Cell culture and reagents
Human colorectal carcinoma cell lines HCT-116 and HT-29, and mouse colon carcinoma cell line CT-26 were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). HCT-116 and HT-29 cells were grown in McCoy's 5A (Sigma-Aldrich, Darmstadt, Germany) and CT-26 was grown in RPMI-1640 (Gibco, Dublin, Ireland) supplemented with 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate. All cells were maintained in humidified air containing 5% CO2 incubator at 37°C and cultured every 2-3 days.
EGFL6 recombinant protein was purchased from Sino Biological (Beijing, China). EGFL6 therapeutic antibody (EGFL6-E5-IgG) was generated and provided by Dr. Yu-Ching Lee using phage display technology. Bevacizumab (Avastin®) was purchased from Genentech (California, USA). 5-FU (Fluorouracil® Injection) was purchased from Pfizer (New York City, USA).
Patients and colon tissue specimens
Tissue biopsy samples used in fig. 1A were collected after prior informed written consent as part of a study (no. 106IRB0417) approved by the human ethics committee of Taipei Medical University Joint Institutional Review Board. No chemotherapy or radiation therapy were given to the enrolled patients before surgical therapy. The TNM stages were determined based on the American Joint Committee on Cancer/International Union Against Cancer TNM staging system. Tissue biopsy used in fig. 1B are approved by Taipei Medical University Joint Biobank (TMU-JBB) (no. N201703080).
In vivo animal models
All experiments on mice were performed in accordance with institutional and national guidelines and regulations. Protocols have been reviewed and approved by Animal Use and Management Committee of Taipei Medical University (IACUC approved No. TMU LAC-2016-0034). Mice were maintained under a 12 h:12 h light: dark cycle and fed with standard diet and water ad libitum.
For Azoxymethane (AOM)-induced mutagenesis assay, a total of 35 eight-week-old A/J mice (Jackson Laboratories, Bar Harvor, ME) were randomly divided into Control or AOM treatment group. Mice were injected with 10 mg/kg AOM intraperitoneally (i.p.) once a week for 6 consecutive weeks as previously described [38, 39]. Mice were sacrificed and colons were collected at 1, 2, 4, 8, and 18 weeks after the last AOM challenge to assess for methylene blue, H&E staining, and immunohistochemistry (IHC) staining of EGFL6.
For anti-cancer activity xenograft model, a total of 24 six-week-old nude mice (National Laboratory Animal Center (NLAC), Taipei, Taiwan) were injected subcutaneously with the same volume of Matrigel (BD bioscience, San Jose, California, USA), and 1×107 of HCT-116 cells into the right flank of each animal. When tumors had grown to around 300 mm3, the treatment started. Tumor size was measured twice weekly and calculated from V = length* width2/2. Tumor growth inhibition (TGI%) = [1-(Tt-T0)/(Ct-C0)x100], where C0 and Ct are mean tumor volumes of Control group by first data point and day t, respectively; while T0 and Tt are mean tumor volumes of treatment group by first data point and day t.
For lung metastasis animal model, eight-week-old balb/c mice (NLAC, Taipei, Taiwan) were injected with CT-26 cells (2x105 cell/mouse) intravenously and then divided into three groups for treatment. After 3 weeks of treatment, animals were sacrificed and lung nodules were counted for data analysis.
For wound healing animal model, after given narcotic, 4.0-mm circular full-thickness skin excision wound were created on the dorsal site of each nude mice using biopsy punch. A Tegaderm film was attached to protect and avoid scab generation. Mice were then randomly divided into three groups for treatments. The wound area and body weight were measured every other day until wound closure.
For angiogenesis animal model, mice were injected with 500 µl matrigel subcutaneously mixed with endothelial growth factor (EGF, 150 ng/ml) and heparin (10 µl), treated anti-EGFL6 antibody (15 mg/kg, q5D) through intravenous injection immediately after subcutaneous injection. After seven-day treatment, animals were sacrificed and the matrigel were carefully dissected. Hemoglobin content was then analyzed by Drabkin’s reagent kit (Sigma Chemical, St. Louis, MO, USA) to quantify the blood vessel formation.
RNA isolation and Quantitative real-time PCR
The RNA was isolated using the TRIzol reagent and the Direct-zol RNA MiniPrep (ZYMO research, Irvine, CA, USA) according to the manufacturer’s instruction. Reverse transcription reactions were performed using the RT Kit (Takara, USA) with 2 µg of total RNA according to the manufacturer’s instruction. Quantitative real-time PCR was performed with ABI StepOnePlus Real-Time PCR Systems using SYBR Green dye (Life Technologies, Grand Island, NY, USA). Relative RNA abundance was calculated using the DDCT formula and normalized to the transcript levels of the housekeeping gene GAPDH. Primer sequences used for quantitative real-time PCR were listed in Additional file 2: Table S1.
Total protein was extracted by homogenization in ice-cold RIPA buffer containing protease and phosphatase inhibitor cocktail. Equal amounts of protein extracts were heated in sample buffer and then separated by SDS-polyacrylamide gel electrophoresis. Separated proteins were then transferred to PVDF membrane. The membranes were subsequently probed using the following primary antibodies listed in Additional file 2: Table S2-3. Immunoreactive bands were visualized with an enhanced chemiluminescence substrate detection kit (Amersham, Buckinghamshire, UK).
Colony Formation Assay
For determination of colony forming units (CFUs), cells were plated at a density of 300 cells/9.01 cm2 culture dish. After 8 days of incubation, the colonies formed were fixed with ice-cold methanol for 10 min, and then stained with Giemsa solution for 15 min. After washing and drying, the colony numbers were calculated.
Cells were transfected with the siRNA targeting EGFL6, HIF-1α and scrambled siRNA (#1299003, #42840, #4390847, Thermo Fisher Scientific) using RNAiMAX Transfection Reagent according to the manufacturer’s protocol.
Migration and invasion assays
Cell migration and invasion abilities were evaluated in transwell with 8-µm pore size polycarbonate membranes in 24-well plates (Corning Inc., Corning, NY, USA). The 1×105 cells were seeded to each transwell insert, and filled each well with culture medium. For invasion assays, the membranes in transwell were pre-coated with 50 µg of Matrigel to form matrix barriers. After incubation for 16 h, the cells remaining on the upper surfaces of the membrane were cleaned. The cells moving to the lower surfaces of the membrane were fixed with ice-cold 10% formalin for 10 min, stained with 0.2% crystal violet for 15 min and counted under a light microscope.
Construction of chicken scFv library and biopanning
Chicken scFv library was constructed according to the published protocol with minor modifications . Detail methods are described in Additional file 3: supplementary materials.
Each experiment was performed independently at least three times and the data were presented as mean ± standard error of the mean. Student t test was used to analyze the data between two groups. Kruskal-Wallis test and multiple comparisons were used to analysis data between more than 3 groups. A P value of less than 0.05 was defined as a statistically significant difference.