1. Materials
The sulfo-cyanine5.5-NHS ester was purchased from Lumiprobe Corporation (USA). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS-ester) was bought from Macrocyclics, Inc. (USA). 4′,6-Diamidino-2-phenylindole (DAPI), ammonium acetate (NH4OAc), and other chemicals were purchased from Sigma Aldrich. 68GaCl3 was eluted from an 68Ge-68Ga generator (ITM, Germany) with 0.05 M super-pure Hydrogen chloride solution (HCl). Yttrium-90 chloride (90YCl3, in 0.05M HCl, about 18.5 TBq/mg) was purchased from PerkinElmer Inc. (USA). Sodium acetate buffer (S7899, pH 5.2 ± 0.1, 3 M, sterilized) purchased from Sigma (USA). Recombined active human CTS-B enzyme (ab90387) and all antibodies were purchased from Abcam (USA). 4–12% Bis-Tris gels were bought from the Shanghai Epizyme Biomedical Technology Co., Ltd. SDS-PAGE protein loading buffer (5X, p0015L) purchased from Beyotime (China).
2. BMX2 synthesis and radiolabelling
The Cbz-Phe-Lys-AOMKs were synthesized following an optimized synthesis route based on the previously reported method[22] Then the amine terminal of the side chain was modified with lysine for sulfo-cyanine5.5-NHS ester and DOTA-NHS-ester conjugating to get the final precursor BMX2. The synthesis scheme and more information can be found in the supporting information (Fig S1).
For 68Ga radiolabeling, the BMX2 solution (20 µg, 1 µg/µL in pure water) was mixed with 500 µL ammonium acetate buffer (NH4OAc,0.5 M, pH = 5.0) and was degassed for 3 min. Then 68GaCl3 (925–1295 MBq) was eluted with 4 mL HCl (0.05 M) into the reaction vial and diluted with 280 µL NH4OAc buffer (1.0 M, pH 5.0). Subsequently, the BMX2 solution was transferred into the reaction vial, and the pH of the resulting mixture was checked as 4.5-5.0. After being incubated at 90 ℃ for 10 min, the resulting solution was first diluted 10-fold with sterilized water and injected through a Sep-Pak C18 Light Cartridge (Waters, USA) followed by a 10 mL water flush. The product trapped on the C18 was slowly eluted with 1 mL 70% ethanol solution and formulated with 10mL saline containing 1% ascorbic acid. Before injecting into the mice, the solution was passed through a 0.22 µm Millipore filter into a sterile vial and aliquoted for many vials for cell study or in vivo study.
For 90Y labeling, 50 µL of 90YCl3 (about 74 MBq in 0.05 M HCl aqueous solution) was constituted with 200 µL of 0.5 M NaOAc and transferred into a reaction vial. Then 20 µL freshly prepared solution of BMX2 (1 µg/µL), dissolved in 10% DMSO containing 0.5 M NH4OAc buffer (pH 5.5), resulting in a total volume of about 300 µL and a final pH of 5.5. After incubating at 75°C for 30 min, 100 µL of 0.5% ethylenediaminetetraacetic acid (EDTA) aqueous solution and then was diluted with 10 mL sterilized distilled water. The solution was purified, sterilized, and conditioned following the same procedure of 68Ga labeling.
To test the stability of the probes in serum, 3.7 MBq 68Ga-BMX2 and 90Y-BMX2 (37 MBq/mL in saline) were respectively incubated with 0.5 mL human serum at 37°C for 2 h and 12 h. Then, the samples were added with cold ethanol (500 µL) and kept in an ice bath to precipitate serum proteins for 10 min, followed by a centrifuge at 6000 rpm for 15 minutes. 200 µL of the supernatant solution was isolated and analyzed on an RP-HPLC.
3. Western Blot analysis
To verify the binding of BMX2 with CTS-B, 0.2 µg and 0.09 µg recombined active human CTS-B enzyme (rh-CTS-B) were incubated with BMX2 (6.25 µM) at 37°C for 2 h. Then, the solution was loaded onto 12% Bis-Tris gels and run at 110 V for 40 min. Proteins were transferred to a PVDF membrane at 0.2 A for 1 hour. Membranes were blocked with 5% BSA in Tris-buffered saline/0.1% v/v Tween 20 (TBS-T) for 1 hour at room temperature. CTS-B was identified using a primary rabbit anti-h-Cathepsin B antibody (ab92955, 0.2 mg/mL, 1:5000, in 5% BSA TBST buffer, 4°C for 12 hours) followed by an Alexa Fluor 488 conjugated secondary antibody (ab150077, goat anti-rabbit, 1:2000, room temperature for 1 h). The fluorescence of the antibody and BMX2 were respectively read at the UV channel (excitation 525nm) and near-infrared (NIR) channel (excitation 685 nm and emission 700 nm) using an Odyssey XF Imaging System (Li-Cor, USA). The images were analysed using ImageJ software Version 1.53a.
To determine the activation and binding specificity of BMX2 with CTS-B, 6.25 µM BMX2 was incubated with different concentrations (0, 0.09, 0.13, 0.2, 0.3, 0.45 µg/µL) of rh-CTS-B in sodium acetate buffer (0.1 M, pH 6.0) at 37°C for 2 hours. And rh-CTS-B was incubated with different concentrations of CTS-B inhibitor (CA074) (0, 0.75, 1.50, 3.125, 6.25, 12.5 µM) for 2 h and subsequentially incubated with 6.25 µM BMX2 for another 2 h. Then, the solutions were loaded onto 12% Bis-Tris gels for fluorescent western blot analysis following the same procedure as described above.
To test the time dependence of BMX2 activation and binding with the enzyme, a fluorescent western blot was done with 6.25 µM BMX2 incubated with 0.2 µg CTS-B enzyme for 30, 60, 120, 240, and 480 min. And we tested the fluorescent western blot of different concentration of BMX2 probe (0, 2, 3, 4, 6, 9 µM) with 0.2 µg enzyme.
We also demonstrated a new radioactive western blot approach for detecting radiolabeled BMX2 binding affinity with CTS-B. Recombined active human CTS-B enzyme (0.2 µg) in 10 µL sodium acetate buffer (0.1 M, pH 6.0) were incubated with 370 KBq 68Ga-BMX2 in 10 µL saline and incubated at 37 ℃ for 30, 60, 120, and 240 min. Then, all samples were added 5 µL 5× loading buffer and denatured at 100 ℃ for 10 min. Following that, all samples were loaded onto 4–12% Bis-Tris gels and run for 40 minutes at 110V. The gel was placed on a flat board and scanned with a PET/CT scanner (Biograph mCT, SIEMENS).
The CTS-B expression and BMX2 binding ability in four different cancer cell lines (HeLa, HepG2, MCF7, and U87MG) were determined by fluorescent western blot analysis. HeLa, HepG2, MCF7, and U87MG cells were respectively seeded in four 6-well plates and cultured overnight to allow the cells to adhere to the bottom of the plate. For each cell line, 3 wells of the 6-well plate were incubated with BMX2 (1 µM), and another 3 wells for BMX2 and CA074 (BMX2 1 µM and CA074 25 µM at 37 ℃) for 2 hours. Subsequently, cells were washed three times with cold 1× PBS and lysed with RIPA. Protein concentration was determined using the BCA method. Then 20 µg cell lysates (1 µg/µL) were injected into 4–12% Bis-Tris gels for fluorescent western blot analysis using the same procedure described above.
To further study the activation and binding specificity of BMX2, HeLa and HepG2 cells were incubated with BMX2 for 0, 30, 60, 120, 240, and 480 min and tested with fluorescent western blots. As well, HeLa and HepG2 cells were treated with different concentrations of CA074 (0, 0.75, 1.5, 3.125, 6.25, 12.5 µM, 37°C, 120 min) and then incubated with 1 µM BMX2 probe (37°C, 120 min). Then cells were washed and lysed. 20 µg cell lysates were loaded onto 4–12% Bis-Tris gels for fluorescent western blot analysis using the same procedure described above.
4. Fluorescence microscope imaging
The binding ability and distribution of the BMX2 with CTS-B in HeLa and HepG2 cells were tested by counter immunofluorescent microscope imaging with an anti-CTS-B antibody. Glass coverslips (0.17 mm thickness, 12 mm diameter) were coated with poly-D-lysine and placed in 24-well plates. HeLa and HepG2 cells were seeded (5000 cells per well) and allowed to attach overnight. Cells were then incubated with BMX2 (1 µM in culture medium) for 4 hours. After incubation, the medium was removed, and the cells were gently washed twice with DPBS (1 mL each). The cells were then fixed with ice-cold 4% paraformaldehyde, stained with fluorescent CTS-B antibodies, and counter-stained with DAPI following the standard procedure. The coverslips were then placed on the microscope slides with a mounting medium. Later, all slides were subjected to a laser scanning confocal microscope for imaging and analysis (BC43, ANDOR, England).
5. Cell Uptake
5×105 HeLa and HepG2 cells were seeded in four 24-well plates and incubated overnight. Cells were then incubated with 68Ga-BMX2 (37 kBq/well) for 30 min, 60 min, 120 min, and 240 min. Then cells were lysate with 200 µL cell lysate and collected in test tubes. The radioactivity was measured respectively to calculate the cell uptake ratio as percent of added radioactivity (%ID).
6. In vivo imaging and biodistribution
To evaluate the in vivo distribution and tumor uptake of the 68Ga-BMX2, the subcutaneous HeLa xenograft mice model were established by inoculating 1×106 HeLa cells into the right forelimb of the mice. Tumor sizes were recorded via digital calliper every day, and tumor volume was calculated according to the function:
Volume (mm3) = (L× W2) / 2.
The value L is the largest diameter of the tumor, and W is the diameter perpendicular to the L.
When tumor size reached 200–300 mm3, mice were randomly divided into two groups for the PET imaging study, a blocked group (68Ga-BMX2 with CA074) and a non-blocked group (68Ga-BMX2 only). Mice were anesthetized and intravenously injected of CA074 (4 mg/kg body weight) or the same volume of saline 60 min before 68Ga-BMX2 injection. Mice were then set on the micro-PET scanner (Inveon small-animal PET/CT, Siemens) for CT imaging first and followed by intravenously injecting 200 µCi of 68Ga-BMX2. Imaging acquisition was immediately started for 1h-dynamic PET scan and a static PET/CT scan at 2 h post-injection. Then quantitative imaging assays were performed. The fluorescence intensity and radioactivity in tumor and major organs were measured. The radioactivity in the specific tissue was expressed as a decay-corrected percentage injected dose per gram of tissue (%ID/g).
The biodistribution of 68Ga-BMX2 at 2 h was performed to evaluate the tumor uptake and metabolism route after PET imaging. Some mice (n = 4 for each group) were euthanized and subjected to tissue collection. All interested organs were weighed and tested for radioactivity with a gamma counter to calculate the biodistribution of the 68Ga-BMX2. The radioactivity in the tumor and specific organs were expressed as a decay-corrected %ID/g.
To evaluate the in vivo distribution and tumor uptake of the BMX2, 8 HeLa xenograft mice were randomly divided into two groups (CA074 blocked group and non-block group) for fluorescence imaging study. Mice were anesthetized and intravenously injected with CA074 (4 mg/kg body weight) or the same volume of saline 60 min prior to BMX2 injection. Then BMX2 was injected through the tail vein, and fluorescence images were acquired at 1, 4, 12, and 24 h post-injection to monitor long time accumulation of BMX2 in the tumor.
7. Radionuclide therapy
The therapeutic efficacy of 90Y-BMX2 was evaluated on HeLa xenograft models. When tumor size reached about 200 mm3, mice were randomized and assigned to 3 treatment groups (n = 4/group) as saline, 90Y-BMX2 (about 3.7 MBq), and 90Y-BMX2 (about 3.7 MBq) with CA074 (4 mg/kg bodyweight) treated groups. All therapeutic reagents were given via tail vein injection. Tumor sizes were monitored with a digital calliper every 2 days, and tumor volume was calculated. Meanwhile, the body weight of mice was also measured. The mice were euthanized when their body weight decreased by more than 20% from day 1, or the tumor volume exceeded 10% of the total body weight, or the tumor size reached about 1.0 cm in the largest diameter. The day the mice were euthanized was defined as the endpoint of the group.
8. Statistical analysis
Data were expressed as mean ± SEM. Unpaired Student t-test was used to determine the statistical significance of differences between groups using Origin pro 2021 (OriginLab). P < 0.05 were considered statistically significant.