Database analysis of gene and protein expression profiles
TIMER2 web (http://timer.cistrome.org/), web server (http://gepia2. cancer-pku.cn/#analysis), the UALCAN portal (http://ualcan.path.uab.edu/analysis-prot.html), and TCGA database were used to analyze the expression level of c-MET in primary tumor and normal tissues.
Human CRC specimens and peripheral blood were obtained from Tongji Hospital, Shanghai, China after obtaining written informed consent from the donors. The study was performed according to the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Tongji Hospital, Shanghai, China (2021-KYSB-061). Excised tumor tissues were paraffin-embedded and sectioned for immunohistochemistry.
Cell lines and cell culture conditions
HCT116, SW480, DLD1, RKO, and THP1 cells were obtained from the ATCC, cultured in DMEM supplemented with 10% FBS (Gibco-Thermo Fisher Scientific) and of 1% penicillin/streptomycin (Gibco). Cells were incubated at 37℃ in a humidified incubator containing 5% CO2.
Reagents and Antibodies
Human recombinant HGF was purchased from GenScript Biotech (Z03229, Nanjing, China). The c-MET kinase inhibitors JNJ38877605 was obtained from Selleckchem (S1114, Houston, TX, USA). Antibodies were purchased from Cell Signaling Technology, Inc. and included antibodies against: c-MET (#8198), phospho-MET (Tyr1234/5) (#3077), phospho-MET (Tyr12349) (#3133), Gab1(#3232), p-Gab1, FAK (#3285), p-FAK (Tyr397) (3283#), PD-1 (#86163), PD-L1 (#85164), GM130 (#12480), Flotillin-1 (#18634), CD9 (#13174), β-actin mAb (#4967). Horse radish peroxidase-conjugated anti-mouse IgG antibody (#7076) was used as a secondary antibody.
RNA isolation and real-time quantitative PCR (qPCR)
Total RNA was isolated from each cell line with TriReagent (Sigma-Aldrich) according to the manufacturer’s instructions. The synthesis of cDNA (Sangon Biotech, Shanghai, China) and qPCR reactions were performed using the SYBR® FAST qPCR Kit Master Mix (KAPA, Biosystems). The primer pairs used were as follows: c-MET, forward 5’-TTCACCGCGGAAACACCCATC-3’, reverse 5’-GTCTTCCAGCCAGG CCCA-3’; PD-L1, forward 5’-TGGCATTTGCTGAACGCATTT-3’, reverse 5’-GTG GTGGTCTTACCACTCAGG-3’; CD163, forward 5’-GACGCATTTGGATGGATC ATGT-3’; reverse 5’-CCCACCGTCCTTGGAATTTGA-3’; CD206 forward 5’-GGG TTGCTATCACTCTCTATGC-3’, reverse 5’-TTTCTTGTCTGTTGCCGTAGT-3’; IL-10 forward 5’-TCAAGGCGCATGTGAACTCC-3’, reverse 5’-GATGTCAAACT CACTCATGGCT-3’; TNF-α forward 5’-CCTCTCTCTAATCAGCCCTCTG-3’ reverse 5’-GAGGACCTGGGAGTAGATGAG-3’; IL-1β forward 5‘-TGATGG CTTATTACAGTGGCAATG-3', reverse 5’-GTAGTGGTGGTCGGAGATTCG-3’; IL-12 forward 5’-GGAAGCACGGCAGCAGAATA-3’ reverse 5’-AACTTGAGG GAGAAGTAGGAATGG-3’; GAPGH forward 5’-GGTGGTCTCCTCTGACTTCAA CAG-3’ reverse 5’-GTTGCTGTAGCCAAATTCGTTGT-3’. ΔCT (cycle threshold) values were calculated based on the mean CT values of the target genes and mean CT values of the reference control gene GAPDH, using the following formula: ΔCT = Mean CT for Target Gene – Mean CT for GAPDH. Relative gene expression levels were calculated using ΔΔCT analysis. ΔΔCT = ΔCT of Sample – ΔCT of Calibrator. Relative Gene Expression = 2-(ΔΔCT).
Exosome isolation from plasma and cell culture supernatants
There were 30 CRC patients enrolled for testing and validation studies between March 2021 and July 2021 as well as 20 healthy individuals who were sex and age matched with patients in the testing and validation sets. Detailed clinical data are summarized in supplementary Table 1. The blood samples included in this study were collected in vacuum blood tubes with anticoagulant (EDTA-K2) prior to surgery and pharmacotherapy and centrifuged at 3000 × g for 15 min at 4ºC. The plasma (250 μL) was added to a new tube, to which Exo-Quick™ solution (EXOQ5A‐1; SBI System Biosciences, USA) (63 μL) was added. The mixture was mixed, kept at room temperature for 30 min, and then centrifuged at 1500 × g for 30 min. The supernatant was discarded and the pellets were resuspended at 1500 × g for 5 min. The pellets containing total exosomes were resuspended in 100 μL of PBS.
The exosome isolation from cell culture supernatant was performed using differential ultracentrifugation. Briefly, culture cells (100 mL) were centrifuged at 4°C to obtain supernatant, which were subject to centrifuge at 10,000 × g for 20 min. The resulting supernatant were transferred to sterile centrifuge tube and then centrifuged at 100,000 × g at 4°C for 70 min. The supernatant was removed, and the sediments resuspended in 1× PBS/TBS, filter through 0.22 μm strainer, and recentrifuged at 100,000×g for 1 h. The previous step was repeated, and the exosomes were collected.
Transmission electron microscopy
Isolated exosomes were re-suspended in PBS and 20 μL of the suspension was placed on a carbon-coated copper grid and incubated together for 10 min at room temperature. Next, the grid was washed by sterile distilled water, placed in contact with 2% uranyl-oxalate solution for 1 min, and dried for several min. Finally, the grid was observed using an electron microscope (JEM1400, JEOL, Tokyo, Japan).
Nanoparticle tracking analysis
In order to identify the exact size and quantity of isolated particles, the suspension with concentration between 1×107/mL and 1×109/mL was examined using ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) equipped with a 405 nm laser. For each suspension, a video of 60 s duration was recorded with a frame rate of 30 frames/s, and particle movement was analyzed using nanoparticle tracking analysis software (ZetaView 8.02.28).
In vitro wound healing assay to measure migration
HCT116 and SW480 cells were added to a 24-well plate. After overnight incubation, a sterile 10 μL pipette tip was used to create a wound across a cell culture monolayer. Cells were incubated in DMEM-0% FBS in the presence of BsAb or the c-MET kinase inhibitor JNJ38877605 for 2 h and were treated with HGF (100 ng/mL). Photographs of the wound were taken immediately after wounding and 24 h using a phase-contrast microscopy. The efficiency of the wound healing process was determined by calculating the area of the cell gap at the indicated times (0 and 24 h) using ImageJ software. Three images were recorded for each wound at each experimental timepoint. The results are expressed as the percentage of healing at 24 h with respect to the zero timepoint.
Cell invasion assay
Cell invasion assays were performed as previously described . HCT116 and SW480 cells were suspended in serum-free DMEM and then 0.1 mL of each cell line was added to the inserts. Next, 0.7 mL DMEM containing serum with or without BsAb was added to each lower chamber. The chemoattractant was 10% fetal calf serum. The chambers were assembled and incubated for 24 h at 37°C. Non-invading cells were removed from the upper surface of the membrane. The invading cells on the lower surface were fixed with 100% methanol, stained with 0.1% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA), and counted using five random microscopic fields. The relative migration was calculated based on the ratio of the number of invading cells in the BsAb or JNJ38877605 treatment group versus the number of invaded cells in the control group.
The cells were lysed with M-PER® Mammalian Protein Extraction (Pierce). Proteins were quantified using the BCA Protein Assay kit (Pierce) in accordance with the manufacturer's instructions. Samples containing a total of 50 μg protein were incubated at 100°C for 5 min, separated by SDS-polyacrylamide gel electrophoresis, and subsequently electrotransferred onto polyvinylidene difluoride membrane. Protein detection was performed by overnight incubation with a specific primary antibody at 4°C, incubation with horse radish peroxidase-conjugated secondary antibody (1:5,000 dilution; Pierce Chemical) was added for 1 h at room temperature, and used of a chemiluminescent detection system. Anti-β-actin antibody was used as the control.
Tumor samples were fixed in 4% formalin, embedded in paraffin, and sectioned into 4-μm-thickness slices. After dewaxing, tissues sections were processed by antigen retrieval, followed by the quenching of endogenous peroxidase activity using hydrogen peroxide. PBS containing 0.1% Tween 20 and goat serum was used to block non-specific binding sites. Slides were incubated with polyclonal antibodies against c-MET. After washing with PBS, slides were incubated with biotinylated anti-rabbit IgG antibody followed by horseradish peroxidase-conjugated streptavidin. After developing in DAB substrates (Invitrogen), 5 high-magnification fields were randomly selected in each slide, and the numbers of positive cells in each field were counted using Image-Pro Plus (Media Cybernetics, Inc.).
Enzyme-linked immunosorbent assay (ELISA)
The quantification of c-MET and PD-L1 in exosomes from cell culture supernatants and plasma was performed by sandwich immunoassay using the Human MET/Hepatocyte Growth Factor Receptor ELISA Kit (RAB0676, Sigma) and Human PD-L1 Sandwich ELISA Kit (KE00074, proteintech). Briefly, 100 µL of each sample was added to the ELISA plate and incubated for 60 min at 37℃. The plate was washed 3 times, and solution B was added. The plate was incubated for 30 min at 37℃, washed 5 times, and 90 µL of substrate was added. After incubating for 15 min at 37℃, 50 µL of termination solution was added, and the plate was immediately analyzed using a spectrophotometer set at a wavelength of 450 nm.
Human peripheral blood mononuclear cell (PBMC) preparation and transplantation
Blood from healthy volunteers was collected in heparinized tubes. The isolation of PBMCs was described as previously . A total of 1×108 PBMC per mouse were injected into 5-to 6-week male NOD/SCID mice (SLAC Laboratory Animal Co., Shanghai, China) through tail vein for the reconstitution of immune system.
Tumor xenograft study
Humanized NOD/SCID mice production method was reported as previously (16). All animals were kept in a specific pathogen-free facility and treated in accordance with the National Institutes of Health Care and Use of Laboratory Animals. This study was approved by the Institutional Animal Care and Use Committee of Shanghai Tongji Hospital. For HCT116 xenograft, mice were treated as follows: BsAb (10 mg/kg, intraperitoneal (IP), twice per week), IgG4 and anti-PD-1 (5 mg/kg, IP, twice per week), JNJ-38877605 (20 mg/kg, itragastric gavage, twice per week). PBMCs (1×108) were injected into the tail vein on the first and the fourth time drugs were administered. Body weight and tumor size were measured using an electronic balance and a vernier caliper respectively. Tumor volume was calculated using the formula: volume = (length × width2) × 0.5.
Neutral red analysis
Monolayers of cells were prepared in 24-well plate incubated with 0.1% neutral red physiological saline for 20 min. The supernatant was discarded, and the cells were washed three times with warm PBS to remove unincorporated neutral red particles. The neutral red dye was released from the cells by incubation with 0.2 mL cytosolic solution (50:50 acetic acid: anhydrous ethanol) at room temperature for 2–3 h. After the cells were dissolved, the absorbance was measured using a microplate reader set at 540 nm wavelength.
Statistical analysis was performed using SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA) and graphs were generated using GraphPad Prism 5.0. Student’s t-test was used in analyzing differences between the two groups. The p-value of <0.05 was used to evaluate significance of the data.