Our study enrolled 434 patients who underwent surgical resection of NSCLC, which comprise of the testing cohort, from 2017 to 2020 in the First Affiliated Hospital of Shandong First Medical University and the Second Affiliated Hospital of Shandong First Medical University. From the testing cohort, a total of 203 patients were selected and their outcomes were followed up. The inclusion standards contain that (i) available formalin-fixed tumor tissues and follow-ups, (ii) no pre-operational adjuvant therapy including chemotherapy or radiotherapy. Patients who died during the perioperative period were excluded from the study. All patients received the standard treatment strategies of NSCLC after lung cancer resection including chemotherapy if tumors were invasive, and target therapy if there existed available mutations (17). The study was approved by the Ethics Committee of the First Affiliated Hospital of Shandong First Medical University, and the Second Affiliated Hospital of Shandong First Medical University (Approval No. 2021-091).

CCKAR expression in NSCLC and BM was detected and semi-qualified with IHC by streptavidin peroxidase complex method as previous described (18). Slides were treated with boiled citrate buffer (pH = 6.0) for antigen retrieval. The inactivity of endogenous peroxidase was completed by 3% hydrogen peroxide, and unspecific antigen binding was blocked by 5% bovine serum albumin. The primary antibody of CCKAR (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or CCK-8 (1:100, Abcam, ab27441, UK) was used to incubate the specimens overnight at 4°, and the corresponding secondary antibody labelled with streptavidin-biotin-peroxidase reagent (Beyotime, Beijing, China) was administrated to incubate the specimens for 30 minutes at room temperature. At last, the antigens were visualized by incubation in the 3,3’-diaminobenzidine solution.

The IHC results were semi-quantified by two aspects, the staining intensity and the positive stained cell percentage, which were the IHC score. The IHC score was assessed by two senior pathologists who were unaware of the clinical data. The IHC score was defined as the score of the staining intensity multiplied by the score of positive stained cell percentage. The scores of staining intensity were defined as: score 0, 1, 2 and 3 for negative staining, weak staining, moderate staining and strong staining, respectively. The scores of positively stained cell percentage were defined as: score 1, 2, 3 and 4 for <25% positive cells, 25%-50% positive cells, 50%-75% positive cells and 75%-100% positive cells respectively. The final IHC score varied from 0 to 12. The cohort was divided into subsets with different CCKAR expression by the cut-off of IHC score, which was identified by receiver operating characteristic (ROC) curve.

qRT-PCR was performed to assess CCKAR expression in fresh NSCLC tissues and para-tumor tissues. The total RNA was extracted by TRIzol agent (Thermo Fisher, Waltham, MA, USA). Reverse transcription of cDNA was accomplished with ReverTra kit (TOYOBO, Japan), and the quantitative real-time polymerase chain reaction (qRT-PCR) was applied with Thermo Fisher 7500 PCR System and SYBR Premix. The quantification of qRT-PCR results was analyzed by the 2^-ΔΔCt method with GAPDH as an internal control. The primers of CCKAR and GAPDH were designed as follows: CCKAR, forward: 5’-ATGGATGTGGTTGACAGCCTT-3’, reverse: 5’-AAGCGTCTCATTTTCGAGCCC-3’; GAPDH: forward: 5’-GAGTCAACGGATTTGGTCGT-3’, reverse: 5’-GACAAGCTTCCCGTTCTCAG-3’.

SPSS 22.0 software (SPSS, Chicago, IL, USA) was used to analyze all the statistical significance. The correlations between CCKAR, BM and other clinicopathological factors were assessed by the chi-square test. The statistical difference between subgroups was assessed by the log-rank test, and overall survival(OS) curves were plotted by Kaplan-Meier method. The Cox-regression hazard model was applied to identify the independent prognostic factors. P value less than 0.05 was regarded as statistically significant.

The expressions of CCKAR in NSCLC and corresponding tumor-adjacent tissues were detected with IHC and qRT-PCR. In 203 cases of NSCLC, IHC was performed to show the expression and localization of CCKAR. In our study, CCKAR was mainly expressed in cytoplasm and membrane ( Figure 1A ). The patients were further divided into subgroups with low or high expression of CCKAR, accounting for 116 and 87 respectively. CCK-8 expression was also detected with IHC in NSCLC. In NSCLC, CCK-8 was hardly detected ( Figure 1B ).

In 12 pairs of NSCLC tissues and para-tumor tissues, CCKAR expression was detected with qRT-PCR. As the result, CCKAR in NSCLC was significantly higher than that in para-tumor tissues ( Figure 1C ), suggesting that CCKAR may play an important role in NSCLC progression.

These 203 patients were followed-up and a total of 64 patients suffered from asynchronous BM. The correlations between CCKAR and clinicopathological factors were analyzed with chi-square test ( Table 1 ). Interestingly, high CCKAR in primary NSCLC was significantly associated with positive asynchronous BM (P<0.001). The number of patients with BM was 58 in high-CCKAR patients but only 6 in low-CCKAR patients. The BM percentages for NSCLC patients with low and high CCKAR were surprisingly 5.2% and 66.6%, respectively ( Figure 1D ). Moreover, CCKAR tended to be significantly associated with advanced TNM stage (P=0.087) and female gender (P=0.067).

The correlation between asynchronous BM and clinicopathological factors was also analyzed ( Table 2 ). Patients with positive lymphatic invasion were more likely to suffer from asynchronous BM (P=0.010). More interestingly, target therapy of NSCLC was associated with low probability of BM (P=0.044).

The prognostic significance of CCKAR and other clinicopathological parameters were analyzed by univariate and multivariate analyses. The univariate analysis was performed with the log-rank test ( Table 3 ). In the univariate analysis, CCKAR was a significant prognostic factor of NSCLC ( Figure 2A ). In addition, large tumor size(P =0.006), lymphatic invasion (P<0.001), advanced T stage (P=0.025) and TNM stage (P<0.001) were all substantially associated with unfavorable outcome ( Figures 2B–E ). Moreover, positive BM was a prognostic indicator of poor prognosis ( Figure 2E ).

The independent prognostic factors of NSCLC were further validated by multivariate analysis ( Table 3 ). The prognostic factors in univariate analysis were enrolled into the Cox-regression model including tumor size, T stage, lymphatic invasion, BM and CCKAR expression. Positive lymphatic invasion was identified as an independent prognostic parameter of NSCLC. CCKAR and BM were not an independent prognostic biomarker of NSCLC mainly because of they had significant correlations.

A total of 43 patients with BM underwent surgical resection, and CCKAR expression in BM lesion was assessed with IHC and WB ( Figure 3A, B ). According to the cut-off of CCKAR IHC score in BM, we divided these 43 patients into subgroups with high or low CCKAR expression, accounting for 23.3% (10/43) and 76.7% (33/43) respectively. These results further indicate that CCKAR may be a driving force of BM from NSCLC.