc-DTPA enabled imaging of the early tumour blood pool soon after injection, but not at 3 h post-injection.

It is not surprising that 99mTc sestamibi and 99mTc glucarate exhibit different washout kinetics from the xenografted tumour models, as they are chemically different radiopharmaceuticals with different biological pathways. The rapid early clearance phase mainly reflects blood perfusion and effusion, in which the T1/2 values did not differ between 99mTc glucarate and 99mTc sestamibi in the MCF7/S tumours. The slow second phase, which reflects cellular efflux of radiotracers, showed shorter T1/2 in 99mTc glucarate than that in 99mTc sestamibi. The greater washout property of 99mTc glucarate may be a disadvantage for tumour targeting, but the larger amount of 99mTc glucarate radioactivity initially delivered to the tumour might compensate for the greater rate of clearance. Either in early phase or later phase washout of 99mTc glucarate, there was no difference in T1/2 between the MCF7/S and MCF7/D40 tumours. This kinetics result in the present study provides evidence that 99mTc glucarate is a promising agent in imaging breast tumours, including those with drug resistant properties. The shorter half-time (faster washout) in both of the early and second phase for 99mTc sestamibi was observed in the MCF7/D40 tumours compared to the MCF7/S tumours. The faster washout of 99mTc sestamibi from the drug resistant tumours can thereby be used as a means of identifying MDR Pgp expression in the patients with breast tumours.

Based on the current data, 99mTc glucarate offers favourable characteristics with significantly lower accumulation in non-tumour soft tissue. The mean tumour-to-muscle ratio for 99mTc glucarate is 4.5 and 6.2 (9.4 and 19.2 times higher than that for 99mTc sestamibi) in both the drug sensitive tumours and drug resistant tumours. In BT20 tumour-bearing mice, Petrov et al. reported that the tumour to non-tumour (shoulder region tissue) was 20.53:1 12 h after intravenous injection of 99mTc glucarate. This favourable tumour targeting property of 99mTc glucarate may make this agent potentially useful for detecting a small malignant lesion. 99mTc sestamibi is not accurate in the detection of axillary lymph node metastasis [3335], but 99mTc glucarate might be superior in the visualization of malignant axillary lymph node. 99mTc glucarate may provide utility in visualizing breast tumours and axillary metastases in cases where the MDR1 Pgp is overexpressed or the tumours become resistant to drugs during chemotherapy.

The rapid sequences of tomographic images obtained in this study demonstrate the capability of FASTSPECT for quantitative 3-dimensional imaging. Such functional images are not practical with conventional rotating SPECTsystems. FASTSPECT provides an ideal approach for tumour targeting and radiopharmaceutical in vivo kinetic studies using small xenografted tumours with less necrosis. It is a dedicated imaging system with stationary camera modules and a stationary multiple pinhole aperture [24,25]. The stationary multiple pinhole collimation overcomes the disadvantages of poor sensitivity and long-time acquisition of projection data in single pinhole or rotating multi-pinhole SPECT cameras, and makes it possible to perform fast dynamic tomographic imaging in small animals. Since no centre-of-rotation corrections are required, artifacts caused by inaccurate centre of rotation are not present in this high-resolution SPECT system.

Conclusion

The results in this study demonstrate that MCF7 human breast tumour xenografts can be detected by 99mTc glucarate in vivo imaging with unequivocal visualization within 10–30 min, and remain visible for at least 2 h after intravenous administration. 99mTc glucarate offers favourable imaging properties of higher uptake in the MCF7 breast tumours compared to 99mTc sestamibi. More importantly, 99mT glucarate can potentially localize drug resistant breast tumours, from which 99mTc sestamibi is rapidly expelled by Pgp, a drug-efflux pump. Thus, 99mTc glucarate warrants further study to determine its utility in screening for breast cancer, characterizing suspicious lesions, detecting axillary lymph node metastasis and evaluating various antitumour therapies. Furthermore, scintigraphic imaging using 99mTc glucarate needs to be studied to determine if this alternative tumour targeting agent can localize in all cell line human breast cancer xenografts, as well as differentiate benign and malignant breast tumours.

FASTSPECT demonstrated the ability to provide high-resolution tomographic images in a mouse model, combined with the capability for fast dynamic SPECT imaging. This novel imaging system allows quantitative dynamic imaging and functional determination of radiotracer kinetics in mice with xenografted tumours.

Acknowledgments

The authors wish to acknowledge Bethany Skovan, Gillian Paine and Henry Allan Toppin for assistance in establishing the animal tumour models, and Brenda K. Baggett for her expertise in preparing tissues for western blotting.

Supported by NIH grants P41 EB002035 and R24CA83148.

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