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Novel Positron Emission Tomography Tracer Distinguishes Normal from Cancerous Cells*

Open AccessPublished:August 08, 2011DOI:https://doi.org/10.1074/jbc.M111.275446
      Development of tumor-specific probes for imaging by positron emission tomography has broad implications in clinical oncology, such as diagnosis, staging, and monitoring therapeutic responses in patients, as well as in biomedical research. Thymidylate synthase (TSase)-based de novo biosynthesis of DNA is an important target for drug development. Increased DNA replication in proliferating cancerous cells requires TSase activity, which catalyzes the reductive methylation of dUMP to dTMP using (R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (MTHF) as a cofactor. In principle, radiolabeled MTHF can be used as a substrate for this reaction to identify rapidly dividing cells. In this proof-of-principle study, actively growing (log phase) breast cancer (MCF7, MDA-MB-231, and hTERT-HME1), normal breast (human mammary epithelial and MCF10A), colon cancer (HT-29), and normal colon (FHC) cells were incubated with [14C]MTHF in culture medium from 30 min to 2 h, and uptake of radiotracer was measured. Cancerous cell lines incorporated significantly more radioactivity than their normal counterparts. The uptake of radioactively labeled MTHF depended upon a combination of cell doubling time, folate receptor status, S phase percentage, and TSase expression in the cells. These findings suggest that the recently synthesized [11C]MTHF may serve as a new positron emission tomography tracer for cancer imaging.

      Introduction

      Molecular imaging technologies are the most widely used clinical tools for diagnosis, staging, and monitoring therapeutic response in cancer patients (
      • van der Meel R.
      • Gallagher W.M.
      • Oliveira S.
      • O'Connor A.E.
      • Schiffelers R.M.
      • Byrne A.T.
      ,
      • Perrone A.
      ,
      • McEwan A.J.
      • Van Brocklin H.F.
      • Divgi C.
      ). Many different technologies have been developed to image the structure and function of systems, such as autoradiography, optical imaging, positron emission tomography (PET),
      The abbreviations used are: PET
      positron emission tomography
      TSase
      thymidylate synthase
      MTHF
      (R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate
      TES
      N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid
      THF
      (S)-5,6,7,8-tetrahydrofolate
      HMEC
      human mammary epithelial cell(s)
      LSC
      liquid scintillation counting
      FR-α
      folate receptor-α.
      magnetic resonance imaging, and x-ray computed tomography (
      • Hayat M.A.
      ). Among those, PET is the only non-invasive technology that can measure metabolic, biochemical, and functional activity in vivo. Because morphological response to chemotherapy or radiation therapy lags behind the course of the treatment, analysis of PET images can potentially detect pathological features and therapeutic response before they are visible on computed tomography and magnetic resonance images (
      • Juweid M.E.
      • Cheson B.D.
      ,
      • Bouchelouche K.
      • Capala J.
      • Oehr P.
      ), and PET is thus emerging as a valuable clinical tool to monitor therapeutic responses in patients.
      PET imaging requires positron-emitting radioisotopes, such as oxygen (14O, 15O) (
      • Sajjad M.
      • Zaini M.R.
      • Liow J.S.
      • Rottenberg D.A.
      • Strother S.C.
      ,
      • Lodge M.A.
      • Jacene H.A.
      • Pili R.
      • Wahl R.L.
      ) nitrogen (13N) (
      • Xiangsong Z.
      • Xinjian W.
      • Yong Z.
      • Weian C.
      ), fluorine-18 (18F) (
      • Timmers H.J.
      • Chen C.C.
      • Carrasquillo J.A.
      • Whatley M.
      • Ling A.
      • Havekes B.
      • Eisenhofer G.
      • Martiniova L.
      • Adams K.T.
      • Pacak K.
      ,
      • Drzezga A.
      ,
      • Yoshida Y.
      • Kurokawa T.
      • Tsujikawa T.
      • Okazawa H.
      • Kotsuji F.
      ), and carbon (11C) (
      • Ullrich R.T.
      • Kracht L.
      • Brunn A.
      • Herholz K.
      • Frommolt P.
      • Miletic H.
      • Deckert M.
      • Heiss W.D.
      • Jacobs A.H.
      ,
      • Song W.S.
      • Nielson B.R.
      • Banks K.P.
      • Bradley Y.C.
      ,
      • Hooker J.M.
      • Schönberger M.
      • Schieferstein H.
      • Fowler J.S.
      ), incorporated into pharmaceutical probes to observe selective accumulation in a tissue of interest (
      • Phelps M.E.
      ,
      • Phelps M.E.
      ). Two of the most extensively used PET probes for cancer are [18F]fluorodeoxyglucose (
      • Nair V.S.
      • Krupitskaya Y.
      • Gould M.K.
      ,
      • Miele E.
      • Spinelli G.P.
      • Tomao F.
      • Zullo A.
      • De Marinis F.
      • Pasciuti G.
      • Rossi L.
      • Zoratto F.
      • Tomao S.
      ,
      • Pelosi E.
      • Deandreis D.
      ) and [18F]fluorothymidine (
      • Bradbury M.S.
      • Hambardzumyan D.
      • Zanzonico P.B.
      • Schwartz J.
      • Cai S.
      • Burnazi E.M.
      • Longo V.
      • Larson S.M.
      • Holland E.C.
      ,
      • Chao K.S.
      ,
      • Saga T.
      • Kawashima H.
      • Araki N.
      • Takahashi J.A.
      • Nakashima Y.
      • Higashi T.
      • Oya N.
      • Mukai T.
      • Hojo M.
      • Hashimoto N.
      • Manabe T.
      • Hiraoka M.
      • Togashi K.
      ,
      • Shields A.F.
      • Briston D.A.
      • Chandupatla S.
      • Douglas K.A.
      • Lawhorn-Crews J.
      • Collins J.M.
      • Mangner T.J.
      • Heilbrun L.K.
      • Muzik O.
      ). The [18F]fluorodeoxyglucose probe targets metabolic activity in a nonspecific way, resulting in high background labeling of normal tissues, such as brain, and areas of inflammation. On the other hand, [18F]fluorothymidine is a proliferation marker and targets thymidylate kinase 1, which is a scavenging pathway used by some cells when dTMP is required for DNA synthesis. The [18F]fluorothymidine activity in tumors is not always reliable in the detection of viable residuals in patients with viable carcinoma or mature teratoma in histology (
      • Lindebjerg J.
      • Nielsen J.N.
      • Hoeffding L.D.
      • Bisgaard C.
      • Brandslund I.
      • Jakobsen A.
      ). False negative and false positive rates for these probes limit their accuracy in monitoring cancer therapy (
      • Saga T.
      • Kawashima H.
      • Araki N.
      • Takahashi J.A.
      • Nakashima Y.
      • Higashi T.
      • Oya N.
      • Mukai T.
      • Hojo M.
      • Hashimoto N.
      • Manabe T.
      • Hiraoka M.
      • Togashi K.
      ,
      • Been L.B.
      • Suurmeijer A.J.
      • Cobben D.C.
      • Jager P.L.
      • Hoekstra H.J.
      • Elsinga P.H.
      ,
      • Troost E.G.
      • Vogel W.V.
      • Merkx M.A.
      • Slootweg P.J.
      • Marres H.A.
      • Peeters W.J.
      • Bussink J.
      • van der Kogel A.J.
      • Oyen W.J.
      • Kaanders J.H.
      ,
      • Honer M.
      • Ebenhan T.
      • Allegrini P.R.
      • Ametamey S.M.
      • Becquet M.
      • Cannet C.
      • Lane H.A.
      • O'Reilly T.M.
      • Schubiger P.A.
      • Sticker-Jantscheff M.
      • Stumm M.
      • McSheehy P.M.
      ,
      • Buck A.K.
      • Hetzel M.
      • Schirrmeister H.
      • Halter G.
      • Möller P.
      • Kratochwil C.
      • Wahl A.
      • Glatting G.
      • Mottaghy F.M.
      • Mattfeldt T.
      • Neumaier B.
      • Reske S.N.
      ,
      • van Westreenen H.L.
      • Cobben D.C.
      • Jager P.L.
      • van Dullemen H.M.
      • Wesseling J.
      • Elsinga P.H.
      • Plukker J.T.
      ,
      • Yap C.S.
      • Czernin J.
      • Fishbein M.C.
      • Cameron R.B.
      • Schiepers C.
      • Phelps M.E.
      • Weber W.A.
      ,
      • Bading J.R.
      • Shields A.F.
      ,
      • Wang W.
      • Cassidy J.
      • O'Brien V.
      • Ryan K.M.
      • Collie-Duguid E.
      ,
      • van Waarde A.
      • Elsinga P.H.
      ). We have therefore undertaken to develop an improved PET proliferative tracer with different uptake and/or therapeutic response prediction.
      Thymidylate synthase (TSase; EC 2.1.1.45) plays a central role in the de novo biosynthesis of the DNA base thymine in humans. It is overexpressed in many cancers, making it a good target for a diagnostic probe to detect rapidly growing cells (
      • Lindebjerg J.
      • Nielsen J.N.
      • Hoeffding L.D.
      • Bisgaard C.
      • Brandslund I.
      • Jakobsen A.
      ,
      • Yu Z.
      • Sun J.
      • Zhen J.
      • Zhang Q.
      • Yang Q.
      ,
      • Yasumatsu R.
      • Nakashima T.
      • Uryu H.
      • Ayada T.
      • Wakasaki T.
      • Kogo R.
      • Masuda M.
      • Fukushima M.
      • Komune S.
      ,
      • Kiss-László Z.
      • Nagy B.
      • Thurzó L.
      • Szabó J.
      ,
      • Shimoda M.
      • Sawada T.
      • Kubota K.
      ). It catalyzes the reductive methylation of dUMP to dTMP, where the methyl group is provided by the methylene from its cofactor, (R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (MTHF) (
      • Hong B.
      • Maley F.
      • Kohen A.
      ,
      • Agrawal N.
      • Hong B.
      • Mihai C.
      • Kohen A.
      ). We incorporated a radionuclide (11C or 14C) into the methylene carbon of MTHF so that the radiolabeled methylene would be incorporated into dTMP and its downstream products toward DNA. If selectively incorporated into cancerous cells, [11C]MTHF could be used as a PET imaging probe (Fig. 1). The synthesis of [11C]MTHF is presented elsewhere,
      M. Saeed, T. J. Tewson, E. Colbin, and A. Kohen, submitted for publication.
      but because of the short half-life of the 11C radionuclide (20.4 min), we have used the chemically equivalent [14C]MTHF for the in vitro studies with cell culture presented here. Our results suggest that the radioactivity is selectively incorporated in cancerous cells, paving the way for further development of MTHF as a cancer detection probe.
      Figure thumbnail gr1
      FIGURE 1Schematic representation of a designed trapping 11C-labeled radiotracer in a typical cancerous cell. [11C]MTHF is sequestered and taken up by FR-α, and the radionuclide (11C) is then transferred to dTMP by TSase and thus retained in the cell by metabolic conversion of dUMP to dTMP.

      REFERENCES

        • van der Meel R.
        • Gallagher W.M.
        • Oliveira S.
        • O'Connor A.E.
        • Schiffelers R.M.
        • Byrne A.T.
        Drug Discov. Today. 2010; 15: 102-114
        • Perrone A.
        J. Nucl. Med. 2008; 49: 25N
        • McEwan A.J.
        • Van Brocklin H.F.
        • Divgi C.
        J. Nucl. Med. 2008; 49: 37N-40N
        • Hayat M.A.
        Cancer Imaging. Volumes 1 and 2. Academic Press, New York2008
        • Juweid M.E.
        • Cheson B.D.
        N. Engl. J. Med. 2006; 354: 496-507
        • Bouchelouche K.
        • Capala J.
        • Oehr P.
        Curr. Opin. Oncol. 2009; 21: 469-474
        • Sajjad M.
        • Zaini M.R.
        • Liow J.S.
        • Rottenberg D.A.
        • Strother S.C.
        Appl. Radiat. Isot. 2002; 57: 607-615
        • Lodge M.A.
        • Jacene H.A.
        • Pili R.
        • Wahl R.L.
        J. Nucl. Med. 2008; 49: 1620-1627
        • Xiangsong Z.
        • Xinjian W.
        • Yong Z.
        • Weian C.
        Nucl. Med. Commun. 2008; 29: 1052-1058
        • Timmers H.J.
        • Chen C.C.
        • Carrasquillo J.A.
        • Whatley M.
        • Ling A.
        • Havekes B.
        • Eisenhofer G.
        • Martiniova L.
        • Adams K.T.
        • Pacak K.
        J. Clin. Endocrinol. Metab. 2009; 94: 4757-4767
        • Drzezga A.
        Behav. Neurol. 2009; 21: 101-115
        • Yoshida Y.
        • Kurokawa T.
        • Tsujikawa T.
        • Okazawa H.
        • Kotsuji F.
        J. Ovarian Res. 2009; 2: 7
        • Ullrich R.T.
        • Kracht L.
        • Brunn A.
        • Herholz K.
        • Frommolt P.
        • Miletic H.
        • Deckert M.
        • Heiss W.D.
        • Jacobs A.H.
        J. Nucl. Med. 2009; 50: 1962-1968
        • Song W.S.
        • Nielson B.R.
        • Banks K.P.
        • Bradley Y.C.
        Nucl. Med. Commun. 2009; 30: 462-465
        • Hooker J.M.
        • Schönberger M.
        • Schieferstein H.
        • Fowler J.S.
        Angew. Chem. Int. Ed. Engl. 2008; 47: 5989-5992
        • Phelps M.E.
        J. Nucl. Med. 2000; 41: 661-681
        • Phelps M.E.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9226-9233
        • Nair V.S.
        • Krupitskaya Y.
        • Gould M.K.
        J. Thorac. Oncol. 2009; 4: 1473-1479
        • Miele E.
        • Spinelli G.P.
        • Tomao F.
        • Zullo A.
        • De Marinis F.
        • Pasciuti G.
        • Rossi L.
        • Zoratto F.
        • Tomao S.
        J. Exp. Clin. Cancer Res. 2008; 27: 52
        • Pelosi E.
        • Deandreis D.
        Eur. J. Surg. Oncol. 2007; 33: 1-6
        • Bradbury M.S.
        • Hambardzumyan D.
        • Zanzonico P.B.
        • Schwartz J.
        • Cai S.
        • Burnazi E.M.
        • Longo V.
        • Larson S.M.
        • Holland E.C.
        J. Nucl. Med. 2008; 49: 422-429
        • Chao K.S.
        Semin. Oncol. 2006; 33: S59-S63
        • Saga T.
        • Kawashima H.
        • Araki N.
        • Takahashi J.A.
        • Nakashima Y.
        • Higashi T.
        • Oya N.
        • Mukai T.
        • Hojo M.
        • Hashimoto N.
        • Manabe T.
        • Hiraoka M.
        • Togashi K.
        Clin. Nucl. Med. 2006; 31: 774-780
        • Shields A.F.
        • Briston D.A.
        • Chandupatla S.
        • Douglas K.A.
        • Lawhorn-Crews J.
        • Collins J.M.
        • Mangner T.J.
        • Heilbrun L.K.
        • Muzik O.
        Eur. J. Nucl. Med. Mol. Imaging. 2005; 32: 1269-1275
        • Been L.B.
        • Suurmeijer A.J.
        • Cobben D.C.
        • Jager P.L.
        • Hoekstra H.J.
        • Elsinga P.H.
        Eur. J. Nucl. Med. Mol. Imaging. 2004; 31: 1659-1672
        • Troost E.G.
        • Vogel W.V.
        • Merkx M.A.
        • Slootweg P.J.
        • Marres H.A.
        • Peeters W.J.
        • Bussink J.
        • van der Kogel A.J.
        • Oyen W.J.
        • Kaanders J.H.
        J. Nucl. Med. 2007; 48: 726-735
        • Honer M.
        • Ebenhan T.
        • Allegrini P.R.
        • Ametamey S.M.
        • Becquet M.
        • Cannet C.
        • Lane H.A.
        • O'Reilly T.M.
        • Schubiger P.A.
        • Sticker-Jantscheff M.
        • Stumm M.
        • McSheehy P.M.
        Transl. Oncol. 2010; 3: 264-275
        • Buck A.K.
        • Hetzel M.
        • Schirrmeister H.
        • Halter G.
        • Möller P.
        • Kratochwil C.
        • Wahl A.
        • Glatting G.
        • Mottaghy F.M.
        • Mattfeldt T.
        • Neumaier B.
        • Reske S.N.
        Eur. J. Nucl. Med. Mol. Imaging. 2005; 32: 525-533
        • van Westreenen H.L.
        • Cobben D.C.
        • Jager P.L.
        • van Dullemen H.M.
        • Wesseling J.
        • Elsinga P.H.
        • Plukker J.T.
        J. Nucl. Med. 2005; 46: 400-404
        • Yap C.S.
        • Czernin J.
        • Fishbein M.C.
        • Cameron R.B.
        • Schiepers C.
        • Phelps M.E.
        • Weber W.A.
        Chest. 2006; 129: 393-401
        • Bading J.R.
        • Shields A.F.
        J. Nucl. Med. 2008; 49: 64S-80S
        • Wang W.
        • Cassidy J.
        • O'Brien V.
        • Ryan K.M.
        • Collie-Duguid E.
        Cancer Res. 2004; 64: 8167-8176
        • van Waarde A.
        • Elsinga P.H.
        Curr. Pharm. Des. 2008; 14: 3326-3339
        • Lindebjerg J.
        • Nielsen J.N.
        • Hoeffding L.D.
        • Bisgaard C.
        • Brandslund I.
        • Jakobsen A.
        Appl. Immunohistochem. Mol. Morphol. 2006; 14: 37-41
        • Yu Z.
        • Sun J.
        • Zhen J.
        • Zhang Q.
        • Yang Q.
        Histol. Histopathol. 2005; 20: 871-878
        • Yasumatsu R.
        • Nakashima T.
        • Uryu H.
        • Ayada T.
        • Wakasaki T.
        • Kogo R.
        • Masuda M.
        • Fukushima M.
        • Komune S.
        Chemotherapy. 2009; 55: 36-41
        • Kiss-László Z.
        • Nagy B.
        • Thurzó L.
        • Szabó J.
        Magy. Onkol. 2006; 50: 33-37
        • Shimoda M.
        • Sawada T.
        • Kubota K.
        Pathobiology. 2009; 76: 193-198
        • Hong B.
        • Maley F.
        • Kohen A.
        Biochemistry. 2007; 46: 14188-14197
        • Agrawal N.
        • Hong B.
        • Mihai C.
        • Kohen A.
        Biochemistry. 2004; 43: 1998-2006
        • Changchien L.M.
        • Garibian A.
        • Frasca V.
        • Lobo A.
        • Maley G.F.
        • Maley F.
        Protein Expr. Purif. 2000; 19: 265-270
        • Agrawal N.
        • Mihai C.
        • Kohen A.
        Anal. Biochem. 2004; 328: 44-50
        • Murphy M.
        • Keating M.
        • Boyle P.
        • Weir D.G.
        • Scott J.M.
        Biochem. Biophys. Res. Commun. 1976; 71: 1017-1024
        • Verlinde P.H.
        • Oey I.
        • Deborggraeve W.M.
        • Hendrickx M.E.
        • Van Loey A.M.
        J. Agric. Food Chem. 2009; 57: 6803-6814