Effect of Napsin A and Circulating Tumor Cells with Mesenchymal Phenotype in Lung Adenocarcinoma

Objective: To evaluate the clinical signicance of Napsin A and circulating tumor cells (cid:0) CTCs (cid:0) with mesenchymal phenotype (M-CTC) in lung adenocarcinoma(LUAD). Materials and Methods: Clinical data of 97 LUAD patients were retrieved. The CanPatrol TM CTC enrichment platform was used to isolate CTCs from the peripheral blood of LUAD patients. The protein expression of Napsin A in the tumor tissues was analyzed by immunohistochemistry. Results: 20 of the 97 patients (20.62%) were negative expression of Napsin A (cid:0) Napsin A- (cid:0) and 60 (61.86%) were M-CTC positive (cid:0) M-CTC+). Both Napsin A expression (P=0.004) and M-CTC+ (P<0.001) showed signicant correlation to lymphatic metastasis, and M-CTC+ was also signicantly correlated with the tumor stage (P=0.009) but was not correlated with gender, age, smoking, tumor size and degree of differentiation. Furthermore, the Napsin A- patients had a higher positive rate of M-CTC. In addition, the recurrence-free survival (RFS; Log-rank P <0.001) and overall survival (OS; Log-rank P <0.001) of the M-CTC+ LUAD patients were signicantly worse. Likewise, Napsin A- was also associated with poor RFS (Log-rank P <0.001) and OS (Log-rank P = 0.0003). Conclusion: LUAD patients with Napsin A- have a higher frequency of M-CTC+, and the Napsin A- and M-CTC+ status portends poor prognosis. revealed that patients with negative A and positive M-CTC had poor prognosis. There was a signicant correlation between M-CTC and cancer stage, and the differences between stage I and stage II, III and were signicant.


Introduction
Lung cancer is associated with high morbidity and mortality rates worldwide and in China, and its incidence rate is increasing annually 1,2 . Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and includes adenocarcinoma, squamous cell carcinoma and large cell carcinoma3,4, of which lung adenocarcinoma (LUAD) is the most common. Despite early detection of LUAD with low-dose spiral computed tomography (CT), patient prognosis has not improved. Therefore, there is an urgent need to identify novel prognostic markers of LUAD in order to treat patients at high risk and reduce their mortality rates.
Ashworth rst proposed the concept of circulating tumor cells (CTCs)5 in 1869. CTCs are shed from the primary tumor and enter circulation, eventually seeding into distant organs and giving rise to metastatic tumors 6 . Although most CTCs are recognized and eliminated by the host immune system, a rare population of highly aggressive CTCs can evade the immune cells and cause distant metastasis 7 . There is also evidence that CTCs enter the blood circulation before metastasis 8 . CTCs can be divided into the epithelial (E-CTC), mixed (E/M-CTC) and mesenchymal (M-CTC) subtypes 9 . During epithelial-mesenchymal transition (EMT), tumor cells lose cell-to-cell contact and polarity, and undergo major cytoskeletal changes that endow them with greater mobility and invasiveness 10,11 . EMT not only increases the survival and metastatic abilities of CTCs 12 but is also crucial to chemoresistance and immune evasion 13 . Studies show that an increased frequency of CTCs with the EMT phenotype portends poor prognosis and greater aggressiveness of gastric cancer 14 , colorectal cancer 15 , liver cancer 16 and esophageal cancer 17 .
Many techniques have been reported for the isolation and characterization of CTCs based on the surface antigen expression or physical properties of CTCs 18 . For example, the commonly used CellSearch system is the only method approved by the FDA 19 . As mentioned above, CTCs with epithelial mesenchymal transition will lose epithelial markers completely or partially 20 . And M-CTC will become more invasive, so it is necessary to identify M-CTC. Previous studies have also con rmed that CanPatrol™ has a strong ability to capture CTCs 21 , and can be classi ed into three subgroups by RNA in situ hybridization. Compared with other techniques, CanPatrol™ can identify and classify all CTCs subpopulations, which have been widely used 22 .
Napsin A is an aspartic protease primarily expressed in the lungs and kidneys 23 . It is an immunohistochemical marker of LUAD along with thyroid transcription factor-1 (TTF-1) 24 , and promotes lung maturation by lysing a preform of surfactant protein B expressed in type II alveolar epithelial cells 25,26 .
Napsin A also inhibits the growth of tumor cells by a mechanism independent of its catalytic activity 27 , and also affects malignant transformation 28, 29 . In addition, Napsin A may have function in the differentiation of epithelial cells 27 . Consistent with this, low Napsin A expression in LUAD patients is associated with poor prognosis, although the speci c mechanism is not clear 30,31 .
The aim of this study was to determine the prognostic value of M-CTC and Napsin A in LUAD, and their relationship. To this end, we isolated and typed CTCs from LUAD patients using the CanPatrol™ enrichment platform 32 and RNA in situ hybridization (ISH) respectively, and also evaluated Napsin A expression in tumor tissues.

Study population and design
Ninety-seven LUAD patients admitted to the First A liated Hospital of Guangxi Medical University (Nanning, China) from March 2014 to July 2015 were enrolled. The inclusion criteria were as follows: (i) pathologically con rmed LUAD post-surgery, (ii) underwent radical lobectomy and systemic lymph node dissection, (iii) no history of targeted therapy, radiotherapy and chemotherapy, (iv) no distant metastasis con rmed before surgery, (v) no history of other tumors, and (vi) availability of complete test results and medical records.
Within three days of surgical resection, 5 ml peripheral blood was collected from the LUAD patients into anticoagulant-coated test tubes for CTCs enrichment and biochemical analysis. The ethics committee of the First A liated Hospital of Guangxi Medical University approved the study, and all patients provided written informed consent.

Isolation of CTCs
The CanPatrol TM33 CTC enrichment platform was used to isolate CTCs from peripheral blood samples. Brie y, the red blood cells (RBCs) were rst removed with an RBC lysis buffer, and the plasma was ltered through an 8μm pore size membrane.
We used 1 ml wash buffer (0.1×SSC; Sigma, St. Louis, USA) to wash samples to remove the un-bound probes. Then we putted 0.5 fmol preampli er and samples in 100μl preampli er solution (1.5% sodium dodecyl sulfate, 30% horse serum from Sigma and 3mM Tris-HCl) at 42°C for 20 minutes. Then, we used 0.1×SSC to wash membrane. Then we putted it in 100μl of the ampli er solution (same composition as the preampli er solution, pH 8) with 1 fmol ampli er. We used Alexa Fluor 647-CD45 (leukocyte), Alexa Fluor 594-CD19 (epithelial cells) and Alexa Fluor 488-Twist (mesenchymal cells) to probe cells at 42°C for 20 minutes. Finally, we used 0.1×SSC to wash the cells. The cell nuclei were stained with 4,6-diamidino-2phenylindole (DAPI, Sigma, USA), and the cells were analyzed with uorescence microscope (Olympus BX53, Tokyo, Japan). CTCs count of 0 was de ned as negative (-), and ≥1 as positive (+).

Immunohistochemistry
Tissue specimens were xed in 10% neutral buffered formalin, embedded with para n and cut into 3 mm thick sections. The tissue sections were immersed in citrate buffer (pH 6) and heated in an incubator for 20 minutes for antigen retrieval, and then incubated with a monoclonal mouse anti-human Napsin antibody (clone IP64, 1:100; Leica). The color was developed using diaminobenzidine, followed by counterstaining using hematoxylin. The sections were scored as Napsin A positive or negative based on cytoplasmic immunostaining 34 .

Follow-up
All patients were followed up through telephone interviews or outpatient review until July 31, 2020. Recurrence-free survival (RFS) was de ned as the time from surgery to disease recurrence or the last followup, and overall survival (OS) from the time from surgery to death for any reason or the last recorded followup visit.

Statistical analysis
Statistical analysis was performed using SPSS version 19.0 (SPSS Inc., Chicago, Illinois, USA), and the gures were drawn using GraphPad Prism version 5.0 (GraphPad software, Inc., La Jolla, CA, USA). Logistic regression was used to estimate odds ratio (OR) and 95% CI in order to evaluate the association between clinical features, M-CTC and Napsin A. RFS and OS in patients with different CTCs phenotypes and Napsin A expression were determined by the Kaplan-Meier method. Univariate and multivariate analysis of M-CTC, Napsin A and clinical features were performed using the cox proportional regression model, and a nomogram was plotted based on the multivariate model using the rms package in R platform (R version 3.5.3, https://www.r-project.org/). P<0.05 was considered statistically signi cant.   Table 1).

Patient characteristics
The number of CTCs increased with tumor stage progression (P = 0.0340 between stage I and IV, Fig. 2A).
The number and positive rate of M-CTC also increased with the stage, and were respectively 41.18%, 65.22%, 76.92% and 78.57% in stages I, II, III and IV (P = 0.0133 between stage I and II, P = 0.0015 between stage I and III, P = 0.0026 between stage I and IV, Fig. 2B). However, the frequencies of E/M-CTC and E-CTC were not affected by LUAD progression. In addition, M-CTC showed signi cant correlation with lymphatic metastasis (P < 0.001, OR = 5.100, 95% CI = 2.094-12.426, Table 2) and stage (P = 0.009, OR = 3.326, 95% CI = 1.344-8.227, Table 2).  Twenty patients (20.62%) did not express Napsin A in the tumor tissues (Fig. 3). As shown in Table 3, Napsin but not with gender, age, tumor size, stage, differentiated degree and smoking. In addition, the Napsin Apatients had a greater frequency of M-CTC compared to the Napsin A + patients (Fig. 4). The positive rate of M-CTC was also higher in the Napsin A-versus Napsin A + patients (P = 0.010, OR = 0.133, 95%CI = 0.028-0.620), whereas that of E/M-CTC and E-CTC were not signi cantly different between the two groups (Table 4).     Abbreviation:M-CTC, CTCs with epithelial-mesenchymal transition phenotype; HR, hazard ratio; CI ,con dence interval ; RFS, Recurrence-free survival; OS, Overall surviva.

Discussion
The occurrence, development and evolution of lung cancer is a multi-gene, multi-step and complex biological process, but its mechanism is not clear. Napsin A is A new member of the aspartic acid protease family.
Napsin A is A single chain protein molecule with A relative molecular weight of about 45 000, consisting of 420 amino acid residues,which is encoded by NSPSA gene located on human chromosome 19ql3.3g 35 .
Napsin A is expressed in normal type II alveolar epithelial cells and plays an important role in maintaining normal lung morphology and physiological function. Lee et al. had shown that the high expression of Napsin A suggested a better prognosis, and the lack of expression might be related to the increase of tumor invasiveness 36 . However, the role of Napsin A in the occurrence and development of lung cancer and its mechanism is not very clear, which needs further research.
To my knowledge, this study is the rst to report the relationship between Napsin A and M-CTC and their relationship with prognosis in patients with lung adenocarcinoma. We revealed that patients with negative Napsin A and positive M-CTC had poor prognosis. There was a signi cant correlation between M-CTC and lung cancer stage, and the differences between stage I and stage II, III and IV were statistically signi cant.
CTCs were crucial to tumor invasiveness and metastasis, therefore, they were associated with patient prognosis. In this study, we found that the numbers of CTCs and M-CTC were increased with tumor stage progression, and the M-CTC positive rate was signi cantly higher in patients with lymphatic metastasis and advanced tumor stage.
The difference of M-CTC between patients with and without lymph node metastasis was statistically signi cant (P 0.001), which was related to EMT's changes in tumor cell characteristics and the microenvironment of tumor growth, and the high expression of lytic enzymes involved in the degradation and destruction of extracellular matrix and basement membrane 12  differences or the small sample size of this study. We expect that more conclusions with clinical signi cance can be drawn from larger sample size.
The numbers and positive rate of M-CTC were higher in the Napsin A negative patients, and the M-CTC+/Napsin A-patients had the worst prognosis. This is consistent with the previous nding that Napsin A overexpression inhibits proliferation and EMT of the epidermal growth factor receptor tyrosine kinase inhibitor (EGFE-TKI) resistant cells, induce apoptosis, and sensitizes them to ge tinib 48 . In addition, Napsin A also blocked the G0/G1 transition in an in vitro model of EMT, and inhibited focal adhesion kinase (FAK), which is a critical regulator of cellular adhesion, motility, metastasis and survival 49 .
There were several limitations in our study that ought to be considered. The sample size was small since the patients were from a single center. Therefore, our ndings will have to be validated with further multi-center prospective studies. Second, the mechanism through which Napsin A affects EMT was not studied, and will have to be determined by functional assays. Nevertheless, we established a correlation between high M-CTC count and lack of Napsin A in LUAD for the rst time. Authors' contributions ZH designed and supervised the study. HW conducted the experiment,collected and analyzed the data and drafted the manuscript.All authors read and approved the nal manuscript.

Funding
No founding was received.

Availability of data and materials
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The experimental procedures were approved by the Ethic Committee of the First A liated Hospital of Guangxi Medical University, and a written informed consent was provided by each participant.

Consent for publication
Written informed consent for publication was obtained from each participant.    M-CTC in Napsin A+ and Napsin A-patients.