Cell culture and animal models
A HepG2 human HCC cell line was purchased from Health Science Research Resources Bank (Osaka, Japan). Male BALB/c nude mice (4 to 5 weeks of age) were purchased from CLEA Japan Inc. (Tokyo, Japan). HepG2 cells were grown in DMEM and cultured in a medium supplemented with 10% (v/v) foetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. For the HepG2 tumour-bearing animal models, the HepG2 cells (2 × 106, 200 μL) were inoculated subcutaneously into the right flanks of male BALB/c nude mice. At 5 weeks after inoculation, the mice were subjected to the studies. All animal studies were approved by the Animal Care Committee of The University of Tokyo.
Sf9 cells were cultured in Grace's supplemented media (Invitrogen) containing 10% foetal calf serum, as described [15].
Antibodies
A MAb against human ROBO1 was generated as previously described [14, 15]. Briefly, human ROBO1 cDNA was polymerase chain reaction (PCR)-amplified from Alexander cells and inserted into the pBlueBac 4.5-TOPO vector. The recombinant baculovirus expressing ROBO1 was immunized directly into gp64 transgenic mice. A positive hybridoma clone, B5209B, was selected by the reactivity to the ROBO1 stable cell line, by flow cytometry. An anti-hemagglutinin (HA) antibody was purchased from Sigma (St. Louis, MO, USA). MAb B5209B was purified by ammonium sulphate precipitation from the ascitic fluid of nude mice, to which the hybridoma cells were implanted intraperitoneally. To raise a MAb, which recognizes cell surface ROBO1, gp64 transgenic mice were immunized subcutaneously with 1 mg of ROBO1-expressing budded baculovirus with pertussis toxin adjuvant, as previously described [15].
Evaluation of ROBO1-binding affinity of the anti-ROBO1 antibody
To evaluate binding affinities of the MAb against ROBO1, we prepared a stable ROBO1-expressing cell line and a soluble form of the ROBO1 (sROBO) protein.
The sROBO protein was affinity-purified from the culture supernatant of Sf9 cells infected with recombinant baculoviruses, which harboured a gene fragment encoding the extracellular domain of the human ROBO1 (1-861 aa) protein with V5 and 6 × His tags at its C-terminus.
A CHO cell line stably expressing ROBO1 fused with an HA tag (ROBO1-HA) was generated using the Flp-In System (Life Technologies Japan Corp., Tokyo, Japan). ROBO1 fused to HA, encoding the tag at the C-terminal, was inserted into the pcDNA5/FRT vector and was co-transfected with the pOG44 vector to Flp-In-CHO cells using Lipofectamine 2000. A highly ROBO1-HA-expressing clone was selected from the 1 mg/mL hygromycin-resistant cells.
Specificity and affinity of the anti-ROBO1 antibody, B5209B, were evaluated using flow cytometry or cell ELISA, as previously described [15, 16]. For flow cytometry, wild type or ROBO1-HA-expressing Flp-In-CHO cells were incubated with primary antibodies for 1 h at a concentration of 1 μg/mL. For the anti-HA antibody, 0.02% saponin was added for permeabilisation. The cells were then washed with dilution buffer (1% bovine serum albumin and 0.1 mM EDTA in PBS) and reacted with R-Phycoerythrin conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted to 1:200 with dilution buffer. Finally, cells were washed twice with dilution buffer and analysed by flow cytometry (GUAVA EasyCyte™ Plus System; Millipore, Billerica, MA, USA).
For the cell ELISA, 105 cells/well were plated on poly-d-lysine-coated, 96-well plates. The plates were centrifuged at 2,000 rpm for 1 min, and supernatants were discarded. Then, the plates were blocked with blocking buffer [40% Block Ace (Dainippon Sumitomo Pharma, Osaka, Japan) in 10 mM Tris-buffered saline (TBS)] for 30 min. Cells were incubated with primary antibody of various concentrations in blocking buffer for 1 h. After washing twice with 0.05% Tween 20 in saline, cells were incubated for 30 min with peroxidase-conjugated anti-mouse IgG Fc-specific (Jackson ImmunoResearch). After washing three times, the enzymatic reaction was visualized with TMB Soluble Reagent (ScyTek Laboratories, Logan, UT, USA). After the reaction was stopped with TMB Stop Buffer (ScyTek Laboratories), the absorbance was measured at 450 nm using a microplate reader (Biotrak II; GE Healthcare, Piscataway, NJ, USA). The data were fitted with a four parameter logistic curve (Figure 1c).
DOTA conjugation and radiolabelling
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was purchased from Macrocyclics (Dallas, TX, USA). 111InCl3 was obtained from Nordion Inc. (Vancouver, Canada). 90YCl3 was obtained from Eckert and Ziegler (Braunschweig, Germany).
The anti-ROBO1 MAb in 0.1 M NaHCO3 buffer (pH 9.0) was conjugated to DOTA at a molar ratio of 1:10 (protein to chelate). After incubation for 1 h at 37°C, the MAb-chelate was purified on an ultra-filtration column (ultra-4; Millipore, Billerica, MA, USA).
90YCl3 was added to the DOTA-anti-ROBO1 MAb in 0.25 M ammonium acetate buffer (pH 5.5). The mixture was incubated for 1 h at 45°C. The 90Y-DOTA-anti-ROBO1 MAb (90Y-anti-ROBO1) was purified on a desalting column equilibrated with PBS (NAP-5 column; GE Healthcare, Buckinghamshire, UK).
Labelling yield and radiochemical purity were estimated by instant thin layer chromatography (Pall Corp., MI, USA). A similar procedure was used to prepare the 111In-DOTA-anti-ROBO1 MAb (111In-anti-ROBO1).
Competitive ELISA was implemented to assess potency of the anti-ROBO1 MAb, DOTA-anti-ROBO1 MAb, and 90Y- and 111In-anti-ROBO1 MAb. sROBO was coated onto 96-well assay plates, and then plates were blocked by Tris buffer containing 1% BSA. The serially diluted antibody solutions were mixed with horse radish peroxidase (HRP)-labelled anti-ROBO1 MAb (HRP-anti-ROBO1; The University of Tokyo). Aliquots of the mixed solution were added to each well of the assay plate and incubated for 2 h at 37°C. The assay plate was washed with TBS-T, and TMB solution was added to each well and incubated for 5 min at room temperature. Absorbance was measured at 450-nm wavelength with a microplate reader (SpectraMax; Molecular Devices, Sunnyvale, CA, USA). IC50 was calculated using GraphPad Prism4 software (GraphPad; San Diego, CA, USA).
Biodistribution study
HepG2 xenograft (858.3 ± 237 mm3) mice were randomly divided into six groups (n = 3 per group). Each mouse was injected with 0.37 MBq of 111In-anti-ROBO1 (10 μg) via the tail vein. The mice were euthanised at 6, 24, 48, 72, 144, and 240 h after injection. Blood, heart, lung, liver, kidney, spleen, stomach, intestine, muscle, femoral bone, sternum, and tumour were collected, weighed, and measured for radioactivity. The percentage of injected dose per gram of tissue (% ID/g) was calculated for each organ.
RIT and immunotherapy
HepG2 xenograft mice were randomly divided into three groups (n = 4 to 5 per group). Mice were injected via the tail vein with a single dose of 6.7 MBq of either 90Y-anti-ROBO1 (70 μg, n = 5 mice), cold-anti-ROBO1 MAb (70 μg, n = 4 mice), or saline (n = 5 mice). Tumour volumes for the mice in the 90Y-anti-ROBO1, cold-anti-ROBO1, and saline groups were 674.9 ± 315, 545.0 ± 342, and 437.6 ± 223 mm3, respectively.
The tumour size, body weight, and blood cell count were measured twice a week until day 28 after injection. The tumour volume was calculated using the following formula: 0.5 × (shortest diameter)2 × (longest diameter). Tumour growth (%) was calculated using the formula: (tumour volume at each time point)/(tumour volume on day0) × 100. Peripheral blood was collected from the tail vein and then tested in an automated haematology analyser, Celltac α (MEK-6400; Nihon Kohden, Tokyo, Japan).
Mice were euthanised when the tumour size was >1,500 mm3 or the body weight decreased by >20% of its original weight.
Histological analysis
Tissue specimens of HepG2 tumour, liver, kidney, intestine, spleen, femoral bone, and sternum were obtained from mice at day 0, 7, 14, and 28 after injection of 90Y-anti-ROBO1 (n = 3 per group). All samples were fixed in 4% paraformaldehyde overnight at 4°C. They were embedded in paraffin, and 3 to 5 μm sections were obtained. The femoral bone and sternum samples were decalcified before embedding in paraffin. Slide sections, excluding the femoral bone and sternum, were deparaffinised, dehydrated, and stained with haematoxylin and eosin (H&E). Femoral bone and sternum sections were stained with Giemsa solution.
Ki-67 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) were carried out on HepG2 sections to investigate the status of tumour cell proliferation and apoptosis, respectively.
For Ki-67 stain, antigen retrieval was performed in 10 mM citrate buffer solution (pH 6.0) in a pressure cooker for 15 min. Endogenous peroxidase activity was quenched using 0.3% hydrogen peroxide-methanol for 30 min, and then the specimens were blocked with 5% normal goat serum for 1 h. Specimens were incubated with Ki-67 MAb (1:100; DAKO, Carpinteria, CA, USA) overnight at 4°C. Next, the specimens were treated with a secondary antibody (simple Stain MAX-PO; Nichirei, Tokyo, Japan) for 30 min. To visualize peroxidase activity, 0.2 mg/mL 3,3′-diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) was used as the substrate in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% H2O2. Nuclear staining was achieved with haematoxylin.
Apoptosis was detected using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon International, Inc., Billerica, MA, USA) for TUNEL, per the manufacturer's instructions.
Proliferative and apoptotic cells were quantified by determining the percentage of Ki-67-positive and TUNEL-positive cells, respectively. Two regions of interest measuring 200 × 200 μm were established on each tumour per one section. The percentage of positive-stained cells among all the counted tumour cells was calculated.
Statistical analysis
Data are expressed as the mean ± standard deviation (SD). Statistical analyses were carried out using JMP Pro 9.0.3 software. Means were compared using one-way ANOVA and Student's t test. p values <0.05 were considered statistically significant.