Positron-Emission Tomography Imaging of the TSPO with [ 18 F]FEPPA in a Preclinical Breast Cancer Model †

Abstract

The present study aims to image the 18-kDa translocator protein (TSPO; formerly known as the peripheral benzodiazepine receptor) in a preclinical human breast cancer (BC) xenograft mouse model with positron-emission tomography (PET). An automated radiosynthesis of [¹⁸F]-N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([¹⁸F]FEPPA) was validated for human use using a commercial synthesis module and resulted in a high radiochemical yield (30%±8%, uncorrected; n=54) and specific activity (6±4 Ci/μmol). Tumor uptake of [¹⁸F]FEPPA in mice bearing subcutaneous MDA-MB-231 BC xenografts was evaluated by PET-computed tomography imaging and ex vivo biodistribution studies. Although the tumor was successfully visualized, ex vivo biodistribution studies revealed low tumor uptake (0.7%ID/g), with the majority of radioactivity distributed in the spleen, muscle, and heart despite high TSPO expression in this cell line. Our laboratory routinely prepares [¹⁸F]FEPPA for human-imaging studies in the central nervous system, and we envision that radiopharmaceuticals that target the TSPO have the potential for imaging macrophages in the tumor microenvironment.

Introduction

The translocator protein (TSPO; molecular weight=18 kDa), formerly known as the peripheral benzodiazepine receptor, is located in the outer mitochondrial membrane,^1,2 and is a part of a larger complex that includes the adenine nucleotide carrier and the voltage-dependant anion channel.³ The TSPO has several putative roles, including involvement in neurosteroid syntheses via cholesterol transport across the membrane.⁴ Examples of increased TSPO production in inflammation models include cerebral ischemia in mice⁵ and human immunodeficiency virus encephalitis, Alzheimer's disease, multiple sclerosis, and stroke in human subjects,⁶ as well as arterial plaques in patients suffering from atherosclerosis.⁷ Moreover, increased production of TSPO often occurs early in the pathology of disease progression.⁸

The TSPO also plays an important role in cancer, including the breast, colon, and brain.⁹ Numerous studies have established that TSPO is overexpressed and participates in the advancement of invasive progression in these diseases, particularly in breast cancer (BC).^10

–15 It is also established that TSPO is expressed at much higher levels in aggressive metastatic human BC tumor biopsies compared with normal breast tissue and noninvasive breast tumors.^10,13 Given the massive burden of BC on society,¹⁶ patients, and their families, and the strong association between TSPO expression and an aggressive BC phenotype, development of a TSPO-targeted imaging agent that reveals the expression of this target could improve noninvasive detection, diagnostic and management options, as well as enable the probing of tumor-associated macrophages (TAMS) in BC.¹⁷

Molecular imaging with positron-emission tomography (PET) is being actively pursued to probe the TSPO in vivo using appropriate radiotracers that bind specifically and selectively to this target. The prototypical and most widely used radiotracer for PET imaging of TSPO is carbon-11- (¹¹C; t _½=20.4 minutes) labeled 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinoline carboxamide, preferably as its R-enantiomer ([¹¹C]PK11195)^18

–24; however, this radiotracer has recognized limitations as a TSPO imaging tool,²⁵ namely, high nonspecific binding and high binding to plasma proteins.^18,23,24 The deficiencies of [¹¹C]PK11195, coupled with the recognized importance of TSPO imaging, have fueled considerable efforts to develop improved carbon-11- or fluorine-18- (¹⁸F; t _½=109.7 minutes) labeled radiotracers, and has been extensively reviewed.^26,27 The greatest advantage of fluorine-18-labeled radiotracers is the half-life of this nuclide (t _½=109.7 minutes), which enables longer imaging times as well as shipment of the nuclide and/or radiotracers to imaging facilities distant from the site of production. Fluorine-18-labeled radiopharmaceuticals that target TSPO have significant potential to influence the assessment, monitoring of treatment response, and management of patients with BC, and would greatly complement existing PET radiopharmaceuticals that have established roles for imaging BC.^28,29

The goal of the present study was to image the TSPO in a preclinical human BC xenograft mouse model with PET and was carried out using a radiopharmaceutical developed by our laboratory, [¹⁸F]-N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([¹⁸F]FEPPA).³⁰ Herein, we describe the automation of the radiosynthesis of [¹⁸F]FEPPA, including validation for human use. The tumor uptake of this radiotracer was assessed in mice bearing subcutaneous MDA-MB-231 BC xenografts and evaluated by PET-(CT) computed tomography imaging in conjunction with ex vivo biodistribution studies.

Materials and Methods

Automated radiosynthesis

[¹⁸F]FEPPA is prepared by the reaction of cyclotron (Scanditronix MC 17) produced [¹⁸F]-fluoride with a tosyloxy precursor as previously described by our laboratory.³⁰ Briefly, [¹⁸F]-potassium cryptand fluoride is azeotropically dried, and then reacted with the N-(2-((n-4-phenoxypyridin-3-yl)acetimido)methyl)phenoxy)ethyl-4-methylbenzenesulfonate in acetonitrile for 10 minutes at 90°C, followed by quenching the reaction mixture before semipreparative high performance liquid chromatography (HPLC) purification (refer to Fig. 1) and solid-phase formulation. The entire radiosynthetic procedure was carried out in a General Electric Medical system FX_FN-sealed module in an analogous fashion to previous automation of ¹⁸F-labeled compounds by our laboratory,³¹ with minor modifications as follows: (1) the tosyloxy-precursor (ca. 5 mg) to [¹⁸F]FEPPA was dissolved in 0.75 mL of anhydrous CH₃CN; (2) the reaction was quenched after the fluorination reaction with 3 mL of 30:70:0.5 MeOH/H₂O/HCOOH and modified HPLC conditions as previously described: (a) semipreparative HPLC: prodigy ODS-prep (250×10 mm, 10 micrometers), 50:50 MeOH/H₂O+0.5% HCOOH, 6 mL/min, λ=254 nm; τ_R≈21 minutes; and (b) analytical HPLC: prodigy C18 ODS (250×4.6 mm, 10 micrometers), 50:50 CH₃CN/H₂O + 0.1 N NH₄ ⁺HCOO^–, 3 mL/min, λ=254 nm; τ_R≈3 minutes. The product was formulated in buffered saline containing 5%–10% ethanol, followed by sterilization by passing the solution through a 0.22-μm filter into a vial containing 1 mL of 8.4% sodium bicarbonate solution.

FIG. 1.

Representative preparative high performance liquid chromatography traces (UV on top and radioactivity on bottom; arbitrary units) of the reaction mixture containing [¹⁸F]-N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([¹⁸F]FEPPA) (t_R =23 minutes). Color images available online at www.liebertpub.com/cbr

Small-animal PET imaging and ex vivo biodistribution studies

The principles of Laboratory Animal Care (NIH Publication No. 86–23, revised 1985) were followed. All studies were conducted under protocols approved by the Animal Care Committee at the University Health Network (Protocol No. 989.10) in accordance with the Canadian Council on Animal Care guidelines.

MDA-MB-231 human BC cells were purchased from the American Type Culture Collection (Manassas, VA). The cells were cultured in the Dulbecco's minimal essential medium (Ontario Cancer Institute, Toronto, ON) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) containing 100 U/mL penicillin and 100 μg/mL streptomycin under a 5% CO₂ atmosphere at 37°C. MDA-MB-231 tumor xenografts were established in female athymic CD1 nu/nu mice. The mice were inoculated subcutaneously (s.c.) in the right hind leg with 5×10⁶ MDA-MB-231 cells in 200 μL of a 1:1 mixture of Matrigel (BD Biosciences, Bedford, MA) and a serum-free culture medium. MDA-MB-231 human BC cells were chosen for this study, as they are known to have high expression of TSPO (8.7±1.4 pmol/mg protein).¹⁰ Once tumors had reached an appropriate size (5–8 mm in diameter), the mice underwent small-animal PET imaging, which was carried out using a Focus 220 microPET (Siemens Preclinical Solutions, Knoxville, TN). Mice were warmed on a heating pad for 30 minutes before injection. Conscious mice were injected intravenously (i.v.) with 270–540 μCi (10–20 MBq) of [¹⁸F]FEPPA using a previously described method.³² Mice (n=3) were anesthetized by inhalation of 2% isoflurane in oxygen and imaged at 90 minutes postinjection (p.i.) using an acquisition time of 10 minutes. A second group of mice (n=3) was injected with 2 mg/kg (in 1% Tween 80 in saline) of unlabeled FEPPA 1 hour before injection of [¹⁸F]FEPPA to assess the specificity of tumor uptake. Images were reconstructed into a 256×256 matrix (reconstructed voxel size 0.80 mm×0.48 mm×0.48 mm) using an ordered subset expectation maximization followed by a maximum a posteriori probability. Tumor uptake of [¹⁸F]FEPPA was quantified by the volume-of-interest (VOI) analysis using Inveon Research Workplace software (Siemens Preclinical Solutions) and expressed as the mean±SD%ID/g. After PET, small-animal CT imaging was performed for anatomical reference on a GE eXplore Locus Ultra Preclinical CT Scanner (GE Healthcare, Chalfont St. Giles, United Kingdom) using standard acquisition parameters (80 kVp, 70 mA, reconstructed voxel size of 150 μm×150 μm×150 μm). Small-animal PET and CT images were coregistered using Inveon Research Workplace software (Siemens Preclinical Solutions). The tumor and normal tissue uptake of [¹⁸F]FEPPA between blocked (mice administered unlabeled FEPPA before imaging) and control mice was compared using Student's t-test (P<0.05).

Endpoint biodistribution was carried out immediately following the studies imaging (90–100 minutes postinjection of the radiotracer). Mice were sacrificed under anesthesia by heart puncture, and ex vivo biodistribution studies were carried out to assess tumor and normal tissue uptake of [¹⁸F]FEPPA. Tumor, blood, and normal tissues were collected and weighed, and their radioactivity was measured in a γ-counter (PerkinElmer, Wellesley, MA). Tumor and normal tissue uptake was expressed as mean±SD percent injected dose per gram (%ID/g), as well as the standardized uptake values (SUVs) were calculated by the formula, SUV=RC/(ID/m), where RC is the decay-corrected tissue concentration of radioactivity (μCi/g); ID is the net injected dose (μCi); and m is the mass of the mouse (g). Tumor-to-normal tissue ratios (T/NT) were calculated.

Results

Automated radiosyntheses of [¹⁸F]FEPPA were routinely and efficiently achieved. The uncorrected radiochemical yield of [¹⁸F]FEPPA was 30%±8% (n=54), and the total synthesis time was 36±1 minutes, including formulation. Coinjection of the radioactive product with an authentic standard of FEPPA under multiple HPLC conditions (solvents, pH, and wavelength) with several analytical columns further established the identity of the radiotracer. The product was prepared consistently with >99% radiochemical purity, and the specific activity at the end of synthesis was 6±4 Ci/μmol (222±148 GBq/μmol; n=54). The integrity of the final filter was demonstrated by a bubble-point procedure (>50 psi). Formulated [⁸F]FEPPA maintained stability, as measured by HPLC, in an inverted dose vial, and maintained clarity and a pH of 8 over a period of 6 hours. The half-life was verified to be 109.7 minutes, as determined by a dose calibrator, and no long-lived isotopes were observed (5 days) and determined by γ-detection after ¹⁸F-decay. Formulated [¹⁸F]FEPPA was free of pyrogens (in house using the Limulus Amebocyte Lysate test), sterile (USP sterility testing at the Microbiology Laboratory, Princess Margaret Hospital, Toronto, ON), and passed the Kryptofix^® spot test (<20 μg/mL). Volatile organic compound analysis, by gas chromatography, showed negligible levels of residual methanol (26 ppm).

Small-animal PET imaging

MDA-MB-231 xenografts in athymic mice were successfully visualized by dynamic small-animal PET imaging at 90–100 minutes p.i. of [¹⁸F]FEPPA (this time point represented optimal uptake determined by dynamic PET imaging; data not shown). Although the tumor was visualized in these studies (Fig. 2), image intensities were adjusted for optimal delineation of VOIs. VOI analysis demonstrated low uptake of [¹⁸F]FEPPA in the tumors (1.5±0.3%ID/g). In agreement with the imaging and biodistribution results, administration of 2 mg/kg of unlabeled FEPPA 1 hour before radiopharmaceutical injection did not reduce accumulation of [¹⁸F]FEPPA in the tumors (2.0±0.6 vs. 1.5±0.3%ID/g, respectively; P=0.28). Additionally, the tumor-to-muscle ratio (T/M) in unblocked mice was 0.4±0.1, indicating that accumulation of [¹⁸F]FEPPA in the tumors was significantly lower than the surrounding muscle (P<0.05).

FIG. 2.

Coronal (upper) and transaxial (lower) small-animal positron-emission tomography coregistered to microCT images of representative athymic mice implanted s.c. in the right hind leg with MDA-MB-231 xenografts (see arrows) at 90 minutes p.i. of [¹⁸F]FEPPA. Color images available online at www.liebertpub.com/cbr

Ex vivo biodistribution studies: tumor and normal tissue uptake of [¹⁸F]FEPPA

The tumor and normal tissue uptake at 100 minutes p.i. of [¹⁸F]FEPPA in athymic mice bearing MDA-MB-231 xenografts is shown in Table 1. There was poor specific accumulation of [¹⁸F]FEPPA in the tumors as demonstrated by the low tumor uptake (0.7±0.6%ID/g) and a tumor-to-blood (T/B) ratio of 0.8±0.6. Further, preinjection of an excess of unlabeled FEPPA (2 mg/kg) 1 hour before [¹⁸F]FEPPA did not decrease tumor uptake (1.5±0.5 vs. 0.7±0.6%ID/g, respectively; P=0.13; Table 1) or the T/B ratio (1.5±0.4 vs. 0.8±0.6, respectively; P=0.15), indicating that the low tumor uptake of [¹⁸F]FEPPA was likely not TSPO specific. The highest normal tissue uptake of radioactivity was found in the heart, lungs, kidneys, and spleen. Administration of unlabeled FEPPA to block TSPO activity resulted in reduced accumulation of [¹⁸F]FEPPA in the heart, lungs, spleen, and muscle; however, these reductions were only statistically significant for changes in the SUVs of the spleen and muscle (P<0.05) and changes in the T/NT of the spleen, heart, and muscle (P<0.05). There was a trend toward significance for reductions in the %ID/g in the spleen (P=0.06), heart (P=0.06), and muscle (P=0.05) compared to the unblocked mice.

Table 1.

Tumor and Normal Tissue Distribution of Radioactivity in Athymic Mice Implanted Subcutaneously with MDA-MB-231 Human Breast Cancer Xenografts Immediately After Imaging with [¹⁸F]FEPPA

	[¹⁸F]FEPPA						[¹⁸F]FEPPA uptake after preadministration of FEPPA ^a
	%ID/g		SUVs		T/NT		%ID/g		SUVs		T/NT
Organs	mean	SD	mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
Blood	0.9	0.2	0.2	0.0	0.8	0.6	1.0	0.1	0.2	0.0	1.5	0.4
Bladder	3.3	1.2	0.8	0.3	0.2	0.2	3.7	1.6	0.9	0.4	0.4	0.1
Large Int.	2.4	0.7	0.6	0.1	0.3	0.3	3.0	0.4	0.7	0.1	0.5	0.2
Small Int.	3.3	1.0	0.8	0.2	0.2	0.2	4.6	1.2	1.1	0.3	0.3	0.1
Stomach	2.5	0.9	0.6	0.2	0.4	0.3	3.2	1.1	0.8	0.3	0.5	0.2
Spleen	16.8^*	4.2	4.0^**	0.8	0.0^**	0.0	10.1	0.9	2.5	0.4	0.1	0.0
Liver	1.4	0.2	0.3	0.0	0.5	0.4	3.3	1.4	0.8	0.3	0.5	0.3
Kidneys	18.7	6.5	4.4	1.3	0.0	0.0	18.8	3.9	4.6	1.2	0.1	0.0
Lungs	20.7	10.6	4.9	2.3	0.1	0.0	11.5	2.2	2.8	0.7	0.1	0.0
Heart	34.0^*	18.7	8.2^*	4.4	0.0^**	0.0	6.1	2.0	1.5	0.6	0.3	0.0
Muscle	4.5^*	1.4	1.1^**	0.3	0.2^**	0.2	2.3	0.2	0.5	0.1	0.7	0.2
Skin	4.1	1.4	1.0	0.3	0.2	0.2	4.0	0.4	1.0	0.1	0.4	0.1
Brain	1.0	0.3	0.2	0.1	0.7	0.6	1.0	0.2	0.3	0.1	1.5	0.4
Tumor	0.7	0.6	0.2	0.1	1.0	0.0	1.5	0.5	0.4	0.1	1.0	0.0
Bone	3.0	0.4	0.7	0.1	0.3	0.2	3.4	0.7	0.8	0.1	0.5	0.2

Results are presented as the mean±SD of three tumor-bearing mice and were carried out at ca. 100 minutes postinjection of the radiotracer.

Mice were injected intraperitoneally with 2 mg/kg FEPPA 1 hour before injection of [¹⁸F]FEPPA.

Trend toward significant difference compared to preadministration of FEPPA (blocked; P=0.05–0.06).

Significant difference compared to blocked (P<0.05).

[¹⁸F]FEPPA, [¹⁸F]-N-(2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide; SUVs, standardized uptake values; T/NT, tumor-to-normal tissue ratio.

Discussion

The automated synthesis of [¹⁸F]FEPPA offers an improvement to our previously reported manual synthesis method,³⁰ as routine production for human imaging studies are now facilitated. The formulated product was efficiently prepared in a high radiochemical yield, specific activity, and radiochemical purity. The cleaning procedure for this module was also automated, thereby minimizing exposure to radioactivity when rapidly preparing the module for subsequent syntheses. Using this new methodology, [¹⁸F]FEPPA was successfully validated for clinical PET studies and has since been applied for first in human-imaging studies at our PET center.³³

MDA-MB-231 human BC cells exhibit TSPO gene amplification¹² and have been shown to express high levels of TSPO protein (8.7±1.4 pmol/mg of protein), measured by binding assays using ³H-PK11195, a ligand with a high binding affinity for TSPO.¹⁰ It is also noteworthy that a recent report that validated TSPO imaging using a near-infrared (NIR) probe, NIR-con-PK11195, also used the same preclinical xenograft model.³⁴ We have previously shown that FEPPA binds to TSPO with 18-fold higher affinity (K_i =0.07 nM) than ³H-PK11195 in displacement binding assays performed using mitochondrial tissue fractions from the rat cortex.³⁰ In the present study, MDA-MB-231 xenografts in athymic mice were successfully visualized by PET imaging at 90 minutes p.i. of [¹⁸F]FEPPA (Fig. 2). Interestingly, however, tumor uptake in MDA-MB-231 xenografts was low despite their high TSPO expression (see Table 1). VOI analysis of the PET images revealed low tumor uptake, which was significantly lower than the surrounding muscle tissue. These results were confirmed by biodistribution studies, which demonstrated low tumor uptake that was not significantly different than circulating blood radioactivity. Further, a preinjection of 2 mg/kg of FEPPA in an attempt to block TSPO activity did not significantly reduce tumor uptake of [¹⁸F]FEPPA, indicating that the low tumor uptake was not TSPO specific (see Table 1). Tumor uptake of [¹⁸F]FEPPA in MDA-MB-231 xenografts was more than sevenfold lower than the tumor uptake of 2-[¹⁸F]fluoro-2-deoxy-d-glucose as reported previously by our laboratories in this same tumor model (0.7%ID/g vs. 5.4%ID/g).³⁵

The highest normal tissue accumulations of [¹⁸F]FEPPA were found in the heart, lungs, spleen, and kidneys in agreement with biodistribution. Indeed, expression of TSPO in the kidneys, lungs, and heart has been previously reported.¹³ Our recent human dosimetry studies with [¹⁸F]FEPPA (unpublished results) reveal that the heart wall and lungs have the highest uptake, and this is absent in the genetic nonresponder or low-affinity binder, and is consistent with previously reported dosimetry studies on nonhuman primates and human subjects with [¹¹C]PBR28³⁶; therefore, the heart wall and lung uptake is likely mediated by TSPO. With exception of the kidneys, uptake of [¹⁸F]FEPPA in these tissues was decreased after preinjection of unlabeled FEPPA to block TSPO activity, indicating that a proportion of the uptake may be mediated by TSPO. Radiotracer accumulation in the kidneys is most likely due to the renal clearance and metabolism of [¹⁸F]FEPPA.

Recent reports have identified that our understanding of the molecular structure of the TSPO, its interaction with ligands via multiple uncharacterized binding sites, and the role of TSPO polymerization are among the fundamental challenges that will need to be resolved before accurately interpreting PET data for this diagnostic target.^37

–40 Significant efforts are continuing to enhance our understanding of the TSPO, via novel and existing PET radiotracers to probe this target in oncology, including gliomas,^23,41
–43 as well as exploration of other imaging modalities, such as NIR³⁴ and PET in conjunction with photodynamic therapy.⁴⁴ We envision that [¹⁸F]FEPPA and related radiotracers will have applications in the imaging of several solid tumors, and our future work aims to explore the application of such radiotracers to probe TAMS in the tumor microenvironment.

Conclusions

The radiosynthesis of [¹⁸F]FEPPA was successfully automated and validated for the use in human studies. Tumor uptake of this radiotracer in MDA-MB-231 human BC xenografts in athymic mice was successfully visualized in the imaging study; however, this accumulation was low and not greater than circulating blood radioactivity. Blocking studies suggested that the uptake of [¹⁸F]FEPPA in MDA-MB-231 tumors was nonspecific, despite the high expression levels of TSPO in this tumor model. Although these results may preclude the use of [¹⁸F]FEPPA for imaging in this BC model, this radiopharmaceutical is currently under investigation in human subjects for other applications, including neuroinflammation, and our future work aims to explore the imaging of TAMS in the tumor microenvironment as well as other solid tumors with TSPO ligands.

Footnotes

Acknowledgments

This research was supported by grants from the Ontario Institute for Cancer Research (One Millimetre Cancer Challenge) with funding provided by the Government of Ontario. The authors gratefully acknowledge Armando Garcia and Winston Stableford for ¹⁸F production. The authors would like to thank Lisa DiDiodato for performing the CT imaging.

Disclosure Statement

No institutional or commercial affiliations would pose a conflict of interest regarding the publication of this article.

References

Verma

, Facchina

, Hirsch

et al. Photodynamic tumor therapy: Mitochondrial benzodiazepine receptors as a therapeutic target. Mol Med, 1998; 4:40.

Schlichter

, Rybalchenko

, Poisbeau

et al. Modulation of GABAergic synaptic transmission by the non-benzodiazepine anxiolytic etifoxine. Neuropharmacology, 2000; 39:1523.

Bono

, Lamarche

, Prabonnaud

et al. Peripheral benzodiazepine receptor agonists exhibit potent antiapoptotic activities. Biochem Biophys Res Commun, 1999; 265:457.

Benavides

, Quarteronet

, Imbault

et al. Labelling of “peripheral-type” benzodiazepine binding sites in the rat brain by using [³H]PK 11195, an isoquinoline carboxamide derivative: Kinetic studies and autoradiographic localization. J Neurochem, 1983; 41:1744.

Tallman

, Thomas

, Gallager

. GABAergic modulation of benzodiazepine binding site sensitivity. Nature, 1978; 274:383.

Anholt

, Murphy

, Mack

et al. Peripheral-type benzodiazepine receptors in the central nervous system: Localization to olfactory nerves. J Neurosci, 1984; 4:593.

Beard

, Waring

, O'Brien

et al. Nonsteroidal anti-inflammatory drug use and Alzheimer's disease: A case-control study in Rochester, Minnesota, 1980–1984. Mayo Clin Proc, 1998; 73:951.

Cymerman

, Pazos

, Palacious

. Evidence for species differences in “peripheral” benzodiazepine receptors: An autoradiographic study. Neurosci Lett, 1986; 66:153.

Maaser

, Sutter

, Scherübl

. The peripheral benzodiazepine receptor: A target for innovative diagnostic and therapeutic approaches in gastrointestinal oncology. Cancer Ther, 2004; 2:345.

10.

Hardwick

, Fertikh

, Culty

et al. Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res, 1999; 4:831.

11.

Beinlich

, Strohmeier

, Kaufmann

et al. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines. Biochem Pharmacol, 2000; 60:397.

12.

Hardwick

, Rone

, Han

et al. Peripheral-type benzodiazepine receptor levels correlate with the ability of human breast cancer MDA-MB-231 cell line to grow in SCID mice. Int J Cancer, 2001; 94:322.

13.

Han

, Slack

, Li

et al. Expression of peripheral benzodiazepine receptor (PBR) in human tumors: Relationship to breast, colorectal, and prostate tumor progression. J Recept Signal Transduct Res, 2003; 23:225.

14.

Zheng

, Boisgard

, Siquier-Pernet

et al. Differential expression of the 18 kDa translocator protein (TSPO) by neoplastic and inflammatory cells in mouse tumors of breast cancer. Mol Pharmacol, 2011; 8:823.

15.

Hunakova

, Bodo

, Chovancova

et al. Expression of new prognostic markers, peripheral-type benzodiazepine receptor and carbonic anhydrase IX, in human breast and ovarian carcinoma cell lines. Neoplasma, 2007; 54:541.

16.

American Cancer Society. Cancer Facts and Figures. Atlanta: American Cancer Society, 2009.

17.

Laoui

, Movahedi

, Overmeire

et al. Tumor-associated macrophages in breast cancer: Distinct subsets, distinct functions. Int J Dev Biol, 2011; 55:861.

18.

Pappata

, Levasseur

, Gunn

et al. Thalamic microglial activation in ischemic stroke detected in vivo by PET and [¹¹C]PK1195. Neurology, 2000; 55:1052.

19.

Banati

, Goerres

, Myers

et al. [¹¹C]-(R)-PK11195 positron emission tomography imaging of activated microglia in vivo in Rasmussen's encephalitis. Neurology, 1999; 53:2199.

20.

Cagnin

, Myers

, Gunn

et al. [¹¹C]-(R)-PK11195-PET following herpes encephalitis reveals projected neuronal damage beyond the primary focal lesion. Brain, 2001; 124:2014.

21.

Turner

, Cagnin

, Turkheimer

et al. Evidence of widespread cerebral microglial activation in amyotrophic lateral sclerosis: An [¹¹C]-(R)-PK11195 positron emission tomography study. Neurobiol Dis, 2004; 15:601.

22.

Gerhard

, Banati

, Goerres

et al. [¹¹C]-(R)-PK11195 PET imaging of microglial activation in multiple system atrophy. Neurology, 2003; 61:686.

23.

Pappata

, Cornu

, Samson

et al. PET study of carbon-11-PK 11195 binding to peripheral type benzodiazepine sites in glioblastoma: A case report. J Nucl Med, 1991; 32:1608.

24.

Sauvageau

, Desjardins

, Lozeva

et al. Increased expression of “peripheral-type” benzodiazepine receptors in human temporal lobe epilepsy: Implications for PET imaging of hippocampal sclerosis. Metab Brain Dis, 2002; 17:3.

25.

Chauveau

, Boutin

, Van Camp

et al. Nuclear imaging of neuroinflammation: A comprehensive review of [¹¹C]PK11195 challengers. Eur J Nucl Med Mol Imaging, 2008; 35:2304.

26.

Dollé

, Luus

, Reynolds

et al. Radiolabelled molecules for imaging the translocator protein (18 kDa) using positron emission tomography. Curr Med Chem, 2009; 16:2899.

27.

Luus

, Hanani

, Reynolds

et al.

The development of PET radioligands for imaging the translocator protein 18 kDa: What have we learned?

J Labelled Compd Radiopharm, 2010; 53:501.

28.

Quon

, Gambhir

. FDG-PET and beyond: Molecular breast cancer imaging. J Clin Oncol, 2005; 23:1664.

29.

Pantaleo

, Nannini

, Maleddu

et al. Conventional and novel PET tracers for imaging in oncology in the era of molecular therapy. Cancer Treat Rev, 2008; 34:103.

30.

Wilson

, Garcia

, Parkes

et al. Radiosynthesis and initial evaluation of [¹⁸F]-FEPPA for PET imaging of peripheral benzodiazepine receptors. Nucl Med Biol, 2008; 35:305.

31.

van Oosten

, Wilson

, Stephenson

et al. An improved radiosynthesis of the muscarinic M₂ radiopharmaceutical, [¹⁸F]FP-TZTP. Appl Radiat Isot, 2009; 67:611.

32.

Vines

, Green

, Kudo

et al. Evaluation of mouse tail-vein injections both qualitatively and quantitatively on small-animal PET tail scans. J Nucl Med Technol, 2011; 39:264.

33.

Rusjan

, Wilson

, Bloomfield

et al. Quantitation of translocator protein binding in human brain with the novel radioligand [¹⁸F]-FEPPA and positron emission tomography. J Cereb Blood Flow Metab, 2011; 31:1807.

34.

Wyatt

, Manning

, Bai

et al. Molecular imaging of the translocator protein (TSPO) in a pre-clinical model of breast cancer. Mol Imaging Biol, 2010; 12:349.

35.

McLarty

, Fasih

, Scollard

et al. ¹⁸F-FDG small-animal PET/CT differentiates trastuzumab-responsive from unresponsive human breast cancer xenografts in athymic mice. J Nucl Med, 2009; 50:1848.

36.

Brown

, Fujimura

, Liow

J-S

et al. Radiation dosimetry and biodistribution in monkey and man of ¹¹C-PBR28: A PET radioligand to image inflammation. J Nucl Med, 2007; 48:2072.

37.

Ching

ASC

, Kuhnast

, Damont

et al. Current paradigm of the 18-kDa translocator protein (TSPO) as a molecular target for PET imaging in neuroinflammation and neurodegenerative diseases. Insights Imaging, 2012; 3:111.

38.

Owen

, Gunn

, Rabiner

et al. Mixed-affinity binding in humans with 18-kDa translocator protein ligands. J Nucl Med, 2011; 52:24.

39.

Scarf

, Kassiou

. The translocator protein. J Nucl Med, 2011; 52:677.

40.

Owen

, Yeo

, Gunn

et al. An 18-kDa translocator protein (TSPO) polymorphism explains differences in binding affinity of the PET radioligand PBR28. J Cereb Blood Flow Metab, 2012; 32:1.

41.

Junck

, Olson

, Ciliax

et al. PET imaging of human gliomas with ligands for the peripheral benzodiazepine binding site. Ann Neurol, 1989; 26:752.

42.

Buck

, McKinley

, Hight

et al. Quantitative, preclinical PET of translocator protein expression in glioma using ¹⁸F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline. J Nucl Med, 2011; 52:107.

43.

Tang

, Hight

, McKinley

et al. Quantitative preclinical imaging of TSPO expression in glioma using N,N-diethyl-2-(2-(4-(2-¹⁸F-fluoroethoxy) phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide. J Nucl Med, 2012; 53:287.

44.

Chen

, Sajjad

, Wang

et al. TSPO 18 kDa (PBR) targeted photosensitizers for cancer imaging (PET) and PDT. ACS Med Chem Lett, 2011; 2:136.

Positron-Emission Tomography Imaging of the TSPO with [ 18 F]FEPPA in a Preclinical Breast Cancer Model †

Abstract

Introduction

Materials and Methods

Automated radiosynthesis

Small-animal PET imaging and ex vivo biodistribution studies

Results

Small-animal PET imaging

Ex vivo biodistribution studies: tumor and normal tissue uptake of [18F]FEPPA

Discussion

Conclusions

Footnotes

Acknowledgments

Disclosure Statement

References

Ex vivo biodistribution studies: tumor and normal tissue uptake of [¹⁸F]FEPPA