Cell culture and establishment of tumor-endothelial cell coculture model
Human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs) were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in M200 basal medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), low serum growth supplement (Cascade Biologics, USA), 100 U/ml penicillin, and 10 mg/ml streptomycin. The cells were maintained in a CO2 incubator (Forma Scientific). Human umbilical vein endothelial cells (HUVECs) were prepared essentially by the method of Jaffe et al. [16]. The MKN45 and HUVEC cells were co-cultured in transwell plates with a pore diameter of 0.4 µm. Briefly, two cells were separated by semi-permeable membranes of transwell plate, where the upper and lower chambers were interlinked and thus HUVEC and MKN45 cells could interact with each other by secreted soluble factors, which simulates the tumor microenvironment.
Immunosuppressed mice model with human tumor xenograft by Subrenal Capsule Assay (SRCA). Following the SRC method described by Bogden et al. [17], four-week-old female nude mice with an average weight of approximately 16 g were selected. The mice were anesthetized by intraperitoneal injection, and under aseptic conditions, the kidney was isolated, the renal capsule was separated. Fresh gastric adenocarcinoma specimens resected surgically were washed several times with serum-free RPMI1640 (Gibco) culture medium to remove connective tissue and necrotic tissue. The tissue was then cut into 1 mm3 pieces and implanted into the subcapsular space of the mouse kidney. Seven days later, the transplanted tumors from the nude mice were dissected, fixed with 10% paraformaldehyde, routinely embedded in paraffin, and then sectioned and stained with HE. The tumor tissue was observed under a microscope to assess the angiogenesis.
Phage displayed peptide library and bacterial strain
The Ph.D.-C7C Phage Display Peptide Library Kit (New England Biolabs, Beverly, USA) was used to screen the specific peptide binding to tumor vascularity in nude mouse subcapsular human gastric cancer xenograft model. The phage display library contains random cyclic heptapeptides constrained at the N terminus of the minor coat protein (cpIII) of M13 phage. The titer of the library is 2×1013 pfu (plaque-forming units). The library contains a complexity of 1.2×109 individual clones, representing the entire obtainable repertoire of cyclic 7-mer peptide sequences, which expresses random seven-amino-acids sequence with a structural constraint imposed by a disulfide bond between two cysteine residues flanking the variable region. Extensive sequencing of the naive library has revealed a wide diversity of sequences with no obvious positional biases. Escherichia coli host strain ER2738 (A robust F + strain with a rapid growth rate, New England Biolabs) was used for M13 phage propagation.
In vitro negative screening of phage peptide library with HUVECs and HMVECs
We took HUVECs and HMVECs as the absorber cells for whole-cell subtractive screening from a phage display C7C peptide library. In vitro selection procedure was performed as described by Ridgway et al. [18] and Poul et al. [19]. HUVEC cells (1×107) and HMVEC (1×107) were harvested using 2.5 mM EDTA solution. The HUVEC cells and phages of approximately 2×1011 pfu were blocked with 500 µl blocking buffer (BF), containing 5 mg/ml bovine serum albumin (BSA, Sigma), and 0.1 M NaHCO3 for 30 min at room temperature, respectively; the same procedure was applied to subsequent cells blocking steps. The blocked phages were added to the blocked HUVEC cells and mixed gently for 1.5 h at 4°C. Cells were then pelleted, at this and subsequent panning steps, by centrifugation at 800 rpm for 5 min. HUVEC cells and the phages bound to them were removed by centrifugation, and the phage-containing supernatant was incubated with the BF blocked 1×107 HMVEC cells for 1.5 h at 4°C. Centrifuge the mixture of HMVEC and phages at 800rpm for 5 minutes to obtain the supernatant of unbound HMVEC phages, which is reserved for in vivo screening.
In vivo positive screening of phage peptide library with tumor bearing nude mice model
In vivo phage selection was performed as described with modifications [20]. Briefly, mice were firstly anesthetized deeply using 70 mg/kg body weight of sodium phenobarbital and then injected intravenously (via tail vein) with 200 µl (1011 pfu) of the phage displayed peptide. After 6 minutes, the heart of the nude mouse was exposed, and puncture the left ventricle with a venous needle to inject preheated 50mIRPMI1640 (Gibco), while cutting a small opening at the right auricle. The xenograft tumor and the control organs were removed and weighed. The cell pellet eluted with 4 ml of 0.2 M Glycine–HCl (pH 2.2) for 10 min at 4°C and neutralized with 0.6 ml of 1 M Tris–HCl (pH 9.1), which is the first round of eluted phages. For each (except for the last) round of in vivo selection, the eluted phages were rescued by infection with E. coli host strain ER2738, and propagated individually in 40 ml of LB medium containing 1 µg/ml tetracycline for 4–6 h at 37°C. Cultures were then pooled and centrifuged. The amplified phage particles were purified using PEG-8000 (polyethylene glycol); 2×1011 pfu of the purified phages particles diluted in 200 µl of PBS were reinjected into tumor xenograft mice for another round of selection. Four rounds of negative and positive selection were performed as above. The final round of phages obtained without undergoing amplification are directly used to infect the host bacterium E. coli ER2738. Approximately 20 phage clones are picked for sequencing and identification.
Sequence analysis of selected phages and peptide synthesis
After the fourth round of panning, 20 phage clones were picked up randomly and their ssDNA were automatically sequenced with ABI Prism kit (Perkin Elmer Applied Biosystems, USA). The primer used for sequencing was 5′- HOCCC TCATAG TTA GCG TAA CG-3′ (-96 gIII sequencing primer, provided in C7C kit, New England Biolabs). Then, homologous analysis and multiple sequence alignment were done using BLAST (http://ncbi.nlm.nih.gov/blast/) and ClustalW (http://www.ebi.ac.uk/clustalw/) programs to determine the groups of related peptides [21]. Several peptides displayed by selected phages were synthesized for subsequent experiments. The synthesis of fluorescein-conjugated peptides was performed as described[22]. In brief, the peptides were synthesized with an automated peptide synthesizer by using standard solid-phase Fmoc chemistry. The products were purified by high-performance liquid chromatography (HPLC) and isolated by lyophilization. Their sequence and structure were characterized by mass spectrometry, and the purity of the peptides (95%) was determined by analytical HPLC. The tarting peptide (CNTGSPYEC) was synthesized by GL Bio-chem (Shanghai) Ltd. A control peptide was created by randomly scrambling the amino acid sequence of the targeting peptide (CNTGSPYEC) while maintaining the disulfide bond to preserve the U-type structure (CNKSPSGNC).
In vitro endothelial tube formation assay
Co-HUVEC cells were seeded at 2 × 104 cells/well in 96-well plates, pre-coated with matrigel (BD Biosciences, New Jersey, USA), and treated with targeting peptide CNTGSPYEC and control peptide. After 24 h, tube formation was visualized using an inverted microscope.
Immunofluorescence microscopy
The cells were cultured on coverslips, then blocked with 10% normal goat serum in PBS. For immunofluorescent staining, the synthetic peptides labeled with Sulfo-Cyanine3 (Cy3) were incubated with Co-HUVECs and control cells overnight at 4°C. PBS and control peptides labeled with Cy3 were used as negative controls. Co-localization of the selected peptides with endothelial marker CD31 was performed to identify the ability of the peptides staining blood vessels in gastric adenocarcinoma tissues as described previously [23]. The Cy3-conjugated peptides were incubated with the sections of gastric adenocarcinoma tissues and nontumor human gastric tissues, and then the sections were stained with anti-CD31 antibody followed by FITC-conjugated anti-mouse IgG. Control peptides labeled with Cy3 were used as negative controls. After washing three times with PBS, the sections were observed in the fluorescence microscope (Nikon EZ-C1, Nikon, Tokyo, Japan).
Competitive and inhibitory assay
The cells in the different groups (Co-HUVECS and wild-type HUVECs) were cultured on coverslips and then fixed with PFA at 4°C for 20–30 min. different concentration unlabeled peptide (0, 50, 100, 150 and 200 µg/ml) diluted in PBS were added onto the slips and incubated at 4°C overnight. After being washed three times with PBS (pH = 7.2), the synthetic peptides labeled with Fluorescein Isothiocyanate (FITC) were incubated with Co-HUVECs or HUVECs for 15 min. control peptides labeled with FITC were used as negative controls. Staining of nuclei was performed with DAPI and sections were mounted with nail polish. Fluorescent signals were detected using a confocal fluorescence microscope (Nikon EZ-C1, Nikon, Tokyo, Japan).
Statistics analysis
Immunofluorescence stain and Competitive and inhibitory assay were repeated more than three times. The staining intensity was evaluated by giving a mark according to the grade standard from our laboratory. All data were reported as mean ± standard deviation (SD). Unpaired two-tailed t-test or one-way analysis of variances (ANOVA) was applied for statistical analysis by SPSS 29.0. A P value below 0.05 was considered to be statistically significant.