Investigation of SP94 Peptide as a Specific Probe for Hepatocellular Carcinoma Imaging and Therapy

SP94 (SFSIIHTPILPL), a novel peptide, has shown specific binding to hepatocellular carcinoma (HCC) cells. We aimed to investigate the capability of SP94 as a targeting probe for HCC imaging and therapy following labeling with technetium-99m (99mTc) and rhenium-188 (188Re). HYNIC-SP94 was prepared by solid phase synthesis and then labeled with 99mTc. Cell competitive binding, internalization assay, in vitro and in vivo stability, biodistribution and micro-single photon emission computed tomography /computed tomography (SPECT/CT) imaging studies were performed to investigate the capability of 99mTc tricine-EDDA/HYNIC-SP94 as a specific HCC imaging probe. Initial promising targeting results inspired evaluation of its therapeutic effect when labeled by 188Re. HYNIC-SP94 was then labeled again with 188Re to perform cell apoptosis, microSPECT/CT imaging evaluation and immunohistochemistry. Huh-7 cells exhibited typical apoptotic changes after 188Re irradiation. According to 99mTc tricine-EDDA/HYNIC-SP94 microSPECT/CT imaging, tumor uptake was significantly decreased compared with that of pre-treatment with 188Re-HYNIC-SP94. The immunohistochemistry also displayed obvious necrosis and apoptosis as well as inhibition of proliferation in the 188Re-HYNIC-SP94 treatment group. The results supported that 99mTc tricine-EDDA/HYNIC-SP94 is able to target HCC cells and 188Re-HYNIC- SP94 holds potential as a therapeutic agent for HCC, making 99mTc/188Re-HYNIC-SP94 a promising targeting probe for HCC imaging and therapy.

Scientific RepoRts | 6:33511 | DOI: 10.1038/srep33511 Since SP94 can specifically bind to HCC, we hypothesized that it could be used as a probe for molecular imaging and even for radioimmunotherapy, after being labeled with a suitable radionuclide such as 99m Tc or 188 Re. 99m Tc labeling remains a striking approach for peptide SPECT/CT imaging because of its 140 keV γ -photon emission and a manageable 6 h half-life. Additionally, 99m Tc is highly cost-effective and can be easily obtained from a commercial 99 Mo-99m Tc generator [12][13][14][15] . 188 Re is an attractive radionuclide for targeted radiotherapy and imaging because of its optimal physical characteristics of high-energy β-particles (E max = 2.12 MeV) and γ -photons (155-keV) in 15% abundance 16,17 . In addition, strong tissue penetration effects (max 11 mm, mean range 3.5 mm), suitable half-life (16.9 hours) and convenient availability from a generator have greatly increased the research and clinical application of 188 Re-labeled radiopharmaceuticals 18,19 .
Although SP94 has been proposed as a targeting peptide to deliver drugs to treat advanced HCC 8,10,20 , SP94 as an imaging and therapeutic agent for HCC using the same pharmacophore but different isotopes has not been investigated so far. Therefore, in this study, SP94 was radiolabeled with 99m Tc and 188 Re, and in vitro and in vivo studies were performed to evaluate its capability as a specific probe for HCC imaging and therapy.
In vitro HCC cell competitive receptor binding assay. Results of a comparative displacement assay in Huh-7 cells were plotted as a sigmoid curve for the displacement of 99m Tc tricine-EDDA/HYNIC-SP94 when concentrations of HYNIC-SP94 increased (from 0.001 nM to 10000 nM). The binding rate was plotted as percentage binding of total activity added. The IC 50 value was 4.49 ± 0.20 nM, which indicated that SP94 peptide showed a high binding affinity on Huh-7 cell membranes. An example of the competitive binding curve is shown in Fig. 2A.
Internalization assay. The uptake rates of 99m Tc tricine-EDDA/HYNIC-SP94 by Huh-7 and Hela cells were determined (Fig. 2B). At 0.5 h after incubation of 99m Tc tricine-EDDA/HYNIC-SP94 with Huh-7 cells, the uptake rate was 8.41 ± 0.31% and increased to 20.92 ± 1.38% at 4 h. However, Hela cells showed a significantly lower uptake rate of only 7.69 ± 0.03% at 4 h (P < 0.001). In the blocking study performed in Huh-7 cells, the tracer uptake rate was 8.46 ± 0.22% at 4 h, which was approximately 2.5-fold less than that of the non-blocked group.  Indeed, statistical analysis revealed that there was a significant difference in uptake between the unblocked and blocked groups (P < 0.001). These data suggested that 99m Tc tricine-EDDA/HYNIC-SP94 specifically binds to and penetrates the Huh-7 cell membranes.
In vivo stability. It was found that 99m Tc tricine-EDDA/HYNIC-SP94 could maintain about 66% of the radiochemical purity (RCP) within 60 min, which indicated that it has a suitable in vivo stability regarding tumor targeting. Furthermore, no sign of free 99m Tc release was found in the following biodistribution study based on low radioactivity uptakes in stomach and thyroid. With comprehensive analyses of blood samples using reversed-phase high-performance liquid chromatography (RP-HPLC) and biodistribution, favorable in vivo stability of 99m Tc tricine-EDDA/HYNIC-SP94 was demonstrated.
Biodistribution. Biodistribution of 99m Tc tricine-EDDA/HYNIC-SP94 was evaluated in Huh-7 tumor-bearing mice. Results are shown in Table 1 as the percentage of injected dose per gram of tissue (%ID/g). Uptake in the kidneys was highest (7.40 ± 2.66% ID/g) because radioactivity was rapidly cleared from the blood via the preferred renal-urinary route. Aside from the bladder and kidneys, labeled peptide showed moderate uptake values in the Huh-7 tumors, which was higher at 0.5 h (1.02 ± 0.26% ID/g) than at 1.5 h post-injection (p.i.) (0.74 ± 0.07% ID/g). The tumor uptake only reduced significantly (0.34 ± 0.09% ID/g; P = 0.024) after injection of the blocking agent, indicating specific receptor targeting of 99m Tc tricine-EDDA/HYNIC-SP94.
MicroSPECT/CT imaging. Figure 3 shows microSPECT/CT images at 0.5 h after injection of 99m Tc tricine-EDDA/HYNIC-SP94 with or without the blocking agent in Huh-7 and Hela tumor-bearing mice. The Huh-7 tumor could be seen clearly at 0.5 h after injection of 99m Tc tricine-EDDA/HYNIC-SP94 (Fig. 3A), while no radioactivity accumulation was observed in the Hela tumors at the same time point (Fig. 3B). In addition, the tumor uptake was not visualized in the blocked mice (Fig. 3C).

Results for 188 Re-HYNIC-SP94
Radiolabeling and stability of 188 Re-HYNIC-SP94. The RCP and the specific activity of 188 Re-HYNIC-SP94 were 96% and 1.2 GBq/μ mol, respectively. The in vitro stability studies showed that when incubated in normal   .07 ± 0.63% to 11.10 ± 1.50% (P = 0.012). In addition, in the SP94, blank and Hela groups, no obvious apoptotic cells were observed (Fig. 5A). As seen in Fig. 5B, 188 Re-HYNIC-SP94 treatment significantly increased apoptosis of Huh-7 cells when compared with the 188 Re, blank and SP94 treatment groups. These results all indicated that SP94 can target Huh-7 cells and induce apoptosis by virtue of radiation from labeled 188 Re.  Hematoxylin and eosin (H&E) staining and immunohistochemistry. For major organs, such as lungs, liver and kidneys, no macroscopic abnormalities and histopathological damage were observed in any of the irradiated and control mice, suggesting no toxicity in the treated mice (Fig. 6B). CD34 is a transmembrane glycoprotein on cells, which can promote the aggregation of endothelial progenitor cells to induce angiogenesis and can be used to evaluate the risk of recurrence and prognosis. After treatment with 188 Re-HYNIC-SP94, the amount of newly generated vessels was significantly reduced when compared with the other groups, indicating that 188 Re-HYNIC-SP94 effectively inhibited the invasion and metastasis of cancer cells. Bax is a pro-apoptotic protein and was highly expressed only in Huh-7 tumor tissue after treatment with 188 Re-HYNIC-SP94, which showed as yellow-brown or brown tissue (Fig. 6C). The expression of bax can be used to evaluate the extent of apoptosis, which reflects the therapeutic efficacy of 188 Re-HYNIC-SP94. Ki-67 is a nuclear antigen that only exists in proliferating cells and is widely used as a marker of cell proliferation. Ki-67 expression in 188 Re-HYNIC-SP94-treated Huh-7 tumor tissue was lower (12.33 ± 0.02%) than that of the control group (41.33 ± 0.04%) (P = 0.001) and the 188 Re treated group (30.67 ± 0.03%) (P = 0.001), suggesting that 188 Re-HYNIC-SP94 could inhibit the excessive proliferation of HCC cells.

Discussion
SP94 peptide, comprising twelve amino acids, can selectively bind to HCC cells and is under development as a targeting peptide for therapeutic purposes 8,10,20 . Recently, polymer coupled with SP94 peptide was found to exhibit a 10 4 -fold greater avidity for HCC than for hepatocytes 10,11 . Based on these results, SP94 may be a good candidate as an imaging or therapeutic probe to detect HCC, when labeled with 99m Tc or 188 Re.
There are many bifunctional chelating agents (such as HYNIC and NHS-MAG3) that could be used for both 99m Tc and 188 Re labeling of biomolecules. Among them, HYNIC has demonstrated a number of advantages such as high RCP and good stability, as well as a predominantly renal excretion 21 . Because HYNIC can only occupy one or two coordination positions on the radionuclide, coligands (such as tricine and EDDA) are necessary to complete the radionuclide coordination sphere 22,23 . Decristoforo et al. 24,25 have used EDDA as a coligand, showing that it has advantages over other coligands in terms of peptide labeling. However, Erfani et al. 26 found that in a group of different coligands, tricine offered the best radiolabeling efficiency. In this study, we tested two coligand systems using tricine and tricine/EDDA. The differences between the two coligand systems did not appear very pronounced; however, because of its better stability, 99m Tc tricine-EDDA/HYNIC-SP94 was selected for further evaluation. 99m Tc tricine-EDDA/HYNIC-SP94 was synthesized with high radiochemical yield (> 96%) and proved highly stable in normal saline, FBS and PBS (Fig. 1).
The IC 50 value for HYNIC-SP94 was 4.49 ± 0.20 nM from competitive binding assay ( Fig. 2A), which suggested that SP94 peptide had a high binding affinity on Huh-7 cells. Percentage of internalization was around 20% for Huh-7 cells (Fig. 2B), indicating that SP94 peptide has the ability to penetrate the HCC cell membranes. Based on in vivo stability assay, the labeled peptide could maintain about 66% of the RCP within 60 min, indicating a fair stability according to its in vivo tumor targeting and pharmacokinetics. In vivo biodistribution experiments showed rapid tumor uptake, higher at 0.5 h (1.02 ± 0.26% ID/g) and dropping slightly at 1.5 h (0.74 ± 0.07% ID/g) p.i. 99m Tc tricine-EDDA/HYNIC-SP94 uptake was highest in the kidneys but 99m Tc tricine-EDDA/HYNIC-SP94 was rapidly cleared from the blood via the preferred renal-urinary route, thus the retention time was quite short, owing to the kidney being the major organ for metabolism. Pre-injection with the blocking agent did not reduce kidney uptake or excretion but significantly decreased tumor uptake of 99m Tc tricine-EDDA/HYNIC-SP94. In other organs such as liver and lungs, a moderate radioactivity accumulation was observed, possibly caused by the saturation effect of SP94 in the distribution mechanisms (Table 1). Using SPECT/CT imaging, Huh-7 tumors were clearly delineated from normal tissues as early as 0.5 h after injection of 99m Tc tricine-EDDA/HYNIC-SP94 (Fig. 3A), but no obvious tracer could be seen in Hela tumors (Fig. 3B) and tumor uptake of the radiolabeled peptide was significantly reduced (P = 0.024) in the blocked group (Fig. 3C). The moderate uptake of 99m Tc tricine-EDDA/HYNIC-SP94 for the liver was due to the lipophilicity of this tracer; however, concentration differences in radioactivity enabled the tumor area to be distinguished from the liver. Imaging and biodistribution studies further revealed that 99m Tc tricine-EDDA/HYNIC-SP94 exhibited clear targeting of HCC.
The current major limitation to use this peptide is its fast metabolism in plasma by endogenous peptidases under physiological conditions 27 . Also, background interference from the liver may hinder the detection of tumors to some degree. However, as discussed earlier, when SP94 peptide is conjugated to polymers, its in vivo behavior is improved, encouraging us to make further modification to this peptide 10,11 . Moreover, in vitro and in vivo results all supported that 99m Tc tricine-EDDA/HYNIC-SP94 can target HCC cells and this finding may have potential application in the design of an internal radiotherapy agent for HCC, when the peptide was labeled again with 188 Re.
During the past decades, radiolabeled peptides have been investigated as potential therapeutic agents, both in research and in clinic; for example, 188 Re-HYNIC-trastuzumab enhanced the effect of apoptosis in HER2-overexpressing breast cancer cells and 188 Re-labeled HEDP could be used for internal radiation therapy to treat painful bone metastases 28,29 . The β ray (mean range 3.5 mm) emitted by 188 Re causes little injury to surrounding tissues and can induce apoptosis for the internal radiation therapy of tumors. In this study, we revealed that 188 Re-HYNIC-SP94 led specifically to apoptosis of HCC cells, in a radioactivity dose-and time-dependent manner. The data suggested that 188 Re-HYNIC-SP94 has the potential for use as a therapeutic radiopharmaceutical in HCC treatment.
In this study, 188 Re-HYNIC-SP94 could be labeled with 96% of RCP and remained at a level > 90% after 48 h of incubation with FBS (Fig. 4); these properties suggested its therapeutic function was worthy of further evaluation. TUNEL staining showed significant apoptosis of Huh-7 cells after 188 Re-HYNIC-SP94 treatment compared with the control groups (Fig. 5). This may has been due to the deep penetration of SP94 peptide and irradiation on the tumor cells by the energetic β-emissions 30 . The 99m Tc tricine-EDDA/HYNIC-SP94 microSPECT/CT images of Huh-7 tumor-bearing mice showed significant differences in tumor uptake before and after injection of 188 Re-HYNIC-SP94 (Fig. 6A), revealing the loss of specific receptors in the cell membranes because of cell apoptosis induced by radiation from 188 Re. This indicated that 188 Re-HYNIC-SP94 is a favorable therapeutic agent in HCC treatment. The above results were consistent with H&E staining and immunohistochemistry analysis of bax, which showed massive necrosis and apoptosis in Huh-7 tumors but not in Hela tumors, after treatment by 188 Re-HYNIC-SP94. The immunohistochemistry analysis of CD34 showed the suppression of angiogenesis after 188 Re-HYNIC-SP94 treatment. Ki-67 expression in 188 Re-HYNIC-SP94-treated Huh-7 tumor tissue was lower than that of other treatment groups (Fig. 6C). These findings demonstrated that 188 Re-HYNIC-SP94 can induce cell apoptosis and inhibit excessive HCC cell proliferation, providing evidence of its potential for the internal radiotherapy of HCC. Although tumor size changes were not observed in this study, it was interesting to find apparent tumor receptor and pathological changes from imaging and other in vitro assays. Furthermore, it was observed from H&E staining and immunohistochemistry analyses of normal organs such as kidneys, liver and lungs, together with the biodistribution studies, that no further damage was observed on the main organs aside from the tumors.
Although some small molecules and peptides labeled by 188 Re have been shown to posses the capacity for treatment of HCC, our imaging and therapeutic probes ( 99m Tc/ 188 Re-HYNIC-SP94) still have potential applications, besides commercial availability, easy labeling, tumor specificity, fast clearance et al., most important is that HYNIC-SP94 could be served as an hybrid imaging and therapeutic agent for HCC by using of the same pharmacophore but different isotopes. Therefore, it will be meaningful that SP94 was radiolabeled with 99m Tc and 188 Re to evaluate its capability as a specific probe for HCC imaging and therapy.

Conclusion
In vitro and in vivo results all supported that 99m Tc tricine-EDDA/HYNIC-SP94 can target HCC cells. However, its in vivo behavior requires further modification, by conjugating the peptide with other carriers such as nanoparticles or polymers to improve tumor targeting and pharmacokinetics. Nevertheless, its high specificity to HCC cells warrants its potential as a HCC radiotherapeutic agent when labeled with 188 Re. TUNEL assay, SPECT/CT imaging and immunohistochemistry results revealed that 188 Re-HYNIC-SP94 has the potential as a therapeutic radiopharmaceutical agent in HCC treatment. The inhibitory effects in Huh-7 cells suggested that 188 Re-HYNIC-SP94 may be therapeutically beneficial for HCC patients in the future. When labeled with 99m Tc or 188 Re, HYNIC-SP94 has the potential for using as a diagnostic or therapeutic agent for HCC.
In vitro HCC cell competitive receptor binding assay. The binding affinity of HYNIC-SP94 was assessed via a competitive displacement assay with 99m Tc tricine-EDDA/HYNIC-SP94 35 . Briefly, Huh-7 cells (1 × 10 6 ) and 99m Tc tricine-EDDA/HYNIC-SP94 (370 kBq) were added to each test tube in the presence of increasing concentrations of the SP94 peptide (from 0.001 nM to 10000 nM) and incubated for 2 h at 37 °C. To remove unbound 99m Tc tricine-EDDA/HYNIC-SP94, the cells were washed three times with cold PBS (1× , pH 7.2) and then centrifuged (600 rpm, 5 min). Radioactivity was determined using a γ -counter. The IC 50 value was calculated by fitting experimental data with nonlinear regression using GraphPad Prism (GraphPad Software, San Diego, CA, USA).
Preparation of animal models. Four-to six-week-old male athymic nude mice were used for preparation of the xenograft tumor models. Huh-7 and Hela cells were subcutaneously injected at a concentration of 5 × 10 6 cells/mouse in the right front flank of the mice. The mice were ready for biodistribution and microSPECT/ CT imaging study when the tumors reached an average diameter of 0.8~1.0 cm (3~4 weeks after inoculation). Animal care and experimental procedures were accordant with the Helsinki Declaration and were approved by the institutional animal care committee.
Biodistribution study. Fifteen Huh-7 tumor-bearing mice were randomly divided into three groups. 99m Tc tricine-EDDA/HYNIC-SP94 (~0.74 MBq/mouse) in 0.1 mL of saline was injected via tail vein. At 0.5 and 1.5 h p.i., the mice were anesthetized via 2% isoflurane and sacrificed by cervical dislocation. Immediately after sacrifice, the blood, tumors and organs of interest (heart, lungs, liver, spleen, stomach, small intestine, large intestine, kidneys, muscle and thyroid) were harvested, washed with saline and weighed. Radioactivity was determined using a γ -counter. To determine the specific uptake of 99m Tc tricine-EDDA/HYNIC-SP94, 5 mice received an intravenous injection of 200 μ g of unlabeled HYNIC-SP94 as a blocking agent 30 min prior to injection of 99m Tc tricine-EDDA/HYNIC-SP94 (~0.74 MBq/mouse). The mice were sacrificed 30 min p.i. and the experiments performed as described above. Absorption of organs and tissues was calculated as a percentage of the injected dose per gram of tissue mass (%ID/g).
SPECT/CT imaging. SPECT/CT images were acquired at 0.5 and 1.5 h after 99m Tc tricine-EDDA/ HYNIC-SP94 (~37 MBq) administration. For the blocking study, Huh-7 tumor-bearing mice were administered an excessive dose (200 μ g/mouse) of non-labeled HYNIC-SP94 0.5 h prior to injection of 99m Tc-labeled peptide. Mice were placed in the prone position under 2% isoflurane anesthesia and whole-body imaging was performed. The CT scanning parameters used were as follows: tube voltage, 45 keV; current, 150 μ A; frame resolution, 256 × 512; and exposure time, 500 ms/frame. Each scan took about 4 min. SPECT was performed after CT scanning with energy peak, 140 keV; matrix, 256 × 256; window width, 10%; resolution, 1 mm/pixel; and scan time, 40 s/projection, with 24 projections in total. The whole-body scan for each mouse took about 20 minutes. Three-dimensional ordered subset expectation maximization images were reconstructed using HiSPECT (Bioscan, Washington DC, USA).

Re-HYNIC-SP94 experiments
Labeling and stability. The labeling of 188 Re-HYNIC-SP94 was carried out as previously described 16,28,30 . Briefly, 50 μ L of GH (100 mg/mL) and 10~80 μ L of SnCl 2 solution (10 mg/mL) were reacted with 188 Re-perrhenate solution (37 MBq) for 1 h at room temperature. An amount of 50 μ g or 200 μ g HYNIC-SP94 (2 mg/mL) peptide was added to the solution and incubated for 1 h at room temperature. The RCP of 188 Re-HYNIC-SP94 was determined by radio-TLC. The analytical system uses chromatography paper as the stationary phase and normal saline separated the 188 ReO 4 − (R f = 0.7~1.0) from 188 Re-HYNIC-SP94 and colloidal 188 Re (R f = 0). ITLC-SG was used as the stationary phase and a solution of ethanol: ammonia: water (2:1:5) separated the colloidal 188 Re (R f = 0) from 188 Re-HYNIC-SP94 and 188 ReO 4 − (R f = 0.7~1.0). If necessary, the 188 Re-HYNIC-SP94 was purified on a Sep-Pak C 18 column 30 . The column was activated with 10 mL methanol followed by equilibration with 3 mL 0.125 M NH 4 OAc. The sample was then loaded onto the column and washed with 10 mL 0.125 M NH 4 OAc, then the radiolabeled HYNIC-SP94 was eluted with methanol. Finally, the methanol was removed by evaporation under a nitrogen stream and the desired product was redissolved in saline.
The in vitro stability of 188 Re-HYNIC-SP94 was evaluated by incubating 188 Re-HYNIC-SP94 in normal saline and FBS at room temperature. RCP was analyzed by radio-TLC at 1, 4, 8, 18, 26 and 48 h after incubation to determine the stability.
188 Re-HYNIC-SP94 cell study and TUNEL assay. Huh-7 and Hela cells were cultured in 24-well plates (0.5 × 10 5 cells/well) 2 days before treatment. To assess 188 Re-HYNIC-SP94-mediated apoptosis, cells were co-incubated with three different doses (0.37, 1.11 and 2.22 MBq/mL) of 188 Re-HYNIC-SP94 in 1 mL cell culture medium in triplicate. Three control studies were performed, with all procedures the same except 188 Re-HYNIC-SP94 was replaced by medium alone, unlabeled SP94 (2 μ g/mL) and 188 Re isotope (2.22 MBq/mL) in each group. To further evaluate cell apoptosis induced specifically by 188 Re-HYNIC-SP94 targeting to its receptors, Hela cell was used as an additional control. The above samples were then incubated in a 5% CO 2 incubator for 15 and 45 h at 37 °C. Cell apoptosis was evaluated using the TUNEL assay with a kit, according to the manufacturer's instructions 28,37 .
H&E staining and immunohistochemistry. To evaluate whether there was a visceral injury, slides of lungs, liver and kidneys were stained with H&E. Tumors and indicated organs were collected and fixed in 4% paraformaldehyde, embedded in paraffin. Routine H&E staining and immunohisto-chemistry for CD34, bax and Ki67 were carried out according to the manufacturer's instructions. Afterwards, slides were viewed with a light microscope and the images were captured. Statistical analysis. All experimental data were represented as mean ± standard deviation (SD). Statistical analysis was performed using Student's t-test and any results with a P-value < 0.05 were considered as statistically significant. The statistical analysis was performed with SPSS 20.0 (IBM, Chicago, Illinois, USA).