3.1 Expression correlation of BPTF, VEGF, VE cadherin(CD144) and CD31 was detected by Western Blot and immunofluorescence at cell and nude mice levels. The results showed that BPTF knockdown inhibited VEGF, CD31 and VE cadherin associated with tumor angiogenesis in non-small cell lung cancer.
By transfecting BPTF siRNA, BPTF was respectively knocked down in A549 and NCI-H460 cells, and VEGF protein expression was also detected to decrease (Fig. 1a). Further, in vivo experiments were conducted in nude mice. A549 cells were implanted subcutaneously in nude mice. After 14 days of tumor growth to 5x5cm, the animals were divided into two groups, one group was injected with control BPTF plasmid and the other group was injected with BPTF shRNA plasmid, once every 4 days for a total of 28 days. Tumors were removed, and the expression of BPTF and VEGF in tumor tissues of mice was detected by immunofluorescence method. The results showed that compared with the control group, the expression level of VEGF decreased in BPTF knockdown group (Fig. 1b). We further detected the expression of VE Cadherin and CD31 (Fig. 1c), and found that following with the down-regulated expression of BPTF, the expressions of VE cadherin and CD31 also decreased.
3.2 Expressions of BPTF, VEGF, VE cadherin and CD31 in postoperative cancer tissues and fresh adjacent tissues of patients with first-stage lung adenocarcinoma were detected by immunofluorescence and Western Blot. The results showed that compared with the adjacent tissues, BPTF, VEGF, VE cadherin and CD31 were highly expressed in cancer tissues.
Cancer and para-cancer specimens of 10 patients with primary adenocarcinoma after operation were randomly selected as controls. Figure 2a showed the immunofluorescence diagram of BPTF and VEGF, which showed higher expression of BPTF and VEGF in cancer tissues compared with para-cancer tissues. Further analysis of vascular endothelial cadherin (VE cadherin, CD144) and endothelial tissue biomarker CD31 showed that CD144 and CD31 were higher in cancer tissues than in para-cancer tissues (Fig. 2b). Figure 2c showed the expression of BPTF, VEGF and CD144 in cancer and para-cancer tissues which collected from 4 patients was detected by Western Blot. The results still showed that the expression of BPTF, VEGF and CD144 in cancer tissues was higher than that in para-cancer tissues.
3.3 Expression, clinicopathological characteristics, prognosis and survival analysis of BPTF and VEGF in postoperative lung adenocarcinoma tissue samples, and statistical analysis of their correlation.
A total of 75 samples (tumor and adjacent tissue) were collected from the lung adenocarcinoma microarray microarray chip of Shanghai Xinchao Compony. The case collection period was 5 years from June 2004 to June 2009. All the patients were from third-class hospitals in Shanghai. They had not received radiotherapy or chemotherapy before surgery, and had complete clinical data. The follow-up ended in September 2014.
In order to study the expression of BPTF and VEGF in non-small cell lung cancer, the above lung adenocarcinoma tissue samples were selected and the expression of BPTF and VEGF was detected by immunohistochemical method. The results were evaluated by two deputy chief physicians and pathologists. (Fig. 3a)
Table 1 showed the related clinicopathological features. There were 75 pathological tissues of lung adenocarcinoma, aged 35–80 years, with an average age of 59 years, including 45 males and 30 females, 35 patients with lymph node metastasis and 40 patients without lymph node metastasis, 37 patients with TNM stage T1 + T2, and 38 patients with T3 + T4. There were 17 patients in clinical stage I, 22 in clinical stage II, 19 in clinical stage III, and 17 in clinical stage IV. The positive rate of VEGF in lung adenocarcinoma was 65% and that in para-cancer was 11.8%, the difference between the two groups was statistically significant (P < 0. 05); the positive rate of BPTF in lung adenocarcinoma was 52%, and the para-cancer positive rate was 3.2%, the difference was statistically significant (P < 0.05). According to Pearson Chi-Square test analysis, BPTF overexpression and VEGF overexpression were statistically significant with lymph node metastasis and clinical stage, respectively, while there was no significant correlation with patient age, gender and tumor size.
Table 1
VEGF and BPTF expression in tumor tissues from 75 patients with lung adenocarcinoma and the correlation between their expression and the clinicopathological features.
Clinicopathological Features | Case Number | VEGF | BPTF |
+ | - | P value | + | - | P value |
Age(years) | <60 | 31 | 20 | 11 | 0.901 | 15 | 16 | 0.599 |
≥ 60 | 44 | 29 | 15 | 24 | 20 |
Sex | Male | 45 | 30 | 15 | 0.766 | 26 | 19 | 0.22 |
Female | 30 | 19 | 11 | 13 | 17 |
Lymphatic metastasis | Yes | 35 | 28 | 7 | 0.013 | 25 | 10 | 0.002 |
No | 40 | 21 | 19 | 14 | 26 |
Clinical Stages | I | 17 | 11 | 6 | 0.033 | 5 | 12 | 0.01 |
II | 22 | 10 | 12 | 10 | 12 |
III | 19 | 17 | 2 | 10 | 9 |
IV | 17 | 11 | 6 | 14 | 3 |
T | T1 + T2 | 37 | 22 | 15 | 0.292 | 22 | 15 | 0.202 |
T3 + T4 | 38 | 27 | 11 | 17 | 21 |
Furthermore, Kaplan-Meier method was used to describe the survival curve, and log-rank test was used for statistical analysis. The test level was P < 0.05 was statistically significant. The results showed that the 5-year survival rate was 78% when both of them were low expression in lung adenocarcinoma, while the 5-year survival rate was only 23% when both of them were high expression in lung adenocarcinoma, the difference between the two groups was statistically significant (P < 0. 05). This indicated that the high expression of both indicated worse prognosis of patients (Fig. 3b). Further analysis of the correlation between BPTF and VEGF showed that < 0.05 was statistically significant (Fig. 3c), and there was a certain correlation between them, with a correlation coefficient of 0.29 (Fig. 3d).
3.4 The clinical significance of the positive correlation between BPTF and VEGF in evaluating the efficacy of bevacizumab in patients with lung adenocarcinoma.
Bevacizumab, as a monoclonal antibody that inhibits angiogenesis, inhibits the bioactive of VEGF by binding to VEGF and blocking its biological activity (Rosen et al. 2017). Therefore, we collected 26 clinical tissue samples from patients with lung adenocarcinoma who were still progressing after surgery, chemotherapy and targeted therapy, and treated with bevacizumab, and tested whether the expressions of BPTF and VEGF were correlated with bevacizumab efficacy. The results showed that BPTF was generally highly expressed in the bevacizumab sensitive group, and lowly expressed in the bevacizumab insensitive group (Fig. 4a). Similarly, VEGF expression was relatively higher in the bevacizumab sensitive group than in the non-sensitive group (Fig. 4b). Further, we statistically analyzed the number of cases with high and low expression of BPTF and VEGF in the bevacizumab sensitive and insensitive groups, respectively. The results showed that in the bevacizumab sensitive group, there were 12 cases with high expression of BPTF and 5 cases with low expression. In the insensitive group, there were 2 cases with high expression of BPTF and 7 cases with low expression, P = 0.019 by Chi-square test, showing statistical difference (Fig. 4c). Similarly, VEGF expression was statistically analyzed in the bevacizumab sensitive and insensitive groups. The results showed that in the bevacizumab sensitive group, there were 11 cases of high VEGF expression and 4 cases of low VEGF expression. In the insensitive group, there were 6 cases of high VEGF expression and 5 cases of low VEGF expression. Although there was a higher VEGF expression trend in the bevacizumab sensitive group, the Chi-square test showed no statistical difference (P = 0.32) (Fig. 4d). The use of bevacizumab is not based on VEGF expression, and the specific mechanism of this phenomenon needs to be further studied.