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[99mTcOAADT]-(CH2)2-NEt2: A Potential Small-Molecule

Single-Photon Emission Computed Tomography

Probe for Imaging Metastatic Melanoma

Zhen Cheng,1Ashfaq Mahmood,

1Huazhi Li,

1Alan Davison,

2and Alun G. Jones

1

1Department of Radiology, Harvard Medical School, Boston, Massachusetts and 2Department of Chemistry, Massachusetts Institute ofTechnology, Cambridge, Massachusetts

Abstract

Evaluation of [99mTc]oxotechnetium(V) complexes of theamine-amide-dithiol (AADT) chelates containing tertiaryamine substituents as small-molecule probes for the diagnos-tic imaging of metastatic melanoma has shown that techne-tium-99m–labeled AADT-(CH2)2-NEt2 (

99mTc-1) has the highesttumor uptake and other favorable biological properties. Wehave, therefore, assessed this agent in a more realisticmetastatic melanoma model in which, after i.v. tail injection,a highly invasive melanoma cell line, B16F10, forms pulmo-nary tumor nodules in normal C57BL6 mice. Small melanoticlesions develop in the lungs and, on histologic examination,appear as small black melanoma colonies, increasing in sizeand number with time after tumor cell injection. Groups ofmice received tumor cell inocula of 2 � 105, 4 � 105, or 8 � 105B16F10 cells; 14 days later, 2 hours after 99mTc-1 administra-tion, lung uptake of 2.83 F 0.21%, 3.63 F 1.07%, and 4.92 F1.61% injected dose per gram of tissue (% ID/g), respectively,was observed, compared with normal lung uptake of 2.13 F0.2% ID/g (P < 0.05). Additionally, a higher level of 99mTc-1accumulation was seen 17 days after tumor cell inoculationas the lung lesions grew. These in vivo studies coupled withadditional in vitro and ex vivo assessment show that 99mTc-1has high and specific uptake in melanoma metastases in lungsand can potentially follow the temporal growth of thesetumors. (Cancer Res 2005; 65(12): 4979-86)

Introduction

Malignant melanoma is a public health challenge with a risingincidence rate and estimates of 47,700 new cases to be diagnosedannually (1–3). The significant mortality of this cancer is associatedwith its high cellular proliferation rate and the early occurrence ofmetastases. Because early detection of the melanoma and itsassociated metastases considerably improves management andprognosis of the disease, there has been intense interest in thesearch for melanoma-specific diagnostic imaging probes andtherapeutic agents (4, 5).2-[18F]Fluoro-2-deoxy-D-glucose ([18F]FDG) is widely used in

positron emission tomographic (PET) imaging of various tumorsincluding melanoma. In patients with stage III or in-transitmelanoma, prospective whole-body [18F]FDG PET studies displaya sensitivity of 87.3% with a positive predictive value of 78.6%,which can be further improved with the help of other pertinent

clinical information (6). Other prospective clinical studies have alsoshown the utility of whole-body [18F]FDG PET imaging in stage IIto IV melanoma with sensitivity and specificity of 94.2% and 83.3%,respectively, for lesions in the soft tissue, lymph nodes, and liver (7);false-positives are due to the accumulation of [18F]FDG in surgicalwounds, pneumonia, and other etiologies including infection/inflammation (6–9).A number of radiolabeled, single-photon emission computed

tomography (SPECT) metabolic tracers, peptides, and monoclonalantibodies have also been investigated for melanoma detection,staging, and follow-up (4, 5). Among these SPECT agents, the mostpromising are the radioiodinated benzamides (10–14). Althoughthe nature of the affinity of iodobenzamides for melanoma is notclearly understood, it has been shown that radiolabeled iodoben-zamides possess a considerable in vivo affinity for melanoticmelanoma lesions compared with amelanotic lesions (10). Animaland human studies of several radioiodobenzamide derivativesindicate a promising profile for their use in melanoma diagnosis. Ascintigraphic phase II clinical study in 110 patients for detectingmalignant melanoma and its metastases with N-(2-diethylami-noethyl)-4-[123I]iodobenzamide ([123I]BZA; Fig. 1) found 81% diag-nostic sensitivity, 87% accuracy, and 100% specificity (11) for thecompound. Additional studies to optimize the pharmacokineticcharacteristics of [123I]BZA derivatives have shown that N-(2-diethylaminoethyl)-3-[123I]iodo-4-methoxybenzamide ([123I]IMBA;Fig. 1) has an 8-fold higher melanoma/nontarget tissue ratiothan [123I]BZA at 1 hour postinjection and a 4-fold higher ratio at4 hours in the C57BL6 mouse model (12, 13). Preliminaryscintigraphic studies in patients with melanoma metastases haveconfirmed the efficacy of this compound (12). More recently, asingle-center clinical trial in 25 patients using another radio-iodinated iodobenzamide derivative, N-(2-diethylaminoethyl)-2-[123I]iodobenzamide ([123I]BZA2; Fig. 1), showed 100% sensitivity,95% specificity, a positive predictive value of 86%, and a negativepredictive value of 100% on a per patient basis (14).Whereas iodine-123 is commercially available and the radio-

iodobenzamide derivatives, such as [123I]IMBA and [123I]BZA2,exhibit promise as potential SPECT agents for imaging metastaticmelanoma, a technetium-99m–based radiopharmaceutical wouldhave considerable advantages. Technetium-99m decays with a6-hour half-life and is widely available on demand in mostnuclear medicine facilities as a 99Mo-99mTc generator. It has a highphoton flux and emits 140 keV g-photons with ideal character-istics for SPECT imaging, making it very cost-effective andthe radiolabel of choice for routine clinical imaging. Technetiumcomplexes that contain structural elements of the benza-mides have been explored, including technetium-99m–labelednitridotechnetium-bis(aminethiol)benzamide (TcN-BAT-BZA) andoxotechnetium-bis(aminethiol)benzamide (TcO-Cf; Fig. 1); the

Requests for reprints: Ashfaq Mahmood, Department of Radiology, HarvardMedical School, Armenise Building, 200 Longwood Avenue, Boston, MA 02115. Phone:617-432-3995; Fax: 617-432-2419; E-mail: [email protected].

I2005 American Association for Cancer Research.

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latter has limited tumor uptake [

rotary evaporator followed by additional drying via high vacuum. The dryresidue was later redissolved in argon-saturated methanol and distributed

into 10 vials that were dried and kept under vacuum for later preparation

of the technetium-99m–labeled compound. 99mTc-1 was synthesized using a

vial containing 0.5 mg of the above thiol-deprotected ligand which wasdissolved in 0.25 mL PBS (5 mmol/L, pH 7.4). To this was added the required99mTc activity in the form of a 99mTc-glucoheptonate solution (0.3 mL), and

the reaction was heated at 75jC for 25 minutes in a water bath. HPLCanalysis of the reaction mixture typically indicated >90% radiochemicalyield of the 99mTc-1 complex (Fig. 2). In addition, coinjection of the

structurally characterized rhenium complex (20) with the analogous 99mTc-1

complex resulted in coelution of the radioactive species with the

corresponding UV-active rhenium complex. All in vitro and in vivoexperiments were conducted with the 99mTc-1 complex isolated via

preparative HPLC. The solution was subsequently evaporated to complete

dryness and the complex reconstituted in sterile distilled water to therequired radioactivity concentration.

In vitro studies with murine B16F10 melanoma cells. B16F10 cells(2 � 106) from the American Type Culture Collection (Manassas, VA) wereplated and grown in three T-175 flasks in 14 mL DMEM (pH 7.4; Gibco

Life Technologies) containing 4,500 mg/L D-glucose, L-glutamine, and

pyridoxine hydrochloride, 110 mg/L sodium pyruvate, 10% fetal bovine

serum, 0.2% gentamicin, and 0.5% penicillin-streptomycin solution.At the time of the cell uptake assay, all cells were harvested from

the culture flasks by trypsinization with 1 mL trypsin-EDTA solution

(0.25% trypsin, 1 mmol/L EDTA�4Na; Gibco Life Technologies). Afterbeing washed twice with 12 mL cold PBS (Gibco Life Technologies),the cells were counted and resuspended in Ca2+-free MEM with reduced

Mg2+ content (S-MEM, Gibco Life Technologies) to a final concentration

of 5.0 � 106 cells/mL. The effect of inhibition of melanin synthesis onthe uptake of 99mTc-1 was studied by substituting DMEM containing

500 Amol/L N-butyldeoxynojirimycin (NBdNJ; ref. 23) as the growthmedium 48 hours before the assay, and replenishing the NBdNJ medium

every 24 hours. Similarly, the effect of increased melanin synthesis/contenton the uptake of 99mTc-1 was assessed by stimulating melanin synthesis in

Figure 3. In vitro cell uptake of 99mTc-1 by intact B16F10 cells at 37jC (A) and4jC (B).

Table 1. Biodistribution of 99mTc-1 at 2, 3, and 6 hourspostadministration in C57BL6 mice bearing s.c. B16F10melanoma

Organ 2 h 3 h 6 h

Blood 0.71 F 0.13* 0.56 F 0.02 0.23 F 0.09Heart 0.67 F 0.13 0.45 F 0.03 0.16 F 0.05Liver 10.03 F 0.62 7.57 F 0.22 4.32 F 0.59Lungs 3.19 F 0.51 1.94 F 0.20 0.70 F 0.23Muscle 0.54 F 0.12 0.22 F 0.02 0.09 F 0.05Kidneys 3.06 F 0.54 2.40 F 0.34 1.39 F 0.85Spleen 1.35 F 0.25 0.69 F 0.05 0.53 F 0.53Brain 0.19 F 0.03 0.11 F 0.00 0.05 F 0.03Intestines 6.51 F 2.18 6.21 F 5.57 2.39 F 0.62Tumor 7.60 F 1.09 6.60 F 2.16 4.72 F 1.93Skin/Fur 0.67 F 0.11 0.57 F 0.31 0.19 F 0.09Tumor/Blood 10.53 F 3.01 11.83 F 3.76 24.01 F 15.90Tumor/Muscle 16.34 F 2.30 30.58 F 11.52 67.54 F 58.30Tumor/Liver 0.77 F 0.07 0.88 F 0.30 1.15 F 0.65Tumor/Lung 2.21 F 0.31 3.41 F 1.04 8.21 F 6.55Tumor/Spleen 5.88 F 0.90 9.53 F 2.79 15.01 F 9.63

*% ID/g F SD; n = 4-6.

Figure 4. Comparison of in vivo tumor accumulation of 99mTc-1 (Tc-1 ),[18F]FDG (FDG), [18F]DOPA (F-DOPA ), and [123I]IMBA (IMBA ) in s.c.B16-melanoma tumor model. Tumor uptake expressed as differential absorptionratio (DAR ) as reported by Ishiwata et al. (28) for [18F]FDG and [18F]DOPA.Data for IMBA calculated from Eisenhut et al. (13).

[99mTcOAADT]-(CH2)2-NEt2 for Melanoma Metastases




the B16F10 cells with DMEM supplemented with 2 mmol/L L-tyrosine24 hours before the assay.

For in vitro cell uptake studies of 99mTc-1, 2.5 � 106 B16F10 tumor cellswere incubated at 37jC or 4jC in polypropylene test tubes with 2 to 5 ACi(10 AL) 99mTc-1 in a total volume of 500 AL S-MEM with intermittentagitation. At appropriate time intervals, the tubes were vortexed and

triplicate samples of 15 AL were layered in individual 400-AL Eppendorfmicrocentrifuge tubes containing 350 AL cold calf serum. After centrifuga-tion at 12,000 rpm for 1 minute, the tubes were frozen in a dry ice-acetonebath and, while still frozen, cut into two sections (a bottom section

containing the cell pellet and an upper section with the supernatant) that

were placed in individual counting tubes. Both fractions were counted for

radioactivity in a g-counter (WALLAC, 1480 WIZARD 3). The percentage of99mTc-1 uptake was calculated as % cell uptake = 100 � [cpm (pellet)] /[cpm (pellet) + cpm (supernatant)].

In vivo studies in murine melanoma C57BL6/B16F10 mouse models.Murine B16F10 cells grown in DMEM were harvested from cell culture

flasks by trypsinization and washed as described above. The cells were

counted and resuspended in PBS to final concentrations of 2.0 � 105, 4.0 �105, or 8.0 � 105 cells/mL and were maintained at 4jC for immediate i.v. ors.c. inoculation in C57BL6 male mice. The cell viability always exceeded

95%, as determined by trypan blue dye exclusion.

Pulmonary melanoma metastases in C57BL6 mice resulting from the

i.v. inoculation of B16F10 melanoma cells were produced usingtwo independent groups of C57BL6 male mice as previously described

(24–26). One group was given 4 � 105 viable B16F10 melanoma cells (0.1mL) via lateral tail vein injection. On 11, 14, and 17 days after tumor cellinoculation, these mice received an i.v. injection of 99mTc-1 (25–40 ACi in0.1 mL). At 2 and 3 hours after injection of 99mTc-1, the mice were

weighed and sacrificed. Various organs and tumor-bearing lungs were

harvested and placed in preweighed tubes, with the excised lungs, liver,spleen, and brain collected in tubes containing 15% formalin buffer.

Radioactivity in the various organs was determined in a g-counter alongwith a known standard of technetium-99m, and subsequently the tissues/

organs were weighed. The excised lung tissues were then prepared forex vivo radiophosphor imaging (see below). In the second group of mice,

similar biodistribution studies were done 14 days after i.v. injection in

three subgroups of 2 � 105, 4 � 105, and 8 � 105 B16F10 melanomacells. After the counting of radioactivity and measurement of organ

weights, the tubes were stored for radioactivity decay. Subsequently the

tissues/organs were processed for histopathologic examination. In vivo

biodistribution studies with 99mTc-1 were also done in normal (control)mice that did not receive an i.v. tumor cell inoculation.

Subcutaneous tumors were created using 2.5 � 105 B16F10 cells (0.1 mL)transplanted s.c. on the left hind flank of C57BL6 male mice. After 10 to

14 days, the animals developed palpable tumor nodules 0.5 to 1.0 cm indiameter. The biodistribution studies were carried out in these mice at 2, 3,

and 6 hours after tail vein injection of 25 to 40 ACi (0.1 mL) 99mTc-1. Theorgans and tumors were harvested and counted in a g-counter along withtechnetium-99m standards of the injected dose. The results are expressed as% ID/g.

All animal experiments were done in compliance with the Principles of

Laboratory Animal Care (NIH publication no. 85-23, revised 1985) andunder approved institutional animal protocols.

Radiophosphor imaging. Excised lung tissue collected from tumor-bearing mice was placed in preweighed tubes containing 15% formalin

buffer solution and subjected to ex vivo radiophosphor imaging. Theformalin-fixed lung tissues were blotted dry on tissue paper and placed on

a clean microscope slide and photographed with a digital camera. The

slides were then placed in a phosphor imaging cassette (Molecular

Dynamics, Inc., Sunnyvale, CA). In the closed imaging cassette, the lungtissues were compressed to a thickness of 1 to 2 mm. The imaging plates

were exposed overnight to the lung tissues to accumulate counts. These

counts were then processed on a Molecular Dynamics Phosphor Imager(Storm 860 Scanner, Scanner Control Version 4.1, Molecular Dynamics) to

obtain the radiophosphor images. Image analysis was carried out with

the software program ImageQuant 1.2 (Molecular Dynamics).

Histology. The excised lung, liver, spleen, and brain tissues collectedin 15% formalin buffer were processed for histopathologic studies after

the complete decay of radioactivity. The tissue was embedded in

paraffin and 5-Am-thin sections were cut on a microtome and stainedwith H&E. Slides of all the sample tissues were examined under lightmicroscopy to determine the presence, extent, and size of melanoma

metastases.

Table 2. In vivo uptake of 99mTc-1 at 2 and 3 hours postadministration in C57BL6 mice bearing 11-, 14-, and 17-day-oldB16F10 melanoma lung metastases

Organ Normal 11-day-old* 14-day-old* 17-day-old*

2 h 3 h 2 h 3 h 2 h 3 h 2 h

Blood 0.3 F 0.03c 0.31 F 0.01 0.75 F 0.15 0.54 F 0.06 0.60 F 0.04 0.49 F 0.02 0.72 F 0.13Heart 0.34 F 0.03 0.28 F 0.07 0.50 F 0.07 0.32 F 0.04 0.41 F 0.04 0.25 F 0.08 0.47 F 0.05Liver 5.52 F 0.35 2.42 F 0.38 7.25 F 0.97 6.48 F 0.93 6.33 F 0.44 5.41 F 0.55 6.46 F 0.85Lungs 2.13 F 0.20 0.89 F 0.26 4.55 F 0.83b 3.44 F 0.94b 6.12 F 0.81b 7.74 F 1.34b 10.84 F 2.03b

Muscle 0.21 F 0.02 0.10 F 0.06 0.31 F 0.05 0.33 F 0.29 0.21 F 0.11 0.17 F 0.01 0.29 F 0.03Kidneys 1.59 F 0.14 1.35 F 0.13 2.35 F 0.39 1.72 F 0.34 1.92 F 0.27 1.44 F 0.10 2.35 F 0.31Spleen 0.72 F 0.04 0.30 F 0.12 0.80 F 0.08 0.56 F 0.10 1.03 F 0.63 2.12 F 1.3 0.78 F 0.11Brain 0.12 F 0.01 0.07 F 0.02 0.16 F 0.01 0.12 F 0.01 0.15 F 0.01 0.11 F 0.01 0.16 F 0.02Upper intestine 6.62 F 1.88 4.68 F 0.97 8.43 F 4.49 4.76 F 4.15 5.95 F 2.69 3.94 F 0.61 3.51 F 2.10Skin 0.69 F 0.57 0.40 F 0.20 0.73 F 0.24 0.60 F 0.30 0.66 F 0.18 0.41 F 0.08 0.73 F 0.27Lung/Blood 7.05 F 0.52 2.84 F 0.78 6.29 F 1.63 6.03 F 1.17 10.23 F 1.77 15.85 F 3.08 15.74 F 4.14Lung/Muscle 9.74 F 0.97 11.02 F 3.57 15.01 F 3.12 15.68 F 4.03 22.77 F 4.51 46.89 F 9.38 39.14 F 8.66Lung/Liver 0.39 F 0.03 0.36 F 0.06 0.64 F 0.20 0.50 F 0.15 0.98 F 0.10 1.43 F 0.19 1.76 F 0.53Lung/Spleen 2.98 F 0.14 3.62 F 2.18 5.73 F 1.2 5.78 F 1.54 6.68 F 2.66 5.30 F 4.05 14.62 F 3.07

*4 � 105 B16F10 cells inoculated i.v.c% ID/g F SD; n = 4-6.bP < 0.05 compared with normal.

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Cancer Res 2005; 65: (12). June 15, 2005 4982 www.aacrjournals.org



Statistical methods. Statistical analysis was done using Student’s t testfor unpaired data. A 95% confidence level was chosen to determine the

significance between days, with P < 0.05 being significantly different.

Results and Discussion

Small technetium-labeled molecules based on the structuralelements of the iodobenzamides currently in clinical trials formelanoma detection (11, 12, 14) have also been identifiedas possessing high in vivo melanoma accumulation.[99mTc]oxotechnetium(V) complexes of tetradentate N2S2 (AADT)chelates containing only tertiary amine substituents haveconsiderable melanoma affinity (18, 20–22), with 99mTc-1 display-ing an in vivo melanoma uptake of 7.6% ID/g (melanoma/nontarget tissue = 9.5) at 1 hour postinjection in the s.c.C57BL6/B16F0 mouse model (20). This in vivo melanomauptake of 99mTc-1 is comparable with that of the radioiodinatedIMBA and BZA molecules, which have 6% to 8% ID/g in thesame s.c. mouse model (13).

In vitro cell uptake studies. B16F10 cell uptake studies over a1-hour incubation period at 37jC (Fig. 3A) show rapid uptake of99mTc-1, with a 30% maximum being reached within 20 minutes.Because L-tyrosine is the main substrate in the synthesis of melaninvia the oxidative enzyme tyrosinase, melanin synthesis/content isenhanced by growing B16F10 cells in DMEM medium supple-mented with additional L-tyrosine (2.0 mmol/L). There is asignificant darkening of the cells/medium compared with controlcells cultured in DMEM alone. These tyrosine-stimulated cells havea rapid and significantly enhanced cellular accumulation of the99mTc complex at 37jC, such that within 5 minutes of incubationthey display a 70% uptake of 99mTc-1, which reaches a maximum of78% in 60 minutes. To distinguish an active accumulationcomponent from passive diffusion, simultaneous experiments weredone at 4jC (Fig. 3B). Under these conditions a significantreduction in cellular uptake is observed in the cellscultured in DMEM alone, with a maximum of only 8% observedover a 60-minute period, indicating that a significant fraction of thecellular uptake of 99mTc-1 by B16F10 melanoma cells is due toactive, temperature-dependent processes. At 4jC, the cellularaccumulation in tyrosine-stimulated cells is reduced from amaximum of 78% to 50%; moreover, the uptake is higher thanthat observed for control cells at 37jC, suggesting that the uptakeand accumulation of the 99mTc-1 complex is related to the melaninsynthesis/content of the cells, a phenomenon also observed withthe radioiodinated benzamides (10, 22, 27).NBdNJ added to the growth medium of melanoma cells pro-

duces functionally inactive tyrosinase, a result of inhibition ofcorrect N -glycan processing, and thereby reduces melaninsynthesis/content within intact melanoma cells. Despite thisincomplete glycan processing, the enzyme is transported to themelanosome; however, it lacks catalytic activity, in effect,generating amelanotic B16F10 cells that appear pale in color andcontain little or no melanin compared with normal control cells

Figure 5. Uptake at 2 hours after administration of 99mTc-1 in the lung ofC57BL6 mice bearing metastatic melanoma lesions as a function of tumorgrowth post i.v. tumor cell inoculation (A) and tumor dose (� 106 B16F10 cells)injected 14 days before 99mTc-1 administration (B ).

Table 3. Biodistribution of 99mTc-1 at 2 hours postadmi-nistration 14 days after inoculation of C57BL6 mice withvarying numbers of B16F10 cells

Organ Normal 2 � 105cells*

4 � 105cells*

8 � 105cells*

Blood 0.30 F 0.03c 0.63 F 0.03 0.60 F 0.10 0.60 F 0.04Heart 0.34 F 0.03 0.49 F 0.03 0.52 F 0.12 0.51 F 0.06Liver 5.52 F 0.35 7.23 F 0.81 7.53 F 2.09 7.32 F 0.86Lungs 2.13 F 0.20 2.83 F 0.21b 3.63 F 1.07b 4.92 F 1.61b

Muscle 0.21 F 0.02 0.33 F 0.05 0.29 F 0.08 0.28 F 0.03Kidneys 1.59 F 0.14 2.16 F 0.20 2.13 F 0.52 2.20 F 0.27Spleen 0.72 F 0.04 0.85 F 0.07 1.67 F 1.75 1.14 F 0.78Brain 0.12 F 0.01 0.15 F 0.01 0.16 F 0.03 0.16 F 0.01Intestines 6.62 F 1.88 6.70 F 3.34 10.46 F 4.42 6.38 F 1.34Skin 0.69 F 0.57 0.68 F 0.11 0.65 F 0.26 0.99 F 0.45Lung/Blood 7.05 F 0.52 4.47 F 0.20 5.99 F 0.94 8.36 F 2.17Lung/Muscle 9.74 F 0.97 8.60 F 1.08 12.60 F 2.25 14.76 F 2.47Lung/Liver 0.39 F 0.03 0.39 F 0.03 0.48 F 0.06 0.69 F 0.15Lung/Spleen 2.98 F 0.14 3.35 F 0.37 3.42 F 1.53 6.20 F 1.60

*Number of B16F10 cells inoculated i.v.c% ID/g F SD; n = 4-6.bP < 0.05.





(18). Cellular uptake studies in these NBdNJ-treated cells show adramatic decrease in accumulation of 99mTc-1, with a maximumuptake of 6% at 37jC throughout the 1-hour incubation period(Fig. 3A). Taken together, these in vitro cell uptake data indicate theeffect of modulating the melanin synthesis/content on theaccumulation of 99mTc-1 by intact melanoma cells and suggestthat mechanistically the accumulation of 99mTc-1 is related to theirmelanin synthesis/content.In vivo biodistribution of [99mTcOAADT]-(CH2)2-NEt2 in

B16F10 metastatic melanoma models. In vivo distribution of99mTc-1 was examined in the s.c. mouse model bearing metastaticB16F10 tumors. The biodistribution values in Table 1 show highin vivo tumor uptake of 99mTc-1 at 2 hours after its administra-tion. This tumor uptake is similar to that observed previously (20)and is also comparable with that obtained with the iodobenza-mides in the B16F0 tumor model (13). The biodistributiondata over an extended 6-hour period also indicate that tumoruptake of 99mTc-1 remains high whereas a relatively fasterwashout from normal organs results in increasing tumor/nontumor ratios (Table 1).For comparison of the in vivo melanoma uptake of 99mTc-1

with that of other small-molecule PET imaging probes such as2-[18F]fluoro-3,4-dihydroxy-L-phenylalanine ([18F]DOPA) and

[18F]FDG in a similar s.c. B16F10-melanoma tumor model, thetumor accumulation has also been calculated as a differentialabsorption ratio [= (counts of tissue/g tissue � g body weight) /total injected counts or (% ID/g � g body weight) / 100] asreported by Ishiwata et al. (28) for [18F]FDG and [18F]DOPA.Figure 4 contains a comparison of the differential absorptionratio of 99mTc-1 with those previously observed for [18F]FDG,[18F]DOPA (28), and [123I]IMBA (13). Because the degree of[18F]FDG accumulation in an organ or lesion is also semiquanti-tatively determined by PET in terms of standardized uptake values[= (decay-corrected activity in tumor/g tumor) / (decay-correctedinjected dose/g body weight of animal)] that serve as a normalizedmeasure of target to background ratios (9, 29), these have also beencalculated for the uptake of 99mTc-1 in the tumor. Values of 2.36 F0.42, 2.37 F 0.87, and 2.73 F 1.09 are obtained for 99mTc-1 tumorstandardized uptake values at 2, 3, and 6 hours, respectively.Corresponding 99mTc-1 standardized uptake values for blood are0.22 F 0.03, 0.26 F 0.06, and 0.13 F 0.05, and those for muscle are0.17 F 0.05, 0.11 F 0.06, and 0.05 F 0.03 at 2, 3, and 6 hours,respectively.As the foremost concern in the evaluation of patients diagnosed

with metastatic melanoma is the detection of the presence andextent of metastasis at distant sites, 99mTc-1 was also assessed in

Figure 6. Histology of lung and braintissues of C57BL6 mice 14 days after i.v.inoculation of B16F10 melanoma cells.A, 2 � 105 B16F10 cells at �10magnification; arrows, tumors. B, 2 � 105B16F10 cells at �50 magnification of A .C, 4 � 105 B16F10 cells at �5magnification. D, 8 � 105 B16F10 cells at�5 magnification. E, 14-day-old tumorbrain tissue after i.v. dose of 4 � 105B16F10 cells at �25 magnification. F,14-day-old tumor brain tissue after i.v. doseof 4 � 105 B16F10 cells at �150magnification.

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mice that received an i.v. inoculation of B16F10 melanoma cells,resulting in the formation of metastatic melanoma lesions in thelungs. When mice are evaluated 11 days after receiving 4 � 105B16F10 cells i.v., they have a significantly higher lung uptake of99mTc-1 2 hours postadministration of the radiopharmaceuticalthan normal mice (4.55 F 0.83% ID/g versus 2.13 F 0.20% ID/g,respectively, P < 0.05; Table 2). This difference in lung uptakeincreases further at 3 hours postinjection of the compound, withthe uptake of 11-day tumor-bearing mice 3.44 F 0.94% ID/g andthat of normal mice 0.89 F 0.26% ID/g, indicating a relativelyfaster washout of 99mTc-1 from normal lung tissue comparedwith lungs bearing metastatic melanoma lesions. When the timeinterval following tumor cell inoculation is prolonged to 14 daysto allow further growth of the metastatic lesions, theaccumulation of 99mTc-1 in the lungs increases to 6.12 F0.81% ID/g and 7.74 F 1.34% ID/g at 2 and 3 hourspostadministration, respectively. Moreover, at 17 days post tumorcell injection, the uptake of 99mTc-1 in the lungs reaches a valueof 10.84 F 2.03% ID/g at 2 hours postadministration. Thisaccumulation of 99mTc-1 as a function of the growth of themetastatic pulmonary melanoma lesions over time is illustratedin Fig. 5A .The uptake of 99mTc-1 was also examined in a group of mice

that received varying doses of B16F10 tumor cells 14 days beforethe assessment. The uptake of 99mTc-1 in the lungs of these tumor-bearing mice has a positive correlation with the number of tumorcells administered, such that mice receiving 2 � 105, 4 � 105,and 8 � 105 B16F10 cells have lung uptake of 2.83 F 0.21% ID/g,3.63 F 1.07% ID/g, and 4.92 F 1.61% ID/g, respectively, comparedwith 2.13 F 0.20% ID/g observed in the lungs of normal mice(Table 3; Fig. 5B). Taken together, these in vivo experimentsillustrate the potential use of 99mTc-1 as an in vivo probe to assessboth temporal growth and extent of metastatic tumor burden inthe lungs.

Histology. Evaluation of numerous histologic sections of thelung, liver, spleen, and brain of animals bearing s.c. B16F10 tumorsdoes not indicate any metastatic lesions in these organs. However,as previously shown (19–21, 24), excised lungs from mice thatreceived i.v. inoculations of B16F10 melanoma cells have distinctsmall black melanoma colonies, which increase both in numberand in size with increasing dose of the tumor inoculum. Paraffin-embedded sections from metastatic tumor–bearing lungs of micereceiving i.v. inoculation of 2 � 105, 4 � 105, and 8 � 105 B16F10melanoma cells 14 days before evaluation are shown in Fig. 6. Theadministration of 2 � 105 tumor cells results in the formation of asignificant number of small distinct metastatic lesions with mostof these extravasated near blood vessels (Fig. 6A and B). Increasingthe tumor dose to 4 � 105 and 8 � 105 cells causes a significantincrease in both the number and size of the metastatic lesions(Fig. 6C and D). As previously observed (24–26, 30), the metastaticlesions not only extravasate and grow near blood vessels but alsogrow near and at the lung surface; this is quite pronounced inthe 14-day lung tissue when 8 � 105 cells have been administered(Fig. 6D).An increase in both the number and size of metastatic

melanoma lesions also occurs in the group where the time posttumor cell injection (4 � 105 cells) is prolonged from 11 to 17 days.This was visually apparent at the time of dissection (Fig. 7). Afterbiodistribution studies with this group of mice at 2 hourspostadministration of 99mTc-1 (Table 2), the excised lung tissuewas photographed and subjected to ex vivo radiophosphorimaging. The distinct and specific localization of 99mTc-1 by themelanotic lesions as observed in the radiophosphor images (Fig. 7)is underscored by the intensity and specificity of the accumulatedactivity in the individual tumor lesions. Also apparent in the corre-lative examination of the radiophosphor images with the digitalimages of the lung tissue displayed in Fig. 7 is the relationship ofthe level of accumulation of 99mTc-1 to the degree of melanin in the

Figure 7. Ex vivo radiophosphor images of 11-, 14-,and 17-day-old tumors.





individual melanotic lesions. Histologic examination of lungsections does not reveal any infiltrating inflammatory cells at ornear the metastatic lesions. In conjunction with the specific uptakeof 99mTc-1 observed in the radiophosphor images, this observationemphasizes that the accumulation is specifically localized in thetumor cells.Whereas examination of this group of mice (i.v. tumor

inoculation) does not show any distant metastasis in the liverand spleen, the sections of the brain display small but noticeableinfarcted areas. A closer and more detailed examination of thebrain sections adjoining the infarcted regions reveals blockade inthe blood vessels by small but distinct tumor metastases (Fig. 5Eand F). Further detailed examination of the brain sections showsno metastatic tumor growth within the brain tissue, and smalltumor metastases confined to the blood vessels supplying theneighboring infarcted areas. The corresponding in vivo brainuptake of 99mTc-1 in the biodistribution studies of these miceindicates a very small increase in uptake compared with normalmice (Table 3). This increase may be relatively small due to anumber of factors: (a) the whole brain is excised and measured andthe number and size of such metastases in the brain vasculatureare very small; (b) a reduction in blood flow due to blockade ofthe vessels by the metastases produces decreased delivery and

extraction of 99mTc-1 in the relatively small tumor metastases; and(c) the presence of small infarcted areas caused by the blockademay also bring about decreased brain uptake of 99mTc-1. A moredetailed study using this model will be required to derive anyconclusions with respect to brain uptake of 99mTc-1 and melanomametastases within the brain vasculature.

99mTc-1, a small, neutral technetium complex, displays highmelanoma uptake in both s.c. and pulmonary metastaticmelanomas. The use of a more realistic model for evaluatingmetastatic melanoma-targeting agents shows that 99mTc-1 not onlyhas high and specific uptake by melanoma metastases in the lungsbut also can potentially provide a means to assess noninvasivelyboth temporal growth and extent of metastatic tumor burdenin vivo .

Acknowledgments

Received 10/1/2003; revised 2/3/2005; accepted 3/16/2005.Grant support: NIH grant R37 CA34970.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Alice D. Carmel for her excellent technical support and Roderick T.Bronson and his staff in the Rodent Histopathology Core, Dana-Farber/HarvardCancer Center.

Cancer Research


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2005;65:4979-4986. Cancer Res Zhen Cheng, Ashfaq Mahmood, Huazhi Li, et al. Imaging Metastatic MelanomaSingle-Photon Emission Computed Tomography Probe for

: A Potential Small-Molecule2-NEt2)2TcOAADT]-(CH99m[

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