Solid tumors often develop an acidic environment due to the Warburg effect. The effectiveness of diagnosis and therapy may therefore be enhanced by the design and use of pH-sensitive agents that target acidic tumors. Recently, a novel technology was introduced to target acidic tumors using pH low insertion peptide (pHLIP), a peptide that inserts across cell membranes as an α-helix when the extracellular pH (pHe) is acidic. In this study, we expanded the application of the pHLIP technology to include positron emission tomography imaging of the acidic environment in prostate tumors using 64Cu conjugated to the pHLIP (64Cu-DOTA-pHLIP). Studies showed that this construct avidly accumulated in LNCaP and PC-3 tumors, with higher uptake and retention in the LNCaP tumors. Uptake correlated with differences in the bulk pHe of PC-3 and LNCaP tumors measured in magnetic resonance spectroscopy experiments by the 31P chemical shift of the pHe marker 3-aminopropylphosphonate. This article introduces a novel class of noninvasive pH-selective positron emission tomography imaging agents and opens new research directions in the diagnosis of acidic solid tumors. [Cancer Res 2009;69(10):4510–6]

Studies of the gene signatures of cancer cells indicate that many different genes are either up-regulated or down-regulated, even within a given type of tumor, so it is problematic to rely on any single biomarker for the diagnosis of even a single type of cancer (1). However, physiologic processes (e.g., hypoxia and acidity), which are present in 90% of tumor microenvironments, are considered promising environmental markers for tumor targeting (2, 3). In particular, an acidic tumor environment plays a significant role in tumor progression and is often associated with increased invasion and metastasis (46) as well as resistance to drug therapies (5, 7, 8). Recently, a correlation was observed between extracellular pH (pHe) and overall survival following treatment with combined hyperthermia and radiation therapy for spontaneous sarcomas in canines (9). Tumor pH has practical importance because most anticancer drugs must be transported either by active transport or by passive diffusion into cells, where they frequently undergo further metabolism. As all of these processes might be pH sensitive, the cytotoxic activity of an anticancer drug could depend on both the intracellular pH and the pHe. In the case of chemotherapeutic agents that are weak acids or bases and enter targeted cells via passive diffusion, it is the nonionized form that crosses the cell membrane. It also has been suggested that modulation of tumor pH, both intracellular pH and pHe, could be used to increase the uptake of therapeutic drugs (7, 1012). Delineation of intracellular pH from pHe is impossible with electrode-based techniques due to the scale of the electrode compared with the tissue. Thus, noninvasive tools for monitoring pHe and the effectiveness of its modulation could be extremely valuable.

Several magnetic resonance-based methods for the estimation of pHe in animal tumor models have been introduced in recent years. These approaches include the use of T1-weighted magnetic resonance imaging with extracellular contrast agents that exhibit pH-dependent 1H relaxation enhancement (13), 1H and 31P pH-dependent chemical shift measurements of exogenous extracellular species (1416), and 13C chemical shift imaging of hyperpolarized 13C-labeled bicarbonate (17). Studies with the 31P pHe indicator 3-aminopropylphosphonate (3-APP) have confirmed that the average pHe of tumors is acidic, with values reaching as low as 6.0 (18). At present, however, these techniques cannot be readily translated to the clinic.

Positron emission tomography (PET) is a widely used molecular imaging modality in both clinical and research settings (19). The use of 64Cu as a PET nuclide, and the basis for new radiopharmaceuticals, is particularly attractive given its 12.74 h half-life (20). 64Cu complexes have been studied as PET agents with the nuclide incorporated into either small molecules or peptides (21, 22). Here we present the 64Cu PET imaging of prostate tumors using the recently discovered acid-targeting peptide, pH low insertion peptide (pHLIP; refs. 2325). The pHLIP contains 37 residues. At neutral pHe, it interacts with the surface of membranes as an unstructured peptide, but at acidic pHe (<7.0), it inserts across the membrane and forms a stable transmembrane α-helix (24). We have shown that pHLIP can target a variety of fluorescent dyes conjugated to its NH2 terminus to a tissue with elevated extracellular acidity in vivo and also that it can deliver chemotherapeutics into cells through interactions with the membrane lipid bilayer, avoiding the need for any specific interaction with membrane proteins (23, 25, 26). The present study was undertaken to evaluate whether it is possible to delineate lower pHe in prostate tumors in vivo with 64Cu conjugated to the pHLIP (64Cu-DOTA-pHLIP), the first compound of a new generation of novel pH-selective tumor PET imaging agents.

All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich. Radioactive samples were counted in a radioisotope calibrator (Capintec) for determination of mCi and an automated-well Beckman 8000 gamma counter (Beckman Coulter) for counts/min. Male athymic nu/nu mice were purchased from the National Cancer Institute. Human prostate carcinoma tumor cell lines PC-3 and LNCaP were obtained from the American Type Culture Collection and maintained by serial passage in cell culture.

Preparation of peptides and 64Cu radiolabeling. All peptides were prepared by solid-phase peptide synthesis using standard 9-fluorenylmethyloxycarbonyl chemistry and purified by reverse-phase chromatography (on a C18 column) at the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Peptide sequences were ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG (pHLIP) and ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG (K-pHLIP). The NH2 terminus of the pHLIP was covalently conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7-Tris-acetic acid-10-maleimidoethylacetamide, a maleimide-containing derivative of DOTA, a chelator used for the effective binding of copper radionuclides (27). The peptides were incubated with 2× molar excess of DOTA-maleimide (Macrocyclics) in the presence of 2 mmol/L EDTA in PBS (pH 7.4) overnight at 4°C. Free DOTA-maleimide and EDTA were removed by a Sephadex G-10 spin column equilibrated with PBS (pH 7.4). The concentration of peptide was determined by absorbance at 280 nm using a molar extinction coefficient of ε280 = 13,940 mol/L−1 cm−1. The composition and degree of conjugation were confirmed by mass spectrometry and high-performance liquid chromatography. The peptides were analyzed by electrospray mass spectrometry [ES+; DOTA-pHLIP: m/z calculated for C215H323N50O66S (M+) = 4693.3, found 1174.74 (M + 4H+) and 1565.4 (M + 3H+)]. Reverse-phase high-performance liquid chromatography involved a Waters X-Bridge (C4, 4.6 × 75 mm) column with a gradient [0-2 min 20% B to 40% B, 2-7 min 40% B to 70% B, and 7-10 min 70% B to 100% B (solvent A: 0.01% trifluoroacetic acid in water and solvent B: 0.01% trifluoroacetic acid in acetonitrile)] at a flow rate of 1 mL/min. Detection was accomplished at 280 nm and the retention times for DOTA-pHLIP and DOTA-K-pHLIP were 5.45 and 4.82 min, respectively. The peptides were stored at −80°C.

64Cu was produced at Washington University School of Medicine and processed by previously reported literature methods (28). Maximal labeling for both constructs was achieved in 0.5 mol/L ammonium acetate (pH 5.5) at 25°C for 30 min at a ratio of 1:1 (μg:mCi). Before purification, 5 mmol/L EDTA was added to scavenge any uncomplexed 64Cu. Purification was achieved by C18 SepPak Light using 100% ethanol as the eluent. Purity and labeling efficiency were determined by radio-TLC on silica using 10% ammonium acetate/methanol (50:50).

Acute biodistribution. All animal experiments were done in compliance with the Guidelines for the Care and Use of Research Animals established by Washington University Animal Studies Committee. Male athymic mice (20-25 g; National Cancer Institute) were implanted in the right flank with either 1 × 107 PC-3 or LNCaP human prostate adenocarcinoma cells in 100 μL cell medium with >90% viability. The LNCaP cells were implanted using Matrigel (BD Biosciences). Tumors were allowed to grow to ∼0.5 to 0.75 cm3 in volume. Mice were randomized before the study. To investigate specific uptake of tumor, organ, and other nontarget tissues, a small amount of 64Cu-DOTA-pHLIP (∼28 μCi, 0.22 μg) was injected intravenously into each of the mice bearing palpable PC-3 or LNCaP tumors. The animals were sacrificed at selected time points after injection (1, 4, 24, and 48 h; n = 4-5) and desired tissues were removed, weighed, and counted for radioactivity accumulation. The %ID/g and %ID/organ were calculated by comparison with a weighed, counted standard solution. An additional group of PC-3 mice were injected with 64Cu-DOTA-K-pHLIP (∼28 μCi, 0.22 μg) and sacrificed at 1 h (n = 5). Due to a technical error, an additional group of mice were injected and just the kidneys were removed. Because the aspartic acid residues have been changed to lysines in the transmembrane portion, the K-pHLIP should not have the ability to form an α-helix and span the membrane of the cells in acidic environments. On a different day, an additional cohort of LNCaP-bearing mice (which were implanted with a different passage of LNCaP cells; n = 8) were split into two groups, with the first group receiving 150 mmol/L bicarbonated water at pH 8.0 (3.1455 g sodium bicarbonate dissolved in 250 mL drinking water) ad libitum for 7 days before the acute biodistribution study (4 h) to modulate tumor pHe (12, 25, 29) and the second group receiving regular drinking water. Magnetic resonance spectroscopy (MRS) studies were done on the tumors in all of these mice before biodistribution.

Small-animal PET. Imaging 64Cu-DOTA-pHLIP was done on two tumor models using a microPET-F220 (30). Imaging was done in 10 min static sessions, with a collection of 600 frames per session. Isoflurane (2%) was used as an inhaled anesthetic to induce and maintain anesthesia during imaging. Male athymic mice were implanted in the right flank with PC-3 (n = 4) or LNCaP (n = 10) human prostate adenocarcinoma cells, which were allowed to grow until palpable. At this point, the mice were injected intravenously with 200 μCi 64Cu-DOTA-pHLIP and then imaged in pairs at 1, 4, and 24 h post-injection. Images were reconstructed by Fourier rebinning followed by two-dimensional Ordered Subset Expectation Maximization. Small-animal PET images were evaluated by analysis of the standardized uptake value (SUV) of the tumor and muscle using the software ASIPRO (Concorde MicroSystems). The average radioactivity concentration within the tumor or tissue was obtained from the average pixel values reported in nCi/mL within a volume of interest drawn around the entire tumor or tissue on multiple, consecutive transaxial image slices. SUVs were calculated by dividing this value, the decay-corrected activity per unit volume of tissue (nCi/mL), by the injected activity per unit of body weight (nCi/g).

MRS.In vivo pHe was determined in LNCaP and PC-3 tumors within the 24 h period immediately before injection of 64Cu-labeled pHLIP. Noninvasive measurements of pHe were based on the 31P magnetic resonance-observable chemical shift of 3-APP (31). Before magnetic resonance measurements, mice bearing PC-3 (n = 3) or LNCaP (n = 6) tumors were anesthetized with isoflurane and maintained on isoflurane/O2 (1.25% v/v) throughout data collection. Each anesthetized mouse received an intraperitoneal injection of 0.35 mL 3-APP (Sigma-Aldrich) solution prepared at a concentration of 75 mg 3-APP/mL in isotonic saline. After injection, mice were placed in a head holder with a nose cone for delivery of anesthetic gas and a custom-built 31P magnetic resonance surface coil was placed around the tumor.

31P magnetic resonance studies were done using a small-animal magnetic resonance scanner based on a Magnex Scientific Instruments 11.74 T (31P resonance frequency 202.3 MHz), 26 cm horizontal-bore magnet interfaced with a Varian NMR Systems INOVA console. Magnetic field homogeneity was optimized by shimming on the free induction decay of the tumor water resonance, observed through the 31P magnetic resonance surface coil. 31P spectral acquisition parameters were: sweep width of 20,000 Hz, 0.3 s acquisition time, 4.06 s repetition time, and a 60° excitation pulse. A total of 512 transients were averaged for each measurement; thus, the total length of each 31P scan was 34 min. Magnetic resonance data were processed with a matched filter (30 Hz line-broadening) and frequency estimates for the 31P MRS components were done using a Bayesian Probability Theory analysis package developed at Washington University (32). The resulting estimated chemical shift difference between 3-APP and the pH-independent α-nucleoside triphosphate resonance (33) was used to determine pHe based on the fitting parameters provided by Gillies and colleagues (31).

Statistics. Statistically significant differences between mean values were determined using ANOVA coupled to Scheffe's test or, for statistical classification, a Student's t test was done. Differences at the 95% confidence level (P < 0.05) were considered significant.

64Cu-DOTA-pHLIP and 64Cu-DOTA-K-pHLIP can be obtained in high yield and good specific activity.64Cu labeling of DOTA-pHLIP was attempted at various pH values between 4.5 and 9.2 using 0.125 to 0.5 mol/L ammonium acetate. Optimal labeling was achieved in 0.5 mol/L ammonium acetate (pH 5.5) at 25°C for 30 min at a ratio of 1:1 (μg:mCi) at a specific activity of 1,591 mCi/μmol. Without heating, the labeled solution remained stable overnight. Radiochemical purity was consistently >95% following EDTA scavenge and SepPak purification as shown by radio-TLC on silica using 10% ammonium acetate/methanol (50:50). As a control, we used a mutant version of pHLIP (K-pHLIP) in which key Asp residues of the transmembrane part of pHLIP were replaced by positively charged Lys residues (Fig. 1). These changes prevent the peptide from inserting across the cell membrane and forming an α-helix at acidic pH (25). The mutant/control peptide (K-pHLIP) was labeled in a similar manner at a specific activity of 1,713 mCi/μmol at >95% radiochemical purity.

Figure 1.

Amino acid sequence of the native peptide compared with the mutant pHLIP (K-pHLIP).

Figure 1.

Amino acid sequence of the native peptide compared with the mutant pHLIP (K-pHLIP).

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Acute biodistribution of 64Cu-DOTA-pHLIP in PC-3 and LNCaP and 64Cu-DOTA-K-pHLIP in PC-3 tumor-bearing mice. Organ, tissue, and tumor uptake was examined by acute biodistribution studies of 64Cu-DOTA-pHLIP after injection into male nude mice bearing either LNCaP or PC-3 tumors (Table 1). One additional group of PC-3–bearing mice received the equivalent mass of 64Cu-DOTA-K-pHLIP as a control. The PC-3 tumor accumulation of 64Cu-DOTA-pHLIP reached a maximum after 4 h (2.78 ± 0.19%ID/g) and the tumor retention was high, with 2.46 ± 0.57%ID/g remaining at 24 h. In the LNCaP mice, tumor accumulation of 64Cu-DOTA-pHLIP reached a maximum after only 1 h (4.50 ± 1.71%ID/g) and the tumor retention was also high, with 3.23 ± 0.55%ID/g remaining at 24 h and 1.74 ± 1.41%ID/g at 48 h post-injection. The biodistribution data for 64Cu-DOTA-pHLIP showed that the radioactive background in the blood and blood-rich organs, such as liver, lung, heart, and spleen, was similar in the two tumor models, whereas differences were noted in the tumors. Examination of tumor-to-tissue ratios (Fig. 2) clearly shows that, for imaging purposes, the 4 h time point is optimal for both tumor models. This can be attributed to better retention of the agent in tumor tissue and more rapid washout of activity from background organs.

Table 1.

Acute biodistribution of 64Cu-DOTA-pHLIP in tissues in PC-3 and LNCaP mice and 64Cu-DOTA-K-pHLIP in PC-3 mice

PC-31 h
4 h
24 h
1 h
64Cu-DOTA-pHLIP64Cu-DOTA-pHLIP64Cu-DOTA-pHLIP64Cu-DOTA-K-pHLIP
Blood 11.27 ± 0.81 7.45 ± 1.76 2.59 ± 0.48 7.09 ± 0.67 
Lung 5.16 ± 0.51 4.25 ± 0.94 2.35 ± 0.46 3.75 ± 0.18 
Liver 8.56 ± 1.14 7.99 ± 1.28 5.86 ± 1.23 5.21 ± 1.75 
Spleen 2.10 ± 0.14 2.02 ± 0.47 1.82 ± 0.44 1.64 ± 0.29 
Kidney 6.32 ± 0.39 6.93 ± 0.83 4.64 ± 1.27 4.63 ± 0.35* 
Muscle 1.11 ± 0.79 0.86 ± 0.16 0.65 ± 0.14 0.56 ± 0.09 
Skin 1.77 ± 0.09 2.07 ± 0.36 1.87 ± 0.33 0.96 ± 0.06 
Fat 0.80 ± 0.39 0.86 ± 0.27 0.53 ± 0.18 1.09 ± 0.80 
Heart 3.95 ± 0.60 3.17 ± 0.83 1.67 ± 0.14 2.50 ± 0.55 
Brain 0.47 ± 0.11 0.35 ± 0.12 0.16 ± 0.04 0.30 ± 0.06 
Bone 1.55 ± 0.19 1.17 ± 0.41 0.81 ± 0.14 1.12 ± 0.41 
PC-3 1.72 ± 0.17 2.78 ± 0.19 2.46 ± 0.57 1.04 ± 0.15 

 

 

 

 

 
LNCaP 1 h
 
4 h
 
24 h
 
48 h
 

 
64Cu-DOTA-pHLIP
 
64Cu-DOTA-pHLIP
 
64Cu-DOTA-pHLIP
 
64Cu-DOTA-pHLIP
 
Blood 14.97 ± 1.11 8.08 ± 0.35 3.96 ± 1.11 1.68 ± 0.35 
Lung 5.63 ± 0.62 4.38 ± 0.39 3.22 ± 0.53 2.22 ± 0.70 
Liver 6.53 ± 1.02 4.80 ± 0.72 4.88 ± 0.98 4.43 ± 0.74 
Spleen 2.75 ± 0.48 2.05 ± 0.09 1.84 ± 0.27 1.41 ± 0.35 
Kidney 8.34 ± 0.66 5.42 ± 0.58 4.48 ± 1.08 2.76 ± 0.50 
Muscle 1.38 ± 0.53 0.72 ± 0.11 0.85 ± 0.25 0.49 ± 0.10 
Skin 4.16 ± 1.72 2.35 ± 0.15 2.87 ± 0.54 1.68 ± 0.20 
Fat 2.40 ± 0.73 0.87 ± 0.17 0.60 ± 0.16 0.26 ± 0.14 
Heart 5.31 ± 1.03 3.60 ± 0.28 2.02 ± 0.31 1.45 ± 0.08 
Brain 0.45 ± 0.06 0.29 ± 0.05 0.24 ± 0.05 0.16 ± 0.04 
Bone 3.77 ± 1.88 1.03 ± 0.10 1.00 ± 0.28 0.60 ± 0.10 
LNCaP 4.50 ± 1.71 3.67 ± 0.56 3.23 ± 0.55 1.74 ± 1.41
 
PC-31 h
4 h
24 h
1 h
64Cu-DOTA-pHLIP64Cu-DOTA-pHLIP64Cu-DOTA-pHLIP64Cu-DOTA-K-pHLIP
Blood 11.27 ± 0.81 7.45 ± 1.76 2.59 ± 0.48 7.09 ± 0.67 
Lung 5.16 ± 0.51 4.25 ± 0.94 2.35 ± 0.46 3.75 ± 0.18 
Liver 8.56 ± 1.14 7.99 ± 1.28 5.86 ± 1.23 5.21 ± 1.75 
Spleen 2.10 ± 0.14 2.02 ± 0.47 1.82 ± 0.44 1.64 ± 0.29 
Kidney 6.32 ± 0.39 6.93 ± 0.83 4.64 ± 1.27 4.63 ± 0.35* 
Muscle 1.11 ± 0.79 0.86 ± 0.16 0.65 ± 0.14 0.56 ± 0.09 
Skin 1.77 ± 0.09 2.07 ± 0.36 1.87 ± 0.33 0.96 ± 0.06 
Fat 0.80 ± 0.39 0.86 ± 0.27 0.53 ± 0.18 1.09 ± 0.80 
Heart 3.95 ± 0.60 3.17 ± 0.83 1.67 ± 0.14 2.50 ± 0.55 
Brain 0.47 ± 0.11 0.35 ± 0.12 0.16 ± 0.04 0.30 ± 0.06 
Bone 1.55 ± 0.19 1.17 ± 0.41 0.81 ± 0.14 1.12 ± 0.41 
PC-3 1.72 ± 0.17 2.78 ± 0.19 2.46 ± 0.57 1.04 ± 0.15 

 

 

 

 

 
LNCaP 1 h
 
4 h
 
24 h
 
48 h
 

 
64Cu-DOTA-pHLIP
 
64Cu-DOTA-pHLIP
 
64Cu-DOTA-pHLIP
 
64Cu-DOTA-pHLIP
 
Blood 14.97 ± 1.11 8.08 ± 0.35 3.96 ± 1.11 1.68 ± 0.35 
Lung 5.63 ± 0.62 4.38 ± 0.39 3.22 ± 0.53 2.22 ± 0.70 
Liver 6.53 ± 1.02 4.80 ± 0.72 4.88 ± 0.98 4.43 ± 0.74 
Spleen 2.75 ± 0.48 2.05 ± 0.09 1.84 ± 0.27 1.41 ± 0.35 
Kidney 8.34 ± 0.66 5.42 ± 0.58 4.48 ± 1.08 2.76 ± 0.50 
Muscle 1.38 ± 0.53 0.72 ± 0.11 0.85 ± 0.25 0.49 ± 0.10 
Skin 4.16 ± 1.72 2.35 ± 0.15 2.87 ± 0.54 1.68 ± 0.20 
Fat 2.40 ± 0.73 0.87 ± 0.17 0.60 ± 0.16 0.26 ± 0.14 
Heart 5.31 ± 1.03 3.60 ± 0.28 2.02 ± 0.31 1.45 ± 0.08 
Brain 0.45 ± 0.06 0.29 ± 0.05 0.24 ± 0.05 0.16 ± 0.04 
Bone 3.77 ± 1.88 1.03 ± 0.10 1.00 ± 0.28 0.60 ± 0.10 
LNCaP 4.50 ± 1.71 3.67 ± 0.56 3.23 ± 0.55 1.74 ± 1.41
 
*

Due to a technical error, an additional group of mice were used to produce these data. Data are %ID/g ± SD in n = 4 to 5 mice per group.

Figure 2.

Comparisons of various tumor-to-tissue ratios from 64Cu-DOTA-pHLIP biodistribution data presented in Table 1. Tumor %ID/g was divided by the corresponding %ID/g of the tissue of interest for (A) blood, (B) liver, (C) kidney, and (D) muscle. *, P < 0.05, statistical significance at the 95% confidence level.

Figure 2.

Comparisons of various tumor-to-tissue ratios from 64Cu-DOTA-pHLIP biodistribution data presented in Table 1. Tumor %ID/g was divided by the corresponding %ID/g of the tissue of interest for (A) blood, (B) liver, (C) kidney, and (D) muscle. *, P < 0.05, statistical significance at the 95% confidence level.

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The administration of the mutant peptide, 64Cu-DOTA-K-pHLIP, resulted in a ∼40% lower PC-3 tumor accumulation at 1 h post-injection, which is impressive in that the subsequent MRS study showed the PC-3 tumors to be less acidic. However, distribution of the control peptide, 64Cu-DOTA-K-pHLIP, was similar in pattern to the parent, 64Cu-DOTA-pHLIP, although with a reduction in accumulation in all tissues. The most likely reason for this universal reduction is a more rapid excretion of the mutant-pHLIP, which has been observed by fluorescence imaging as well, and, as a consequence, less retention in the blood and therefore the tissues. Our biophysical studies indicate that pHLIP binds to the surfaces of membranes at normal pH (26), which might lead to the slow kinetics of peptide distribution in vivo. Most probably, the mutant pHLIP has reduced binding affinity to the membrane and, therefore, a faster kinetic profile. It is important to note that the mutant peptide was not taken up to the same extent by the skin or the kidney, which are tissues known to be more acidic than all other healthy tissues and organs.

The final groups of animals studied were LNCaP-bearing mice that were given either normal drinking water or water spiked with sodium bicarbonate to modulate the tumor pHe. As shown by MRS, the bicarbonate water resulted in the modulated tumor having a more basic pHe (see below). The results of the biodistribution in these mice (Table 2) were similar to those for LNCaP tumors presented in Table 1, but it is important to note that these studies were done on different dates with mice that had been implanted with a different passage of LNCaP cells. Except for uptake in the tumor and kidney, no significant differences were observed between the two groups in the 17 collected organs and tissues. 64Cu-DOTA-pHLIP uptake in the nonmodulated LNCaP tumors was “greater” than the uptake in the tumors in mice that received 7 days of bicarbonated water (4.50 ± 1.71 versus 1.31 ± 0.60, respectively; P = 0.005).

Table 2.

Acute biodistribution in LNCaP-bearing mice split into two groups: (a) mice that received 150 mmol/L bicarbonated water at pH 8.0 ad libitum for 7 d before the study and (b) mice fed regular drinking water

4 h (untreated)4 h (modulated)
Blood 7.21 ± 1.39 7.27 ± 1.00 
Lung 3.63 ± 0.63 3.85 ± 0.61 
Liver 5.29 ± 1.64 5.79 ± 0.66 
Spleen 1.49 ± 0.32 1.53 ± 0.25 
Kidney* 5.20 ± 1.06 3.78 ± 0.29 
Muscle 0.69 ± 0.40 1.05 ± 0.80 
Skin 1.12 ± 0.29 1.02 ± 0.27 
Fat 1.26 ± 1.42 1.16 ± 0.50 
Heart 2.07 ± 0.28 2.39 ± 0.44 
Brain 0.59 ± 0.69 0.27 ± 0.06 
Bone 1.22 ± 1.15 2.22 ± 2.51 
Pancreas 1.48 ± 0.77 1.56 ± 0.67 
Stomach 0.88 ± 0.58 1.01 ± 0.26 
Small intestine 2.05 ± 1.04 2.48 ± 0.59 
Upper large intestine 2.35 ± 1.25 3.55 ± 1.94 
Lower large intestine 1.28 ± 0.79 1.23 ± 0.54 
LNCaP* 4.50 ± 1.71 1.31 ± 0.60 
4 h (untreated)4 h (modulated)
Blood 7.21 ± 1.39 7.27 ± 1.00 
Lung 3.63 ± 0.63 3.85 ± 0.61 
Liver 5.29 ± 1.64 5.79 ± 0.66 
Spleen 1.49 ± 0.32 1.53 ± 0.25 
Kidney* 5.20 ± 1.06 3.78 ± 0.29 
Muscle 0.69 ± 0.40 1.05 ± 0.80 
Skin 1.12 ± 0.29 1.02 ± 0.27 
Fat 1.26 ± 1.42 1.16 ± 0.50 
Heart 2.07 ± 0.28 2.39 ± 0.44 
Brain 0.59 ± 0.69 0.27 ± 0.06 
Bone 1.22 ± 1.15 2.22 ± 2.51 
Pancreas 1.48 ± 0.77 1.56 ± 0.67 
Stomach 0.88 ± 0.58 1.01 ± 0.26 
Small intestine 2.05 ± 1.04 2.48 ± 0.59 
Upper large intestine 2.35 ± 1.25 3.55 ± 1.94 
Lower large intestine 1.28 ± 0.79 1.23 ± 0.54 
LNCaP* 4.50 ± 1.71 1.31 ± 0.60 

NOTE: Data are %ID/g ± SD in n = 4 mice per group.

*

P < 0.005, statistical significance between treated and nontreated groups.

Small-animal PET imaging of 64Cu-DOTA-pHLIP in PC-3 and LNCaP prostate tumors. Delineation of the tumor by uptake of 64Cu-DOTA-pHLIP was evident in both tumor models (Fig. 3) but to a greater extent in the LNCaP-bearing mice, whereas 64Cu-DOTA-K-pHLIP did not exhibit any targeting ability (data not shown). In the PC-3 model, the tumor-to-muscle ratios derived from the SUVs for uptake of native peptide at 1, 4, and 24 h were 1.45 ± 0.09, 2.67 ± 0.40, and 4.64 ± 1.08, showing a gradual increase in tumor uptake with concurrent washout from nontarget organs. The tumor-to-muscle ratios derived from the SUVs in the LNCaP tumor model were significantly higher (P = 0.0001), with values of 3.44 ± 0.50, 5.56 ± 0.21, and 6.55 ± 1.98 for the 1, 4, and 24 h time points, respectively.

Figure 3.

Representative small-animal PET image slices of 64Cu-DOTA-pHLIP (200 μCi/animal) in LNCaP (measured tumor pHe, 6.78 ± 0.29) and PC-3 (measured tumor pHe, 7.23 ± 0.10) tumor-bearing mice at various post-injection time points. Differences in tumor SUVs can be visualized by comparing the LNCaP (A) coronal and (B) transaxial slices to the PC-3 (C) transaxial and (D) coronal slices. White circle, tumor.

Figure 3.

Representative small-animal PET image slices of 64Cu-DOTA-pHLIP (200 μCi/animal) in LNCaP (measured tumor pHe, 6.78 ± 0.29) and PC-3 (measured tumor pHe, 7.23 ± 0.10) tumor-bearing mice at various post-injection time points. Differences in tumor SUVs can be visualized by comparing the LNCaP (A) coronal and (B) transaxial slices to the PC-3 (C) transaxial and (D) coronal slices. White circle, tumor.

Close modal

MRS directly measures the volume-average pHe of tumors. The acute biodistribution data and imaging results presented above show higher uptake and retention of 64Cu-DOTA-pHLIP in the nonalkalinized LNCaP tumors and suggest that the pHe of LNCaP tumors is more acidic than that of PC-3 tumors. To determine tumor-average pHe directly, 31P MRS measurements with the exogenous pHe marker 3-APP were used (Fig. 4). PC-3 and LNCaP tumor models were selected for pH measurements based on their significantly different SUVs as measured by PET, with LNCaP showing more uptake of the pHLIP than PC-3. This difference was mirrored in the pH values determined by magnetic resonance, with the LNCaP tumors showing a significantly more acidic average pHe (6.78 ± 0.29) when compared with the PC-3 tumors (7.23 ± 0.10; P = 0.039). Additional evidence that the 64Cu-DOTA-pHLIP targeted pHe was provided in the study in LNCaP tumors in which half of the mice were fed bicarbonated water before the biodistribution. As expected, in this experiment, the average pHe in the nonmodulated LNCaP tumors was more acidic than the average pHe of the tumors in mice having received 7 days of bicarbonated water (6.62 ± 0.35 versus 6.94 ± 0.56, respectively; P = 0.28). This correlates well with the data from the biodistribution where 64Cu-DOTA-pHLIP uptake in the nonmodulated LNCaP tumors was greater than the uptake in the tumors in mice having received 7 days of bicarbonated water (4.50 ± 1.71 versus 1.31 ± 0.60, respectively; P = 0.005). It should also be noted that the pHe measured in our MRS experiments is a volume-averaged pHe. Although this measured pHe provides a good indication of the acidity outside of the cell, it may not reflect the exact pH on the exterior surface of the cells and does not account for tumor heterogeneity. Thus, pHe as measured by MRS is an indicator, but not a direct measure, of the pH causing insertion.

Figure 4.

In vivo31P MRS of a PC-3 tumor. pHe is estimated from the chemical shift difference (in ppm) between the pHe indicator 3-APP and the pH-independent chemical shift of α-nucleoside triphosphate (α-NTP). The scale bar above the 3-APP resonance indicates a range of chemical shifts (δ) from 24.0 to 26.0 ppm, corresponding to the physiologic range of pHe (6.17-7.43).

Figure 4.

In vivo31P MRS of a PC-3 tumor. pHe is estimated from the chemical shift difference (in ppm) between the pHe indicator 3-APP and the pH-independent chemical shift of α-nucleoside triphosphate (α-NTP). The scale bar above the 3-APP resonance indicates a range of chemical shifts (δ) from 24.0 to 26.0 ppm, corresponding to the physiologic range of pHe (6.17-7.43).

Close modal

The physiologic differences between normal and tumor tissues provide an opportunity for the development of novel diagnostic and therapeutic agents specifically targeting cancer cells. However, the acidic extracellular environment in tumors has not been properly exploited, probably due to a lack of compounds whose properties change dramatically in the range of pH 6.0 to 7.5. The significance of the current work is that it proposes an innovative and novel method to target tumors based on an intrinsic physiologic property, the acidic extracellular environment, and thereby addresses an important problem in the diagnosis and monitoring or response to therapy of cancer. The method is based on the pH-selective interaction of the pHLIP with cell membranes. In this study, we have shown and validated a novel, pH-selective PET tracer, 64Cu-DOTA-pHLIP. This is the first time a peptide-based PET agent has been employed for the delineation of the pHe of tumors.

Our data show that 64Cu-DOTA-pHLIP is stable in tumors. None of the animals in any of the studies showed any adverse effects due to the administration of any of the pHLIP constructs. The biodistribution and PET imaging data showed retention of radioactivity in the tumors over 24 h. The tumor uptake of 64Cu-DOTA-pHLIP correlates well with in vivo fluorescence imaging studies, which showed that Cy5.5-pHLIP and Alexa 750-pHLIP stayed in tumors for several (>4) days (25). This long retention might be explained by the fact that, when the peptide is inserted into the cell membrane, it is protected from attack by proteases, allowing it to accumulate in tumor tissue in significant amounts.

There were two main factors that might contribute to the accumulation of pHLIP in nontumor tissue. While at normal, physiologic pH, the probability of pHLIP insertion into the cell membrane is low, the peptide still interacts with the surface of the membrane through its hydrophobic motif. Although this produces a small background signal, it also allows for a longer blood circulation of pHLIP, enhancing the probability of delivering functional imaging moieties or therapeutic cargo to the site of disease. The background signal decreases with time, whereas the signal in tumors remains static or is enhanced. A second reason for unwanted background is the relative instability of the 64Cu-DOTA chelation. 64Cu has been shown to dissociate in vivo from DOTA and DOTA-conjugates, undergoing subsequent metabolism and trans-chelation to superoxide dismutase and other proteins, resulting in increased accumulation in the blood and liver (27, 34, 35). This could account, in part, for the main differences in biodistribution between Cy5-pHLIP and 64Cu-DOTA-pHLIP, particularly the difference in liver uptake. In contrast to the PET results, the fluorescence data showed a very low uptake of Cy5.5-pHLIP by the liver (25), because the NIR dyes were conjugated covalently to the NH2 terminus of the peptide. We therefore believe that the apparent liver uptake of 64Cu-DOTA-pHLIP could be significantly decreased by optimizing the copper-chelating moiety. The cross-bridged cyclam chelator, CBTE2A, has shown improved in vivo stability and consequently a reduction in transchelation (35, 36) but requires elevated temperatures for copper complexation that may not compatible with the pHLIP construct.

The control peptide, 64Cu-DOTA-K-pHLIP, which had just two amino acid residues replaced, showed ∼40% less PC-3 tumor uptake as early as 1 h post-injection; we did anticipate that the mutant peptide would have lower uptake in the PC-3 model (the less acidic tumor) than the parent pHLIP. Also, small-animal PET imaging experiments showed that control peptide did not accumulate in tumors, consistent with previous fluorescence studies (25). Also, we observed lowering of K-pHLIP level in blood and tissues, although the equivalent mass of labeled K-pHLIP was administered and the data are normalized to the injected dose (%ID/g). We assume that rapid excretion of the K-pHLIP could be associated with its reduced affinity to the membrane at normal pH, in contrast to the parent pHLIP. Our thermodynamic studies indicated that pHLIP has high affinity to the membrane at normal pH (ΔGbinding is about −7 kcal/mol at 37°C; ref. 26), which is probably associated with the prolongated peptide circulation in the blood. The pHLIP affinity to the cell membrane increases in an environment of low pH, at which point pHLIP inserts into the membrane and adopts a stable transmembrane configuration. In contrast to pHLIP, the mutant peptide K-pHLIP cannot insert into the cell membrane to form a transmembrane α-helix (25), so it can be assumed that the tumor uptake we observed for 64Cu-DOTA-K-pHLIP in the acute biodistribution studies must be due to the passive diffusion of the construct into the tumor interstitium. Also, as stated, we cannot exclude the possibility that the creation of 64Cu bound to serum proteins by exchange with 64Cu-DOTA-pHLIP contributes to the background, especially at early time points. Therefore, although it is known that K-pHLIP does not target acidity, the use of K-pHLIP may not be an appropriate control system.

The selectivity of the pHLIP was shown by the modulation of pHe in LNCaP-bearing mice in which more acidic LNCaP tumors had greater uptake of 64Cu-DOTA-pHLIP than the less acidic (bicarb-modulated) LNCaP tumors. As can be seen from Table 2, it is clear that administering sodium bicarbonate significantly altered the uptake of the peptide only in the tumor and kidney. Unlike with K-pHLIP (Table 1), there were no significant differences in the uptake by the skin, which cannot be explained at this time. The use of bicarbonate is known to have a profound effect on pHe (12, 25, 29). For example, Raghunand and colleagues showed with 31P MRS that the pHe of MCF-7 human breast cancer xenografts can be effectively and significantly raised with sodium bicarbonate in drinking water (12). This was achieved with the mice drinking ad libitum water containing 200 mmol/L NaHCO3 for periods up to 90 days continuously, without any changes in subjective parameters and weight gain compared with control mice. In this current study, the average pHe in the nonmodulated LNCaP tumors was more acidic than the average pHe of the tumors in mice having received 7 days of bicarbonated water (6.62 ± 0.35 versus 6.94 ± 0.56, respectively; P = 0.28). This correlated well with the data from the acute biodistribution where 64Cu-DOTA-pHLIP uptake in the nonmodulated LNCaP tumors was significantly greater that the uptake in the tumors in mice having received 7 days of bicarbonated water (4.50 ± 1.71 versus 1.31 ± 0.60, respectively; P = 0.005).

Apart from the liver and blood uptake, the quantitative PET data and images are in general agreement with the biodistribution studies and the previous reported fluorescence studies (25), including uptake of the peptide by the kidney. The kidney has acidic regions and is a major site of catabolism of low molecular weight proteins. The kidney uptake can be reduced by providing mice with bicarbonate-buffered drinking water at pH 8.0 (12, 25, 29) or drugs such as acetozalomide, a carbonic anhydrase inhibitor that causes urinary alkalization. This reduction in kidney uptake with bicarbonate-buffered drinking water was confirmed in our own study presented in Table 2.

In summary, we have synthesized and evaluated the first generation of novel pHe-sensitive peptide PET agents for the delineation of low pHe in tumors. This is the first report of this novel class of PET imaging agents. Although the biokinetics of the agent are not optimal, additional strategies are currently under development to enhance tumor accumulation while reducing unwanted background uptake. This first-generation agent offers the possibility of designing a new class of noninvasive pH-selective PET imaging agents that will be useful for the imaging of a broad range of disease states. Multimodal (diagnostic + therapeutic) pHLIP with a NH2-terminal imaging label and COOH-terminal chemotherapeutic cargo (23) would afford the opportunity to monitor drug delivery, providing a key tool in efforts to predict therapeutic outcome.

No potential conflicts of interest were disclosed.

Note: Current address for A.L. Vāvere: St. Jude Children's Research Hospital, Memphis, TN 38105.

Grant support: NIH grants R24 CA83060 and P30 CA91842 and Department of Defense grant PC040435 (J.S. Lewis), NIH grant R01 GM073857 (D.M. Engelman), NIH grant R01 133890 (O.A. Andreev, D.M. Engelman, and Y.K. Reshetnyak), Department of Defense grant PC050351 (Y.K. Reshetnyak), Department of Defense grant BC061356 and NIH grant R21 CA125280 (O.A. Andreev), and NIH salary support F32 CA110422-03 (A.L. Vāvere).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Artem Lebedev for help and Chris Sherman, Amanda Roth, Nicole Fettig, Margaret Morris, Lori Strong, and Susan Adams for technical support.

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