Ectopic expression of LAG‐3 in non–small‐cell lung cancer cells and its clinical significance

Abstract Background Lymphocyte activation gene 3 (LAG‐3, also known as CD223) is an immune checkpoint molecule expressed on various types of lymphocytes, and it is mainly involved in maintaining immune homeostasis. However, there are currently no data on LAG‐3 expression in non–small‐cell lung cancer cells. Methods Human lung cancer cells were cultured using conventional methods. The expression of LAG‐3 was measured by Western blot and flow cytometry. Between April 2018 and May 2019, we collected 52 surgical specimens of stage I‐III non–small‐cell lung cancer (NSCLC). Fourteen samples of benign lung tissue lesions were collected as the control group, and the expression levels of LAG‐3 in the lung cancer cells and tissue samples were measured via immunohistochemistry. Results Western blots showed that LAG‐3 was expressed in lung cancer cell lines. There was significant difference in the LAG‐3 expression levels in the NSCLC cells and benign lung tissue (χ 2 = 13.055, P = .0003). The LAG‐3 expression level was significantly associated with the NSCLC clinical stage, and LAG‐3 expression was significantly higher in stage III patients (P < .05). Conclusion LAG‐3 is expressed in NSCLC tumor cells. Furthermore, LAG‐3 not only is expressed in tumor‐infiltrating lymphocytes in NSCLC patients but also is ectopically expressed in tumor cells and associated with TNM stage.

others. Although these conventional therapies have achieved some success, the prognosis of NSCLC patients has not improved significantly. Since the development of tumor immunology, tumor immunotherapy has become a new treatment method. The latest immunotherapy methods aim to target immune tolerance mechanisms by blocking immune checkpoints, thus reversing the functional suppression of the immune response, reactivating T cells, and promoting anti-tumor immunity. In the past few years, therapies targeting the CTLA-4 and PD-1 inhibitory receptors have shown clinical efficacy in lung cancer patients. In addition to CTLA-4 and PD-1, there are many other immune checkpoint molecules, including TIM-3, LAG-3, and TIGIT, 3 which provide alternative targets that could be utilized to induce an anti-tumor immune response.
Lymphocyte activation gene 3 (LAG-3, also known as CD223) is a type I transmembrane protein consisting of 498 amino acids.
LAG-3 is an immune checkpoint molecule expressed on various types of lymphocytes, and it is mainly involved in maintaining immune homeostasis; furthermore, LAG-3 can play a synergistic role with other immune checkpoint molecules. 4 LAG-3 is located on chromosome 12 (12p13.32) adjacent to the CD4 gene.
Approximately 20% of its sequence is homologous with that of CD4, and it mainly binds to the major histocompatibility complex (MHCII) molecule on antigen-presenting cells (APC), promoting cell apoptosis, and limiting proliferation. 5 LAG-3 has been shown to bind to four ligands: MHCII, 6 LSECtin, 7 galectin-3, 8 and fibrinogen-like protein 1 (FGL1); however, its main inhibitory ligand is FGL1, which can induce T-cell inhibition and promote immune evasion by blocking the FGL1-LAG-3 pathway. 9 LAG-3 is involved in the inhibition of T-cell function, which can lead to the failure of T cells, while synergistic blocking of LAG-3 and PD-1 can restore the function of T cells. 10 Combination immunotherapy with an anti-LAG-3 antibody and an anti-PD-1 antibody can stimulate a tumor-specific response, which includes an increased number of effector T cells 11 ; furthermore, this treatment is less toxic than single antibody treatment and can improve prognosis. 12 Therefore, LAG-3 is not only a valuable marker for evaluating prognosis, and it is also a promising therapeutic target. To strengthen its usefulness in these respects, we measured the expression of LAG-3 in lung cancer and further analyzed its clinical significance.

| Cell culture
Human lung squamous cell carcinoma H226 cells, human lung adenocarcinoma H1299, A549 cells, and human lung epithelial cell BEAS-2B were stored by the Central Laboratory of Changzhou Second People's Hospital, which is affiliated with Nanjing Medical University. The cells were routinely cultured in RPMI 1640 medium containing 10% fetal bovine serum and grown in an incubator at 37°C and 5% CO 2 .

| LAG-3 protein level detection
Western blotting was used to measure the LAG-3 protein levels in NSCLC cells. Protein was extracted from cells, and its concentration was measured. SDS-PAGE protein loading buffer (5×) was added to the protein samples, and the mixture was denatured at 100°C for 5 minutes. The protein samples were subjected to SDS-PAGE electrophoresis, and the proteins were then transferred to a PVDF membrane by a wet transfer method. The membranes were blocked in a solution containing skim milk powder for 1.5 hours, washed 3 times with PBST, and then incubated with mouse anti-LAG-3 antibody (LAG-3, 5 μg/mL, 11E3, ab40465, Abcam) overnight at 4°C with shaking. On the second day, the membranes were washed 3 times with PBST. Next, the corresponding secondary antibody was added, and the membranes were incubated at room temperature with shaking for 1.5 hours. Finally, the membranes were washed and imaged with an instrument.  Inclusion criteria were as follows: 1. The patients did not receive radiotherapy or chemotherapy, and 2. the patients had no other malignant tumors. The patients were staged according to the eighth edition of the International Anti-Cancer Alliance TNM Staging. 13 All specimens were fixed in 4% formaldehyde solution, embedded in paraffin, and then cut into 4-mm-thick sections. Two specimens were cut for each specimen, and they used as a positive control and a blank control.

| Immunohistochemistry
Tissue sections were incubated in an oven at 70°C, dewaxed in xylene and ethanol, and then places into citrate buffer (pH 6.0) for antigen retrieval by heating with an induction heat source. Primary antibody (LAG-3, 1:100, 11E3, ab40465, Abcam) was added, and the samples were incubated overnight at 4°C in a humidified box. Next, secondary antibody (Ultra View Universal HRP Multimer, Ventana) corresponding to the primary antibody was added, and the samples were incubated at 37°C for 30 minutes. DAB color development, which involves soaking in 1% hydrochloric acid to induce lithium carbonate to transition from back to blue, was performed after ethanol gradient dehydration, xylene dehydration, oven drying, and application of a neutral gum seal. Hematoxylin counterstaining was also performed.
The two senior pathologists independently scored the samples under a double-blind method according to the degree of staining: The cells were essentially not colored, (−); the percentage of positive cell staining was ≤10% or the cell staining was weakly positive but the percentage was ≤30%, (+); the cell staining intensity was moderate to strongly positive, the percentage of positive staining was 10%-30%, or the staining intensity was weak to moderate, but the percentage of positive staining was between 30% and 50%, (++); and the cell staining was strong in ≥30% of the cells or the cell staining was positive in ≥50% of the cells, (+++). 14 To determine whether LAG-3 expression is associated with clinicopathological features, all of the NSCLC patients were divided into LAG-3 expression-positive and -negative groups. NSCLC patients with LAG-3 staining intensity scores of (−) and (+) were assigned to the LAG-3-negative group, and NSCLC patients with LAG-3 staining intensity scores of (++) and (+++) were defined as LAG-3-positive.

| Statistical processing
The flow data were analyzed by the FlowJo10.2 software, and the statistical analysis was performed with SPSS 22.0 software. The count data are expressed by case (%), and intergroup comparison was performed via the chi-squared test. P < .05 was considered significant.

| Expression of LAG-3 in NSCLC cell lines
Western blot assays with H226, H1299, A549, and BEAS-2B cells revealed that LAG-3 was expressed in both cell lines (Figure 2A).
The location of the LAG-3 expression in these cell lines was further assessed via flow cytometry. It was found that LAG-3 was expressed not only on the cell membrane but also in the cytoplasm ( Figure 2B).

| LAG-3 expression level in NSCLC patients
There was a significant difference in the positive rate of LAG-3 expression in the NSCLC surgical specimens, including 20 negative cases and 32 positive cases. The benign lesions were negative for LAG-3 expression in 13 cases and positive in 1 case. There was a significant difference in the LAG-3 expression levels between the NSCLC and benign lung specimens (χ 2 = 13.055, P = .0003). The relationship between the clinicopathological features and LAG-3 expression level in NSCLC patients is shown in Table 1. The median age was 65 years, and 33% of the patients received postoperative F I G U R E 2 Ectopic expression of LAG-3 in NSCLC cells. A, LAG-3 is expressed in the H226, H1299, A549 lung cancer cell lines, and BEAS-2B lung epithelial cells. Cells were collected from conventional cultures, and the protein levels were measured by Western blotting of whole-cell lysates. β-actin was used as a loading control. B, LAG-3 was expressed not only on the cell membrane but also in the cytoplasm. Cells were collected from conventional cultures, and LAG-

| Relationship between LAG-3 expression and clinicopathological features of patients
LAG-3 was mainly expressed in lymphocytes. In this study, high ectopic LAG-3 expression was observed in 61.5% of the NSCLC patients, and LAG-3 expression was also observed in tumor metastatic lymph nodes.
No LAG-3 expression was observed in non-metastatic lymph nodes.
LAG-3 expression was significantly associated with clinical stage, and LAG-3 expression was significantly higher in stage III patients (P < .05).
The expression level of LAG-3 in patients with squamous cell carcinoma was higher than that in patients with adenocarcinoma, although this difference was not significant (P > .05). There were no significant differences in the LAG-3 expression levels between tumor cells collected from NSCLC patients of different sex, tissue differentiation degree, T stage, or N stage (P > .05) (Table 1, Figure 3). In summary, this study found that LAG-3 was not only expressed in tumor-infiltrating lymphocytes in NSCLC patients, it was also ectopically expressed in tumor cells, and this expression was associated with TNM staging.

ACK N OWLED G M ENTS
This work was supported by grants from the National Natural Science