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CEST and PARACEST MRI Contrast Agents for Imaging Cancer Biomarkers
Vipul R. Sheth and Mark D. Pagel*,,
Chapter
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1. Introduction 2. Chemical Exchange Saturation Transfer 2.1. Physicochemical properties 2.2. LIPOCEST 2.3. Detection of CEST agents 3. Examples of Detecting Cancer Biomarkers 3.1. Cell receptor biomarkers 3.2. Enzyme biomarkers 3.3. Gene biomarkers 3.4. Metabolite biomarkers 3.5. pH as a biomarker 3.6. Temperature as a theranostic biomarker 4. Refinements for Routine in vivo CEST MRI 4.1. Quantification of concentration 4.2. Temporal resolution 4.3 An example References
* Corresponding author: Arizona Cancer Center, room 4949, 1515 N. Campbell Avenue, Tucson, AZ 85724-5024, USA. Email: mpagel@u.arizona.edu Department of Biomedical Engineering, Case Western Reserve University, Cleveland OH, USA. Department of Biomedical Engineering, University of Arizona, Tucson AZ, USA. Department of Chemistry and Biochemistry, University of Arizona, Tucson AZ, USA.
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1. Introduction
Magnetic Resonance Imaging (MRI) can provide a non-invasive assessment of soft tissues throughout the body. MRI is routinely used to localize and monitor the progression of solid tumors, especially for cancers of the brain, spinal cord, musculoskeletal system, breast, prostate, liver, pancreas, kidney, and reproductive organs.1 As an example of the utility of MRI for clinical cancer diagnoses, MRI scans are recommended for women with high risk factors for breast cancer to augment standard mammography screening.2,3 The fine spatial resolution of MRI can identify solid tumors as small as a few millimeters in diameter, which provides additional advantages over some other non-invasive imaging modalities. Although MRI has excellent sensitivity for identifying tissue anomalies, anatomical MR images suffer from relatively poor specificity in distinguishing cancerous tumors from non-cancer lesions, or between different cancer types. MRI methods that provide information about the molecular composition within a putative tumor may improve the specificity of cancer diagnoses. In addition, more advanced MRI methods are currently being developed for the emerging field of theranostics, which consists of diagnostic methods that can direct the choice of therapy.4 MRI theranostics may provide information to tailor a therapy to an individual patient, in order to provide personalized medicine for each patient. For example, MRI methods that measure molecular composition, such as the pH of the tumor tissue, may predict the type of therapy that will be most effective for an individual patient before the therapy is applied. MRI methods that track the delivery of chemotherapeutics, especially new types of drug nanocarriers, may monitor the pharmacokinetics of drug delivery to the tumor in order to select the best nanocarrier for an individual patient. MRI methods that measure molecular function, such as tumor enzyme activity, may evaluate an early response to tumor chemotherapy as soon as 24 hours after initiating therapy. These MRI methods contribute to the paradigm of molecular imaging that augments anatomical imaging of cancer. Many MR molecular imaging methods require the use of an exogenous chemical agent that can change MR image contrast to reflect the molecular composition of the tumor. Contrast agents are used in roughly 1/3 of all clinical MRI exams, and are particularly useful for identifying tumors that show enhanced uptake of contrast agents relative to surrounding tissues. All current clinically approved MRI contrast agents change the T1 or T2* relaxation rate of water in tissues, and MRI methods can easily detect different MRI signals from water
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molecules that have different relaxation rates.5 Unfortunately, two or more relaxivity-based MRI contrast agents simply cause an additive change in the relaxation rate of water, so that each of these MRI contrast agents cannot be selectively detected in the same tissue at the same time. Limiting each MRI study to the use of just one contrast agent compromises the interpretation of the imaging results, and therefore has limited MRI studies of cancer to qualitative or semi-quantitative interpretations. A new type of MRI contrast agent has been developed that can be detected through the mechanism of Chemical Exchange Saturation Transfer (CEST).6 The incorporation of a PARAmagnetic lanthanide ion into a CEST agent (a.k.a. a PARACEST agent) can improve the CEST effect in ways that may be exploited for cancer imaging. This chapter describes the CEST mechanism and MRI methods for detecting (PARA)CEST agents, with an emphasis on advantages and disadvantages for molecular imaging and theranostics. This chapter also includes a survey of CEST and PARACEST agents that have been developed to detect cancer biomarkers, and a discussion of current challenges that must be overcome to translate CEST MRI for clinical cancer diagnostics and theranostics.
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the saturation pulse is close to the water frequency. The CEST effect can be expressed as: Ms /Mo = 1/(1 + T1Wsat * k1) [1]
where k1 is the pseudo first-order exchange rate constant and T1Wsat is the T1 relaxation time constant of water protons in the sample when the exchangeable proton frequency is saturated.11,12 For simple two-site exchange, k1 = kCA * (nA * [Agent] / nH O * [H2O])
2
[2]
where kCA is the rate constant of the exchange site, nA and nH O are the number of 2 exchangeable protons on the contrast agent and water molecule, respectively, and [Agent] and [H2O] are the local concentrations of the CEST agent and free water, respectively.13 The dependence of the CEST effect on these physicochemical properties is shown by combining Eqs. [1] and [2], Ms /Mo = 1/(1 + T1Wsat * kCA * (nA * [Agent] / nH O * [H2O]))
2
[3]
The CEST effect increases with an increasing chemical exchange rate, until exchange becomes so fast that the two exchanging sites become indistinguishable to the MRI scanner. The fast exchange limit is defined as: kCA < CA [4]
where CA is the chemical shift difference between the two exchanging sites.6,11 A larger chemical shift difference allows for a faster maximum exchange rate and thus can generate a greater CEST effect. A larger chemical shift difference also reduces the direct saturation of water. PARAmagnetic CEST (PARACEST) agents incorporate lanthanide ions that shift the chemical shift of the exchangeable protons of the agent to MR frequencies that are very far from the bulk water proton pool. These PARACEST agents can generate a detectable CEST effect from protons of functional groups that are near the lanthanide ion, such as hydroxyls,14,15 amines,1618 and amides,11,16,19,20 and the protons of the metal-bound water molecule,6,12,21 which may have chemical exchange rates that are much greater than the exchange rates of diamagnetic CEST agents (Fig. 1). The importance of the proton exchange rate is central to the difference between the CEST agents and traditional relaxivity-based T1, T2, or T2* MRI contrast agents. Because unique chemical groups (in this case, we define the bound water to be part of the agent) on the contrast agent cause the CEST effect, there is
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Fig. 1. PARACEST agents, representations of PARACEST, and detection of enzyme activity. (a) The proposed mechanism of uPA cleavage of the agent shows that an amide proton (red) is converted to an amine (blue) after uPA cleaves the Z-GGR peptide ligand from the agent. In addition to the amide and amine, the water molecule that is transiently bound to the lanthanide ion can also generate a CEST effect, which demonstates that a variety of chemical designs can be used to create PARACEST agents. (b) A CEST spectrum, a.k.a. a Z-spectrum, shows the water MR signal amplitude when selective saturation is applied at each MR frequency throughout a frequency range. The CEST spectrum showed a CEST effect from the amide at -52 ppm before uPA was added (red). The disappearance of this CEST effect after uPA was added (blue) was used to detect uPA. An enzyme-unresponsive agent, Yb-DOTA-Gly, shows a CEST effect at 16 ppm before and after uPA was added, which served as an internal control. (c) A MTRasy spectrum, a.k.a. a Magnetization Transfer Ratio asymmetry spectrum, shows the difference between the MR signal amplitudes with selective saturation applied at positive and negative MR frequencies that are symmetrical about 0 ppm. The MTRasy spectrum removes the symmetrical features of the CEST spectrum, such as the direct saturation of water in order to better visualize the CEST effect that is an asymmetric feature of the CEST spectrum. The MTRasy spectrum shows a disappearance of the CEST effect at 52 ppm and no change in the CEST effect at 16 ppm after adding uPA to a solution of the contrast agent. (Reproduced with permission from Elsevier Publishers, from Yoo et al. Ref. 47).
great flexibility and creativity in designing CEST agents that can detect cancer biomarkers. Many environmental conditions can affect the chemical exchange rate for CEST agents, including temperature22 and pH.13,19 In the case of PARACEST agents, the chelation of paramagnetic ions provides a host of other chemical parameters that impact the exchange rate including coordination geometry, ligand side chain geometry and electronegativity, the type of lanthanide ion, and exchangeable chemical group.6 Thus, an environmental or molecular biomarker that changes the
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chemical exchange rate can potentially be detected by the CEST contrast. For comparison, the MR relaxation caused by a relaxivity-based MRI contrast agent is caused by the direct interaction between the surrounding water and the metal ion and the chemical groups on the contrast agent only indirectly affect this relaxation mechanism, which limits the chemical designs and sensitivity of the response to biomarkers.23 CEST also differs from relaxivity-based contrast agents in that the CEST contrast from each CEST agent can be turned on and off at will. CEST agents are essentially invisible in the absence of saturation (assuming that their T1 and T2 relativities are negligible, which is usually a good assumption). Because CEST agents have unique frequencies for their exchanging groups, multiple CEST agents can be detected in a single scan session, unlike relaxitivity-based contrast agents. This is an important advantage, because one responsive CEST agent and one unresponsive control agent can be co-injected. The responsive agent changes image contrast in response to interacting with the biomarker, while the control agent accounts for all other effects that may cause an agent to change image contrast, such as dynamic changes in the concentration of the agent during an in vivo tissue study. This advantage is important for quantitative measurements of molecular information. CEST agents have some limitations. Despite amplification by exchange, small-molecule CEST agents are typically detected at 120 mM concentrations, which is relatively poor compared to the 1100 M sensitivity threshold of relaxivity-based contrast agents. B0 and B1 inhomogeneity in the magnetic field of the MRI scanner can create artifacts or interfere with the detection of CEST agents.24 PARACEST agents are less sensitive to B0 inhomogeneity because of their high chemical shift difference from the bulk water.6 For in vivo applications, CEST agents must compete with the underlying Magnetization Transfer (MT) effect from exogenous proteins. The B0 inhomogeneity and MT effects can be separated from the CEST effects by analyzing the CEST spectrum (Fig. 1b).2527
2.2. LIPOCEST
As shown by Eqn. 2, the number of exchanging protons per PARACEST agent can be increased in order to improve the CEST effect. A water molecule has a very high density of exchangeable protons, and therefore chemical designs that associate many water molecules with paramagnetic lanthanide ions have the potential to create the most sensitive PARACEST agents. Liposomes have been used to encapsulate lanthanide chelates with water molecules, and the lipid bilayer of the liposome sufficiently slows the exchange of intra-liposomal water with
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Fig. 2. LIPOCEST MRI. Two LIPOCEST agents, one spherical and one osmotically shrunken, were co-injected in a bovine muscle. The different resonance frequencies, 3 and 18 ppm, of the intraliposomal water protons in the two agents allowed the MRI co-visualization of the two CEST agents in the same image voxels. (Reproduced with permission of John Wiley & Sons, Inc., from Terreno et al. Ref. 31).
extra-liposomal water to generate a CEST effect.28 Encapsulating the water with a high concentration of lanthanide chelates causes the MR frequency of the intraliposomal water to shift from the MR frequency of the extra-liposomal water, due to the pseudocontact shift between the water molecules and lanthanide ions. Liposomes can be made to be non-spherical through osmotic shrinking, which increases the shift of the MR frequency of the intrasomal water through the bulk magnetic susceptibility effect.29,30 By optimizing the size, shape, and membrane composition, LIPOsomal CEST (LIPOCEST), agents have been created that can be detected at concentrations in the pM concentration range. Two LIPOCEST agents each been selectively detected in the same ex vivo tissue and at the same time, which demonstrates one of the primary advantages of CEST MRI (Fig. 2).31
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exchanging protons. The total saturation time usually varies between 2.510 seconds to ensure full steady-state saturation during the MRI scans. The CEST effect can be assessed by incrementally varying the offset frequency of irradiation, and plotting the signal of the bulk water versus saturation frequency to create a CEST spectrum (also known as a Z-spectrum) (Fig. 1b). The CEST spectrum is typically centered about the water frequency (conventionally defined to be at 0 ppm for MRI studies), and therefore the effect of directly saturating the water is symmetrical in the CEST spectrum, while the CEST effect is asymmetrical in this spectrum. A Magnetization Transfer Ratio asymmetry (MTRasy) spectrum compares the water signal with saturations applied at positive and negative frequencies, which removes the symmetrical features of the CEST spectrum (i.e., direct saturation of water) in order to better visualize the asymmetric features (i.e., the CEST effect) (Fig. 1c). For clinical applications, a continuous wave RF pulse is rarely used, because of hardware limitations and concerns for depositing excessive energy into human tissues (a.k.a. the Specific Activity Ratio or SAR). Instead, short repeated pulses are used, which typically have pulse shapes that reduce the SAR. CEST MRI with Gaussian shaped pulses has been shown to achieve 95% of the CW-CEST MRI contrast for similar RF power levels.32 Additional studies are warranted to ensure that clinical translation of CEST MRI is conducted within clinical safety limits.
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Fig. 3. OPARACHEE images of the kidney. Images are taken right before (a) and after injection of a PARACEST MRI contrast agent at 2 min 40 sec (b); 5 min 20 sec (c); 10 min 40 sec (d); 16 min (e); and 18 min 40 sec (f). The upper row corresponds to the 20 mM bolus concentration of the agent and the bottom to the 2 mM bolus. The maxb imum intensity decrease is observed around 3 min (b). As the agent clears through the kidney, the intensity starts to return to the levels seen prior to the agent injection (d-f). (Reproduced with permission of John Wiley & Sons, Inc., from Vinogradov et al. Ref. 34).
presence of a CEST agent reduced the water MRI signal more than 7%, and a comparison of OPARACHEE MR images before and after injecting a CEST agent was used to detect the agent in the kidney of a live mouse (Fig. 3).34 The OARACHEE method is more sensitive than the off-resonance CEST detection method because the chemical exchange rate can exceed the fast exchange limit shown in Eqn. 4. Similar to traditional CEST, large chemical shifts of PARACEST agents should show a greater effect with OPARACHEE. In experiments to date, 12 M of a PARACEST agent was detected with OPARACHEE, and as little as 30100 M of a PARACEST agent may be necessary for in vivo studies. However, this method is also sensitive to the T2 relaxation time of the bulk water, so that a pre-contrast image should be obtained to correct for intrinsic T2 relaxation effects. Also, unlike traditional CEST, a short T1 relaxation time will cause a greater decrease in Mz since rapid T1 relaxation will cause more magnetization to leave the WALTZ trajectory. Thus PARACEST agents which have very effective relaxation properties like Dy3+ and Tb3+ can be used with this sequence.33 One major disadvantage of this technique is that one can no longer detect multiple CEST agents in a single scan, because OPARACHEE applies RF pulses only at the chemical shift of water and frequencyselective saturation is no longer used.
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relative to their monomer concentrations.4043 This provides the potential to develop nanosized PARACEST agents with a lower minimum detection sensitivity threshold compared to relaxivity-based contrast agents, which may be able to detect cancer cell receptors at physiologically relevant concentrations. Some of these agents have already reached the concentration range for detecting cancer cell receptors. These PARACEST agents with outstanding detection sensitivities have not yet been employed to detect cell receptors, but this biomedical application may soon be realized once CEST MRI methods become routine.
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nature of this chemical design provides the capability of positioning the trigger far from the chemical group that generates the CEST effect. This may provide molecular designs of PARACEST agents that can more efficiently interact with the enzyme, and may also simplify the chemical synthesis of these agents.
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Fig. 4. CEST imaging of a reporter polypeptide. (a) Anatomical MR image of rat glioma cells transfected with a lysine-rich protein (LRP) or control glioma cells without LRP. (b) CEST signal intensitydifference map overlaid on the anatomical image distinguishes LRP-expressing and control xenografts; (c) signal-intensity differences (mean s.d.; six mice, each containing two xenografts; *, P = 0.03, two-tailed, unpaired t-test); to compare different mice, signal-intensity changes were normalized to make signal-intensity change of normal brain equal to zero. The proper adjustment of field homogeneity could only be done inside the brain, leading to some artifacts at brain edges. (d) RTPCR of xenografts from rat brains, showing expression of LRP only in the LRP xenograft. (e) Anatomical photo of the same tissue during postmortem analysis. (f) Eosin-hematoxylin stain of a frozen section off the tissue corresponding to LRP tumor in E. (g) Magnification of F shows a uniform tumor mass. Scale bars: 2 mm for A, B, and E, 500 M for F and 100 M for G. (Reproduced with permission of Nature Publishing Group from Gilad et al. Ref. 50).
bind to lactic acid, which changes the chemical exchange rate and the MR frequency of the agent.51 Lactic acid may be a useful cancer biomarker, because tumor cells often rely on glycolysis especially under hypoxic conditions, which generates high lactic acid concentrations in the extracellular tumor environment.52
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3.4.1. Glucose
The increased glycolytic metabolism in anarobic and aerobic tumor microenvironements causes a greater consumption of glucose than surrounding normal tissues.52,53 Biomedical imaging of glucose content has been valuable for diagnosing tumor locations and evaluating the effects of anti-cancer therapies. MR spectroscopy methods have been developed to measure the 1H, 31P, or hyperpolarized 13C MR signals of glucose, but these methods have been difficult to implement on clinical MR scanners. The 1H-MR spectrum of glucose overlaps with the spectrum of many other sugars, obfuscating spectral interpretation. MR spectroscopy of hyperpolarized 13C requires expensive, specialized equipment, and 31P-MR spectroscopy requires specialized detection coils that are not usually available in clinical settings. Therefore, other MRI methods for imaging glucose content may improve cancer detection. Boronic acids are known to bind selectively and reversibly to sugars. A family of PARACEST agents has been developed with phenylboronate ligands that exhibit a slower chemical exchange rate of bound water upon binding to glucose.5456 The resulting change in the CEST effect has been used to measure relative glucose concentrations in perfused mouse livers (Fig. 5). This family of PARACEST agents can bind to sugars other than glucose. Although the specificity for detecting glucose relative to other sugars can be refined by incorporating certain ligands and lanthanide ions into the PARACEST agent, it is unlikely that a PARACEST agent will achieve absolute detection specificity for glucose. The detection of total sugar content may still be a helpful biomarker for evaluating the effects of anti-cancer therapies, because the total sugar content is dominated by glucose content in tumors.55
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Fig. 5. Detection of glucose with PARACEST MRI. (a) CEST spectra of fresh effluent from a perfused fed-mouse liver and a 24-hr fasted mouse liver showing a glucose-induced CEST peak at 42 ppm. Both perfusates contained the PARACEST agent, 10 mM EuDOTAM-2M-2PB, pH = 7.4. (b, c) CEST images of a fed mouse liver (bottom) and a 24-hr fasted mouse liver (top) perfused with the same concentration of the PARACEST agent in the presence (fed liver) and absence (fasted liver) of 10 mM glucose. The control image acquired with selective saturation at 42 ppm (b) showed no contrast between the two livers while the image acquired with selective saturation at 42 ppm (c) showed image darkening of fed liver versus the fasted mouse liver. (d ) The CEST parametric map generated from images (b) and (c) showed the glucose-induced CEST contrast between the fed and fasted mouse livers. (Reproduced with permission of John Wiley & Sons, Inc., from Ren et al. Ref. 56).
exploited to create a PARACEST agent that reacts with an oxidative byproduct of nitric oxide, which causes an irreversible change in the covalent chemical structure of the PARACEST agent and alters the chemical exchange rates of the agent (Fig. 6).16 Similar irreversible, responsive contrast agents may provide similar advantages for detecting metabolite biomarkers of cancer with other molecular imaging modalities.
3.5. pH as a biomarker
Solid tumors are often more acidic than normal tissues due to increased glycolysis that produces lactic acid.52,53 Emerging evidence suggests that an acidic tumor microenvironment conditions tumor cells to aggressively extravasate into normal tissues, which promotes tumor growth into adjacent tissues and potentiates metastasis to distant tissues.57 In addition, an acidic tumor environment can
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Fig. 6. Detection of nitric oxide with PARACEST MRI. (a) MR images of a PARACEST MRI contrast agent, Yb(DO3A-oAA), before and after adding nitric oxide with selective saturation at 11 ppm. The PARACEST map is generated by subtracting these images from an image of PBS without the contrast agent. Each PARACEST map was independently scaled to demonstrate that only susceptibility artifacts are present in the PARACEST maps after reaction. (b) The PARACEST map of the same contrast agent before and after reaction with nitric oxide with selective saturation at +8 ppm. The results shown in panels a and b demonstrate that a decrease in CEST contrast from Yb(DO3A-oAA) indicates detection of nitric oxide. (c) The PARACEST map of 10 mM Tm-(DOTAM-Gly) before and after applying the same reaction conditions with selective saturation at 51 ppm. This result indicates that this unresponsive agent can be used as an internal control. (Reproduced with permission of John Wiley & Sons, Inc., from Terreno et al. Ref. 16).
cause many anti-cancer chemotherapies to become positively charged, which inhibits their transport into tumor cells and reduces their therapeutic effect.58,59 More recently, pH-altering therapies have been shown to alkalinize the tumor tissue and prevent metastasis.60 Therefore, measurements of the extracellular pH of tumor tissues may be used to diagnose tumor grades, predict the effect of pHdependent therapies before the therapies are applied, and evaluate the early response of pH-altering therapies in cancer patients. The first biological application of CEST MRI was for the measurement of 13 pH. The chemical exchange rate between water and the amine, amide, or hydroxyl groups on CEST agents is either acid- or base-catalyzed, and therefore is sensitive to pH. CEST can be used to measure the chemical exchange rate in order to determine pH. However, as shown in Eqn. 3, the concentrations of the agent and water in the tissue must be known, and the T1 relaxation rate of the tissue with the agent and in the presence of selective saturation must also be known. A clever alternative exploits the ratio of two CEST effects from the same agent that have different pH-dependent chemical exchange rates, but necessarily have the same concentration, interact with the same water concentration in tissue, and experience the same T1 relaxation rate.13 This ratiometric approach can accurately
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measure pH independent of the effects of concentration and T1Wsat. By using Eqn. 4, the ratio of two CEST effects from the same agent can be described as: [(M01-MS1)/MS1]/[(M02-MS2)/MS2] = kCA1*n1/kCA2*n2. where kCA = k0 + ka*10-pH +kb*(10-(pKw-pH)) [6] and pKw = 15.4 at 37 C 61,62 As an example, a comparison of the CEST effects of 5-hydroxytryptophan and 2-imidazolidinethione can be used to accurately measure pH throughout the physiological pH range.13 A similar comparison of the CEST effects of two PARACEST agents has also been used to measure pH, which has improved selective saturation of each agent due to greater chemical shift differences of these paramagnetic agents relative to diamagnetic agents.13,19 In both examples, the concentration of both agents must be equivalent to measure pH. To overcome this potential problem, CEST and PARACEST agents have been created that generate two CEST effects from a single agent, so that the concentrations of the chemical groups that generate each CEST effect are inherently equivalent (Fig. 7).13,63,64 The amides endogenously located in the body can also be used to generate a pH-sensitive CEST effect without the need for an exogenous agent. The aggregate of the amide groups from proteins in most tissues showed a CEST effect at 3.5 ppm that was used detect changes in pH caused by ischemia in a rat brain. Also known as the Attached Proton Transfer (APT) test, this method is best used to [5]
Fig. 7. The parametric pH map of a mouse tumor model. A solution of 60 mM of Yb-(DO3A-oAA) in 50 L volume was directly injected to the center of tumor. MRI scans were performed before injection and at 2 minutes after the injection. Only pixels that showed at least a 2.0% PARACEST effect from saturation at +8 ppm and 11 ppm are shown in this pH map, because a 2.0% change in image contrast relative to image noise equated to a 95% probability that the contrast was real. This threshold was only reached by pixels that represented the tumor and phantom with the agent, which had a change in contrast from 2.07.2%. The parametric pH map is overlayed on an anatomical MR image. The result showed the acidic environment of the tumor region. The phantom showed a pH gradient, which was caused by a temperature gradient across the phantom. (Reproduced with permission from Decker, from Liu et al. Ref. 64.)
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measure relative pH changes in the same tissue, because this CEST effect is also dependent on the proteinaceous content of the tissue (i.e. the concentration of the endogenous proteins that act as CEST agents). Yet absolute pH measurements may be made with the APT test if the pH is also calibrated with phosphate MR spectroscopy or MR spectroscopic imaging.65
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been performed with in vivo tissues, which have demonstrated major hurdles that must be overcome before in vivo CEST MRI can be routinely employed for cancer imaging studies.42 Recent studies of ex vivo tissues and biochemical samples that model the extracellular tissue environment have further substantiated the importance of these issues.25,30
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temporal resolution, using single-echo, multi-echo, and steady-state free precession methods.39,67 The method that generates the best CEST sensitivity depends on the T1 relaxation time of the tissue with the agent. Under the best conditions, CEST MR images can be acquired within a few seconds. This provides the practical opportunity to acquire a series of MR images with a range of selective saturation frequencies, to generate a CEST spectrum (Fig. 1b) at each pixel location within the spatial image. MR CEST spectroscopic imaging can detect the CEST effects of two or more contrast agents while also accounting for many artifacts that can affect CEST detection. The further development of fast CEST MR imaging methods has strong potential to accelerate the translation of (PARA)CEST MRI for detecting clinical cancer biomarkers.
4.3 An example
Considerations for sensitivity (Section 3.1), quantification (Section 4.1) and temporal resolution (Section 4.2) were critical for the recent development of a method that detected two PARACEST agents within the same tumor tissue in a mouse model of breast cancer.39 The dendritic PARACEST agents (Eu-DOTAGly-pBnNCS) 41-G5PAMAM and (Yb-DOTA-Gly-pBnNCS) 6-G2PAMAM were injected i.v. into a mouse model of MCF-7 mammary carcinoma. Because the tumor tissue has a relatively long T1 relaxation time of approximately 2.5 seconds, a multiple-echo CEST-RARE MRI acquisition scheme was used to detect each agent within the tumor (Fig. 8a). The decrease in MR signal of the tumor after injection relative to the average MR signal before injection was used to measure the temporal change in the CEST effect for each agent (Fig. 8b). The concentration ratio correctly showed that the lower tissue permeability of the larger dendrimer causes a fraction of (Eu-DOTA-GlypBnNCS) 41-G5PAMAM to accumulate in the tumor tissue relative to (Yb-DOTA-Gly-pBnNCS)6-G2PAMAM, and the lower elimination rate of the larger dendrimer caused this fraction to increase over time. This study demonstrated that two nanocarriers that may be further developed to deliver anti-cancer chemotherapies to tumor tissues could be monitored within the tumor tissue, in order to select the best nanocarrier for each individual tumor. With similar considerations for quantification, sensitivity, and temporal resolution, other examples of two CEST MRI contrast agents may be developed for preclinical molecular imaging studies and translation of quantitative molecular imaging to the clinic.
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Fig. 8. Detection of two PARACEST agents within the same tumor. A large Generation-5 PAMAM dendrimer labeled with a Eu(III) chelate (EuG5) and a relatively smaller Generation-2 PAMAM dendrimer labeled with a Yb(III) chelate (YbG2) can generate selectively detectable CEST effects at +50 ppm and -16 ppm, respectively. (a) Both agents were simultaneously injected i.v. into a xenograft flank mouse model of MCF-7 mammary carcinoma. Axial MR images were acquired by prepending a 20 T saturation period for 2.25 s before a RARE-16 MR signal acquisition period. Selective saturation was applied at the PARACEST frequency of each agent. (b) The decrease in MR signal of the tumor after injection relative to the average MR signal before injection was used to measure the temporal changes of each CEST effect. (Reproduced with permission of ACS Publications, Inc., from Ali et al. Ref. 39).
References
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