Imaging tumor acidosis: a survey of the available techniques for mapping in vivo tumor pH
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Cancer cells are characterized by a metabolic shift in cellular energy production, orchestrated by the transcription factor HIF-1α, from mitochondrial oxidative phosphorylation to increased glycolysis, regardless of oxygen availability (Warburg effect). The constitutive upregulation of glycolysis leads to an overproduction of acidic metabolic products, resulting in enhanced acidification of the extracellular pH (pHe ~ 6.5), which is a salient feature of the tumor microenvironment. Despite the importance of pH and tumor acidosis, there is currently no established clinical tool available to image the spatial distribution of tumor pHe. The purpose of this review is to describe various imaging modalities for measuring intracellular and extracellular tumor pH. For each technique, we will discuss main advantages and limitations, pH accuracy and sensitivity of the applied pH-responsive probes and potential translatability to the clinic. Particular attention is devoted to methods that can provide pH measurements at high spatial resolution useful to address the task of tumor heterogeneity and to studies that explored tumor pH imaging for assessing treatment response to anticancer therapies.
KeywordspH imaging Tumor acidosis Magnetic resonance imaging Chemical Exchange Saturation Transfer (CEST) imaging Iopamidol pH-responsive probes
Solid tumors are characterized by a highly heterogeneous microenvironment, resulting from the combination of poor vascular perfusion and regional hypoxia . Metabolic adaptation represents a canonical response of tumor cells to survive, orchestrated by the transcription factor HIF-1α, which modulates genes involved in angiogenesis, glycolysis, proliferation, and metastasis. This metabolic shift is characterized by elevated glycolysis and lactate production, regardless of oxygen availability (Warburg effect) [2, 3]. The constitutive upregulation of glycolysis leads to the exaggerated generation of metabolites, including acidic products such as lactate and protons that, upon accumulation in the cytoplasm, might result in intracellular acidosis. Therefore, tumor cells require additional activities in order to maintain an intracellular pH (pHi) compatible with the biochemical processes typical of cells characterized by high proliferation rates [4, 5]. This task is taken up by several and redundant families of proton transporters that excrete lactate molecules and protons into the extracellular-extravascular compartment, which induces extracellular acidification, resulting in the reversed extracellular pH (pHe) gradient in tumors in comparison to healthy tissues .
Clinical investigations support the view that the acidic microenvironment in tumors results in less favorable prognosis associated with increased metastatic potential and resistance to chemo- and radiotherapy . Therefore, acid production (or extracellular acidification) is even more important than the altered glucose metabolism [8, 9]. Because survival in the tumor microenvironment depends on the control of pH, interference with pH regulating systems is considered a relevant therapeutic goal . Thus, extracellular acidosis of solid tumors acquires a prognostic value and therapeutic manipulation of tumor acidosis might represent a novel strategy for a successful treatment of cancer [11, 12].
On the basis of the above considerations, the need for advanced medical imaging approaches, based on mapping of tumor pHe that allows early detection of treatment effect upon the impairment of tumor pH dynamics, is urgently needed. The development/exploitation of imaging-based procedures able to quantitatively measure tumor pHe may provide noninvasive additional information to currently used nuclear medicine techniques (18F-FDG/PET) for cancer assessment. If pH regulation is an essential component of tumor cell survival and its impairment may halt proliferation of the primary tumor and formation of metastases, a tool able to noninvasively quantify tumor pHe appears to have optimal chances to be translated to the clinic.
This article surveys the imaging-related methods that have been proposed, and validated in vivo, for measuring tumor acidosis, including magnetic resonance imaging (MRI) and spectroscopy (MRS), nuclear medicine (positron emission tomography, PET), electron paramagnetic resonance (EPR), optical imaging (OI), and photoacoustic imaging (PAI). For each method, the basic principles are described together with the most representative pH-sensitive probes and approaches that have been exploited for attaining in vivo tumor pH maps.
2 Magnetic resonance imaging methods
2.1 Magnetic resonance spectroscopy
Magnetic resonance spectroscopy (MRS) has been proposed in the early days of in vivo nuclear magnetic resonance (NMR) applications as a direct approach to the detection and quantification of metabolites in living tissues. In the oncological field, MRS allows the assessment of abnormal metabolic profiles that may act as useful prognostic biomarkers. In addition to metabolites, MRS has also been exploited for assessing pHi and pHe compartments of tumors cells by combining acceptable sensitivity threshold with spatial resolution. Intense efforts have been devoted to design suitable pH reporters with the aim of satisfying the criteria of favorable pharmacokinetics, pKa suitable for the physiological pH range, good sensitivity, and low toxicity. The measurement of pH by MRS is based on the chemical shift difference between a pH-dependent resonance and a pH-independent peak taken as reference. 31P, 1H, and 19F have been the most investigated NMR active nuclei for selecting pH-dependent resonances containing species.
31P MRS has been the first technique applied for measuring pH in tumors. Phosphorus-31 is 100% abundant and several phosphorus-based metabolites are intracellularly present at a concentration of 0.1–5.0 mM. Among them, inorganic phosphate (Pi) is routinely used for measuring pH in vivo due to the dependence of its chemical shift on pH changes in the physiological range (pKa ~ 6.8). As pH-independent reference peaks, the signals from endogenous nucleoside triphosphates (NTP) and phosphocreatine (PCr) are usually considered. Concerns regarding the use of different reference spectra on pH determination have been raised; however, a recent comparative study involving healthy volunteers and patients with non-Hodgkin’s lymphoma demonstrated that the direct Pi-αATP method is more reliable for measuring pH in tumors, showing low variation among patients and reasonable repeatability . Considering that intracellular Pi is present at a concentration of 2–3 mM, whereas in the extracellular compartment is present at a concentration of 1 mM; it can be calculated that, for an extracellular volume below 55%, most of the Pi-MRS-based signal is coming from the intracellular compartment, thus indicating that the observed shift of Pi is essentially a reporter of intracellular pH . This statement has been validated in vivo by exploiting the intracellular pH reporter 2-deoxyglucose (2DG), that is phosphorylated to 2DG6P and accumulates within cells overcoming the glycolytic process . 31P-MRS of fibrosarcoma xenograft tumors revealed a good correspondence between pH values obtained from Pi and from 2DG6P measurements, confirming that Pi-MRS measurements definitely report on intracellular pH. To supply the lack of extracellular pH reporter probes for 31P-MRS, exogenous phosphonate agents have also been developed. Despite the fact that several extracellular phosphonate-based probes showed good characteristics , most in vivo applications have historically involved the use of 3-aminopropylphosphonate (3-APP). This compound shows a pKa in the physiological range and accurately reports the pHe with little influence of temperature and ion effect . Therefore, simultaneous acquisition of Pi and 3-APP can be combined for assessing the pH values within tissue compartments and quantitative parameters can be extrapolated for an extensive characterization . 31P-MRS measurements elucidated in vivo the concept of the cellular pH gradient of tumors, indicating that intracellular pH in tumor is usually more alkaline in comparison to normal tissue, whereas extracellular pH is generally more acidic. This peculiar information has been exploited in several studies aiming at reverting the acidic-base pH gradient as a potential approach for treating cancer. This idea is based on the fact that the kinetic uptake of drugs strongly depends on their ionization state in relation to a specific pHi/pHe condition . Several in vivo investigations showed increased cytotoxic activity of chemotherapeutic drugs as mitoxantrone and doxorubicin upon induced tumor alkalinization with sodium bicarbonate, which raises the extracellular pH of 0.4–0.8 units. [20, 21] Moreover, inhibitors of mitochondrial metabolism in combination with hyperglycemic conditions induced selective acidification of human melanoma xenografts, with a significant decrease of both intra and extracellular pH . Furthermore, 31P-MRS approach was recently used in a mouse model to evaluate early intracellular pH changes upon antiangiogenic treatment of recurrent glioblastoma . This approach can therefore provide assessments of both intra- and extracellular tumor pH by combining endogenous and exogenous 31P-containing molecules. However, the potential neurotoxicity of 3-APP (analog of the γ-aminobutyric acid neurotransmitter) in the presence of compromised blood brain barriers is a concern for human use and the low spatial resolution and long acquisition times combined with the requirement of dedicated coils limit its application in vivo.
31P-MRS has 15 times less sensitivity in comparison to 1H spectroscopy; conversely, sensitivity of 19F is reasonably close to proton spectroscopy (83%) and its isotopic abundance is 100%. The main advantage of 19F-MRS for in vivo application relies on the minimal NMR background interference from endogenous signal and the large chemical shift range (~ 300 ppm) that allowed the development of several fluorinated probes able to report microenvironment changes of pO2, hypoxia, enzyme activity, and pH . Aromatic molecules, such as the vitamin B6 analogue fluoropyridoxol, were reported for assessing pH in vivo thanks to the larger chemical shift response (~ 9.5 ppm) to changes in pH in comparison to fluoroalanine-based probes (~ 2 ppm) . Early studies demonstrated the capability of 6-fluoropyridoxol (6-FPOL) to simultaneously measure the dynamic changes of pHe/pHi in perfused rat hearth with a time resolution of 2 min . As the pKa (~ 8.2) of 6-FPOL is not ideal, a novel membrane-impermeant CF3-modified 6-trifluoromethylpyridoxine with a pKa in the physiological range was designed. This new molecule was successfully detected in mammary and prostate tumor xenografts allowing the measurement of the extracellular pH with a sensitivity of 0.40 ppm/pH unit . However, fluorophenols have the drawback of ion-binding, thus limiting their application in vivo. An additional 19F-MRS probe, the fluoroaniline sulfonamide ZK-150471, has been validated as a valid pH reporter in different tumor xenograft models. The aromatic fluorine signal of ZK-150471 showed a chemical shift highly dependent on pH, whereas the trifluoromethyl group served as the intramolecular pH-independent reference. In vivo, ZK-150471 demonstrated to distribute only within the extracellular space of the perfused regions, showing good correspondence with microelectrodes pHe measurements . Further, the capability of ZK-150471 to report pHe was compared with the 31P-MRS probe 3-APP in tumors. Although pHe values were not significantly different, the fluorinated probe has the advantage of a lower toxicity, thus allowing the administration of higher doses for investigating pH heterogeneity in tumors. The main limitations for in vivo applications are the relative instability of fluorinated probes and their nonspecific accumulation in tissues due to their hydrophobicity. To overcome these issues, new formulations based on the encapsulation of 19F compounds have been proposed. Promising results were obtained with PEGylated nanogels that showed variation in size in accordance with pH changes, in the range of 6.8–7.3, indicating that extracellular pH can be indirectly estimated from measuring the diameter of nanoprobes by 19F-MRS . Similarly, an on/off strategy based on micelles encapsulating 19F containing species allowed the encoding of narrow pH transition (0.25 pH unit) through barcode map generated from specific 19F signatures . Despite the absence of a background signal that improves the specificity of this approach, the moderate sensitivity still limits the spatial resolution achievable by 19F-MRS, even at higher magnetic fields.
To overcome both 31P- and 19F-MRS issues, proton spectroscopy represents a valid alternative in terms of increased sensitivity due to its highest gyromagnetic ratio and a natural abundance of 99.98%. These properties represent a great advantage for gaining in spatial resolution with reduced acquisition time but a technical challenge since the broad and intense peak arising from the bulk water molecules need to be nullified for observing the smaller resonances of the exploited probes. For instance, a 1H-NMR pH-sensitive probe was reported by Aime and coworkers, exploiting a paramagnetic complex of Ytterbium, a lanthanide ion able to induce large paramagnetic shifts of nearby nuclei . This compound was shown in vitro to represent an excellent NMR pH indicator since the chemical shift separation between a selected pair of resonances is strongly pH dependent and not affected by changes in concentration or in ionic strength. Moreover, the NMR resonances of the paramagnetic complex cover a much wider chemical shift region than those of diamagnetic systems, providing higher pH sensitivity since the NMR resonances are easily distinguishable from that of endogenous molecules. Further details of the in vivo exploitation of the large 1H-chemical shift of paramagnetic metal complexes as pH indicators can be found in the PARACEST paragraph.
Although the accuracy and sensitivity on reporting pH changes offered by this method, the poor spatial resolution and the long acquisition times markedly affect pHe estimation and represent a limitation for accurate quantification of pHe heterogeneity. Another concern on using imidazole-based compound was raised due to the alkalization effect of non-volatile buffer that might affect pH measurements. Indeed, IEPA demonstrated buffering capabilities and chronic administration of IEPA influenced the extracellular acidity of tumors and reduced the formation of lung metastasis in an experimental model of prostate cancer . In addition, the rapid elimination by renal clearance and the presence of only one H2 proton in IEPA and ISUCA limit the sensitivity of this approach for in vivo application. To overcome this issue, the design of diimidazole probes with higher polarity and double H2 resonance intensity is currently under investigation, with promising results for in vivo pHe measurements .
2.2 MRI relaxometry-based pH-sensitive probes
Magnetic resonance imaging (MRI) has been widely used as a routine diagnostic tool in modern clinical medicine. The majority of clinically used MRI contrast agents are paramagnetic gadolinium (Gd3+) chelates, able to increase the signal intensity by shortening the longitudinal (T1) or the transversal (T2) relaxation times of water protons close to the region where the metal complex distributes [38, 39, 40, 41].
2.3 MRI chemical exchange saturation transfer imaging
Chemical exchange saturation transfer (CEST) imaging is a MRI contrast mechanism that allows noninvasive detection of molecules or microenvironmental tumor properties such as pH, redox status, and enzymatic activity . CEST method is based on the detection of changes of the water protons signal that decreases upon saturation magnetization at specific frequencies from an exchanging pool of protons, which can be from endogenous or exogenous sources. When RF pulses are applied, exchangeable protons from the contrast agent molecule become saturated and transfer their saturation to bulk water protons according to the chemical exchange rate, inducing a partial loss of net water signal. By applying a series of pulses, the saturation is transferred continuously to the water protons and is possible to detect the presence of molecules with mobile protons within MR images [52, 53, 54]. By recording the changes in the water signal intensity as a function of the applied RF pulses at different resonances (frequencies), one can obtain the so-called Z-spectrum that provides information on the exchanging molecule.
Proton exchange process depends on several factors, including concentration, temperature and, very often, pH. Many agents show an exchange rate that is usually slower at low pH than at high pH due to the occurrence of base-catalyzed proton exchange . To date, several pH-responsive CEST MRI agents have been developed and in general, CEST has some advantages compared to other imaging modalities. First, CEST MRI has a great spatial resolution (less than 1 mm) that can provide tomographic images exploiting standard 1H coils available in clinical MRI scanners. In addition, in contrast to MRI-relaxivity methods, CEST can take advantage of the ratiometric approach for ruling out the concentration provided that in the same agent there are two different pools of exchanging protons. On the other hand, it suffers of limited sensitivity as it needs millimolar concentrations of mobile protons. The MRI-CEST approach can be further subdivided according to the chemistry of the molecule containing the exchanging protons in DIACEST (diamagnetic molecules, including both endogenous and exogenous molecules) or PARACEST (paramagnetic molecules, mainly represented by metal complexes) agents [56, 57]. The versatility of the CEST approach and probes enables different ways for measuring in vivo tumor acidosis.
2.3.1 Endogenous DIACEST pH methods
2.3.2 Exogenous DIACEST pH-responsive probes
Exogenous DIACEST agents have mobile protons with a chemical shift in the range 0–10 ppm from the water resonance. The main advantage in using exogenous agents relies on their capability to provide a net quantification of the extracellular tumor pH, since following the extravasation from the leaky tumor vasculature they remain in the extracellular extravascular space and do not enter the intracellular compartment. Iodinated or X-ray contrast agents, following the clever intuition of Silvio Aime , gained a lot of attention as DIACEST pH-sensitive agents for several reasons, namely, (i) the chemical structure of these molecules contains exchangeable amide protons that can be selectively saturated and provide CEST contrast in the 4–6 ppm range, far enough from water for a selective irradiation even at the magnetic field of 3 T; (ii) the CEST contrast is pH dependent and by applying a ratiometric approach, the pH measurement can be made concentration-independent; (iii) they are FDA-approved contrast agents used in the last 40 years for computed tomography (CT) investigations and have demonstrated a very high safety profile. Ongoing studies are also evaluating these molecules as alternatives to Gd-based agents for studying tumor perfusion [72, 73].
Other pH sensors such as imidazole-4-5-dicarboxamides (I45DCs) have also been explored to provide CEST signal at frequencies quite far from water, up to 9 ppm, that can guarantee better detection. These systems have shown a good pH sensitivity and accuracy, but so far they have been exploited only for measuring pH in kidneys and not yet in tumors .
2.3.3 Paramagnetic pH-responsive probes (PARACEST)
Since PARACEST agents display resonances with large chemical shifts, the position of the resonating peak(s) can be exploited as an alternative approach for reporting on pH values. Sherry and coworkers developed a Europium-based contrast agent possessing a quite large shift in frequency of the metal-bound water molecule due to the delocalization of negative charge coming from the deprotonation of phenolic residue . This process is driven by pH changes, where a change from 6.0 to 7.6 at 298 K leads to a shift for the water 1H-signal of 4 ppm, i.e., from 50.5 to 54.5 ppm respectively. By recording the CEST spectrum on a 9.4-T scanner, the measurement of the chemical shift of the absorbance peak provides direct estimates of tissue pH in a concentration-independent manner without the need to set up a ratiometric approach . Although promising, it has not yet been validated for measuring tumor pHe. Moreover, since PARACEST agents are sensitive to temperature changes, accurate maintenance of temperature homogeneity is necessary for a correct pH estimation.
As the PARACEST method detects proton exchange between bulk water and any exchangeable sites (on the ligand itself or the inner sphere of bound water) that are shifted by the paramagnetic Ln3+ ion, another approach has been exploited interrogating non-exchangeable protons, in line with the early report of Aime et al. . This method, dubbed biosensor imaging of redundant deviation in shifts (BIRDS), utilizes shifts of non-exchangeable protons from macrocyclic chelates complexed with paramagnetic lanthanide ions to generate pHe maps [102, 103]. The most recent application of the BIRD approach used TmDOTP− to investigate tumor pHe differences in two glioma rat models, showing more acidic peritumoral pHe values in the more aggressive tumors . A major concern regarding this approach was that renal ligation was needed to stop the renal clearance of the agent in order to allow a higher accumulation inside the tumor and, consequently, get higher sensitivity. In addition, very high magnetic fields (11.7 T) were needed to finely resolve small shifts in the resonances, limiting the attainable pH accuracy.
As a final remark on PARACEST agents, one may note that they are more sensitive than DIACEST agents as they are not influenced by the water-indirect saturation or by concomitant effects arising from the endogenous semisolid protein-based components. However, high doses are still required to reach sufficient concentrations at the region of interest for attaining detectable CEST signals, as well as high power levels are required for an efficient saturation of the fast-exchanging protons, which is not readily available on clinical scanners.
Dynamic nuclear polarization (DNP) transfers polarization from electrons to nuclei, which provides a dramatic increase of sensitivity in MRS, particularly for low-γ nuclei, such as 13C . A mix of 13C-labeled molecules with small quantities of a stable free radical cooled to ~ 1 K in a magnetic field and continuously irradiated at the electron paramagnetic resonance (EPR) absorption frequency of the radical species. This results in the transfer of polarization from the unpaired electron to the 13C nuclei . Because the resonance frequencies of electrons are thousands of times higher than those of NMR nuclei, this transfer dramatically increases the polarization of the NMR nuclei, directly increasing the sensitivity theoretically up to 50,000-fold. In practice, increases in the signal-to-noise ratio of 10,000 are routinely achieved for DNP-hyperpolarized 13C-labeled molecules, allowing their direct detection with spectroscopic imaging, MRSI [113, 114]. The development of hyperpolarized molecules whose chemical shift is pH dependent can provide another method for in vivo pH imaging with high sensitivity.
Brindle et al. firstly described the injection of hyperpolarized 13C-bicarbonate as a pH-responsive agent, exploiting the acid-base equilibrium between H13CO3− and 13CO2 that show distinct chemical shifts. According to the Henderson–Hasselbalch equation, the ratio of H13CO3− and 13CO2 resonance intensities can be exploited for assessing pH. The method has been tested for mapping the extracellular pH in a lymphoma tumor murine model . The pH map calculated after the intravenous injection of 100 mM hyperpolarized 13C-bicarbonate gave an average calculated pH of 6.71 ± 0.14. Scholl and coworkers applied this technique for studying temporal changes of tumor pHi and pHe during tumor growth in a rat glioma model . Despite 13C-labeled bicarbonate has been initially proposed as a probe for tumor pHe imaging, the transport of CO2 and bicarbonate in and out the cells results in a mixed contribution of intra- and extracellular values to the measured pH.
3 Electron paramagnetic resonance imaging
Electron paramagnetic resonance (EPR) is a spectroscopic technique based on the detection of unpaired electrons (radicals) in paramagnetic species, exposing the sample to a sweep of microwave frequency irradiation. Because of the insufficient amount of radical species in viable tissues combined with their fast relaxation, the use of paramagnetic probes to reach a sufficient concentration at the site of interest is required . Whereas direct detection of paramagnetic probes guarantees high specificity, development of probes endowed with good biocompatibility, long stability during the measurement, low toxicity, and optimal spectral sensitivity is not a straightforward task. Moreover, unlike MRI measurements, EPR experiments are performed in a continuous wave mode, resulting in long acquisition times, small sample size, and restricted to surface tissues. EPR imaging (EPRI) is challenging for anatomic co-localization since, at low magnetic field, the small polarization of the 1H spin is not sufficient to generate MR images. Two approaches have been developed for imaging purposes, namely (i) the use of pulsed irradiations (pulsed-EPRI), which shortens the acquisition time , but still lacking of anatomical information, and (ii) the exploitation of the Overhauser effect. This technique, called OMRI (Overhauser enhanced magnetic resonance imaging), also known as proton electron double resonance imaging (PEDRI) was developed by Lurie et al. in 1988 . In this case, the electron spin polarization is transferred to 1H polarization with subsequent MR image acquisition, resulting in an indirect detection of the radical probe . Unlike EPRI, higher resolution can be achieved.
Khramtsov and coworkers have designed a pH-sensitive nitroxide R1 and its deutero-substituted analog, R2, for EPR monitoring of pHe in breast cancer models . The enhanced stability of the proposed probe conjugated with its extracellular localization and pH sensitivity in the range of 6.5–7.5 allowed ready detection of pHe. The same group investigated a dual probe pTAM (deuterated derivative of the phosphonated triaryl methyl or TAM) for simultaneous pH and oxygen monitoring thanks to the extraordinary stability toward tissue redox processes, longer relaxation time, and narrower line width of this probe compared to nitroxide-based probes . Suffering of the lack of EPR spectral information, the conventional PEDRI approach was recently improved with the variable field and variable radio frequency (VRF) PEDRI, where the pH map was obtained from two PEDRI acquisitions performed at the EPR frequencies of protonated and unprotonated form of a deuterated pH-sensitive probe, Im6, in Met-1 tumor-bearing mice . The VRF PEDRI technique was applied to obtain the pH map distribution in tumor with an average value of extracellular pH of 6.8 ± 0.1 in agreement with the averaged pH values measured with the pH microelectrode.
EPR approaches are promising in offering accurate pH measurements and multiple functional information, beside tumor pH, simultaneously. However, several drawbacks, including low spatial resolution (2–3 mm3 voxels, comparable to MRS), possible toxicity of the radical probes at the high required doses, demanding technical requirements, and high power radiofrequency irradiations, need to be solved before a large adoption of this approach can be foreseen.
4 Positron emission tomography imaging
5 Optical imaging
Optical imaging is characterized by high in-plane resolution and sensitivity, and excellent sensitivity and temporal resolution. However, it is limited in depth penetration and quantification, due to the well-known issues of absorption, reflection, and refraction processes when the emitted photons travel within biological tissues . Importantly, in the NIR range (650–900 nm), photons travel through tissue deeper than photons in the visible range, and this wavelength range, called in vivo optical window, has been extensively exploited for addressing biological questions following the development of fluorescent-responsive probes .
SNARF-1 (semi-napthorhodafluorescein-1) is a fluorescent molecule that exhibits a pH-dependent emission spectral shift from 580 to 640 nm. The ratio of the measured fluorescent signals between these two wavelengths allows accurate pH measurements. Since the excitation and emission wavelengths are in the visible range, this probe can be applied in cell cultures or in superficial tumors like in rabbit ear chambers  or in intravital microscopy with the support of a window chamber . To date, several studies using the window chamber tumor model have addressed important questions regarding the role of tumor acidosis in driving tumor invasion. In particular, by using this probe, Gillies and coworkers demonstrated that regions of highest tumor invasion correspond to areas of lowest pH . On this basis, the administration of sodium bicarbonate to reduce tumor acidity (as demonstrated by the increase of the measured tumor pH values by this approach) was proposed as a novel therapeutic strategy to reduce tumor metastases . Although the dorsal skinfold window chamber is commonly used to evaluate the microvasculature in various settings in vivo, it has to be stressed that a surgical operation is needed for implanting the window chamber and this operation could influence angiogenic processes. Moreover, pH measurements via SNARF-1 can be affected by difference in temperature between the calibration curve and the imaged tissue, as well as upon interaction of the fluorescent probe with extracellular proteins that can affect the pH dependence of the fluorescent spectra .
To measure in vivo tumor pH without the support of a window chamber, different probes with excitation and emission wavelength in the NIR region have been developed. Most of these approaches require two fluorescent probes, one pH sensitive and one pH insensitive, to set up a ratiometric approach for measuring pH values independently from the probes’ concentration. The conjugation of the two fluorescent dyes to a nanosized carrier guarantees the same concentration of the two molecules, solving the limitations associated with the assumption of the similar pharmacokinetic and biodistribution. However, the increase in the size of the resulting adducts, according to the final size and charge of the nanosystem, may limit the extravasation in poor perfused tumors or promote macrophage uptake and reduce the elimination rate, raising obvious toxicity issues. As an example, a pH-sensitive cyanine dye (CypHer5E), and a pH-insensitive fluorescent dye (Oyster800) have been conjugated on the surface of a biocompatible sphere with a diameter of 100 nm. CypHer5E has minimal fluorescence at neutral pH but becomes highly fluorescent with an emission peak at about 670 nm in an acidic environment. By taking the ratio of fluorescence intensities at different wavelengths, it was possible to detect tumor pH changes during tumor progression in a melanoma (B16f10) tumor murine model .
Finally, a different class of NIR pH-sensitive probes composed of systems that are activated by acidic pH has been proposed. For example, Gao and coworkers synthetized a 30-nm nanoparticle made of a pH-sensitive copolymer and loaded with the Cy5.5 dye. At blood pH values (7.4), the fluorescence is quenched, but at acidic pH values (6.9), the micelles dissociate, resulting in a marked increase of the fluorescence signal that allows detection of the acidic microenvironment of tumors . A similar approach has been adopted to detect tumor acidosis by exploiting two NIR fluorophores (IR783) conjugated via a flexible and acid liable linkage. At neutral pH conditions, the fluorescence of this probe is quenched due to intramolecular dimeric aggregate. In the acidic conditions of the tumor microenvironment, the cleavage of pH liable linkage with the concomitant disruption of aggregates results in a remarkable fluorescence enhancement with a high tumor/background ratio. The ratio changed more dramatically upon time in a more metastatic hepatic tumor model (HCCLM3-GFP) in comparison to a less metastatic one (HepG2) .
6 Photoacoustic imaging
The major limitations of these probes are related to the stability of the self-assembled nanoparticles that usually require further conjugation via glutaraldehyde to induce covalent cross-linking and to the toxicity profiles and biodistribution properties that need to be further optimized for their clinical translation. In addition, the nonlinear response of the ratiometric curve to changes in pH, coupled to the nonlinear optical properties of the photoacoustic effect, makes them more useful for detecting rapid or relative pH changes, rather than pH values. Despite these limitations, optoacoustic pH imaging overcomes the penetration depth limit of optical imaging, so it has the potential to measure tumor pH in vivo in superficial tumors up to 5–7-mm depth or in endoscopically accessible tumors at clinical level.
A wide range of imaging-based techniques have been investigated to monitor tumor pH. In the last decade, several approaches have solved the limitations associated with first studies, including spatial and temporal resolution, pH sensitivity, and clinical translatability. To date, among the proposed techniques, MRI and in particular MRI-CEST methods have emerged as those endowed with good pH sensitivity for assessing tumor acidosis and pH changes following therapeutic treatments and with high spatial resolution for evaluating heterogeneity of the extracellular acidification. In addition, MRI-CEST using Iopamidol has been used to map tumor pH at clinical level, thus providing a novel imaging protocol for assessing fundamental questions in tumor biology, for evaluating the relationship between tumor acidosis and aggressiveness, and for monitoring treatment response to novel anticancer therapies. However, critical evaluation of this approach in single-site and multi-site studies should be encouraged to validate the clinical utility of imaging tumor acidosis as a novel noninvasive diagnostic tool.
We thank Professor Silvio Aime for his helpful suggestions.
This work was supported by grants from the Associazione Italiana Ricerca Cancro (AIRC MFAG 2017 - ID. 20153 project – P.I. Longo Dario Livio), from Compagnia San Paolo project (Regione Piemonte, Grant No. CSTO165925), and from the European Union’s Horizon 2020 research and innovation programme (Grant Agreement No. 667510). Lorena Consolino was supported by the Fondazione Umberto Veronesi fellowship program.
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Conflict of interest
The authors declare that they have no conflict of interest.
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