Retrospective study design
A retrospective study of patients diagnosed with mCRC who were treated with Bev and chemotherapeutics from January 2013 to January 2020 at the General Hospital of Northern Theater Command, China, was performed. The inclusion criteria were the presence of mCRC, ≥ 1 site of metastasis with no surgery, treatment with a combination of chemotherapy and Bev for ≥ 2 cycles, detailed medical records showing blood pressure, and antihypertensive treatment. The exclusion criteria were treatment with chemotherapy regimens that did not meet the inclusion criteria; an insufficient number of chemotherapy and targeted therapy cycles; incomplete medical records with no response evaluation or blood pressure measurement; treatment termination for psychological reasons; drug intolerance or other factors; and surgical resection during chemotherapy and antiangiogenic targeted therapy, local radiofrequency ablation or hepatic artery embolization. Eligible mCRC patients received 5 mg/kg Bev every 2 weeks or 7.5 mg/kg every 3 weeks in combination with 5-Fu or capecitabine ± oxaliplatin or irinotecan-based chemotherapy. The details of the retrospective study assessments, including patient enrollment, evaluation of treatment response, and hypertension, are shown in the Supplementary Material. The study was conducted according to the Declaration of Helsinki and approved by the Ethics Committee of the General Hospital of Northern Theater Command, China with informed consent waiver (no: Y2020046).
Reagents and antibodies
Bev injection (100 mg/4 mL, Avastin, Roche Pharma, Switzerland);Lis hydrochloride tablets (10 mg, Zhongfu, China); 5-Fu injection (0.25 g/mL, Xudong, China); 0.9% sodium chloride injection (100 mL, Baxter, China);Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) (HyClone, UT, USA); a BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China); an ECL detection kit, anti-TGF-β1 and anti-Collagen I (Wanleibio, Shenyang, China); anti-Smad2/3, anti-phospho-Smad2/3 and anti-Smad4 (Affinity Biosciences, OH, USA); anti-CD31 and anti-α-SMA (Servicebio, Wuhan, China); anti-GAPDH and secondary antibodies for Western blot analysis (Proteintech, Chicago, USA) were used.
Cell lines and cell culture
Colon cancer cell lines HCT116 were obtained from American Type Culture Collection (ATCC, Manassas, Virginia). The cells were cultured in DMEM supplemented with 1% penicillin-streptomycin and 10% FBS in a humidified environment at 37°C in a 5% CO2 atmosphere.
Human colorectal cancer xenograft model
Female BALB/c-nu mice (4–6 weeks, weight 18 ± 2 g) were purchased from Beijing HFK Bioscience Co., Ltd., (China) and maintained in a special pathogen-free environment. The food and water were available ad libitum. The mice were maintained under a 12-h light / dark cycle at relative humidity 40–60% and room temperature 24–26 ℃. All experimental procedures were carried out in accordance with the guidelines of the Animal Experimental Ethics Committee of the General Hospital of Northern Theater Command. After 5 days of acclimatization to the environment, each mouse was implanted with 0.2 mL of 5×106 HCT116 cells / mL by subcutaneous injection into the unilateral axilla to establish one tumor. Tumor length and width were measured by calipers every two days, and the volumes were calculated with the following formula: V = (L × W2)/2, where V = the tumor volume (mm3), L = the length of the tumor along its major axis (mm), and W = the width of the tumor along its minor axis (mm). Drug administration began when the tumor volume was 50 − 70 mm3.
In vivo study design
The mice were randomly divided into six groups (n = 12 in every group): the Con group (0.2mL PBS, po, per day), Lis group (2.5 mg/kg Lis, po, per day), low-dose Bev group (5 mg/kg Bev, ip, per 3 days), high-dose Bev group (10 mg/kg Bev, ip, per 3 days), Lis + low-dose Bev group (2.5 mg/kg Lis, po, per day combined with 5 mg/kg Bev, ip, per 3 days) and Lis + high-dose Bev group (2.5 mg/kg Lis, po, per day combined with 10 mg/kg Bev, ip, per 3 days). Dosage of Lis and Bev was calculated based on clinically dose conversion and previous studies [25, 26]. Tumor volume and mouse weight were measured every 2 days until the end of the experiment on day 12. Tumors were randomly extracted on days 3 and 6 for the quantitative detection of VEGFA (n = 3 per time point). On day 12, the mice were intraperitoneally injected with 20 mg/kg 5-Fu and sacrificed 10 minutes later, after which the tumors were photographed and weighed (n = 6). Tumor samples were frozen at -80°C or fixed in paraformaldehyde for further analysis of 5-Fu concentration determination, immunofluorescence, ELISA, Sirius Red staining and western blot.
Determination of the 5-Fu concentration in tumor tissues
The 5-Fu concentration in tumor tissues was measured with a LC-MS/MS (LC21A, Shimadzu, Japan; Triple Quad™ 5500, SCIEX, USA) system. The 5-Fu reference standard and internal standard, 5-bromoutacil (5-Bu), were purchased from Victory Biological Technology Co., Ltd. (Sichuan, China). LC was carried with a Thermo Scientific™ Hypersil GOLD™ column (5 µm, 4.6 mm × 150 mm) and the following conditions: mobile phase: methanol-formic acid-deionized water (50: 0.1: 49.9), isocratic elution mode,10-minute elution time, 0.3 mL/min flow rate, 35°C column temperature, and a 5-µL injection volume. The mass spectrometer was equipped with an electro spray ionization (ESI) probe. The multiple reaction monitoring (MRM) m/z transitions monitored were 128.9–42.1 (CE – 33 V) for 5-Fu and 188.8–42.1 (CE − 37 V) for 5-Bu.
Immunofluorescence staining and analyses
The paraformaldehyde-fixed tumor tissues were embedded in paraffin and cut into sections (5 µm) with a slicer (KD-2508, Zhejiang Jinhua Kedi Instrumental Equipment CO., LTD, China). The paraffin sections underwent dewaxing, hydration, antigen retrieval, and serum blocking and were then incubated with anti-CD31 antibody (1:100) and anti-α-SMA antibody (1:500) at 4°C overnight. Cy3 goat anti-rabbit IgG (1:300) and 488 goat anti-mouse IgG (1:400) were then added. After the nuclei were counterstained with DAPI and auto fluorescence had been quenched, the stained tissues were observed and imaged by inverted fluorescence microscopy (Olympus BX53). Morphological observation and quantitative analysis were conducted with Image J software (version 1.49, National Institutes of Health, Bethesda, MD, USA). Nine optical fields of CD31+hot spots per tumor section were selected in high-power vision (×200). The MVD was calculated by counting the number of individual CD31-positive luminal structures in each field. The VMI was calculated by determining the ratio of areas doubly positive for CD31 and α-SMA in each field according to the previous studies [27–29].
VEGFA and HA ELISAs of tumor tissues
Tumor tissues were weighed, cut into small pieces, homogenized in cold PBS (pH 7.4) and centrifuged at 3,000 ×g for 20 minutes at 4°C to obtain supernatant samples. Protein expression in the tumor tissues was measured with ELISA kits for VEGFA (Cloud-Clone Corp, China) and HA (Mskbio, China) according to the manufacturers’ instructions.
Evaluation of collagen in tumor tissues
Collagen deposition in tumor tissues was investigated by Sirius Red staining. Paraffin sections were dewaxed and hydrated via graded ethanol (70%, 85%, 95%, and 100%, v/v) before being stained with Weigert’s iron hematoxylin solution for 10–20 minutes. Following differentiation with acidic differentiation solution and washing with distilled water, the sections were stained with Sirius red staining solution for 1 hour, gently rinsed, dehydrated with ethanol, cleared with xylene, and gradually mounted with neutral gum. Finally, the slices were imaged with an ordinary optical microscope and a polarized optical microscope.
Protein extraction and Western blot analysis
Tumor tissues were lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). Protein concentrations were detected by BCA protein assay kits (Beyotime Biotechnology, Shanghai, China). Proteins (50 µg / lane) were electrophoretically separated on 12% SDS-PAGE gels and transferred to PVDF membranes. The membranes were immuneblotted with specific primary antibodies and then incubated with HRP-conjugated secondary antibody. HRP was detected with an ECL system (Beyotime Biotechnology, Shanghai, China). The resultant bands were imaged by a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc., USA) and quantified by Image J software.
Statistical analyses
Datasets were analyzed by Student’s t-test or ANOVA, and the results are shown as the mean ± SEM. Statistical significance was defined at P < 0.05. For the retrospective analysis, the Pearson's chi-square test or Fisher's exact test was used to assess differences between the groups. progression-free survival (PFS) and overall survival (OS) curves were estimated using the Kaplan-Meier method, while the associations between survival time and predictor variables were statistically investigated using the Cox proportional hazards regression model. All calculations were performed using IBM SPSS Statistics 25.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA, USA).