In Vivo Distribution and Therapeutic Efficacy of Radioiodine-Labeled pH-Low Insertion Peptide Variant 3 in a Mouse Model of Breast Cancer

Purpose Extracellular acidity is a marker of highly aggressive breast cancer (BC). pH-low insertion peptides (pHLIPs) target the acidic tumor microenvironment. This study evaluates the distribution and therapeutic efficacy of radioiodine-labeled pHLIP variant 3 (Var3) in a mouse model of BC. Methods The binding of fluorescein isothiocyanate (FITC)- or radioiodine-125 (125I) labeled Var3-pHLIP to MDA-MB-231, 4T1, and SK-BR-3 BC cell lines under different pH values was evaluated in vitro. The distribution of 125I-labeled Var3-pHLIP and wild-type- (WT-) pHLIP in tumor-bearing mice was analyzed in vivo using micro-SPECT/CT imaging. The therapeutic efficacy of radioiodine-131 (131I)-labeled Var3-pHLIP in MDA-MB-231 xenografts was evaluated by relative tumor volume measurement and immunohistochemical analysis. Results The binding ability of FITC- or 125I-labeled Var3-pHLIP to tumor cells increased with the decrease in pH. The tumor-to-background ratio of 125I-Var3-pHLIP in BC xenografts showed the best imaging contrast at 24 h or 48 h postinjection. The uptake of 125I-Var3-pHLIP in MDA-MB-231 xenografts at 2 h postinjection was significantly higher than that of 125I-WT-pHLIP (3.76 ± 0.37 vs. 2.87 ± 0.60%ID/g, p = 0.046). The relative tumor volume in MDA-MB-231 xenografts was significantly lower in the 131I-Var3-pHLIP-treated group than in the groups treated with Var3-pHLIP (p = 0.027), 131I (p = 0.001), and saline (p < 0.001). The 131I-Var 3-pHLIP group presented a lower expression of Ki67 and a higher expression of caspase 3. Conclusion Radioiodine-labeled Var3-pHLIP effectively targeted BC cells in an acidic environment and inhibited the growth of MDA-MB-231 xenografts by ionizing radiation.


Introduction
Breast cancer (BC) is the leading cause of cancer death among women [1]. Triple-negative BC (TNBC) is a highly aggressive BC subtype with a high rate of relapse and metastasis and a low survival rate [2]. Chemotherapy resistance is common in TNBC, and the efficacy of endocrine and antihuman epidermal growth factor receptor-2 (HER2) therapies is limited [3]. Therefore, new effective targeted therapies are needed.
Acidosis in the tumor microenvironment promoted by the Warburg effect is a hallmark of cancer. The extracellular pH in this environment is below 6.5 because of H + produc-tion and excretion, whereas extracellular pH in healthy tissue under physiologic conditions is 7. 2-7.4. In addition, the acidic microenvironment is not affected by clonal selection and thus is promising for diagnostic imaging and targeted therapy [4].
This study compared the in vivo distribution of 125 I-WT-pHLIP and 125 I-Var3-pHLIP in mice-bearing human BC xenografts and investigated the therapeutic efficacy of 131 I-Var3-pHLIP in MDA-MB-231 xenografts.  WT-pHLIP and Var3-pHLIP were labeled with 125 I for in vivo imaging experiments, and Var3-pHLIP was labeled with 131 I for therapeutic efficacy experiments. Radioactive iodine binds to tyrosine residues in pHLIPs. Peptides were dissolved in 200 μL of 0.02 M PBS (pH 7.4) to reach a concentration of 1 mg/mL and mixed with 10 μL of Na 125 I (74 MBq) or 20 μL of Na 131 I (925 MBq). Chloramine-T (20 μL, 5 mg/mL) was added to the test tubes and mixed for 70 s at room temperature. The reaction was stopped by adding 200 μL of sodium metabisulfite (5 mg/mL). The peptides were eluted in ethanol through a C18 column equilibrated with 20 mL of 100% ethanol and 20 mL of distilled water. The radioactive purity of 125 I-WT-pHLIP, 125 I-Var3-pHLIP, and 131 I-Var3-pHLIP was determined using thin-layer chromatography. Furthermore, the radiochemical purity of 125 I-WT-pHLIP, 125 I-Var3-pHLIP, and 131 I-Var3-pHLIP was measured, and chemical stability was determined at 24 and 48 h at room temperature (25°C) in PBS and at 37°C in serum.

Fluorescence
Imaging of FITC-Labeled Peptides In Vitro. DMEM medium and MES buffer were mixed at different volume ratios (100 : 0, 50 : 1, 25 : 2, 25 : 6, and 5 : 2) to simulate extracellular environments with a pH of 7.8, 7.4, 7.0, 6.6, and 6.2, respectively. MDA-MB-231, 4T1, and SK-BR-3 cells were seeded in 24-well plates at a density of 5 × 10 4 . After cell attachment, FITC-WT-pHLIP and FITC-Var3-pHLIP (50 μg/mL) were added to the wells in triplicate. Cultures were incubated on a rotary shaker for 30 min. The culture medium was aspirated, and cells were washed three times with a solution of the respective pH without FITClabeled peptides. Subsequently, 250 μL of DAPI staining solution (100×) (Beyotime Biotechnology, Shanghai, China) diluted with DMEM medium at the respective pH was added to each well. The cells were incubated at 37°C in a humidified incubator with 5% CO 2 for 10 min, washed three times with PBS at the respective pH, and imaged on a fluorescence microscope (×100).

2.5.
In Vitro Cell Binding Assay. Cells were incubated with 125 I-WT-pHLIP or 125 I-Var3-pHLIP for 1 h at 37°C in the medium at different pH values ranging from 6.2 to 7.8 (6.2, 6.6, 7.0, 7.4, 7.8). At the same time, cells in the medium at pH 6.2 were further incubated with 125 I-labeled peptide with an excess of unlabeled peptide (25 μg/well). The supernatant was aspirated into a tube. The cells were then washed twice with PBS of the corresponding pH to remove unbound radioactivity. The wash solution was also added to the supernatant tube containing free radioactivity (F). The cells with bound radioactivity (B) were completely lysed with 300 μl sodium hydroxide and aspirated into a cell tube. The radioactive counts of the cell and supernatant tube were measured by a γ counter. The cell binding fraction of the 125 I-labeled peptides was calculated as B/ðB + FÞ × 100%. All experiments were performed in triplicate.

Measurement of Cell Viability Using the Cell Counting
Kit-8 (CCK-8) Assay. MDA-MB-231, 4T1, and SK-BR-3 cells were seeded in 96-well plates (5 × 10 3 cells per well) and cultured for 12 hours. Cultures were divided into a treatment group (pHLIP [50 μg/mL] in 100 μL of culture medium at different pH values [7.8, 7.4, 7.0, 6.6, 6.2]) and control group (100 μL of culture medium at the respective pH). The assays were performed in triplicate and repeated independently three times. The cells were incubated at 37°C in an incubator for 30 min, and the culture medium was aspirated. The CCK-8 solution was mixed with DMEM at a ratio of 1 : 9, and 100 μL of the solution was added to each well. Absorbance was measured at 450 nm in a microplate reader. Absorbance in wells containing medium at pH 7.8 without pHLIP was defined as 100% cell viability, and the percentage of cell viability was calculated. Three days before micro-SPECT/CT imaging, 300 μL of 1% Kl solution was administered to mice by oral gavage once a day, and 0.1% Kl was added into the drinking water to block thyroid gland activity. Mice-bearing MDA-MB-231, 4T1, or SK-BR-3 tumors were intravenously injected with 3.7 MBq of 125 I-WT-pHLIP or 125 I-Var3-pHLIP. Each group contained four mice. The mice were anesthetized by isoflurane  3 Molecular Imaging inhalation, placed in the prone position, and scanned using a four-head SPECT/CT system (U-SPECT/CT, MI-Lab, Netherlands) and dedicated multipinhole apertures with a diameter of 1.5 mm at 1, 2, 4, 24, and 48 h after injection of 125 Ilabeled peptides. CT images were acquired after static SPECT imaging (10 min/frame). The images were reconstructed using U-SPECT REC software and analyzed using PMOD software version 3.9 (Switzerland). Regions of interest (ROIs) were drawn in the tumors and organs, including the brain, heart, liver, lungs, kidneys, intestine, and bladder, at various time points. The radioactivity per gram (ID%/g) in the ROIs was measured to obtain the dynamic tumorto-background ratio (TBR, muscle tissue was used as the background) and the in vivo distribution and kinetics of 125 I-WT-pHLIP and 125 I-Var3-pHLIP in the xenograft models of BC.
2.9. Therapeutic Efficacy of 131 I-Var3-pHLIP in MDA-MB-231 Xenografts. Twenty mice-bearing MDA-MB-231 human BC xenografts were divided into four groups of five animals. Group 1 was injected with 131 I-Var3-pHLIP in the tail vein on days 0 and 3 (29.6 MBq each day). Groups 2, 3, and 4 were injected with the same volume of Var3-pHLIP, 131 I (29.6 MBq), and saline solution, respectively. Tumor size was measured every 2 to 4 days postinjection (pi) for 26 days using a caliper. Tumor volumes (V, mm [3]) were calculated using the formula: V = ða × b 2 Þ/2, where a and b are tumor length and width, respectively. Relative tumor volumes were calculated as V/V 0 (V 0 , tumor volume before treatment). The body weight of mice was also determined.
2.10. Immunohistochemical Analysis. The animals were sacrificed by cervical dislocation. The tumors were excised, fixed in paraformaldehyde, embedded in paraffin, and sec-tioned. The sections were mounted on glass slides, dewaxed in xylene, dehydrated through a graded ethanol series, and incubated in 3% hydrogen peroxide for 25 min. The samples were incubated with rabbit anti-human caspase 3 (1 : 100, Servicebio, Wuhan, China) and rabbit anti-human Ki67 (1 : 500, Servicebio, Wuhan, China) antibodies for 1 h at room temperature. Slices were washed with PBS thoroughly and incubated with HRP-labeled secondary antibody for 1 h at room temperature. Then, DAB kit was used for visualization and examined by light microscopy, and cells containing brown pigment were considered positively stained.
2.11. Statistical Analysis. Data were analyzed using the SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) and GraphPad PRISM version 7.0 (GraphPad Software, La Jolla, CA, USA) and represented as means ± SD. Statistical significance was analyzed by one-way analysis of variance (ANOVA) with Bonferroni correction and was established at p < 0:05.

Fluorescence Imaging and Cell Viability at Different pH
Values. Fluorescence imaging showed that the in vitro distribution of FITC-Var3-pHLIP and FITC-WT-pHLIP in the cell membrane decreased from pH 6.2 to pH 7.8 in the cell lines MDA-MB-231 (Figures 1(a) and 1(b)), 4T1, and SK-BR-3 (Supplemental Figures 1(a)-1(b)). The CCK8 results showed no significant difference in cell viability between MDA-MB-231 (Figures 2(a) and 2(b)), 4T1, and SK-BR-3 (Supplemental Figure 2) treated with or without these two types of pHLIP at the same pH value. Furthermore, there was no significant difference in cell viability between pH treatments.
The radioactive count in major organs or tissues of MDA-MB-231 tumor-bearing nude mice (Tables 1 and 2) and other two BC mice models (Supplemental Table 1-4) decreased over time. The uptake of 125 I-Var3-pHLIP and 125 I-WT-pHLIP was high in the heart, liver, lungs, bladder, and kidneys.

Discussion
The increased acidity in the extracellular environment due to the Warburg effect in highly aggressive cancers, including TNBC, where endocrine and molecular therapies are ineffective, is a new therapeutic target. pHLIPs sense the pH in the vicinity of the plasma membrane. The protonation of two aspartate residues in the spanning domain of pHLIP in an acidic environment allows the insertion of this peptide across the plasma membrane [12].
WT-pHLIP, Var3-pHLIP, and Var7-pHLIP are currently the most promising peptides of the pHLIP family. Positron emission tomography and fluorescence studies showed that, compared with WT-pHLIP, the distribution of Var3-pHLIP and Var7-pHLIP in the liver was lower probably because of their lower hydrophobicity at physiological pH. Var7-pHLIP is shorter and less hydrophobic than Var3-pHLIP, increasing clearance from the blood and liver but potentially decreasing its uptake by tumors [9,14]. Therefore, Var3-pHLIP was selected for this study. A previous study showed that the uptake of [17]F and 64 Culabeled Var3-pHLIP in 4 T1 xenografts was 10:6 ± 2:3%ID/ g at 4 h pi and 19:6 ± 2:3%ID/g at 24 h pi, respectively, and the TBR was 6:9 ± 1:9 and 16:0 ± 3:0, respectively, showing the highest tumoral uptake and tumor-to-background contrast among the three pHLIPs [9]. Our results showed that the tumor uptake and TBR of 125 I-Var3-pHLIP were higher than those of 125 I-WT-pHLIP; however, the significance differed among the three cell lines. The maximum uptake of 125 I-Var3-pHLIP was earlier (2 h vs. 24-48 h) and lower than that of 64 Cu-Var3-pHLIP. Similarly, the maximum uptake of 125 I-Var7-pHLIP was at 1 h pi [16], and the near-infrared fluorescence of this pHLIP was the highest at 2-4 h pi. The discrepancy in maximum uptake may be due to differences in the labeling methods. The labeling of pHLIPs with positron-emitting nuclides requires the addition of a chelating agent, which may reduce hydrophobicity and increase peptide aggregation around tumor cell membranes. The replacement of the hydrogen atom on the phenyl ring of the tyrosine residue with 125 I or 131 I during the iodination of Var3-pHLIP using the chloramine-T method has little effect on peptide hydrophobicity.
The ability of pHLIP to target the acidic microenvironment of tumors allows its use as drug carriers. Studies have covalently bound therapeutic drugs that cannot cross the plasma membrane (phallacidin [18], amanitin [17], proteaseactivated receptor 1-activating peptide [19], antimiR-155 [11]) to the hydroxyl terminus of pHLIP, allowing drug release to tumors. 131 I labeling is simpler than conjugating the above drugs to pHLIP. The present study showed that 131 I-Var3-pHLIP inhibited tumor growth in MDA-MB-231 xenografts through ionizing radiation damage. However, treatment had some limitations. First, 131 I-Var3-pHLIP was widely distributed in blood-rich organs such as the liver, heart, and lungs, increasing radiation damage to these normal organs. Second, 131 I-Var3-pHLIP was cleared quickly from tumors, and its distribution in tumor tissue at 24 h pi was 12.5% of that at 2 h pi; thus, repeated injections are necessary to achieve the radiation dose required for effective treatment. Therefore, developing pHLIP variants with higher retention in tumor and faster blood clearance is necessary to improve the therapeutic efficacy and safety of 131 I-labeled pHLIPs. Third, previous studies have shown that local inflammation was closely associated with increased acidity of extracellular microenvironment in the involved organs and tissues, which makes pHLIPs accumulate in the inflammatory diseases such as arthritis [7], pneumonia [20], and active vulnerable plaques [21]. Thus, inflammatory disease may increase the interpretation complexity of pHLIP for cancer imaging and tumor selectivity of pHLIP as a therapeutic vehicle.

Conclusions
Radioiodine-labeled Var3-pHLIP effectively targeted breast cancer cells in an acidic environment and inhibited the growth of MDA-MB-231 xenografts by ionizing radiation.

Data Availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.  . Tumor samples were immediately snap-frozen and stored at -80°C, and the specimen collection was approved by the Medical Ethical Committee of the hospital.