All human CRC cells were kindly provided by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China) and maintained in a humidified incubator at 37℃ in 5% CO2. SW620 was cultured in RPMI 1640 medium (BI, CT, USA) supplemented with 10% fetal bovine serum (FBS; BI, CT, USA) and 1% penicillin-– Streptomycin (P/S). LS74T and LoVo cells were cultured in DMEM medium (Gibco, NY, USA) with 10% FBS and 1% P/S. RKO cells were maintained in MEM medium (Gibco, NY, USA) supplemented with 10% FBS, 1 mmol/L sodium pyruvate (Gibco, NY, USA) and 1% P/S.
Western Blot analysis
LoVo, SW620, LS174T and RKO were lysed in RIPA buffer with 1 mmol/L PMSF on ice for 30 min. Equal amounts of total cellular protein (30 μg) were dissolved in SDS-PAGE gel and then transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Germany). The PVDF membranes were blocked with PBST containing 5% BSA for 1 h followed by incubation overnight at 4 ℃ with anti-PD-L1 antibody (#ab205921, Abcam, Tkoyo, Japan) at 1/200 dilution and anti-β-actin (Proteintech, Wuhan, China) at 1/5000 dilution. After washing with PBST, the membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (ZSGB-BO, Beijing, China) for 1 h at room temperature (RT). The bands were then detected by ChemiScope 6000 Exp chemiluminescence imaging system (Clinx, Shanghai, China) and β-actin quantitative normalization was performed using ImageJ 1.60 (NIH).
Briefly, the cells were washed thrice with staining buffer (PBS containing 2 mmol/L EDTA and 0.5% FBS), and then the cells were incubated with recombinant anti-PD-L1 antibody ( #ab205921, Abcam, Tkoyo, Japan) or control IgG (#ab17273, Abcam, Tkoyo, Japan) at 1/100 dilution for 30 min at RT after blocking with goat serum for 1 h. Next, the cells were washed and resuspended, stained with Alexa Fluor® 647 conjugated Donkey Anti-Rabbit IgG secondary antibody (Biolegend, CA, USA) at 1/1000 dilution for 30 min at RT without light, and analyzed on a Cyto-FLex flow cytometer (Beckman, CA, USA). At least 20,000 events were recorded and analyzed using FlowJo software VX0.7 (BD, NJ, USA).
The cells at a density of 5×105 per cell line were cultured in 6-well plates with sterile glass slides until the cells were grown on the glass slides. The slides were then washed thrice with PBS and fixed in 4% paraformaldehyde for 15 min. Non-specific binding of antibodies was blocked by 10% goat serum for 30 min. The cells were incubated with anti-PD-L1 monoclonal antibody (#ab205921, Abcam, Tkoyo, Japan) for overnight at 4 ℃. A secondary Goat Anti-Rabbit antibody (Auragene, Changsha, China) conjugated with Dylight-488 was applied for 1 h at RT in a wet box. The nuclei were counterstained with DAPI medium. The images were obtained using an inverted fluorescence microscope (Nikon, Tokyo, Japan).
Radiolabeling of antibody
For 131I radiolabeling, anti-PD-L1 monoclonal antibodies were used with Na131I (Chengdu Gaotong Isotope Co, China) and 1,3,4,6-Tetrachloro-3α,6α-diphenylglycouril ( Iodogen®, Sigma, MO, USA) as previously done in . Briefly, in a tube coated with 50 μg Iodogen, 100μl 6.91nmol/L anti-PD-L1 monoclonal antibody (#HY-P9904, MCE, NJ, USA) 0.25 mol/L phosphate buffer (pH 7.6), and 18.5 MBq of Na131I were added into the tube. All components of the tube were blended frequently, and the reaction was carried out at RT for 15 min. Finally, the reaction system was add 150 μl phosphate buffer and mixed well, and left standing for 10 min to stop reaction. Subsequently, the contents were transferred onto a PD-10 column (GE Healthcare, MA, USA) and purified by using 20 mmol/L PBS containing 0.5% BSA as eluent (pH 7.4). l ml fraction of the eluate was collected and the radioactivity in 1μl aliquots of each fraction was measured by using a PerkinElmer 2480 automatic γ-counter, wherein the radiochemical purity was > 95%.
In vitro assay
Human CRC cell lines LoVo, SW620, LS174T and RKO were adjusted to a concentration of 5×106/ml. RPMI-1640 containing 0.5% BSA was used as the binding buffer, followed by incubation of 100μl fractions of cell suspension with 100μl 47.5pmol/L 131I-PD-L1-Mab (1.3kBq) at 37℃ for 1 h as the test group (X group). The residual group (O group) and the total dose group (T group) for the controls were added with the same amount of 131I-PD-L1-Mab, and binding buffer was added to make the liquid volume equal. To determine the non-specific binding (NSB), 131I-PD-L1-Mab was incubated with RKO cells in the presence of a 2000-fold excess of unlabeled PD-L1 antibody. After cell incubation, the X group, O group and NSB group were separated by centrifugation (1300g, 10min) to obtain the protein-binding fraction (pellet) and then the supernatants were removed. After centrifugation, the counts per minute (cpm) were measured in each group with a γ-counter (PerkinElmer). These were conducted in triplicate, and the cell-binding ratio was calculated using the formula,
[Please see the supplementary files section to view the equation.]
In a competitive inhibition assay, 131I-PD-L1-Mab (750Bq) was incubated with RKO cells in the presence of varying concentrations of the unlabeled antibody (11.5-2300pmol/L) at 37℃ for 1 h within 1ml of binding buffer. After incubation, the cell components were obtained by centrifugation, and the cell-associated activities were measured in a shielded well-type γ-counter. The IC50 value is defined as the concentration of unlabeled antibody that is required for 50% inhibition of radiolabeled antibody. In saturation binding assay, increasing concentrations of 131I-PD-L1-Mab (65-3333Bq) were incubated with 5 ×105 RKO cells in 1 ml binding buffer at 37ºC for 1 h. To detect non-specific binding, a 200-molar ratio of unlabeled antibody was used for co-incubation. Specific binding is defined as the binding of PD-L1 antibody and PD-1 antigen expressed on tumor cells membrane, which is equivalent to the difference between total binding and non-specific binding. The GraphPad Prism 7.00 software fits the curve on the relationship between specific binding and non-specific binding to determine PD-L1 receptor density of each cell and the dissociation constant (Kd) of the 131I-PD-L1-Mab.
Female BALB/C nu-/nu- mice (6-8 weeks old) were obtained from Changzhou Cavens Laboratory Animal Co., Ltd. All animal protocols have been approved by the Animal Ethics Committee Board of Second Affiliated Hospital of Harbin Medical University (KY2018-215), and all procedures are under the National Institutes of Health guide for the care and use of Laboratory animals. Mice were housed in sterile cages with specific pathogen-free (SPF)-class animal facility on a 12 h light/dark cycle at 18 ℃ - 23 ℃ in 50% - 60% relative humidity. The mice had free access to food and drinking water, and were transfected with 200μl 2.5×107/ml human CRC cells subcutaneously in their right flank. Tumors were grown for more than 21 days until the average tumor volume reaches to approximately 500 mm3.
Ex vivo biodistribution
To determine the biodistribution and specificity of binding of 131I-PD-L1-Mab, two sets of study were performed in subcutaneous CRC cell line xenograft mice. Forty-eight hours before each study injection of 131I-PD-L1-Mab, 0.5% sodium iodide solution was used instead of drinking water to prevent the enrichment of 131I in mouse thyroids.
In the first study, subcutaneous LoVo, SW620 and LS174T xenograft mice were divided into three groups to receive intravenous injections of 7.5kBq 131I-PD-L1-Mab, respectively. At 24 h, 48 h and 120 h after 131I-PD-L1-Mab injection, the mice were euthanized, and 100 μl of blood was drawn from the carotid artery. The major tissues, organs and tumor tissues of the mice were dissected and weighed, including the brain, heart, liver, spleen, lung, kidney, stomach, small intestine, colon, pancreas, muscle, adipose, bladder and bone. All are placed in a γ-counting tube, and their radioactivity was measured using a γ-counter.
In the second study, subcutaneous RKO xenograft mice were divided into two groups. The control group was injected with 7.5kBq 131I-PD-L1-Mab, in the blocking group the subcutaneous RKO xenograft mice were injected with an excess of 300 μg of unlabeled PD- L1 antibody to block PD-L1 in vivo. The mice were euthanized at 48 h and 120 h after injection of 131I-PD-L1-Mab, and further processing was carried out according to the above steps. All ex vivo biodistribution results are expressed as percentage of injected activity per gram of tissue (%IA/g).
Cerenkov luminescence imaging (CLI)
Before scanning Cerenkov luminescence imaging, 0.5% sodium iodide solution was used as described above. The mice that inoculated with subcutaneous xenografts of LoVo, SW620, LS174T and RKO cells received intravenous injections of 37MBq 131I-PD-L1-Mab (protein dose 29.26 μg). At 24 h, 48 h, and 120 h after injection, the mice were anesthetized with 2% isoflurane. After that, the mice were subjected to CLI using the IVIS Spectrum Imaging System (PerkinElmer, MA, USA), and the parameters were set to Binning Factor 8, FOV 13.4cm, Exposure Time 300s. The mice were placed in supine position, with the tumors facing the lens during scanning, and were continuously anesthetized. The images obtained from scanning were passed through the Living Image® 4.5 Software to determine the fluorescence intensity of tumors and background.
All data were expressed as means±SD and statistical methods refer to “guidelines for reporting of statistics for clinical research in urology” provided by Assel et al.The difference in the uptake of 131I-PD-L1-Mab was assessed using ANOVA with Student–Newman–Keuls method multiple comparison test. The receiver operating characteristic (ROC) curves were drawn to evaluate the diagnostic efficacy of tumor uptake at different time points on tumors PD-L1 expression, and the areas under the curves (AUCs) at different time points were compared using the U tests. Statistical analysis was performed using GraphPad Prism version 7.00 and SPSS version 19.0 for Windows. A statistically significant difference was defined as p value of <0.05.