Development of a Radiolabeled Peptide-Based Probe Targeting MT1-MMP for Breast Cancer Detection

Breast cancer is one of the most frequent and aggressive primary tumors among women of all races. Matrix metalloproteinase (MMPs), a family of zinc- and calcium-dependent secreted or membrane anchored endopeptidases, is overexpressed in varieties of diseases including breast cancer. Therefore, noninvasive visualization and quantification of MMP in vivo are of great interest in basic research and clinical application for breast cancer early diagnosis. Herein, we developed a 99mTc labeled membrane type I matrix metalloproteinase (MT1-MMP) specific binding peptide, [99mTc]-(HYNIC-AF7p)(tricine)(TPPTS), for in vivo detection of MDA-MB-231 breast tumor by single photon emission computed tomography (SPECT). [99mTc]-(HYNIC-AF7p)(tricine)(TPPTS) demonstrated nice biostability and high MT1-MMP binding affinity in vitro and in vivo. Tumor-to-muscle ratio was found to reach to the highest (4.17±0.49) at 2 hour after intravenously administration of [99mTc]-(HYNIC-AF7P)(tricine)(TPPTS) into MDA-MB-231 tumor bearing mice. Overall, [99mTc]-(HYNIC-AF7P)(tricine)(TPPTS) demonstrated great potential for MT1-MMP targeted detection in vivo and it would be a promising molecular imaging probe that are probably beneficial to breast cancer early diagnoses.


Introduction
Breast cancer is one of the leading causes of cancer death among women of all races. The life time risky for individuals to suffer breast cancer could be as high as 13% [1,2]. Accurate diagnosis of breast cancer is very important and desirable, by which various therapeutic regimens could be given before the primary tumors become metastatic.
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade most of extra-cellular matrix (ECM) proteins. It plays an important role in the development of various diseases including cancer [3], inflammation, neurological and cardiovascular diseases. In the case of cancer, MMPs not only have distinct roles in tumor angiogenesis, but also affect multiple signaling pathways to control the balance between growth and antigrowth signals in the tumor microenvironment. Membrane type-1 matrix metalloproteinase (MT1-MMP) has been shown to be a key member of the MMP family with much biological and pathological significance [4,5]. Specifically, MT1-MMP is intrinsically associated with the plasma membrane of normal and tumor cells and remodels the extracellular matrix (ECM). In patients with breast cancer, MT1-MMP has been reported with high expression in both serum and local lesions [6,7]. Although the failures of applications of MMP inhibitors in tumor prevention and therapy have been reported [8,9], there are accumulating reports about the potential of utilization MMP as target for cancer diagnosis [10][11][12].
So far, MT1-MMP targeting peptides for breast cancer imaging and therapy are being widely studied in both experimental and clinical settings [13][14][15][16]. The first reason of development of small molecules capable to track MT1-MMP in vivo is to help investigating the nature of MT1-MMP expression, distribution, and its biological and pathological functions. Secondly, a specific MT1-MMP targeting molecule would also provide the chance of earlier detection and characterization of disease and evaluation of treatment in diseases where MT1-MMP is overexpressed. Recently, we have developed a high MT1-MMP affinity peptide, named AF7p, by phage display peptide library screening technique. MT1-MMP high expressed tumor was successfully imaged in vivo by a near infrared dye labeled AF7p, suggesting the potential of utilizing AF7p for MT1-MMP targeted tumor detection [12]. Compared with optical imaging, radioisotope mediated imaging is more sensitive and accurate in clinic with less tissue penetration and quantification limitation. Currently both single photon emissions computed tomography (SPECT) and positron emission tomography (PET) are well developed [17][18][19]. Generally, PET imaging holds great promise in visualization of biology activities, but to some extent the short half-life of positron emitting radionuclide, high cost of instruments and the complicated preparation of tracers slow down the application of PET in clinic. On the other hand, SPECT can offer a simple and inexpensive procedure with readily available 99m Tc through inexpensive 99 Mo-99m Tc generator [20][21][22]. Overall, SPECT is more likely to be a cost-effective technique in early detection of breast cancer in clinical practice [23].

Reagents
Chemicals were purchased from Sigma-Aldrich (USA) and were used without further purification. Side chain protected peptide with the sequence of His-Trp-Lys(Dde)-His-Leu-His-Asn-Thr-Lys(Dde)-Thr-Phe-Leu was custom-made by the GL Biochem, Ltd. (Shanghai, China). Na 99m TcO4 was obtained from a commercial 99 Mo/ 99m Tc generator (Beijing Atom High Tech Co., Ltd.).

Synthesis of [ 99m Tc]-(HYNIC-AF7p) (tricine) (TPPTS)
HYNIC-AF7p solution (20 μL, 60 μg/mL in H 2 O), of tricine solution (100 μL, 100 mg/mL in 25 mM succinate buffer, pH 5.0), SnCl 2 solution (10 μL, 3 mg/mL in 0.1 M HCl) and Na 99m TcO 4 (100 μL, 370 MBq) in saline were added into a 5 mL Eppendorf tube. The reaction mixture was kept at room temperature for 10 min. To the reaction mixture above was added 100 μL of the TPPTS solution (50 mg/mL in 25 mM succinate buffer, pH 5.0). The vial containing the reaction mixture was sealed, cramped, and heated at 90°C for 30 min. After cooling to room temperature, sample was analyzed by radio-HPLC. The radio-HPLC method used a HP Hewlett Packard Series 1100 HPLC system equipped with a Radioflow Detector LB509 and a reversed-phase Zorbax SB-C18 column (4.6 mm × 250 mm, 5 μm). The flow rate was 1 mL/ min. The gradient mobile phase started with 90% solvent A (0.1% TFA in water) and 10% solvent B (0.1% TFA in acetonitrile) to 30% solvent A and 70% solvent B at 24 min to 0% solvent A and 100% solvent B at 27 min, followed with 90% solvent A and 10% solvent B at 30 min. Gelman Sciences silica-gel paper strips was used in ITLC method. A 1:1 mixture of acetone and saline were used as eluent. The 99m Tc complexes migrated with the solvent front (Rf = 1.0) while 99m TcO 4 and 99m Tc-AF7p remained at origin (Rf = 0.0).

Tumor immunohistochemistry
MDA-MB-435 and A549 tumor bearing mice were sacrificed and tumors were frozen in OCT embedding medium. Cryosections were cut into 4 μm and subjected to staining. Briefly, tumor slides were dried in the air and fixed with cold acetone for 20 min and dried again in the air for 30 min at room temperature. After blocking with 10% BSA for 30 min, the sections were incubated with rabbit anti-MT1-MMP antibody (10 μg/mL, Abcam, MA) for 60 min at room temperature in the dark, and then visualized with FITC-conjugated donkey anti-rabbit secondary antibody. Finally, the slices were mounted with DAPI-containing mounting medium under a fluorescence microscope (Olympus, X81). Fluorescence pictures were taken using FITC filter settings (excitation = 490 nm, emission = 520 nm).

In vitro cytotoxicity of AF7p analogs
A standard Cell Counting Kit-8 (CCK-8) was utilized to analyze the cytotoxicity of AF7p analogs following a general protocol. Briefly, MDA-MB-231 cells were seeded in a 96-well plate with the concentration of 5×10 4 cells/well. After incubation at 37°C for 24 h, AF7p analogs with a final concentration of 10, 50, 100, 200, 500 or 1000 nM were incubated with cells for 24 h, after which 10 μL of CCK-8 solution was added to each well of the 96-well plate and incubated for another 4 h. The amount of an orange formazan dye, produced by the reduction of WST-8 (active gradient in CCK-8) by dehydrogenases in live cells, is directly proportional to the quantity of live cells in the well. Therefore, by measuring the absorbance of each well at 450 nm using a microplate reader, cell viability could be determined with the calculation of the ratio of absorbance of experimental well to that of the cell control well. All experiments were triplicated and results were averaged.

In vivo biodistribution study
Animals were divided into 4 groups (n = 3) for 4 time points with approximately equal distribution of tumor sizes on the day before the study. At 0.5, 1, 2, and 4 h after intravenous administration of [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS), MDA-MB-231 tumor bearing mice were euthanized. Blood, heart, lung, liver, kidney, stomach, intestine, spleen, pancreas, brain, muscle and tumor were excised, weighed and counted for radioactivity. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g).

Statistical analysis
Results were expressed as mean ± SD. Two-tailed paired and unpaired Student's t tests were used to test differences within groups and between groups, respectively. P values < 0.05 were considered statistically significant.

Stability
For in vivo application, we have to make sure the probe keeps intact before it arrives at the tumor area. We then compared the in vitro stability of [ 99m Tc]-(HYNIC-AF7P)(tricine) (TPPTS) in saline and saline with excess amount of cysteine. As shown in Fig 2, during the 6 hours incubation, the original form of [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) in saline only decreased slightly from 96.8 ± 1.12% at the beginning to 93.5 ± 2.27% (Fig 2A). Similarly, the amount decreased from 96.8 ± 1.52% to 94.2 ±1.72% in the solution containing excess cysteine during the 6 hours incubation (Fig 2B). It is quite clear that [ 99m Tc]-(HYNIC-AF7P)(tricine) (TPPTS) is able to maintain its stability over6 h in saline and in the presence of excess cysteine.
Before performing in vivo imaging, we also evaluated the overexpression of MT1-MMP in MDA-MB-231 tumors xenograft by immunohistochemistry. As expected, MT1-MMP was highly expressed in MDA-MB-231 tumor sections, verified by fluorescent immunostainingusing a MT1-MMP antibody (Fig 3).      It should be noted that besides tumor, kidneys, and bladder also demonstrated strong signals which is consistent with ex vivo distribution data ( Table 1), indicating that the tracer is mainly excreted through the renal-urinary routes.   Table 2).

Discussion
In this study, we used HYNIC as the BFC and tricine/TPPTS as coligands to prepare [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) for in vivo SPECT imaging of breast cancer. The MT1-MMP targetability of AF7p was evaluated as a breast cancer SPECT imaging agent. Radiolabelling of (HYNIC-AF7P)(tricine)(TPPTS) was successful with radiochemical purity higher than 95%. The solution stability data shows that [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) is stable for 6 h in PBS and even in the presence of excess cysteine. Further evaluations showed that the kit formulation was stable, which is important for future clinical translation [14,15]. In particular, breast tumor analysis showed good tumor uptake and T/N ratios. Based on its biodistribution and in vivo SPECT imaging results, [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) would be useful for imaging thorax, head and neck and extremities tumors with highly expressed MT1-MMP.
We also found that [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) was excreted rapidly via liver and renal route with very little radioactivity accumulation in the blood and muscle at 1 h post injection. In spite of the slow clearance from bone observed in the biodistribution data (Table 1), the images obtained at 2 h post injection showed a good tumor to background uptake ratios. This may be caused by the difference of [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) injected activity between the biodistribution mice and the planar imaged mice. It is possible that increased mass of (HYNIC-AF7p) (tricine)(TPPTS) injected in the higher imaging doses could mask nonspecific binding to blood proteins and other normal tissues [15,16]. The administration of higher doses of [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) in the imaged mice appeared to achieve better tumor to normal organ ratios. These results support the use of [ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) for the detection of thoracic tumors, including breast cancer.

Conclusions
[ 99m Tc]-(HYNIC-AF7P)(tricine)(TPPTS) was successfully prepared and its biodistribution and tumor imaging properties were evaluated in BALB/c nude mice bearing MDA-MB-231 human breast cancer xenograft. The relative low-cost and available supply of 99m Tc coupled with selective tumor uptake and rapid clearance from non-targeting organs makes it a promising agent for breast cancer detection, which remains to be validated preclinical. Supporting Information S1 Fig. Characterization of the purity and molecular weight of Cy5.5-AF7P by HPLC and mass spectrum. Analytical HPLC of Cy5.5-AF7P, the retention time of Cy5.5-AF7P is 11.83 min ( Figure A). Molecular weight of Cy5.5-AF7P confirmed by mass spectrometer of, which is 1839.6 ( Figure B). (TIF)