2.1 Cell lines and cell culture
Human non-small cell lung cancer A549 and NCI-H460 cells were purchased from ATCC (American Type Culture Collection). A549 and NCI-H460 were cultured in DMEM and RPMI-1640 medium supplemented with 10% FBS, respectively. All cells were placed in an incubator （Thermo Fisher Scientific）at 37°C containing 5% CO2.
Clinical tissue sections of patients with non-small cell lung cancer or tumor tissue subcutaneously implanted in nude mice were taken, and the sterilized cover glass was placed in a six-well plate, and the sample was fixed with 4% paraformaldehyde for 20min after the cell fusion rate reached more than 80%. After washing with PBS for 3 times, discard PBS, then added 1ml 0.2% Triton-100.Later washed with PBS for 3 times, added 1ml 10%BSA for 30min after permeating at room temperature for 5 minutes. The primary antibody was diluted with PBS and incubated with the sample at 4°C overnight. After washing with PBS, wash solution was drained with absorbent paper, and then fluorescence secondary antibody coupled with fluorescein isothiocyanate and rhodamine was added and incubated at room temperature for 1 h, away from light. After rinsed with PBS for two times, 5mg/ mL DAPI was diluted at a ratio of 1:10,000 and then dropped onto the slide to re-stain the nuclear. After 5 minutes, the slides were washed and sealed, and then the samples were observed and photograghed under a Leica confocal microscope.
2.3 Western blot analysis
Lysates containing protease inhibitors were added to the collected cells or tissues. After wholly lysing, the supernatant was collected after centrifuging at a speed of 14000rcf in a low-temperature high-speed centrifuge for 10min, and 5ul was removed for protein quantification. Firstly, we added sample 20μg to 10% SDS-PAGE gel, and performed electrophoresis at 70V constant pressure to bromocresol blue indicator to the junction of concentrated gel and separated gel, then used 90V constant pressure until bromocresol blue indicator to the bottom of gel. Secondly, we taken out the gel, cut the target band according to the protein marker, cut the polyvinylidene fluoride with the same size as the gel and activated it with methanol.Finally, we putted it in the sequence of black plate - fiber pad - filter paper - gel-polyvinylidene fluorid - filter paper - fiber pad - black plate, clamped the plate and put it in the transfer tank, filled the transfer liquid and started the transfer. After sealing polyvinylidene fluorid with 8% skim milk at room temperature for 2 h, the membrane were separately combined with specific antibodies BPTF(1:2000, Abcam), VEGF(1:1000, Abcam), VE cadherin(1:1000, Abcam), CD31(1:1000, Abcam), GADPH(1:20000,Proteintech) and β actin(1:500,Proteintech) and incubated at 4°C overnight. TBST was rinsed for 3 times and incubated with HRP labeled secondary antibody at room temperature for 2 h. After full washing of TBST, protein bands were detected by enhanced chemiluminescence solution.
2.4 Transient transfection of siRNA
SiRNA was synthesized by Shanghai Genepharma (China). The company designed non-specific siRNA and three BPTF-siRNAs. Among them, two of the BPTF si-RNAs knocked down BPTF were obvious. Their sequences were: nonspecific siRNA: 5’-UUC UCC GAA CGU GUC ACG UTT-3’ and 5’-ACG UGA CAC GUU CGG AGA ATT-3’; BPTF-siRNA-1 (BPTF-homo-1550): 5’-GGU CCA ACU UGC AGA AUU ATT-3’ and 5’-UAA UUC UGC AAG UUG GAC CTT-3’; BPTF-siRNA-2 (BPTF-homo-6959): 5’-GAC CCA AAC AAC UGU UUC ATT-3’ and 5’-UGA AAC AGU UGU UUG GGU CTT-3’. The transfection process was as follows: First, cells were implanted into each well of a 6-well tissue culture plate, and the cell fusion rate was estimated at 60-80% on the next day. Meanwhile, reagents mixed with RNAiMax (Invitrogen, Carlsbad, USA) and BPTF siRNA were placed in the cells that were still suspended according to the instructions of RNAiMax. The next day, when the cells adhered to the culture plate, we replaced the medium containing 10% FBS but no antibiotics with a new medium supplemented with 10% FBS but containing antibiotics. After 48 or 72 hours, cells were collected.
2.5 In vivo tumor model
Animal experiments were carried out in accordance with the National Hygienic Guidelines for the Use of Experimental Animal Care approved by APF Experimental Animal Center of Dalian Medical University. The following study also adhere to the ARRIVE guidelines. Male nude mice from 4 to 6 weeks old were used in this study. Their average weight was 20 grams. They were fed in SPF animal laboratory where food and water were freely available. A549 cells (5106) were placed subcutaneously under the left arm of nude mice. About 14 days later, the tumor had grown to 5mm5mm(length width), and the nude mice were randomly divided into two groups. There were five nude mice in each group. There was no difference in tumor volume and size of nude mice between the two groups. Group 1 was the control shRNA and group 2 was the BPTF shRNA. According to the Lipofectamine 3000 specification, shRNA plasmids were transfected into tumors. 25ug shRNA was injected in 100 μl mixture every 4 days for 28 consecutive days. Used a digital caliper, we calculated the tumor volume by "volume = (width2length)/2". Finally, nude mice were sacrificed in the operating room of SPF animal laboratory by cervical dislocation for further detection of related proteins by Western blot.
2.6. Clinical tissue sample experiment
2.6.1 Case data
Collected 75 cases of lung adenocarcinoma were directly purchased tissue microarray chips from Shanghai Xinchao Company . Normal adjacent lung tissue was used as control. All the patients were patients with lung adenocarcinoma who received surgical treatment from January 2004 to December 2010, and did not receive radiotherapy or chemotherapy before surgery. All the patients had relatively complete clinicopathological data, including age, gender, family history, and postoperative pathological information.
A total of 26 patients receiving bevacizumab in Dalian Central Hospital from January 2018 to January 2021 were collected. Approval was obtained from the ethics committee of Dalian Municipal Central Hospital. The procedures used in this study adhere to the tenets of the Declaration of Helsinki. Informed consent was obtained from the participants.
For 75 patients of Xinchao Company, follow-up was conducted through telephone follow-up and outpatient review, and the follow-up contents were mainly survival, postoperative adjuvant treatment, recurrence, imaging examination and tumor marker level. The follow-up period ended 5 years after the onset of the disease, and the final deadline was December 2016. The end point event was defined as death from any cause. Overall survival was from the first postoperative day to the time of death from any cause.
For the follow-up of 26 patients treated with bevacizumab, we followed them up through out-patient and in-patient review, including survival, postoperative adjuvant treatment, recurrence, imaging examination and tumor marker level. Follow-up ended in March 2022.
Criteria for efficacy evaluation of bevacizumab: Efficacy evaluation was performed after two cycles (6-8 weeks) of bevacizumab, and efficacy evaluation was performed according to RECIST 1.1 criteria, which were divided into complete response (CR), partial response (PR), stable (SD) or progressive disease (PD). (1) Complete remission (CR): Except for nodular disease, all target lesions disappeared completely. All target nodules must be reduced to normal size (short axis < 10 mm). All target lesions should be evaluated. (2) Partial response (PR): The total diameter of all measurable target lesions was ≥30% below baseline. The sum of the target nodules used the short diameter, while the sum of all other target lesions used the longest diameter. All target lesions should be evaluated. (3) Disease progression (PD): Take the minimum sum of the diameter of all target lesions measured during the whole experimental study as the reference, and increase the diameter and relative value by at least 20% (If the baseline measurement value is the minimum, the baseline value is the reference); In addition, an absolute increase of at least 5 mm in diameter and diameter must be met (The presence of one or more new lesions is also considered disease progression). (4) Stable disease (SD): The decrease degree of target lesions does not reach PR, and the increase degree does not reach PD level, which is between the two. The minimum sum of diameter can be used as a reference in the study. Patients were divided into two groups based on their response to treatment: those with good response (complete response, partial response, or stable disease) and those with poor response (progression).
2.6.3 IHC detection methods and evaluation criteria for BPTF and VEGF
Immunohistochemical staining (IHC): After xylene dewaxing, gradient hydration, removal of endogenous Peroxidase activity, high-pressure antigen repair, PBS buffer washing, IHC oil marker circled around the section, and then sheep serum was dropped to seal the section for 30min. Primary antibody incubation: BPTF (purchased from Abcam) was diluted with 1: 500 and VEGF (purchased from Proteintech) at 1:800. The whole section was covered and incubated overnight at 4℃. Then washed with PBS buffer 4 times, 3min each. Secondary antibody incubation: added secondary antibody drops and incubated at room temperature for 1h. Washed with PBS buffer 4 times, 3min each. DAB was used for 2-3min, and the reaction was terminated by washing with water. After 1-2min, the differentiation of alcohol hydrochloride was 1-3s. Dehydrated and dried, sealed with neutral resin, and observed.
Positive results were assessed by two senior pathologists based on the degree of staining. According to the dyeing depth, the results were divided into four grades."-" is rated as 0 points, "+" as 1 point, "++" as 2 points, "+++" as 3 points. According to the staining area of the tissue:25% is rated as 1 point, 25%-50% as 2 points, 50%-75% as 3 points, and over 75% as 4 points. The total score was the staining depth score staining area score, and the highest score was 12. We defined high expression with a score above 7, and low expression with a score below 6.
2.6.4 Statistical analysis
The correlation between BPTF high expression and N (number of lymph node metastasis) and clinical stage was analyzed according to Pearson Chi-Square test. Kaplan-Meier test and log-rank test were used to analyze the difference in overall survival of BPTF and VEGF with both high and low expression. The relationship between BPTF or VEGF expression and bevacizumab efficacy was analyzed by Pearson Chi-Square test. The subject involved all statistics, and the test level was 0.05, P<0.05 was statistically significant.