Molecular Imaging of Tumor Metabolism and Apoptosis

U Haberkorn1,2, A Markert1,2, W Mier1, V Askoxylakis1,3 and A Altmann1,2

1Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany; 2Clinical Cooperation Unit Nuclear Medicine, DKFZ and University of Heidelberg, Heidelberg, Germany and 3Department of Radiation Therapy, University of Heidelberg, Heidelberg, Germany

Increased metabolism in a number of cellular pathways is enzymatic activity, a combination of both or binding to a common feature of malignant tumors. This metabolic membrane-associated structures (Figure 1). Examples hallmark of neoplastic tissue led to the development of are amino acids (transport), caspase inhibitors or radiopharmaceuticals for the assessment of transport and substrates (enzymatic activity), fluorodeoxyglucose and enzymatic activity for tumor diagnosis and staging. The fluoroethyl-choline (transport and enzymatic activity), malignant transformation causes the activation of onco- and Annexin-V (binding to membrane structures). For genic proteins and signaling pathways that stimulate measurement of enzymatic activity, substrates are glycolysis. The resulting high-glucose metabolism of cancer preferred to inhibitors because of signal amplification, cells allows PET imaging using FDG. Other molecules which can be obtained using the former. Signal frequently applied in preclinical and clinical studies are amplification means that an enzyme converts many 11C-methionine, tyrosine analogs and choline-based tracers. substrate molecules producing to multiple metabolites. Using quantitative procedures they enable therapy monitor- This is not the case for an inhibitor, where one inhibitor ing by assessment of changes in transport and metaboliza- molecule binds irreversibly to one enzyme molecule. tion. As apoptosis is an important mechanism of cell death It has to be stressed that nuclear medicine methods in tumors responding to treatment, non-invasive assess- measure a radioactive signal and do not discriminate ment of apoptosis using tracers for detection of phospha- different metabolites. As metabolites may occur, tracers tidyl-serine presentation and/or caspase activation could are preferred with one or two steps of metabolization. be used as a surrogate marker for therapeutic efficacy. In combination with dynamic studies allowing the Oncogene (2011) 30, 4141–4151; doi:10.1038/onc.2011.169; generation of time–activity curves and the use of published online 16 May 2011 mathematical models, pharmacokinetic modeling can be applied for the assessment of kinetic constants, which Keywords: glucose metabolism; amino-acid transport; describe the biochemistry of malignant tissues in more PET; imaging; tumor; apoptosis detail. Therefore, signals obtained after administration of fluorodeoxyglucose may be further analyzed for transport as well as for phosphorylation. In a similar manner, molecules binding to plasma membrane struc- Principles of metabolism and apoptosis imaging tures may be described by kinetic constants for binding and internalization. For visualization and/or assessment of biochemical changes in tumors, procedures are needed that (1) result in a high contrast between malignant and non-malig- Imaging glucose metabolism nant tissues, or between responding and non-responding lesions during therapy, and (2) deliver quantitative data Malignant tumors show increased glycolytic activity, about a specific disease-associated process. As there is which is associated to changes in the expression of no ‘all or nothing’ difference between tumor and normal glycolysis-associated genes occurring during malignant tissue, differences in quantity or quality may be transformation (Flier et al., 1987; Shawver et al., 1987). important, such as an increase in expression or activity, Especially the gene encoding the glucose transporter or a shift to different protein isoforms. Usually imaging subtype-1 (GLUT1) is activated early after transforma- is based on a substantially higher activity and/or tion of cells with oncogenes such as src, ras or fps. expression of tumors as compared with normal tissue. An increase in the mRNA of GLUT1 is observed as The underlying processes are transport of substrates, early as 4–6 h after induction of the p21 c-H-ras oncoprotein, whereas changes in cell morphology occur Correspondence: Professor U Haberkorn, Department of Nuclear after 72–76 h. Furthermore, the increase in GLUT1 Medicine, University Hospital Heidelberg, and Clinical Cooperation mRNA after ras transfection was independent of the Unit Nuclear Medicine, DKFZ, Im Neuenheimer Feld 400, Baden- growth rate. In vivo overexpression of GLUT1 and Wu¨rttemberg, Heidelberg 69120, Germany. E-mail: [email protected] GLUT3 was found in a series of different human and Received 20 February 2011; revised and accepted 5 April 2011; published experimental tumors. The increase in GLUT1 transcrip- online 16 May 2011 tion can be used for imaging or therapy by cloning of a Molecular imaging of tumor metabolism and apoptosis U Haberkorn et al 4142 O et al. - binding to the enzyme hexokinase (Gallagher , I 1977). In contrast to glucose-6-phosphate, FDG-6-

1 phosphate and dGlc-6-phosphate are not further metabolized in significant amounts during PET study. I- 2 dGlc-6-phosphate is not metabolized to the fructose- O 6-phosphate derivative and, therefore, is not a substrate for glucose-6-phosphate dehydrogenase (Sols and trapping Crane, 1954). dGlc-6-phosphate may be converted to dGlc-1-phosphate and uridine-diphosphate-dGlc, followed by incorporation into glycogen, glycolipids NH2 COOH 4 and glycoproteins. However, these reactions are very 3 slow in mammalian tissues. Furthermore, in the brain, NH2 COOH the organ where the deoxyglucose method was applied for the first time, as well as in malignant tumors glucose- Figure 1 Typical examples of radiotracers illustrating the 6-phosphatase activity is downregulated. In contrast to principles of selective uptake. (1) 18F-FDG is taken up into cells auto-inhibition of glucose phosphorylation, FDG- by glucose transporters and subsequently trapped after enzymatic 6-phosphate does not show inhibition of hexokinase phosphorylation by hexokinase; (2) radioactive isotopes of iodide activity (Machado de Domenech and Sols, 1980). are taken up by the human sodium iodide symporter on thyroid cancer tissues; (3) the amino acid 18F-O-(2-fluoroethyl)-tyrosine Compared with 2-deoxyglucose FDG is incorporated (FET) is taken up into cells by the L-type amino-acid trans- very slowly into macromolecules as has been shown in porter system; (4) radiolabeled antibodies such as Zevalin bind to yeasts as well as in chick fibroblasts. The negative charge antigens specifically expressed on the surface of tumor cells. on FDG-6-phosphate and dGlc-6-phosphate prevents 18 18 F-FDG, F-fluorodeoxyglucose. penetration of the negatively charged inner surface of the plasma membrane and causes their accumulation in reporter gene or a therapeutic gene, such as suicide genes cells. A further advantage is the rapid clearance of downstream from the GLUT1 promoter/enhancer ele- the tracer: similar to glucose FDG shows glomerular ments (Sieger et al., 2004; Haberkorn et al., 2005). filtration. However, unlike glucose this is not followed Examples are the Herpes Simplex Virus thymidine by tubular re-absorption, because FDG is not a kinase gene or the sodium iodide symporter gene, where substrate for the tubular sodium glucose symporter, adeno-associated virus or retroviral vectors have been which transports normal glucose back to he blood. This used to transfect tumor cells and measure the uptake of fact is responsible for the rapid renal clearance of the specific substrates, or to treat animals with genetically tracer. modified tumors (Sieger et al., 2003, 2004). In these PET studies in different animal models showed a studies reporter gene expression (green fluorescent correlation between FDG uptake and content of protein, HSVtk or sodium iodide symporter) was GLUT1 and hexokinase mRNA (Haberkorn et al., specific for tumor cells or spontaneously immortalized 1994). Differences in FDG uptake in different lung human keratinocytes (HaCaT) with expression of an carcinomas, with lower values for adenocarcinomas as activated ras oncogene. compared with squamous cell carcinomas, corresponded Besides increased glucose transport, the malignant to the histologically determined expression of GLUT1, transformation goes along with changes in the activity which was higher in squamous cell carcinomas than in of glycolytic enzymes. For imaging, hexokinase is the adenocarcinomas (Brown et al., 1999). Therefore, the most important enzyme. Here, a change in the isoform genetic program in malignant tumors leads to the pattern occurs as well, with a predominant expression of corresponding FDG uptake values as measured by hexokinase-II in all tumors, except for brain tumors PET. Similar results were obtained in bronchioalveolar where hexokinase-I may be overexpressed. In rapidly adenocarcinomas, with significantly lower values for growing tumor cells, hexokinase activity is greatly number of GLUT1-positive cells and FDG uptake, and enhanced, and up to 80% of the enzyme molecules are a correlation between histological grade and amount of bound to the outer mitochondrial membrane, which GLUT1-positive cells and FDG uptake. ensures privileged access to the mitochondrial ATP Originally, clinical application of 18F-FDG predomi- pool. nantly focused on tumor diagnosis and in the staging of These changes in transport and phosphorylation are a variety of tumor entities such as lung; colon; breast; exploited for tumor imaging by positron emission head and neck, and esophageal cancer; melanoma and tomography (PET) using 18F-fluorodeoxyglucose lymphoma. Besides staging, the prognostic value of (18F-FDG). 18F-FDG for PET studies of glucose 18F-FDG-PET has also been evaluated. The relation of metabolism was introduced as a consequence of auto- high pre-therapeutic FDG uptake to a decrease in radiographic and biochemical studies of glucose analogs prognosis was observed by different groups in patients in different tissues. Similar to glucose, 2-deoxyglucose with lung cancer (Vansteenkiste et al., 1999). (dGlc) and fluorodeoxyglucose (FDG) are transported PET using 18F-FDG has been used for the evaluation bi-directionally and are phosphorylated by the enzyme of treatment response during chemotherapy (Figure 2), hexokinase. This is possible because the C-2 position gene therapy and radiotherapy in a variety of tumors, unlike the C-1, C-3 and C-6 positions is uncritical for indicating that FDG delivers useful parameters for the

Oncogene Molecular imaging of tumor metabolism and apoptosis U Haberkorn et al 4143

Figure 2 PET/CT images (coronal sections, display as virtual anatomy) using FDG of a patient with non-Hodgkin lymphoma before (left) and after (right) chemotherapy, showing complete response. CT, computed tomography; FDG, fluorodeoxyglucose; PET, positron emission tomography. early assessment of therapeutic efficacy (Rozental et al., increased the amount of apoptotic cells (Haberkorn 1989; Haberkorn et al., 1991, 1992, 1993, 1997b, 1998; et al., 2001a, b), whereas monotherapy with these drugs Bassa et al., 1996). The idea is mainly that a decrease in had no effect. Enhanced glycolysis may be used for a FDG uptake in the tumor reflects a decrease in viable metabolic design of combination therapy as has been cell number. However, tumors may show inflammatory done for chemotherapy (Haberkorn et al., 1992) or responses at some time period after therapeutic inter- radiotherapy (Singh et al., 2005). These strategies intend ventions such as radiation therapy, or stress reactions, to disturb possible repair processes, which are in need of both leading to a transient increase in glucose meta- energy, by interfering with glycolysis. Besides deoxyglu- bolism, especially during the very early phase during or cose, a couple of compounds are available such as 6- after treatment. aminonicotinamide, 3-bromopyruvate, oxythiamine, 5- In general, increased FDG transport rates, which may thioglucose or genistein, where at least deoxyglucose occur early after treatment, are suggested as evidence of shows rather selective toxicity for cells showing che- stress reactions in tumors after chemotherapy, gene motherapy resistance (Haberkorn et al., 1992). The therapy or radiation therapy (Haberkorn et al., 1998, design of such a combination treatment requires data 2001a, b). The glucose carrier shows a complex regula- about the changes in the metabolic pathways with tion: Glucose transport may be altered by phosphoryla- respect to dose and time dependence, which may be tion of the transport protein (Hayes et al., 1993), obtained by 18F-FDG-PET. decreased degradation (Shawver et al., 1987), transloca- tion from intracellular pools to the plasma membrane (Widnell et al., 1990) or increased expression of the gene Imaging amino-acid transport (Flier et al., 1987). Increase in glucose transport after exposure of cells to damaging agents has been Although PET using 18F-FDG has been proven to be ascribed mainly to a redistribution of the glucose useful for diagnosis and therapy monitoring in a large transport protein from intracellular pools to the plasma variety of tumors, there is need for complementary membrane. Such reactions have been found in cells information of tumor biology. FDG is not tumor- exposed to arsenite, the calcium ionophore A23187 or selective and also shows accumulation in inflammatory 2-mercaptoethanol (Hughes et al., 1989; Widnell et al., lesions. Furthermore, tissues with a high FDG uptake 1990; Wertheimer et al., 1991). Furthermore, increased background such as the brain may cause difficulties in glucose metabolism has been observed after chemo- image interpretation. Further problems arise for slow- therapy or gene therapy of hepatoma using HSV growing tumors or differentiated tumors such as thymidine kinase (Haberkorn et al., 1998, 2001a, b). prostate cancer or neuroendocrine tumors. Incubation with deoxyglucose or the glucose transport In addition to enhanced glucose metabolism, inhibitor cytochalasin-B after the end of treatment malignant tumors may show changes in amino-acid

Oncogene Molecular imaging of tumor metabolism and apoptosis U Haberkorn et al 4144 transport and protein synthesis. Therefore, many been found in transformed and malignant cells as a efforts have been made to establish tracers based on result of oncogene action (Saier et al., 1988), and in amino acids. clinical studies a correlation between amino-acid trans- Radiolabeled amino acids may be used to assess the port and cellular proliferation has been described rate of protein synthesis and amino-acid transport. (Kuwert et al., 1997; Jager et al., 2001). The system However, besides protein synthesis, amino acids are pre- ASCT2 (SLC1A5), which accepts neutral amino acids cursors for many other biomolecules, such as adenine, and prefers glutamine as substrate, has been shown to be cytosine, histamine, thyroxine, epinephrine, melanin and upregulated in several types of tumors and cell lines of serotonin, and are important in other metabolic cycles, colorectal carcinoma and prostate carcinoma. LAT1/ including transamination and transmethylation; methio- 4F2hc is upregulated in brain, colon, lung, liver and skin nine has a specific role in the initiation of protein tumors. Its expression seems to correlate with cell synthesis and amino acids such as glutamine are used for proliferation and cancer growth. This transporter production of energy. As all these pathways create a also mediates the influx of melphalan. Finally, the dependence on amino-acid uptake, it is evident that the xCT transporter (SLC3A2) has been found to be values obtained by measurement of amino-acid trans- increased in gliomas and lymphoma cells. port do not faithfully represent protein synthesis alone. For assessment of protein synthesis rate, relatively Rather they provide a general measure of the cellular complex kinetic models are necessary. Although need for amino acids. 11C-leucine appears to be the best amino acid for Amino acids enter cells mainly through specific trans- measuring the rate of protein synthesis (Vaalburg et al., port systems (Christensen, 1990; Ganapathy et al., 1992), most studies have used methionine because of the 2009). These systems can be sodium-dependent or ease of tracer synthesis. The drawbacks of methionine are -independent. Sodium-dependent transport relies on its use in metabolic cycles other than protein synthesis, the sodium chemical gradient and the electric potential which results in a variety of metabolites and difficulties across the plasma membrane, as well as on the activity in quantification (Ishiwata et al., 1996). Conflicting of the Na þ /K þ -ATPase. In general, a change in affinity reports have been published about the specificity of occurs when a sodium ion binds to the transporter carrier-mediated transport of methionine into brain protein. Subsequent binding of an amino acid results in tumors in studies comparing D-andL-methionine using a conformational change of the transporter protein, an overload of branched amino acids. Furthermore, at which in turn leads to an influx of the attached sodium least part of the tracer uptake seems to be a result of ion and the amino acid into the cell. Sodium-indepen- passive diffusion. Cellular uptake in vitro is mainly dent systems depend on the amino-acid concentration accomplished through the L-system, with minor contribu- gradient across the cell membrane and are often coupled tions from systems A and ASC. to counter-transport (that is, in the opposite direction) Patient studies have shown high uptake of methionine of K þ . in the pituitary gland and pancreas, moderate uptake in Kinetic studies have identified several sodium-depen- salivary glands, lacrimal glands and bone marrow, and dent transport systems, A, ASC and Gly that transport low uptake in the normal brain (Jager et al., 2001). amino acids with short polar or linear side chains, for The tracer has been used mainly in brain tumors, example, alanine, serine and glycine. System-A is trans- where it shows excellent contrast between normal inhibited by intracellular substrates (that is, the presence brain and tumors, and has high sensitivity for tumor of intracellular substrates slows the uptake of amino detection (Langen et al., 1997), but also for the diagnosis acids), whereas system-ASC is trans-stimulated by the and staging of a variety of other tumor entities such as presence of intracellular substrates (that is, the presence head and neck cancer, lung cancer, breast cancer, of intracellular substrates increases activity). sarcoma and lymphoma. The sodium-independent systems, L (ubiquitously An alternative to 11C-methionine are tyrosine deriva- found), B0, þ (SLC6A14) and y þ , are carriers for tives such as 11C-tyrosine, L-2-18F-fluoro-tyrosine, branched-chain and aromatic amino acids, for example, 123I-a-methyl-tyrosine (123I-IMT), 124I-IMT, L-3-18F- leucine, valine, tyrosine and phenylalanine. System-L a-methyl-tyrosine and O-(2-18F-fluoroethyl)-L-tyrosine (LAT1/4F2hc) shows trans-stimulation by intracellular (L-FET). These tracers have been used in experimental substrates such as leucine, valine etc. Most amino-acid (Figure 3) as well as in clinical studies. carrier systems can also transport synthetic, non-meta- A high sensitivity for the detection of primary and bolizable amino-acid analogs. metastatic brain tumors was found using either Regulation of amino-acid transport is complex and is 11C-tyrosine or L-2-18F-fluoro-tyrosine (Wienhard et al., influenced by hormones, cytokines, changes in cell 1991; Willemsen et al., 1995). Analysis of the plasma 11 11 volume and the availability of nutrients (Christensen, metabolites of C-tyrosine showed that C-CO2, 1990). For example, the number of system-A active 11C-labelled proteins and 11C-L-DOPA constituted carriers increases during starvation; hence imaging 450% of total plasma radioactivity at 40 min after studies should be performed preferentially while patients injection, making a complex pharmakokinetic model for are fasting. further analysis necessary. Using a five-compartment Malignant cells were found to have increased amino- model, it was shown that, whereas the net protein acid transport (Busch et al., 1959; Isselbacher, 1972; synthesis rate was dependent on the recycling of amino Saier et al., 1988). Strong expression of system-A has acids from the protein, tracer influx into the cell was not.

Oncogene Molecular imaging of tumor metabolism and apoptosis U Haberkorn et al 4145

Figure 3 L-FET-PET in a rat with Morris hepatoma transplanted subcutaneously on the right thigh. Coronal slice (left) with tracer accumulation in the bowel and the tumor (circle), and time–activity curves (right) showing time-dependent accumulation of the tracer in the left ventricle and the tumor. L-FET, O-(2-18F-ﬂuoroethyl)-L-tyrosine; PET, positron emission tomography.

Figure 4 FET-PET in a patient with recurrent glioblastoma: transaxial slices (PET on the right side, fusion image of PET and CT on the left side) demonstrating tracer accumulation in the periphery and necrosis in the center of the lesion. FET, 18F-O-(2-ﬂuoroethyl)- tyrosine; PET, positron emission tomography.

The curve-fitting results of dynamic scans were unreli- performance liquid chromatography analysis of brain, able owing to the exchange of 11C-tyrosine between pancreas and tumor homogenates, as well as plasma plasma and erythrocytes, whereas the graphical Patlak– samples of mice at 10, 40 or 60 min after injection, Gjedde analysis was not influenced by this. L-2- showed only unchanged L-FET, indicating high stability 18F-fluorotyrosine was studied in 15 patients with and lack of metabolization of the tracer. The tracer has brain tumors and showed rapid uptake, which was mainly been applied in brain tumors for planning of mainly attributed to an increased transport rate. Also, radiation therapy, detection of tumor recurrence after improved localization of tumor tissue for biopsy has therapy (Figure 4) and differentiation between inflam- been described for both methionine and tyrosine. mation and tumor (Po¨pperl et al., 2004; Pauleit et al., L-FET, which is not incorporated into proteins, has 2005; Salber et al., 2007; Vees et al., 2009). been evaluated in mammary carcinoma-bearing mice A variety of synthetic amino acids, including a-amino- and in mice transplanted with the colon carcinoma cell isobutyric acid (AIB), 1-aminocyclopentane carboxylic line SW707 (Heiss et al., 1999; Wester et al., 1999). (ACPC) acid, 2-amino-3-fluoro-2-methylpropanoic acid Results of transport inhibition experiments using (FAMP), 3-fluoro-2-methyl-2-(methylamino)propanoic specific competitive inhibitors have shown that uptake acid (N-MeFAMP) and 1-amino-3-fluorocyclobutane- of L-FET into SW707 cells is caused mainly by system- 1-carboxylic acid (FACBC), have been synthesized and L. In vivo studies showed a plasma half-life of 94 min for evaluated, mostly in cell culture and animal systems. L-FET and increasing brain uptake up to 120 min with a AIB is thought to be actively accumulated in viable cells brain:blood ratio of 0.9. Xenotransplanted tumors have primarily by the A-type amino-acid transport system shown a higher uptake of L-FET (46% injected dose/g) and has shown avid uptake in a melanoma model. than all other organs, except the pancreas. High- Additionally, ACPC and AIB imaging was found to be

Oncogene Molecular imaging of tumor metabolism and apoptosis U Haberkorn et al 4146 superior to FDG in C6 gliomas and Walker 256 rat nine uptake experiments showed a decrease in tracer carcinosarcoma, especially for identifying tumor infil- accumulation in the acid-insoluble fraction (represent- tration of adjacent brain tissue beyond the macroscopic ing nucleic acids and proteins), indicating impaired border of the tumor, and in low-grade tumors with an protein synthesis and an increase in the acid-soluble intact bloodÀbrain barrier. The contrast-enhancing fraction. The increase of radioactivity found in the regions of the tumors were visualized more clearly with acid-soluble fraction may be caused by enhanced AIB than with FDG; viable and necrotic-appearing transmethylation processes, which usually are observed tumor regions could be distinguished more readily with during oncogenic transformation and after exposure to AIB than with FDG (Uehara et al., 1997). As for AIB, DNA-damaging agents. amino-acid transport assays using 9L gliosarcoma cells Clinical studies in brain tumors have been conducted showed that FAMP and N-MeFAMP are substrates for early evaluation of treatment response and differ- for the A-type amino-acid transport system and show entiation between recurrence and radiation necrosis. very high tumor:normal brain ratios of 36:1 and 104:1, In 10 patients with low-grade gliomas, a dose-dependent respectively (McConathy et al., 2002). In a rat brain reduction in methionine uptake was seen after bra- tumor model, maximum tumor uptake of 18F-FACBC chytherapy (Wurker et al., 1996). Differentiation was seen at 60 min, with a tumor:normal brain ratio of between radiation necrosis and tumor recurrence was 5.6 at 5 min and 6.6 at 60 min after tracer administration possible by methionine-PET (Ogawa et al., 1991). After (Shoup et al., 1999). radiotherapy of head and neck cancer, a lower post- Measurement of the effects of therapy on tumor therapeutic methionine uptake was shown to correlate metabolism may be useful in predicting therapy out- with therapy response (Lindholm et al., 1998). Similar come at an early stage of treatment. This principle may results were obtained in patients after radiotherapy or be applied not only to glucose metabolism but also to chemotherapy of lung, breast and rectal cancer (Daemen amino-acid transport and metabolism. Studies of et al., 1991; Jansson et al., 1995). However, the pre- various human tumors treated with a variety of dictive value of methionine-PET remains questionable. therapies and of the rat AH109A tumor model after Amino acids have been suggested to be useful for radiotherapy showed a rapid post-therapeutic reduction differentiation between inflammation and malignancy. in methionine uptake, reflecting inactivation of protein Experimental studies have shown that amino acids synthesis and damage to the membrane transport system accumulate less in inflamed tissue than FDG. However, (Bergstrom et al., 1987; Jansson et al., 1995; Schaider amino-acid uptake may occur in benign lesions such as et al., 1996). Furthermore, uptake of L-1-11C-tyrosine in ischemic brain, infarction, scar, abscesses and sarcoido- the rhabdomyosarcoma of Wag/Rij rats was dose sis, and also in irradiated areas. Therefore, active dependently reduced after local hyperthermia (Daemen inflammatory cells may need amino acids, and the et al., 1991). Moreover, accumulation of AIB is specificity of amino acids for tumor imaging is not decreased in rat prostate tumors after long-term absolute. However, in mice with tumor-infiltrated or treatment with stilbestrol (Dunzendorfer et al., 1981). inflammatory lymph nodes, accumulation of L-FET These changes were followed later by a reduction in showed significant differences, with no overlap between tumor mass. inflammatory and tumorous nodes (Rau et al., 2002). In vitro studies have shown that methotrexate and In summary, amino acids may have a potential role in cisplatin induce a decline in AIB and methionine the characterization of the biological properties of accumulation in L1210 murine leukemia cells (Scanlon tumors as increased amino-acid transport or protein et al., 1983, 1987), leading to the speculation that synthesis. Advantages over FDG imaging can be inhibition of methionine uptake by methotrexate may expected in the imaging of brain tumors, because the be because of the drug binding to a specific membrane background of tracer accumulation is lower as com- carrier, or reduction in the sodium gradient across the pared with FDG. The role of amino acids for monitor- plasma membrane, which is necessary for uptake of ing the treatment response of tumors as well as the amino acids, or effects on intracellular processes, which differentiation between inflammation and tumor tissue support the uptake of amino acids. Higashi et al. (1993) has to be established in further studies. showed an increase in methionine and FDG uptake in human ovarian carcinoma cells after radiotherapy, which was accompanied by an increase in cell volume. Imaging of choline phospholipid metabolism These phenomena were interpreted as giant cell formation with enlarged cellular volume and continued Elevated levels of phosphocholine, phosphoethanola- protein synthesis, but accelerated repair was also mine and total choline have been detected by in vivo and suggested. Another in vitro study combined information in vitro 1H and 31PMR spectroscopic studies in a variety obtained from experiments using a transport tracer of tumors, including breast, prostate and brain tumors (AIB) and a tracer, which is transported and metabo- (Negendank, 1992; de Certaines et al., 1993; Leach et al., lized (methionine), and found a decrease in neutral 1998). Phosphocholine is a precursor as well as a amino-acid transport after gene therapy of hepatoma breakdown product of phosphatidylcholine, the most cells using HSV thymidine kinase and ganciclovir, abundant phospholipid found in biological membranes. indicating treatment effects on the energy-dependent The molecule together with other phospholipids such as transport systems (Haberkorn et al., 1997a). Methio- phosphatidylethanolamine is involved in the formation

Oncogene Molecular imaging of tumor metabolism and apoptosis U Haberkorn et al 4147 of the bilayer structure of the cell membrane and (Hernandez-Alcoceba et al., 1999). Incubation of regulates membrane integrity (Ackerstaff et al., 2003). Ehrlich ascites tumor cells with 3H- or 14C-choline In addition, synthesis and hydrolysis of phosphatidyl- showed that choline was taken up at low concentrations choline are essential for mitogenic signal transduction by an active transport mechanism and converted into (Cai et al., 1993). In that respect, the products of choline phosphorylcholine within 1 h, followed by degradation phospholipid metabolism, such as phosphocholine and to phosphatidylcholine (Haeffner, 1975). diacylglycerol, may function as second messengers The mentioned changes in choline content and for the mitogenic activity in the activation of the metabolism have been used for tumor diagnosis by ras–raf-1–mitogen-activated protein kinase (MAPK) proton magnetic resonance spectroscopy, which is cascade and the protein kinase-C pathway (Cai et al., usually performed in combination with anatomical 1993). A couple of studies showed that activation of MRI. Pre- and post-therapy studies showed that choline uptake and phosphorylation is rather a late combined MRI and magnetic resonance spectroscopic event involved in a cascade of intracellular signal imaging was able to assess the presence and localization transduction events that results in cellular transforma- of cancer, as well as the effects of treatment (Leach tion. This was seen for activation of the ras-GTPase- et al., 1998; Kurhanewicz et al., 2000). This has been activating protein, the phosphatidylinositol-3-kinase done for a variety of tumors, including prostate, brain and other tyrosine kinases (Molloy et al., 1989; Kypta and breast cancer (Negendank, 1992; Gribbestad et al., et al., 1990; Cuadrado et al., 1994; Galetic et al., 1999). 1999; Kurhanewicz et al., 2000; Li et al., 2002). In quiescent NIH3T3 fibroblasts, DNA synthesis was Furthermore, radiolabeled choline analogs have stimulated by phosphocholine, with no effect for been used for tumor detection by PET in many tumor choline, phosphorylserine and phosphoethanolamine. entities such as brain tumors, lung cancer, esophageal Furthermore, incubation with the choline kinase cancer, colon cancer, bladder cancer and prostate cancer inhibitor HC-3 blocked growth factor induced prolif- (Hara et al., 1998, 2002; Kobori et al., 1999; DeGrado eration, an effect that could be reversed by addition of et al., 2000, 2001; Kwee et al., 2004, 2006). As phosphocholine. Therefore, phosphocholine is seen as mentioned above, choline is transported into cells. an important second messenger for mitogenic activity, Thereafter, it is rapidly metabolized to phosphocholine with choline kinase activity as a critical step for the or oxidized by choline dehydrogenase and betaine- regulation of cell proliferation (Cuadrado et al., 1993). aldehyde dehydrogenase to betaine. The latter occurs Besides growth factors, choline phospholipid meta- mainly in the liver and kidneys. Phosphorylation of bolism is regulated through a variety of mechanisms, choline by choline kinase represents the first and including stimulation by cytokines (Bogin et al., 1998), obligatory step for the incorporation of choline into oncogenes (Aboagye and Bhujwalla, 1999; Ronen et al., phosphatidylcholine. The resulting first metabolite, 2001), hypoxia and chemical carcinogens (Kiss et al., phosphocholine, is negatively charged and, therefore, 1993; Galons et al., 1995). trapped within the cell. The mechanisms responsible for the increased phos- Several choline-based tracers exist that have been phocholine levels in malignant cells are an increase in synthesized and evaluated pre-clinically as well as in the expression and activity of choline kinase (Ramirez patients, for example, 11C-choline, 18F-fluoro-choline de Molina et al., 2002a, b), choline transport activity and 18F-fluoroethyl-choline (Figure 5). Comparative (Haeffner, 1975; Katz-Brull and Degani, 1996) and studies in vitro showed that fluoro-choline uptake in elevated levels of enzyme activity for phospholipase-D PC-3 prostate cancer cells was similar to choline uptake. (Noh et al., 2000) and phospholipase-A2 (Guthridge Furthermore, a 90% decrease of tracer uptake was et al., 1994). Choline kinase is the first enzyme involved seen after pretreatment with HC-3, a specific inhibitor of in the Kennedy pathway and transforms choline into choline uptake and phosphorylation (DeGrado et al., phosphocholine. Its enzyme activity is upregulated by 2000, 2001). Experiments in tumor bearing mice showed growth factors, chemical carcinogens and oncogenes a similar bio-distribution as with 11C-labeled choline.

Figure 5 PET/CT (transaxial slices) using 18F-ﬂuoroethyl-choline in a patient with rising PSA levels 2 years after surgery. Rapid accumulation (2 min, left) of the tracer occurs in a lymph node metastasis (shown in the center of the crosshairs) and remains in the lesion also at later time periods (60 min, right). CT, computed tomography; FDG, ﬂuorodeoxyglucose.

Oncogene Molecular imaging of tumor metabolism and apoptosis U Haberkorn et al 4148 At 1 h after administration the tumor:blood ratio was 5:1, of phosphatidylserine on the outer surface of the suggesting trapping of tracer in the tumor cells, consistent membrane. This effect has been used to develop an with phosphorylation by choline kinase activity. imaging agent for apoptosis (Blankenberg et al., 1998, The blood clearance of 18F-fluoro-choline was very 1999): Annexin-V, a 35-kDa human protein with high rapid in patients, leading to an excellent contrast affinity for cell membrane-bound phosphatidylserine, between tumor and background in PET images at was labeled with 99mTc and investigated for its uptake in 3 min after tracer administration. In comparison with apoptotic cells. An increased accumulation was found in 11C-choline, 18F-fluoro-choline and 18F-fluoroethyl-cho- Jurkat cells, where programmed cell death was initiated line showed a higher renal clearance. It is known that by growth factor deprivation, anti-CD95 antibody and choline is efficiently reabsorbed by the renal proximal doxorubicin treatment. Also anti-CD95-treated mice tubular cells. Thus, the main route of clearance relies on showed a threefold rise in hepatic 99mTc-Annexin-V the oxidation of the molecule by the enzymes choline accumulation in response to severe liver damage, with dehydrogenase and betaine-aldehyde dehydrogenase, histological evidence of apoptosis. Finally, increased which convert choline into betaine. Thereafter, betaine uptake was detected in animal models using acute is excreted in the urine. Therefore, it is likely that the rejection of transplanted heterotopic cardiac allografts very rapid appearance of fluorinated choline tracers or transplanted murine B-cell lymphomas treated with is because of incomplete tubular re-absorption of cyclophosphamide (Blankenberg et al., 1999). Figure 6 the intact tracer or owing to enhanced excretion of shows a patient studied with 99mTc-Annexin-V after oxidative metabolites (DeGrado et al., 2000, 2001). treatment. In benign prostate tissues, a decrease of tracer accumu- As caspases have a key role during the early period of lation was observed with time, also leading to a higher the intracellular signal cascade of cells undergoing apo- contrast between tumor and normal tissue. It has been ptosis, benzyloxycarbonyl-Val-Ala-DL-Asp(O-methyl)- suggested that this is because of dephosphorylation of fluoromethyl ketone (Z-VAD-fmk), a pan-caspase 18F-phosphorylfluoro-choline by prostatic acid phos- inhibitor, was evaluated as a potential apoptosis- phatase, an enzyme specific for prostate tissues imaging agent (Haberkorn et al., 2001c). Uptake (DeGrado et al., 2000, 2001). Normal prostate tissue measurements were performed using Morris hepatoma as well as prostate hyperplasia contain higher levels of cells (MH3924Atk8), which showed expression of the prostatic acid phosphatase than prostate carcinoma, Herpes Simplex Virus thymidine kinase (HSVtk) gene. which accepts phosphocholine and possibly also Apoptosis was induced by treating cells with ganciclovir 18F-phosphorylfluoro-choline as a substrate. and a twofold increase in [131I]I-Z-VAD-fmk uptake was found at the end of treatment with the HSVtk/suicide system, which constantly remained elevated for the Imaging apoptosis following 4 h. The slow cellular influx and lack of uptake For in vivo detection of apoptosis mainly two targets in saturation of [131I]IZ-VAD-fmk are evidences for simple the apoptotic pathway are of interest: (1) The presenta- diffusion as uptake mechanism. In addition, absolute tion of phosphatidylserine residues at the outer surface cellular uptake of [131I]IZ-VAD-fmk was found to be of the plasma membrane and (2) the appearance of low. Besides peptidic molecules, a couple of isatin activated caspases (Martin et al., 1995; Villa et al., sulfonamide analogs have been identified as potent 1997). Phosphatidylserine is maintained at the inner inhibitors of executioner caspases such as caspase-3 surface of the plasma membrane by the ATP-dependent and caspase-7 and, therefore, have been radiolabeled for enzymes floppase and translocase (Zwaal and Schroit, use in apoptosis imaging (Zhou et al., 2009). 1997). Apoptosis induced inactivation of these enzymes, As inhibitors suffer from the drawback that one and activation of a scramblase leads to the appearance inhibitor molecule binds to one enzyme molecule, the

Figure 6 Transaxial SPECT/CT images of a patient with non-small-cell lung cancer after chemotherapy: CT (left), SPECT (middle) and fusion image (right). The tumor (at the center of the crosshairs) and the central lymph node metastasis show accumulation of 99mTc-Annexin-V after chemotherapy, indicating apoptosis. Unspeciﬁc accumulation is seen in the sternum and the vertebra. CT, computed tomography.

Oncogene Molecular imaging of tumor metabolism and apoptosis U Haberkorn et al 4149 signal obtained is rather weak. Instead of using an including radiometal complexes, to further improve inhibitor, synthetic caspase substrates are investigated, these characteristics. which may accumulate in an apoptotic cell by metabolic In summary, all these imaging procedures may be trapping, thereby enhancing the imaging signal. In one used to characterize the biological features of tumors of these studies, 10 radiolabeled peptides containing the and their metastases with respect to metabolism, DEVDG sequence, selective for downstream caspases apoptosis and microenvironment. The information such as caspase-3, were synthesized and evaluated for obtained with these techniques can be expected to their uptake kinetics using an apoptosis test system individualize treatment, and makes radioisotope-based (Bauer et al., 2005).Within this series of peptides, methods promising tools for tumor detection, therapy radioiodinated Tat49–57-yDEVDG-NH2 and Tat57– planning and therapy monitoring. 49-yDEVDG-NH2, both containing an additional HIV Tat sequence, were taken up by apoptotic cells to a significant higher extent as compared with controls. This enhanced uptake was interpreted as interaction of Conflict of interest the labeled peptide or fragment with activated caspases. Current efforts are focused on alternative radioisotopes, The authors declare no conflict of interest.

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