A total of 235 patients with lung adenocarcinoma were collected from one hospital in Wuhan city in China from March 2010 to September 2015 and followed up to June 2018. All the patients underwent lung cancer surgery and were finally diagnosed with lung adenocarcinoma by pathological biopsy. This study was approved by the Ethnics and Human Subject Committees of Tongji hospital at Huazhong University of Science and Technology. All participants enrolled in this study signed written informed consent for participation, storage and use of surgically-removed cancer and adjacent tissues. And all methods of this study were carried out in accordance with relevant guidelines and regulations.
Data and tissue samples collection
We collected basic information such as age, gender, smoking status, smoking amount, and clinicopathological features including differentiated types of tumor cells, tumor size, tumor node metastasis (TNM) stage, lymphatic or distant metastasis, cell proliferation index (Ki67) and chemoradiotherapy. The differentiated types of lung adenocarcinoma were classified into micropapillary and solid, acinar and papillary, and lepidic types, which are identified as poorly, moderately and well differentiated tumors according to the criteria established by World Health Organization . The TNM stage of patients with lung adenocarcinoma was diagnosed based on the 7th edition (2009) of the American Joint Committee on Cancer for Lung Cancer . And the cancerous tissue and matched adjacent tissue of each patient were collected for histological analysis.
Tissue chips preparation
Small pieces of tissue samples were fixed in 4% paraformaldehyde for 24 h, and then dehydrated, paraffin-embedded, sliced and stained with hematoxylin and eosin (HE), which were examined by microscope to determine the sampling location of the chip. After heating the paraffin block, the target samples taken out by a sampler were inserted into the prepared paraffin block receptor hole in sequence. The samples were merged at 60~65℃ for 20 min using tissue chip fusion instrument (BP0100, Biossci Company). The melted block was then put into a wax mold, embedded in paraffin and cut into 4 µm thick sections.
The sections were deparaffinized, rehydrated, rinsed with distilled water, and repaired at high temperature and pressure for 1~2 min. After cooled to room temperature and washed with Tris-buffer solution (TBS) three times, the sections were incubated with fresh 3% hydrogen peroxide at room temperature for 20 min and blocked with 10% normal goat serum for 20 min. The sections were then incubated with primary antibodies (IFT20, 1:200, proteintech, 13615-1-AP; GM130, 1:200, abcam, ab52649) overnight at 4℃. After incubated at room temperature for 15 min, the sections were washed by TBS three times and then incubated with secondary antibodies (50 µl DAKO) for another 25 min at room temperature. Then the sections were washed three times, stained with diaminobenzidine and counterstained by hematoxylin.
The expressions of positive staining IFT20 and GM130 protein were indicated by the mean optical density (IOD/area), the rate and intensity of positive staining cells. The mean IOD/area and the rate of positively staining cells in five representative views (original objective 400×) from each section were analyzed by Image J software version 1.2.4 (the National Institutes of Health, NIH free software, Bethesda, MD, USA). The mean IOD/area of these views represented the relative expression of positive staining IFT20 and GM130 protein, and the rate of positive stained cells was equal to positive cells number/total cells number×100%. The staining intensity of positive cells of each section was denoted by staining intensity score (0= no staining; 1= weak staining; 2= intermediate staining; 3= strong staining), which was independently evaluated by five professionally trained persons without knowledge of the clinicopathological data of patients. The final score was determined as the consistent results made by three or more persons.
Continuous variables were divided into normal and non-normal distribution, which were compared by Student’s t test and Wilcoxon rank sum test, respectively. Categorical variables were analyzed by chi-square test or Fisher’s exact test. Associations of IFT20 and GM130 protein expressions with clinicopathological features of lung adenocarcinoma patients were assessed by multivariate logistic regression models, adjusting for multiple potential confounders including age, gender, smoking status, smoking amount. The survival analysis of patients with lung adenocarcinoma after lung cancer surgery was performed by a Cox proportional hazards regression model and the median OS was analyzed by Kaplan-Meier survival analyses. All statistical analyses were carried out with SPSS version 26.0 (SPSS Inc., Chicago, IL), and P<0.05 was considered statistically significant.