Cell culture
The human colorectal carcinoma HCT116 cell line was purchased from ATCC and cultured in McCoy’s Modified Eagle Medium with 10% fetal bovine serum (FBS), 1% l-Glutamine and 1% antibiotics (100 IU penicillin and 100 μg/ml streptomycin). The human squamous cell carcinoma cell line UM-SCC-74B, kindly provided by Professor TE Carey (University of Michigan, MI, USA), was cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS, 1% l-Glutamine, 1% antibiotics (100 IU penicillin and 100 μg/ml streptomycin) as well as 1% non-essential amino acids. Starvation medium contained the above additives with the exception of FBS. Previous studies by our group have shown that HCT116 can be considered a moderate CD44v6-expressing cell line and UM-SCC-74B a low CD44v6-expressing cell line [11]. Cells were incubated at 37 °C with 5% CO2 and cultured for no longer than 3 months.
Antibodies and PM2
AbN44v6, a fully human recombinant, full-length antibody targeting CD44v6, was developed from the CD44v6-targeting Fab-fragment AbD15179 and has previously been described [11, 25]. It was supplied in borate buffer at 3 mg/ml by Bio-Rad AbD Serotec (Puchheim, Germany). The commercially available anti-CD20 antibody, rituximab (MabThera), was purchased from Apoteket AB (Stockholm, Sweden) and used as a negative control. U36 was kindly provided by Professor G.A.M.S. van Dongen (VU University Medical Center, Amsterdam, the Netherlands). For Western blotting, primary antibodies, rabbit anti-phosphoErk and rabbit anti-Erk (Cell Signaling Technology, MA, USA), and mouse anti-β-catenin (BD biosciences, CA, USA) as well as secondary antibodies horse-radish peroxidase-conjugated secondary antibodies (GE Healthcare, UK) were used. The novel, stapled MDM2/X-p53 antagonist, PM2 (Mw = 1462.75 Da), was produced at the p53 Laboratory (A*STAR, Singapore) and dissolved in DMSO to a stock concentration of 10 mM and stored at − 20 °C.
Western blot
Approximately 7.5 × 104 HCT116 or 5 × 104 UM-SCC-74B cells were seeded in 6-well plates and incubated at 37 °C with 5% CO2 to a confluency of 50–60% before switching to starvation medium. Cells starved overnight were treated or not with fibroblast growth factor 2 (FGF2), 10 ng/ml for 5 min, and antibodies, as indicated, thereafter lysed in Nonidet P-40 (NP-40) lysis buffer: 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EGTA, 1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 100 μM Na3VO4, and 1% NP-40. Lysates were subjected to SDSPAGE (Invitrogen by Thermo Fisher, CA, USA), followed by transfer to Hybond-C extra membranes (Amersham Biosciences, Uppsala, Sweden). Membranes were incubated with indicated primary antibodies followed by horse-radish peroxidase-conjugated secondary antibodies. Immune reactivity was visualized using the enhanced chemiluminescence plus detection system (GE healthcare, UK). Quantification of immunoblotting signals was done using the Image Lab software (Bio-Rad Laboratories, CA, USA).
Radiolabeling and EDTA challenge
177Lu was purchased from PerkinElmer (Waltham, MA, USA) as LuCl3. AbN44v6 (4 mg/ml) and rituximab (5 mg/ml) were chelated for 4 h at 37 °C in 0.07 M sodium borate, pH 9.2, with CHX-A”DTPA containing a molar ratio of 1:5 between antibody and CHX-A”DTPA. Prior to radiolabeling, excess CHX-A”DTPA was separated from the antibodies using NAP-5 size exclusion chromatography columns (GE Healthcare, Uppsala, Sweden) equilibrated with filtered, metal-free 0.2 M ammonium acetate (pH 5.5, stored over Chelex 100). For AbN44v6, 11-30 MBq of 177LuCl3 was added to between 200 and 375 μg of AbN44v6 chelated with CHX-A”DTPA and incubated for 1 h at room temperature. For Rituximab, 5-40 MBq of 177LuCl3 was added to 100-200 μg of antibody and incubated for 1 h at room temperature. Labeled antibodies were separated from radionuclide and low-molecular-weight reaction components by using a NAP-5 column equilibrated with PBS. To determine the yield, purity, and stability of the final product, Instant Thin Layer Chromatography (ITLC) analyses were performed. Approximately 0.5 μl of the mixture was placed on a chromatography strip (Biodex, Shirley, NY, USA), using 0.2 M citric acid as the mobile phase, followed by measurements on a Cyclone Storage Phosphor System (PerkinElmer). The data were analyzed using the OptiQuant image analysis software (PerkinElmer). Yields were defined as fraction (percentage) of radiolabeled antibody versus unlabeled, free 177Lu. NAP-5 purification resulted in > 95% purity for all samples post labeling. For 3D cell culture assays, the amount of free 177Lu was adjusted for all samples in order to ensure equal experimental conditions.
An ethylenediaminetetraacetic acid (EDTA) challenge assessed the stability of the 177Lu-labeled antibody by ITLC, using a 500:1-M ratio with antibody and incubated at 37 °C for up to 48 h.
Specificity assay
Approximately 3 × 104 HCT116 or UM-SCC-74B cells per well were seeded in 48-well plates and incubated for 24 h at 37 °C with 5% CO2 before 20 kBq of 177Lu-AbN44v6 (27 nM) was added to each well. Additionally, 100-fold excess of unlabeled AbN44v6 was added to selected wells in order to block specific binding. The 48-well plates were incubated at 37 °C with 5% CO2 for 24 h and then washed, harvested, and measured in a 1480 WIZARD gamma well-counter (Wallac Oy, Turku, Finland).
LigandTracer analyses
All LigandTracer experiments were performed on LigandTracer Yellow (Ridgeview Instruments, Vänge, Sweden). Approximately 5 × 105 HCT116 or UM-SCC-74B cells were seeded on tilted petridishes 24 h prior to the start of each assay and incubated at 37 °C with 5% CO2. 177Lu-AbN44v6 was added in two concentrations (10 and 30 nM), and the uptake was measured at room temperature for 90 min for each concentration followed by a dissociation phase of at least 10 h. Analysis was performed using TraceDrawer version 1.7 (Ridgeview Instruments, Vänge, Sweden).
3D cell culture assay
For liquid overlay of two flat-bottomed 96-well plates, 0.15 g of agarose was dissolved in 10 ml of PBS, pH 7.4, with 1% penicillin/streptomycin and 5% incomplete, serum-free DMEM. Fifty microliters of the solution was added to each well prior to seeding 103 HCT116 or UM-SCC-74B cells. The plates were incubated at 37 °C with 5% CO2 for at least 3 days prior to start of treatment with 177Lu-labeled antibodies and/or PM2. The 3-day incubation allowed spheroids to form and grow to a preferred size with a diameter of approximately 400 μm. 30 kBq or 100 kBq of 177Lu-labeled antibodies were added per well (40–140 nM and 27–79 nM of AbN44v6 and rituximab, respectively), whereas a previously determined, fixed concentration of PM2 (20 μM) was used for all drug treated wells. For treatment with unlabeled AbN44v6, 10 nM, 100 nM, or 1 μM of AbN44v6 was added thrice at 48 h intervals. Spheroid images were obtained at start of treatment and at 2-to-4-day intervals during growth monitoring using a Canon EOS 700D digital camera mounted on an inverted Nikon Diaphot-TMD microscope. Spheroid volumes were calculated by measuring the surface areas using the ImageJ software, version 1.48 (NIH, Bethesda, MD, USA), and assuming all spheroids retained a spherical form, calculated the volumes accordingly using the formula: \( V=\frac{4}{3}\pi {r}^3 \). For each time point, the growth ratio was calculated by normalizing the volume of the spheroid to the original volume at the start of treatment.
Statistical analyses
For statistical analyses, one-way ANOVA followed by Tukey’s multiple comparisons test using the Graphpad Prism software version 6.0 (San Diego, CA, USA) was used for comparisons between multiple groups. Student’s t test was used for comparisons between two groups. A p value < 0.05 was considered significant.