European Review for Medical and Pharmacological Sciences 2012; 16: 1925-1933 From molecular imaging in preclinical/clinical oncology to theranostic applications in targeted tumor therapy

C. ALBERTI

L.D. of Surgical Semeiotics, Parma, Italy

Abstract. – BACKGROUND: The impact of con- Key Words: stantly developing molecular sciences on the vari- Nuclear imaging, MRI, Optical imaging, SAIFS, ous imaging modalities (particularly nuclear, mag- xenografts, Tissue engineered models, Nanotechnology. netic resonance-based, optical techniques) has produced the beginning of a new complex science – the molecular imaging – that, through the ex- ploitation of specific molecule-probes instead of nonspecific conventional contrast materials, is Introduction aimed at the characterization of the tumor-related molecular abnormalities, to adopt innovative target- As result of an intersection of molecular biolo- ed therapeutic measures, even at the genetic level. gy, bioinformatics and in vivo imaging, the mole- OBJECTIVES: Aim for this review is to focus cular imaging allows the detection of specific on recent significant accomplishments of differ- molecular targets, from those pertaining to gene ent molecular imaging modalities moreover out- expression to that regarding protein products and lining the challenges of current theranostic de- 1,2 velopments. their specific functions . EMERGING KNOWLEDGES: The spatial reso- Among different diagnostic imaging modali- lution of almost all imaging techniques is more ties – computed tomography (CT), planar scintig- and more increasing, so that some experimental raphy, positron emission tomography (PET), sin- in vivo imaging modalities can allow an extreme- gle photon emission computed tomography ly detailed three-dimensional resolution. From (SPECT), magnetic resonance imaging (MRI), the constant developments of molecular biology it follows that, instead of relatively gross con- (US) and optical imaging (OI) – par- ventional diagnostic criteria on malignancies ticularly PET, SPECT, MRI, and OI are currently (anatomic location and size, surrounding tissue used for noninvasive molecular imaging, given involvement, distant spread), more specific mol- their capability to exactly identify molecular tar- ecular imaging parameters might be adopted – gets inside living organisms3. Such above imag- such as tumor kinetics, genetic alterations, ing modalities differ mainly in these following variety of involved growth factors – to reach, by innovative targeted drugs and biological agents, features: spatial/temporal resolution, sensitivity, therapeutic effects at the molecular level. In ani- tissue penetration depth, imaging energy expend- mal models – particularly in cancer xenografts – ed (Table I). the molecular imaging, through the resort to What’s more, the nanotechnologies, by the use SIAFS (small animal imaging facilities), allows in of nanoparticles as multi-functional/multi-stage vivo thorough investigations on the tumor devel- nanovectors of both molecular imaging nanotools opment-related mechanisms, furthermore im- and therapeutic nanoagents, allow them to avoid proving the research on pharmacokinetics and pharmacodynamics of newly developed drugs. various tissue-related immune reactions and cross CONCLUSIONS: Current applications of mole- cell-membrane barriers, so they might directly in- cular imaging are due to its capability of both in teract, at the same size regime, with intracell select vivo identifying tumor early molecular abnormal- targets as single proteins and single genes. ities and monitoring personalized therapies. Foreseeably the research advances will tremen- Positron Emission Tomography (PET) dously expand in the near future, particularly considering that simultaneous both imaging- PET allows the in vivo three-dimensional visu- and therapy implications of the theranostics can alization of molecular targets and related meta- improve, to the highest degree, the potential of bolic-functional processes, by the use of pharma- molecular imaging. ceuticals labelled with positron-emitting radionu-

Corresponding Author: Contardo Alberti, MD; e-mail: [email protected] 1925 C. Alberti

Table I. Different molecular imaging modalities: advantages and disadvantages.

Energy source Probe Imaging technology Favourable properties Drawbacks typology

• Nuclear (ionizing radiations) − PET High sensitiviy (≈ 10-13 mol/L) Low spatial resolution Positron-emitting Metabolic-dynamic informations (1÷2 mm) radionuclides − SPECT High sensitiviy (≈ 10-14 mol/L) Low spatial resolution γ-emitting Metabolic-dynamic informations (1÷2 mm) radionuclides • RM-based (magnetic High spatial resolution (50 µm) Low sensitivity Free MR, field+radiofrequencies) Metabolic-dynamic informations (enhanced by selected paramagnetic or atom hyperpolarization) superparamagnetic contrast materials, USPIO • Optical (nonionizing Capability of differentiating Low tissue penetration, Bioluminescent or radiations, in the field a variety of probes improved by resort fluorescent materials of light wave-lengths to NIR or near-infrared, NIR) clides, produced by particle accelerators (cy- PET oncological studies concern the glucose me- clotrons). Positrons (β+) annihilate through an tabolism by the use of glucose analog 2-de- impact with nearby microenvironmental elec- oxyglucose labelled with 18F (half-life = 109.8 trons (e-), so emitting two 0.511 MeV phothons min). This radiotracer, [18F]FDG, once inside the that, thrown 180 degrees apart in opposite direc- cells, is phosphorylated, like native glucose, by tions, are then detected, in coincidence manner, hexokinase-2, to [18F]FDG-6-P, that, because of by scintillation crystals (bismuth germanate, presence of a fuorine-atom instead of the hydrox- lutetium or gadolinium orthosilicate, barium flu- yl group in position 2, is unable to go on glycolyt- oride, etc), which are full-ring arranged and cou- ic cascade, hence gathering inside the cells and, pled with photomultipliers. The images, resulting therefore, enhancing the visualization of radio- from reconstruction algorithm-based post-pro- tracer-targeted site4. cessing, reproduce the spatial biodistribution of Unfortunately, [18F]FDG-PET suffers from lack positron-emitting tracers (Table I). of specificity in tumor studies because such radio- First and foremost advantage of PET is the tracer is also uptaken from inflammatory cells, so very high sensitivity, given the ability to in vivo that the use of specific tumor growth molecules tar- measure concentrations of a radiotracer, in a well- geting radiopharmaceuticals – such as [18F]-thymi- defined area, in the order of picomolars (10-11 ÷ dine, [11C]-thymidine, [11C]-methionine, [11C]- or 10-15 mol/L). The spatial resolution, instead, by it- [18F] choline – results more advantageous1. self low, may be improved by a suitable image re- Interestingly, PET molecular imaging is one of construction post-processing. Several positron leading tools to show proteomic changes – particu- emitters unfortunately have an extremely low larly the hypoxia-inducible factor-1 (HIF) expres- half-life (e.g., 20.38 min for 11C, 9.96 min for 13N, sion in cancer cells, thus allowing them to be resis- 2.04 min for 15O), therefore, requiring on-site cy- tant to radio-/chemiotherapy under hypoxic condi- clotron together with neighbouring radiopharma- tions – by using suitable radiopharmaceuticals such cy for the synthesis of radiopharmaceuticals. Oth- as [18F]-FETNIM (fluoroerythronitroimidazole), ers, indeed, are endowed with higher half-life, [18F]-FMISO (fluoromisonidazole), [18F]-FAZA such as 94mTc (52 min), 124I (4.2 days), 68Ga (67.6 (fluoroazomycinarabinoside), [62Cu]-ATSM (di- min), 64Cu (12.700 hours). acetil-methylthiosemicarbazone)2. As far as the tumor molecular imaging is con- As for PET with radiolabelled analogs of cerned, PET can properly target either some choline – metabolic precursor of membrane metabolic-functional cell growth-related peculiar- phospholipids – [11C]-choline-PET, though ex- ities, such as enhanced glucose metabolism and hibiting very high sensitivity and specificity, thymidine-kinase activity, or cell death-associated even higher than MR-spectroscopy, nevertheless, dynamic events or even various ligands/cell-re- has the drawback of too short half-life of 11Car- ceptors interactions. Indeed, the most commonly bon, its use remaining restricted to PET centres

1926 Molecular imaging and theranostics equipped with an on-site cyclotron5-7. Thankfully, netium (99mTc), 111 Indium (111In), 123 Iodine [11C]-choline-PET-like results may be achieved (123I), 133 Xenon (133Xe), 201 Thallium (201Tl) – by the use of [18F]-choline-PET, as 18Fluorine, emit γ-rays and, compared to positron emitters, given its longer half-live, proves to be available have the advantage of relatively longer half-life, to any PET unit, though unprovided with cy- thus not requiring on-site cyclotron for their pro- clotron. Anyhow radiolabelled, the choline, be- duction (Figure 1). sides its higher tumor-targeting specificity versus The SPECT uses a large number of γ-emitting [18F]FDG, is negligibly excreted in urine, thus radiopharmaceuticals, from small molecules to not impairing the image interpretation of either (imuno-SPECT), some of which wide- bladder or prostate tumors8-12. ly used in clinical practice. Such molecular imag- Other PET agents, such as radiolabelled ing technique allows to monitor different func- aminoacids or nucleotides, can play an important tional-metabolic processes, quantify various re- role in tumor molecular imaging, given their capa- ceptors density, define particular cell events such bility to show either protein or nucleic acid synthe- as apoptosis, moreover reaching, when integrated sis pathway8. Among the radiolabelled aminoacids, with CT (fusion-imaging), reliable morphologi- fusion-imaging of 11C-methionine-PET/MRI may cal findings, so overcoming its intrinsic limits as 3-dimensionally localize, with high specificity and for spatial resolution16. without inflammatory interferences, the increased In the field of oncology, because of high re- protein synthesis from tumors13,14. quirement of aminoacids from uncontrolled In the field of pure neuroendocrine tumors/ growth of cancer cells and, hence, a tumoral up- neuroendocrine differentiation of tumors, the relia- regulation of aminoacid-transporters expression, bility of PET-molecular imaging by use of afore- such cell-carriers represent an interesting target said radiofarmaceuticals is very poor because of for tumor molecular imaging, so that several ra- nonsignificant hypermetabolic/hyperproliferative diolabelled both natural and synthetic aminoacid- behaviour of such tumors while useful resulting, based probes may be used for SPECT-, besides instead, the resort to a specifically neuroendocrine PET-, tumor molecular imaging17. tissue-uptaken radiotracer [11C]-5-hydroxy- As for immuno-SPECT, murine monoclonal tryptophan – as precursor of 5-hydroxy- antibodies directed against specific cancer mole- triptamine, serotonin – that is able to supply spe- cular targets (e.g., regarding the prostate carcino- cific informations on the primary site of neuroen- ma, PSMA, prostate-specific membrane antigen, docrine tumors and their metastatic spread15. over-expressed by malignant prostate epithelial cells), radiolabelled with 111In-pentetide, are Single Photon Emission Computed specifically uptaken by primary tumor focus – Tomography (SPECT) moreover, also showing a possible local recur- As opposed to positron emitting agents, the ra- rence – together with by involved lymph nodes dionuclides used in SPECT – such as 99m Tech- and distant metastases8,18-20.

OH O OH O OH O \ // \ // \ // 13 13 13 ((( C ((( C ((( C || |

H –––– C –––– NH2 ALT C = O LDH H –––– C –––– OH ||(Main methabolic pathway | in ) CH3 CH3 CH3 Hyperpolarized Hyperpolarized Hyperpolarized alanine pyruvate lactate (High levels, peculiar to prostate cancer, significantly correlate with tumor grading)

ALT = Alanine transaminase LDH = Lactate dehydrogenase 13C = 13-carbon hyperpolarized

Figure 1. Hyperpolarized 13C pyruvate and its metabolic products, in 3D-MRSI (acc. to 22).

1927 C. Alberti

Magnetic Resonance Imaging that can in vivo target specific cell-molecules26,27. MRI, as a versatile molecular imaging modali- Paramagnetic lanthanide (Gd-based) complexes, ty, can show metabolic-functional processes to- endowed with anisotropic electronic configura- gether with producing morphological images with tion, can induce strong effects on MR frequency very high resolution. Unfortunately, MRI has a of the coupled spins dipolarity28. lower sensitivity (10-3 ÷10-5 mol/L) compared Furthermore, multimodal contrast materials al- with other imaging techniques, what’s due to non- low their simultaneous detection by more than significant difference of various atoms energy sta- one imaging modality, so that, by attaching an tus. Hence, the opportunity of improving MR sen- optical probe to a MR contrast material, it is pos- sitivity by either increasing the magnetic field or sible, after the whole body in vivo molecular RM hyperpolarizing selected atoms through dynamic imaging-research, validate RM findings by histo- nuclear polarization (DNP) or optical pumping logical studies50. technique, to reach a highly significant enhance- ment of MR signals from different atoms21,22. Optical Imaging (OI) Magnetic resonance spectroscopy (MRS) can non- Different approaches of optical imaging (OI), invasively provide usefull informations reflecting especially depending upon either biolumines- biochemical changes within different pathological cence or , besides applications in processes – particularly in tumors – to comple- cell biology (fluorescence microscopy), allow in ment MRI morphological findings. vivo surface molecular imaging studies, even re- Hyperpolarized 13C-based three-dimensional cently developed with the optical coherence to- molecular MR spectroscopic imaging (3D-MR- mography29 (Table I). SI) may be utilized to detect and characterize a The OI “beacons”, with high molecular specifici- variety of tumors – particularly the prostate car- ty, may suitably in vivo provide images of enzyme cinoma – by differentiating hyperpolarized 13C- activity together with metabolic pathways, and vi- pyruvate from its metabolic derivatives 13C-ala- sualize both tumor protein (e.g., cathepsin D, a nine and 13C-lactate, the last product resulting es- lysosomal glycoprotein)- and nucleic acid targets. pecially increased given the high tumoral LDH Fluorescence OI-related advantages are both the (lactate-dehydrogenase) activity21,22 (Figure 1). high sensitivity and the specificity as well as the in- Consequently, hyperpolarized 13C-bicarbonate- volvement of low-energy electromagnetic radia- based MRI can show a tumor average interstitial tions (nonionizing radiations) in the field of visible pH significantly lower compared to surrounding light spectrum and near-infrared (NIR). The disad- normal tissue23. vantage of OI is the lack of penetration dept, espe- Even though without the resort to previous ad- cially at visible wave-lenghts, depending on tissue- ministration of 13C-choline, the three-dimension- related light absorption and scattering. Because of al-chemical shift imaging-magnetic resonance considerably lower tissue absorption coefficient in spectroscopy (3D-CSI-MRS) allows the detec- the region of NIR (0.7 ÷ 3 µm), the most common- tion of malignancy-related both high levels of ly molecular OI studies are carried-out resorting to choline and low concentrations of citrate com- NIR-fluorescent probes. pared with those shown in normal tissues24. Fluorochromes or fluorophores, like cell/tissue To differentiate malignancies from fibrotic natural chromophores – such as β-carotene, ly- processes (e.g., hepatocarcinoma from cirrhosis), copene, anthocyanins, metal-complexes as hemo- the molecular MRI, by resorting to intravenously globin/hemocyanin/chlorophyl – are fluorescent administered type I-collagen-targeted probe EP- chemical compounds which absorb light energy of 3533, may noninvasively recognize, within the fi- a specific wavelength and re-emit that at a longer brous tissue, a significant accumulation of extra- wavelength with a lower energy. Besides the fluo- cellular matrix proteins, particularly of type I- roscein, an isothiocyanate, other fluorophores are, collagen-derived hydroxyproline25. among nonprotein compounds, rhodomine, cya- Recently, magnetic liposomes – phospholipid nine, coumarin, acridine-orange, while, among the vesicles encapsulating magnetic or paramagnetic protein-based ones, GFP (green fluorescent pro- ultrasmall particles such as ultrasmall superpara- tein), YFP (yellow fluorescent protein) and RFD magnetic iron oxides (USPIOS) – have been used (red fluorescent protein), all such types generally as MRI contrast materials whose surface may be bounded, as markers (dyes, tags, reporters, labels), advantageously provided, compared to free mag- to macromolecules (nucleic acids, peptides, anti- netic particles, with different functional groups bodies). Particularly, GFP has been proposed as

1928 Molecular imaging and theranostics tag not only for studies of cellular processes and Molecular Imaging to various drug pharmacodynamics/pharmacokynet- Monitor Gene Therapy ics but also for in vivo optical imaging30. More- Aim of gene therapy is to achieve permanent over, RDG (arginine, glycine-aspartate peptide), changes into gene-constitution through various covalently conjugated with an organic dye (e.g., mechanisms, among which corrective gene re- Cy 5.5) and bound to human serum albumin, al- pair, gene suppression or gene addition. More lows to in vivo show high tumor accumulation, and more taken into consideration also the anti- given the specific binding of RDG peptide with sense therapy – administration of short oligonu- αVβ3 integrin receptor protein, that is highly ex- cleotides to block gene expression – and the use 31 pressed in tumoral tissues . Just regarding it, of short inferfering RNAS (siRNAS) to target spe- RDG-Cy 5.5 dye complex-based molecular OI can cific RNA messengers (mRNAS) to arrest genetic reliably show, in animal models, the growth information pathway. course in cancer xenografts, together with their re- Gene oncotherapy includes both the im- sponse to therapy33. Also 2-deoxy-glucose, besides munotherapeutic strategies to deliver genes en- its use in FDG-PET, may be labelled, for in vivo coding for cytokines and the suicide gene thera- OI studies in cancer xenografts, with a NIR fluo- py, an antiproliferative-cytoreductive treatment rophore (e.g., IR-dye 800CW)34. modality based on the use of so-called suicide In large animals approaching the human body genes that are able to induce sensitivity of cells size, NIR fluorescent quantum dots (QDS), en- to nontoxic prodrugs. So, e.g., the herpes sim- dowed with semiconductive properties together plex virus1-derived thymidine kinase (HSV1- with peculiar bright emission, have been used, as TK) suicide gene can convert, by phosphorila- tumor targeting agents, to map sentinel lymph tion, nontoxic Ganciclovir (arabinosine-fureno- node32 but, because of their nano-size-dependent syl-uracil) into toxic Ganciclovir-triphosphate, insufficient renal elimination and, therefore, their thus reaching antiproliferative effects38,39. What’s potential nephrotoxicity – apart from novel den- more, instead of a necrotic tumor cell death, the dron-coated QDS, provided with suitable both apoptosis may be obtained through pro-apoptot- size and renal clearance – are clinically unadvis- ic gene transfer, e.g., by using dominant negative able31,33. Instead, for such purpose, other OI con- mutant of cyclin G- or p53 gene38. trast materials may be used, such as either hep- In vivo molecular imaging of gene expression tamethine-indocyanine-based-fluorophore or, still aims to detect successfully produced gene-depen- better, the fluorescent human serum albumin giv- dent specific proteins. The most commonly used en its high performance to penetrate lymphatics molecular imaging technique to monitor gene and reach sentinel lymph nodes where it is therapy is PET, by using, as a case in point, [18F]- specifically retained32. F-ganciclovir or penciclovir reporter probe, show- High sensitivity of bioluminescence imaging, ing viral thymidine kinase gene reporter expres- involving luciferase enzyme – that, in presence sion40,41. Furthermore, β-galactosidase genere- of ATP (adenosine triphosphate), magnesium and porter may be indirectly identified on the basis of oxygen, produces light emission – may be used, its action on galactosylated chelator substrates, in animal models, to monitor tumor onset, pro- whose cleavage-derived galactose residues are, in gression and response to various treatment strate- turn, detected, on the basis of relaxivity changes, gies35,36. Interestingly, in tumor animal models, a by paramagnetic gadolinium (Gd) probe-based transfected and adequately silenced luciferase MRI40. What’s more, bioluminescent molecular gene reporter, may be activated, so becoming OI, via noninvasive luciferase reporter gene, can, noninvasively detectable in bioluminescence in cancer animal models, show p53 activity molecular imaging as soon as caspase-3 enzyme changes under chemotherapy, so representing a is produced, particularly under pro-apoptotic useful tool to in vivo study, besides in cell culture, agent-mediated conditions (proapoptotic drugs; the effects of anticancer drugs37. TRAIL, TNF-α-related apoptosis-inducing lig- and), within apoptotic cells36. Molecular Imaging in Small Animal What’s more, effects of chemoteraphy on can- Models and Cancer Xenografts cer animal models (e.,g., cisplatin on lung cancer The need to carry out longitudinal research animal model) have been accurately studied via studies in small animal models reproducing hu- noninvasive luciferase reporter gene-related biolu- man diseases (genetic disorders, malignancies, minescent molecular OI focused on p53 activity37. etc) – by repeatedly studying the same animal to

1929 C. Alberti monitor the disease course and the response to po- laser excitation-induced increase in temperature tential new pharmaceuticals – has recently led to – in oncological surgery50,51. With full particulars, develop a variety of molecular imaging tools, that phtalocyanine-based chromophores, given their might be suitable for small animals (SAIF, small potential as high-affinity guanine-quadruplex animal imaging facilities), particularly micro-PET, (DNA belonging-guanine tetrads) ligands – that micro-SPECT, micro-MRI and micro (biolumis- in telomeric DNA inhibit telomerase activity – cence or fluorescence)-OI16. Each among such may represent promising anticancer drugs51. preclinical imaging modalities aims to optimize Moreover, photosensitive nanoparticles (gold- the research directed to identify metabolic-func- nanoshells; goldrods, AuNPS), as provided with tional processes, track drug-kinetics and -dynam- photothermal property of both strongly adsorbing ics, quantify receptor-protein expression, monitor e-m bands in NIR range, particularly NIR laser gene-therapy. Recently, in animal models, molecu- beams, and converting the adsorbed energy to in- lar imaging has been applied to study patient-de- tense heat, may be incorporated into a novel rived cancer xenografts, so in vivo providing in- mesoporous silica-made up material whose sur- formations on both different gene expression and face has been functionalized with an aptamer tar- molecular profiling, receptorial protein density, tu- geting DNA agent (or an anticancer aptamer mor growth, cell apoptosis, together with respons- AS1411). Upon application to NIR laser beam, 42-45 es to anticancer treatments . As for evidence re- the phototermal effect of AuNPS induces tout garding it, αVβ3-integrin-positive melanoma court rise in local temperature allowing the con- xenografts may be suitably identified and studied trolled release of the anticancer agent in cancer by RDG-labelled NIR fluorophore31,33. cells, so that integrate into one multifunctional Intriguingly, the molecular imaging research, platform both molecular imaging and photother- rather than on cancer xenografts in animal mod- maltherapy (burning of malignant cells) with, in els, could be accomplished on cancer xenografts addition, a drug anticancer therapy52,53. implanted on to three-dimensional tissue-engi- What’s more, multifunctional ethylene/acry- neered organ-like structures (liver, skin, etc), late co-polymer-based vesicles, endowed with supplied by a biological vascularized scaffold USPIO together with DOX-HCl anticancer drug, generated from decellularized animal organ (e.g., can exhibit both ultrasensitivity for MRI and tar- segment of porcine small bowel) with preserving geted anti-tumor drug effectiveness54. vascular network within extracellular matrix. Interestingly, in the field of theranostics, strong Bioreactors can sustain physiological tissue con- developments are more and more emerging from ditions by providing 3D-engineered organ like the concurrently combination, within a single plat- structure, together with implanted patient-derived form of molecular imaging specific PET probes tumor, with proper regulatory cell growth factors. with appropriate radionuclide-based personalized Thus, without resorting to experimental animals, radiometabolic effects-reaching therapeuticals55. the molecular imaging could provide, in such Among the multimodal theranostic applications, cancer grafts, usefull informations – like those in the molecular imaging-guided findings may play animal models – on gene expression profile and an important role, besides through showing the ma- different protein production46-49. lignancy-related molecular peculiarities, also by targeting and monitoring tumor therapy56-57. Theranostic Applications in Targeted Tumor Therapy Conclusions on Current Research and Quite recently, in the field of theranostics (fu- New Directions sion-term of ϑεραπεια, therapy, with γνωσις, Molecular imaging has emerged from joining dia-gnosis) – a new technology that concurrently molecular biology to different in vivo imaging combines molecular imaging and personalized technologies, so allowing to in vivo visualize therapeutic capability within a single platform – molecular processes and various cell events and multi-dye silica nanoparticles, composed of both obtain imaging acquisitions on gene expression fluorescent (heptamethine cyanine) and pho- and genetic alterations58-60. In addition, molecular tothermal (metallo-naphtalocyanine) agents with- imaging may provide, in preclinical studies, use- in a single nanoconstruct, have been proposed to ful data on pharmacokinetics and pharmacody- allow tumor NIR fluorescence visualization to- namics of newly developed drugs, particularly re- gether with providing a modality of simultaneous garding the innovative targeted therapies. On this photothermal ablation – tumor necrosis by NIR- subject, in the last two decades, different small

1930 Molecular imaging and theranostics

RASSI AGALLA OTONDO animal imaging facilities (SAIFS) have been de- 3) G R, L R, R A. Genomics, pro- veloped – such as micro-PET, -SPECT, -MRI, - teomics, MEMS and SAIF: which role for diagnos- OI – together with widening their applications to tic imaging? Radiol Med 2008; 113: 775-778. researh on cancer xenografts in animal models, 4) GIORGETTI A, VOLTERRANI D, MARIANI G. Clinical on- cological applications of PET using Fluorine-18- which could be favourably replaced – at least as Fluoro-2-deoxy-D-glucose. Radiol Med 2002; 103: for studies on gene expression and tumor protein 293-318. products – by those on cancer xenografts in tis- 5) JANA S, BLAUFOX MD. Nuclear medicine studies of sue engineered models16,47,48,61. the prostate, testes and bladder. Semin Nucl Med Intriguing developments of other molecular 2006; 36: 51-72. imaging techniques are today in progress, includ- 6) LANGSTEGER W, H EINISCH M, FOGELMAN I. The role of ing those based on radiofrequency- conditioned , 18F-dehydroxyphenilalanine, biological behaviour, wherein a selected biomol- 18F-choline and 18F-fluoride in bone imaging with enphasis on prostate and breast. Semin Nucl ecule (protein, peptide, nucleic acid), covalently Med 2006; 36: 73-92. linked to a metal nanocrystal-based antenna, be- 7) PELOSI E, ARENA V, S KANJETI A, PIRRO V, D OUROUKAS A, comes responsive to external specific radiofre- PUPI A, MANCINI M. Role of whole-body 18F- quency signals, to that to be in vivo precisely lo- choline PET/CT in disease detection in patients cated through molecular imaging and timely with biochemical relapse after radical treatment guided46,62. Among such techniques the MENS, for prostate cancer. Radiol Med 2008; 113: 895- micro-electronic mechanical system, allowing to 904. directly show cell activity also in living animals, 8) OYEN WJG, WITJES JA, CORSTENS FHM. Nuclear is more and more taken into consideration63. medicine techniques for diagnosis and therapy of prostate carcinoma. Eur Urol 2001; 40: 294-299. Recent advances in nanotechnology are open- 9) BRUSH JP. Positron emission tomography in urologi- ing new thorough knowledges on interactions be- cal malignancies. Curr Opin Urol 2001; 11: 175- tween different nanobiomaterials and biosystems, 179. so improving both molecular imaging and thera- 10) VEES H, BUCHEGGERT F, A LBRECHT S, KHAN H, HUSARIK peutic measures. D, ZAIDIT H, SOLOVIEV D, HANY TF, MIRABELL R. 18F- Moreover, molecular imaging, by offering the choline and/or 11C-acetate PET: detection of advantage of a very early diagnosis, allows mini- residual or progressive subclinical disease at very mally invasive surgical approaches, such as, expe- low PSA values (<1 ng/mL) after radical prostate- ctomy. BJU Internat 2007; 99: 1415-1420. cially, image-guided tumor ablation targeted endo- scopic procedures64. Indeed, the most valuable 11) PICCHIO M, LANDONI C, MESSA C, GIANOLLI L, MATAR- RESE M, DE COBELLI F, M ASCHIO AD, FAZIO F. Positive modalities to treat the malignancies lie in both ear- 11C-choline and negative 18F-FDG with PET in ly detecting- and, subsequently, quite resecting recurrence of prostate cancer. AJR 2002; 179: them. With this respect, NIR-fluorescent probes, 482-484. such as tumor-specific NIR emitting quantum dot- 12) RINNAB L, BLUMSTEIN NM, MOTTAGHY FM, HAUTMANN bioconjugates, when intravenously injected, can RE, KÜFER R, HOHL K, RESKE SN. 11-Choline intraoperatively produce a definite tumor-targeted PET/CT and transrectal ultrasonography for stag- ing localized prostate cancer. BJU Internat 2007; imaging, preliminarily to prompt tumor resection 99: 1421-1426. that can be performed via NIR guidance resorting 65 13) HAIN SF, MAISEY MN. Positron emission tomography to the Fluobeam-700 technique . for urological tumours. BJU Internat 2003; 92: Very interestingly, simultaneous imaging-and 159-164. therapy implications of the theranostics can im- 14) TOTH G, LENGYEL Z, BALKAY L, SALAH MA, TRON L, prove, to the highest level, the potential of mole- TOTH C. Detection of prostate cancer with 11C- cular imaging. methionine PET. J Urol 2005; 173: 66-69. 15) GIORGETTI A, RUFINI V, C ALCAGNI ML, BAUM R. Imag- ing of neuroendocrine tumors. Semin Nucl Med References 2006; 36: 228-247. 16) GRASSI R, CAVALIERE C, COZZOLINO S, MANSI L, CIRILLO 1) DE SAINT-HUBERT M, BAUWENS M, VERBRUGGEN A, S, TEDESCHI G, FRANCHI R, RUSSO P, C ORNACCHIA S, RO- MOTTAGHY FM. Apoptosis imaging to monitor can- TONDO A. Small animal imaging facilities: new per- cer therapy: the read to fast treatment evaluation? spectives for the radiologist. Radiol Med 2009; Curr Pharm Biotechnol 2012; 13: 571-583. 114: 152-167. 2) KURIHARA H, HONDA N, KONO Y, A RAY Y. Radiola- 17) KONG FL, YANG DJ. Amino-acid transporter-target- belled agents for PET imaging of tumor hypoxia. ed radiotracers for molecular imaging in oncology. Curr Med Chem 2012; 19: 3282-3289. Curr Med Chem 2012; 19: 3271-3281.

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