Positron emission tomography (PET) can provide noninvasive molecular, functional and metabolic information. Thus, it is playing an increasingly important role in the diagnosis and staging of tumors, image-guided therapy planning, and treatment monitoring. 2-¹⁸F-fluoro-2-deoxy-D-glucose (¹⁸F-FDG) is a commonly used tracer for PET imaging. Based on the increased rate of glucose transport and glycolysis, the uptake of ¹⁸F-FDG in tumors cells is greater than that in normal cells. ¹⁸F-FDG has provided valuable information about tumors diagnosing, staging, and prognosis after surgery and therapy, but it has some limitations. On the one hand, due to the high uptake of ¹⁸F-FDG in the normal brain, it is difficult to obtain images with adequate contrast compared to primary or metastatic brain tumors (Zhao et al., 2014). On the other hand, some tumors, such as neuroendocrine tumors, renal cell carcinoma, prostate cancer and hepatocellular carcinoma, show low or nonspecific uptake, which may lead to false negative or positive results (Powles et al., 2007; Rioja et al., 2010; Bouchelouche and Choyke, 2015). Additionally, ¹⁸F-FDG PET is ambiguous for differentiating tumor from inflammation (Rau et al., 2002; Tang et al., 2003).

Besides glucose, certain AAs also serve as increasing energy sources and anabolic precursors for tumors. Positron nuclide-labeled AA tracers can overcome limitations of ¹⁸F-FDG for tumors imaging, and give information about AA metabolism in tumor. The uptake of AA PET tracers in the normal brain is significantly less than that of ¹⁸F-FDG, but the uptake of them in tumor is high. Thus, images with adequate contrast can be obtained using AA PET tracers for primary and metastatic brain tumors. Also, some AA PET tracers have an advantage over ¹⁸F-FDG in the differentiation of tumor from inflammation (Rau et al., 2002; Tang et al., 2003; Stober et al., 2006). It was reported that O-(2-¹⁸F-fluoroethyl)-L-tyrosine (¹⁸F-FET) and (S-¹¹C-methyl)-L-methionine (¹¹C-MET) have a significantly higher uptake in tumor cells than that in inflammatory cells. This different appearance can be contributed to major AAs transporter system L (Stober et al., 2006). They can also differentiate recurrent brain tumors from pseudo-progression or radiation necrosis among patients after surgery and radiotherapy (Niyazi et al., 2012; Galldiks et al., 2015a,b). In addition, some AA PET tracers with relatively little renal excretion can accurately detect prostate cancer and show high specificity and sensitivity, superior to ¹⁸F-FDG (Toth et al., 2005; Jana and Blaufox, 2006). Last, ¹⁸F-FDG is a nonspecific substrate for neuroendocrine tumors, but a few AA PET tracers are substrates of the enzyme aromatic AA decarboxylase (AADC), which are specific for neuroendocrine tumors imaging, such as 3,4-dihydroxy-6-¹⁸F-fluoro-L-phenylalanine (¹⁸F-FDOPA) and 5-hydroxy-L-[β-¹¹C] tryptophan (¹¹C-HTP) (Jager et al., 2008; Oberg and Castellano, 2011). This review focuses on the fundamental concepts of AAs and the uptake mechanism of AAs, AA PET tracers and their clinical applications.

L-AAs, as essential small-molecule nutrient substances, are crucial for maintaining cell growth and nitrogen balance. Their biological functions are involved in metabolism, protein synthesis, cell signaling transduction, regulating gene expression. They are also precursors for the synthesis of hormones, neurotransmitter, and nitrogenous substances. L-AAs are commonly found in proteins and are either obtained from intracellular protein recycling or are transported into the cell from the extracellular surroundings (Stryer, 1995).

The transporters mediate AA transport across plasma membranes in mammals and are divided into several “systems.” The systems present various transporting mechanisms, including dependence on sodium and independence on sodium, tissue expression patterns, substrate specificity and sensitivity to pH or hormones (Utsunomiya-Tate et al., 1996; Castagna et al., 1997). Cells possess different transport systems in their plasma membranes, consisting of generally existed transport systems (such as systems A, ASC, L, y⁺ and X_AG−, X_C−), and tissue-specific transport systems (such as systems B⁰, and b^{0, +}) (Palacin et al., 1998). Here, we focus on describing their general features and transport mechanism of AAs, as shown in Table 1 and Figure 1.

System A is Na⁺-dependent transporter for serving mainly small aliphatic AAs, such as serine, alanine, and glutamine. It is a member of the solute carrier 38 (SLC38) gene family. Three subtypes of system A have been isolated: sodium-coupled neutral AA transporter 1 (SNAT1), 2, and 4. SNAT 3 and 5 belong to the system N (glutamine preferring) AA transport family, which is also a member of the SLC38 gene family (Broer, 2014). System A and system N are all directly concentrative and function essentially with a monodirectional efflux. System A transports AAs with the N-methyl group and N-methyl aminoisobutyric acid (MeAIB) is a specific inhibitor that can inhibit system A transport activity due to competitive saturation effects. Meanwhile, the activity of transporters is affected by many factors (Shotwell et al., 1983). The activity of system A is sensitive to pH alterations, highly down-regulated by acidic extracellular surroundings, and up-regulated by glucagon, insulin, and growth factors (Castagna et al., 1997).

The ASC system is Na⁺-dependent exchanger capable of mediating net influx or efflux, with substrates (L-alanine, L-serine, L-cysteine, and L-glutamine) and a member of solute the carrier family 1(SLC1) (Castagna et al., 1997). Two subtypes have been isolated: ASC-Type AA transporter 1 (ASCT1) and ASC-Type AA transporter 2 (ASCT2). ASCT2 utilizes an intracellular gradient of AAs, efflux of intracellular AAs in exchange for extracellular AAs. Glutamine is a key substrate of ASCT2 with important roles in tumor metabolism (Fuchs et al., 2007). ASCT2 is over-expressed in many human tumor cell lines including hepatocellular carcinoma, prostate, breast, glioma, and colon tumor cell lines (Li et al., 2003; Fuchs and Bode, 2005). L-γ-glutamyl-p-nitroanilide (GPNA) is used as a specific inhibitor of ASCT2 transporter activity (Schulte et al., 2015). The activity of system ASC is pH-insensitive within a range of pH 5.65–8.2 (Fuchs and Bode, 2005; Kanai et al., 2013).

The Na⁺-independent system L is the major route that takes up branched and aromatic AAs from the extracellular space, such as phenylalanine, isoleucine, tryptophan, valine, methionine and histidine (Castagna et al., 1997). Four subtypes of system L have been isolated: L-type AA transporters 1 (LAT1), LAT2, LAT3, and LAT4. LAT1 and LAT2 are members of the SLC7 gene family, while LAT3 and LAT4 are members of the SLC43 gene family. LAT1 and LAT2 possess “4F2 light chains” containing 12 putative membrane-spanning domains, which covalently bind a type-II membrane glycoprotein heavy chain (4F2hc) with disulfide bridges to produce a functional heterodimeric transporter. LAT3 and LAT4, without 4F2hc, facilitate the transport of AAs (Fuchs and Bode, 2005; Aiko et al., 2014). System L plays an important role for AAs crossing the placenta barrier and the blood-brain barrier (Christensen, 1990). 2-amino-2-norbornane-carboxylic acid (BCH) is a specific inhibitor for system L transporter activity (Palacin et al., 1998; Babu et al., 2003).

The cationic AA transporters include systems B^{0, +}, y⁺, and y⁺L, and the anionic AA transporters contain systems X_AG− and X_C−. Systems B, B⁰, B^{0, +} y⁺, and y⁺L are related Na⁺-dependent transporter systems. They mediate the absorption of branched-chain, aliphatic and aromatic AAs. Systems B and B⁰ are tissue-specific transport systems and present in renal proximal tubular and intestinal epithelial brush-border membranes. Both systems are more broadly specific for neutral AAs than systems A and ASC. System y⁺ transporters are members of the SLC7 gene family. Four subtypes, CAT-1, CAT-2 (A and B), CAT-3, and CAT-4, have been recognized from a subfamily of the SLC7 gene family. CAT-1 is a exchanger targeting unessential AAs, and the action of CAT-4 remains unknown (Hammermann et al., 2001). System y⁺ transports cationic AAs and some neutral AAs, such as lysine and arginine, resulting in electrogenic transport (Castagna et al., 1997; Palacin et al., 1998). System y⁺L transporters are members of the SLC7 gene family as well. Two subtypes (y⁺LAT1 and y⁺LAT2) have been identified, and they create heterodimers with the 4F2hc glycoprotein to be functional AA transporters, such as the LAT1 and LAT2 transporters from system L. System y⁺L serves large neutral and cationic AAs with an exchange mechanism. ATB^{0, +} belongs to the SLC6 gene family and serves cationic and neutral AAs in the presence of sodium and chloride. b^{0, +}AT belongs to the SLC7 gene family, which constitutes a functional heterodimer with the glycoprotein D2/rBAT/NBAT and serves cationic and neutral AAs via an exchange mechanism in the absence of sodium (Torrents et al., 1998; Hammermann et al., 2001).

System X_C− is Na⁺-independent and Cl⁻-dependent heterodimeric AA transporter (Baker et al., 2002; Lewerenz et al., 2012, 2013), an obligate, electroneutral, cysteine/glutamate antiporter, exchanges extracellular cystine for intracellular glutamate (Lo et al., 2008; Lewerenz et al., 2012). It is composed of a subunit xCT light chain and a subunit 4F2 heavy chain (4F2hc). xCT is a member of SLC7, member 11 (SLC7A11), and phosphorylation of xCT can modulate the activity of system X_C− (Baker et al., 2002; Lo et al., 2008; Lewerenz et al., 2012). It is not only a potential target for therapy but also a potential PET biomarker for imaging the system X_C− activity of cancer and other diseases (Lo et al., 2008; Reissner and Kalivas, 2010; Koglin et al., 2011).

System X_AG− is Na⁺-dependent and K⁺-dependent and transports acidic AAs, such as glutamate and aspartate (Dall'Asta et al., 1983; Pan et al., 1995b). Excitatory AA transporters EAAT1 (GLAST), EAAT2 (GLT-1), EAAT3 (EAAC1), and EAAT4 are members of the system X_AG− AA transport family (Howell et al., 2001) and are neuronal/epithelial high affinity glutamate transporters (Yin et al., 2014). They are encoded by the SLC1A1, SLC1A2, SLC1A3, SLC1A6, SLC1A7, respectively (Kanai et al., 2013; Bianchi et al., 2014).

The transporter systems mentioned above are the main targets for AA metabolism PET imaging of tumors (Jager et al., 2001). Tumor cells utilize more AAs compared with normal cells to satisfy their rapid proliferation and invasion demands. And studies indicated that the expression of AA transporters is higher in tumor cells than that in normal tissue, especially LAT1, ASCT2, xCT, and ATB^{0, +} and so on (Karunakaran et al., 2011; Toyoda et al., 2014; Schulte et al., 2015). Both ASCT2 and LAT1 are upregulated three-fold in the most of cancerous tissues. LAT1 has been proven to be associated with tumor growth (Kaira et al., 2013), for example ¹¹C-MET, ¹⁸F-FET, and ¹⁸F-FDOPA are the most widely used AA PET tracers for imaging brain tumors. System A and cationic or anionic AA transporters are over-expressed in dividing cells in certain human cancers (Bussolati et al., 1996). Many examples are showed in Table 2. Tumor cell accumulation of AA PET tracers mainly depends on the rate and mechanism of AAs transport. Based on the over-expression of AA transporters, the uptake of AA PET tracers in tumor cells is greater than that in normal cells (Mossine et al., 2016).

Most AA PET tracers are labeled with positron radionuclides ¹¹C and ¹⁸F. Theoretically, almost all AAs be labeled with ¹¹C, however, their short half-life (20 min, 100% of beta positron decay) is not suitable for delayed PET imaging. To overcome this shortcoming of ¹¹C and to facilitate the utility of AA PET tracers in hospitals without on-site cyclotron and labeling equipment, a series of ¹⁸F labeled AAs (half-life of 110 min, 97% of beta positron decay) were developed (Mossine et al., 2016). Based on that AAs have a common molecular formula [R-CH-(NH₂)-COOH], with a carboxylic acid group (-COOH), an amino group (-NH₂) linking to the alpha-carbon atom (-CH-), and branched-chain group (-R). Thus, ¹¹C and ¹⁸F labeled AAs are divided into [1-¹¹C] AAs ([1-¹¹C]AAs), alpha-C labeled AAs (alpha-C labeled AAs), labeled branched-chain AAs (branched-chain AAs), and N-substituted labeled AAs (N-substituted labeled AAs), which include natural and non-natural AAs.

Labeled natural AAs associated with structure-changed and structure-unchanged labeled AAs. Structure-unchanged labeled natural AAs, such as [1-¹¹C] AAs and ¹¹C-Met, do not chemically change the structure of AAs and can maintain the prototype structure and the fundamental pharmacodynamics and pharmacokinetics characteristics of AAs. So, they are mainly incorporated into protein synthesis, with minor AA transport. On the contrary, structure-changed labeled AAs (such as ¹⁸F-FET, (S-¹¹C-methyl)-L-cysteine) do chemically change the structure of AAs, which are slightly incorporated into protein synthesis. Like structure-changed labeled AAs, labeled non-natural AAs (such as ¹⁸F-FDOPA, ¹¹C-HTP) are mainly involved into AA transport. Most important ¹¹C- and ¹⁸F-labeled AA tracers are shown in Table 2.

[1-¹¹C]AAs have ¹¹C-labeled at the alpha-carboxylate (-COOH) position, [1-¹¹C]-labeled natural AAs such as L-[1-¹¹C]-leucine (¹¹C-Leu) (Veronese et al., 2012), L-[1-¹¹C]tyrosine (¹¹C-Tyr) (de Boer et al., 2003), L-[1-¹¹C]phenylalanine (¹¹C-Phe) (Lebarre et al., 1991) and L-[1-¹¹C]methionine (¹¹C-Met) (Ishiwata et al., 1988) are mainly incorporated into protein synthesis, and can be used to measure the rates of the protein synthesis. [1-¹¹C]-labeled non-natural AAs, such as carboxyl-¹¹C-1-α-aminoisobutyric acid (¹¹C-AIB), carboxyl-¹¹C-1-aminocyclopentanecarboxylic acid (¹¹C-ACPC), and carboxyl-¹¹C-1-aminocyclopentane carboxylic acid (¹¹C-ACBC), etc., are not incorporated into protein synthesis and have been used for imaging of tumor AA transport in several studies (Washburn et al., 1978; De Vis et al., 1987).

Labeled alpha-carbon AAs have radiolabeled at alpha-carbon (-CH-) position of AAs, which are rarely reported. α-[¹¹C-methyl]-L-tryptophan (¹¹C-AMT) and α-[¹¹C-methyl]-aminoisobutyric acid (¹¹CH₃-AIB) are typical examples that have been used for tumors imaging by measuring the rate of AA transport (Juhasz et al., 2011).

Labeled branched-chain AAs have radiolabeled at branched-chain group (-R) of AAs. Labeled branched-chain natural AAs with unchanged structure are rare, for example (S-[¹¹C]methyl)-L-methionine (¹¹C-MET). Most labeled branched-chain natural AAs are changed into different structure labeled AAs from natural AAs, such as ¹⁸F-FET, 2-¹⁸F-fluoro-L-tyrosine (2-FTYR), 6-¹⁸F-L-m-tyrosine (¹⁸F-FMT), O-(3-¹⁸F-fluoropropyl)-L-tyrosine (¹⁸F-FPT), 2-¹⁸F-L-phenylalanine, cis-¹⁸F-fluoroproline (cis-Fpro), (4S)-4-(3-¹⁸F-fluoropropyl)-L-glutamate (BAY 94-9392,¹⁸F-FSPG), (2S,4R)-4-¹⁸F-L-glutamate (BAY85-8050, 4F-GLU), L-(5-¹¹C)-glutamine, (2S,4R)-4-¹⁸F-L-glutamine (¹⁸F-(2S,4R)4F-GLN), (2S,4S)-4-(3-¹⁸F-fluoropropyl) glutamine (¹⁸F-FPGln), and (S-¹¹C-methyl)-L-cysteine (¹¹C-MCYS) (Deng et al., 2011; Huang et al., 2015). Labeled branched-chain non-natural AAs include labeled branched-chain D-AAs and labeled branched-chain L-non-natural AAs. The former includes D-¹¹C-fluoromethyltyrosine, D-¹⁸F-fluoromethyltyrosine (¹⁸F-D-FMT) (Burger et al., 2014) and (S-¹¹C-methyl)-D-cysteine (¹¹C-DMCYS) (Huang et al., 2015). The latter includes 3-¹⁸F-α-methyltyrosine (¹⁸F-FAMT), 1-amino-3-¹⁸F-fluorocyclobutane-1-carboxylic acid (¹⁸F-FACBC), 3-O-methyl-6-¹⁸F-L-3, 4-dihydroxyphenylalanine (¹⁸F-OMFD), (S)-2-amino-3-[1-(2-¹⁸F-fluoroethyl)-1H-[1,2,3]triazol-4-yl]propanoic acid (¹⁸F-AFETP), 3-¹⁸F-2-methyl-2-(methylamino)propanoic acid (¹⁸F-MeFAMP), 3,4-dihydroxy-6-¹⁸F-L-phenylalanine (¹⁸F-FDOPA), anti-1-amino-2-¹⁸F-fluorocyclopentane-1-carboxylic acid (anti-2-¹⁸F-FACPC), 5-¹⁸F-L-aminosuberic acid (¹⁸F-FASu), ¹¹C-HTP, L-[β-¹¹C]DOPA (¹¹C-DOPA), L-[β-¹¹C] dopamine, and ¹⁸F-fluoropropyl-L-tryptophan (¹⁸F-FPTP) (Jager et al., 2001; McConathy and Goodman, 2008; McConathy et al., 2012; He et al., 2013; Huang and McConathy, 2013b; Webster et al., 2014). Among these, ¹⁸F-FAMT, ¹⁸F-FET, ¹⁸F-D-FMT, 2-FTYR, ¹⁸F-FDOPA, ¹⁸F-FMT, ¹⁸F-Cis-FPro, ¹⁸F-OMFD, ¹⁸F-FACBC, ¹⁸F-FACPC, ¹¹C-HTP, ¹¹C-DOPA, BAY 94-9392, BAY85-8050 and ¹⁸F-(2S, 4R)4F-GLN have been used in clinical PET imaging of tumors. Most of labeled branched-chain non-natural AAs are involved in AA transport and a few are incorporated into protein synthesis. However, 2-FTYR and ¹⁸F-Cis-Fpro are involved in AA transport and protein synthesis (Jager et al., 2001; Laverman et al., 2002).

N-substituted labeled AAs have radiolabeled at -NH₂ group of AAs. α-[N-methyl-¹¹C]-methylaminoisobutyric acid (¹¹C-MeAIB) and α-(N-[1-¹¹C]acetyl)-aminoisobutyric acid (Prenant et al., 1996) are N-substituted labeled non-natural AAs targeting transport system A. ¹¹C-MeAIB has been used for clinical PET imaging of tumor (Sutinen et al., 2003). Although several N-substituted labeled natural AAs, such as p-¹⁸F-fluorohippurate (¹⁸F-PFH) as a glycine analog, have been reported, their transport mechanisms remain unknown (Awasthi et al., 2011). N-substituted labeled natural AAs targeting different AA transport systems, such as N-(2-¹⁸F-fluoropropionyl)-L-methionine (¹⁸F-FPMET), N-(2-¹⁸F-fluoropropionyl)-L-glutamic acid (¹⁸F-FPGLU), N-(2-¹¹C-methyl)-L-glutamic acid (¹¹C-MGLU), were first reported by our research group (Hu et al., 2013, 2014). ¹⁸F-FPGLU is a potential AA PET tracer for tumor imaging and can be used for clinical tumor imaging in the near future. Our studies showed that ¹⁸F-FPGLU is mainly transported via X_AG− and X_C− (shown in Figure 1) (Hu et al., 2014; Tang et al., 2015).

AA PET tracers were first used to measure the rate of protein synthesis in vivo (Vaalburg et al., 1992; Ishiwata et al., 1993; Paans et al., 1996). For example, ¹¹C-labeled natural AAs, such as L-leucine, L-methionine, L-phenylalanine and L-tyrosine, are used to measure the protein synthesis rate since they incorporate into proteins or wash out with decarboxylation and oxidation (Ishiwata et al., 1996; Langen et al., 2006). Nowadays, AA transports seem to be more important than protein synthesis for the imaging of tumor metabolism in vivo (Ploessl et al., 2012; Lewis et al., 2015). A wide range of ¹¹C and ¹⁸F AAs have been developed as PET tracers for clinical tumor imaging, as shown in Table 2 and Figure 2. The established AA tracers are used for imaging of brain tumors, neuroendocrine tumors, and prostate cancer, and other tumors.

AA PET tracers can overcome the shortcomings of ¹⁸F-FDG and provide more information for imaging tumors. Uptake mechanism of AA PET tracers involves protein synthesis or AA transport. For PET imaging, AA transport tracers appear more valuable than protein synthesis tracers in clinical applications. Targeting AA transporter system A, ASC, L and X_C−, have been used in the clinical imaging of the biological behaviors of various tumors. Transporter system L has been a major focus of tracer development for imaging tumors (such as ¹¹C-MET, ¹⁸F-FET, ¹⁸F-FDOPA) and has also led to several AA tracers that are effective for imaging neuroendocrine tumors (¹⁸F-FDOPA) and prostate cancer (¹⁸F-FACBC) (Huang and McConathy, 2013a). ¹⁸F-FSPG (BAY 94-9392), which is specific for system X_C− transporters (Koglin et al., 2011; Smolarz et al., 2013a), has been used for imaging patients with hepatocellular carcinoma (Baek et al., 2013), NSCLC (Smolarz et al., 2013a) and breast cancer (Chopra, 2004; Baek et al., 2012). Recently, new ¹⁸F-labeled branched-chain AAs have been developed that target cationic AA transporter and excitatory AA transporters X_AG−, which are potential targets of AA PET tracers for tumor imaging. O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine (¹⁸F-FMAET) is specific for cationic AA transporter (Chiotellis et al., 2014). BAY 85-8050, a glutamate derivative, is specific for transport system X_C− and systems XAG-, which is used to study healthy volunteers (Krasikova et al., 2011; Ploessl et al., 2012).

Besides branched-chain AAs, novel N-substituted labeled AAs and AA mimetics, have also been developed. ¹⁸F-FPGLU is N-methylsubstitutebeled amino glutamic acid as a potential AA tracer for PET imaging of transporter X_AG− and X_C− in tumor, and can be used for clinical tumor imaging in the near future. ¹⁸F-Phe-BF₃ (an exotic replacement of the carboxylate with -BF₃) is a new class of AA mimetics-boramino acid tracer for PET imaging of transporter LAT1 in tumor, with specific accumulation in U87MG xenografts and low uptake in normal brain and an inflammatory region (Liu et al., 2015). Also, synthesis of novel AAs with conformationally constrained side chains will lead to developing a series of new radiolabeled AA mimetics for imaging disease, with good prospect (Mollica et al., 2010, 2012; Stefanucci et al., 2011; Way et al., 2014).

Novel radiolabeling techniques are developing for radiosynthesis of AA PET tracers, resulting in routine high-dose production of AA tracers for clinical PET imaging. Recently, the no-carrier-added (NCA) enantioselective synthesis using a chiral phase-transfer catalyst has been used for automated synthesis of NCA ¹⁸F-FDOPA with the Curie Level (Libert et al., 2013), and simple and efficient two-step synthesis of ¹⁸F-FDOPA with short synthesis times can supply adequate radioactivity for clinical imaging (Tredwell et al., 2014). Thus, ¹⁸F-FDOPA is easily available and will become widely used AA PET tracer for the detection of brain tumors, neuroendocrine tumors, Parkinson's disease (PD), and mental illness (Darcourt et al., 2014; Eggers et al., 2014; Li et al., 2014). Simple and practical click reaction and ⁶⁸Ga labeling methods are also used for preparing new AA tracers for imaging tumors, which will further boost translational application of AA tracers in clinics.

GT is the corresponding author for summarize amino acids PET tracers and the future about amino acids PET. He also reviewed this paper. AS searched literature and wrote the manuscript. XL searched literature and drew the figure and table.