The impact of neuron-related cell adhesion molecule on cellular biological functions and its clinical significance in small-cell lung cancer

Introduction

Small-cell lung cancer (SCLC) represents a highly aggressive neuroendocrine tumor, constituting roughly 15–20% of all lung cancer cases (1,2). This condition is defined by accelerated growth, a significant tendency for metastasis, and a lack of distinct symptoms during its initial phases. The 5-year survival rate is less than 10% since SCLC is typically detected at an advanced stage (3,4). Although recent advances in immunotherapy have improved the prognosis for some patients, current treatment options remain wholly unsatisfactory in meeting clinical needs due to the high recurrence rate and drug resistance associated with the disease (5). The clinical samples for SCLC are relatively scarce, and research progress is slow, which poses significant challenges to advances in treatment. Consequently, investigating more accurate diagnostic and treatment approaches and enhancing the comprehension of the molecular pathways causing SCLC are vital to improving patient survival rates and quality of life.

Neuron-related cell adhesion molecule (NrCAM) is a transmembrane protein belonging to the L1 family of the immunoglobulin superfamily (6,7). NrCAM plays a crucial role in the nervous system, with its key functions being the promotion of neuronal connectivity, guidance of axonal growth, formation of the Ranvier nodes, and regulation cell migration (8). The bulk of research in this field suggests that NrCAM is associated with various neurological disorders, such as schizophrenia and neuropathic pain (9,10); moreover, its genetic variations may affect disease susceptibility and thus potentially serve as a predictive molecule or therapeutic target (11,12). Recent data indicate that NrCAM is overexpressed in tumor tissues and may contribute to tumor invasion and metastasis (13,14). This study analyzed lung cancer transcriptome sequencing data from The Cancer Genome Atlas (TCGA) database and determined that NrCAM was among the 32 most significantly upregulated genes in SCLC. Considering that SCLC is classified as a high-grade neuroendocrine carcinoma and NrCAM is primarily expressed in neural tissues and involved in axonal guidance and synaptic stabilization, we hypothesized that NrCAM may be associated with the neuroendocrine features of SCLC and potentially influence tumor behavior through neurodevelopmental pathways. However, there are currently no reports on the role of this gene in SCLC. In order to provide a theoretical foundation for future investigations into the origins and treatment of SCLC, we sought to examine the clinical significance of NrCAM in SCLC and its influence on cellular biological activities. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1273/rc).

Methods

Cell culture

The American Type Culture Collection (Manassas, VI, USA) cell bank provided the normal human bronchial epithelial (HBE) cells and SCLC cell lines (H446, H69, SHP-77, and H1688) used in this investigation. HBE cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), whereas the SCLC cell lines were cultivated in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin. Every cell was maintained in a normal 5% CO₂ environment at 37 ℃.

TCGA analysis

Differential gene expression in SCLC was assessed by analyzing the transcriptomic data from three major datasets published in TCGA database: GSE6044, GSE111044, and GSE44447. The DEGs were selected based on the following criteria: P<0.05 and |log fold change (FC)| >2 (15).

Clinical tissue and pathological data collection

Between January 2017 and December 2019, 43 patients with pathologically confirmed SCLC who received treatment at Nantong Tumor Hospital were recruited. Formalin-fixed paraffin-embedded tumor tissues and paired adjacent noncancerous tissues were collected, along with corresponding clinical and pathological data. The inclusion criteria were as follows: (I) histologically confirmed diagnosis of SCLC; (II) no prior radiotherapy, chemotherapy, or immunotherapy before tissue collection; and (III) availability of complete clinical and follow-up data. The exclusion criteria were: (I) mixed histological types of lung cancer (e.g., combined SCLC and non-SCLC); and (II) presence of other concurrent malignancies. Tumor-node-metastasis (TNM) staging, tumor size, and other pathological features were among the additional clinical data examined. All procedures in this study involving human participants were performed in accordance with the Declaration of Helsinki and its subsequent amendments, and the study was approved by the Ethics Committee of Nantong Tumor Hospital (No. 2021060). Informed consent was obtained from all the patients.

Immunohistochemistry

Tissue sections (2 µm) that were paraffin-embedded and fixed with formalin were deparaffinized using xylene and hydrated using varying percentages of alcohol (100%, 95%, 80%, and 70%). Citrate buffer (pH 6.0) was used for antigen retrieval, and the sections were heated in the buffer for 10 to 15 minutes at 95 to 100 ℃ before being allowed to cool to room temperature. The slices were incubated for 30 minutes at room temperature with either normal goat serum or 5% nonfat milk to inhibit nonspecific binding sites. Subsequently, the sections were incubated with an anti-NrCAM antibody (cat. No. ab191418; Abcam, Cambridge, UK) overnight at 4 ℃. The following day, the sections were incubated with biotinylated secondary antibody for 30 minutes at room temperature following three 5-minute phosphate-buffered saline (PBS) washes. A 3,3’-diaminobenzidine (DAB) substrate was employed for signal development. After 5–10 minutes, the reaction was halted, and the sections were cleaned with distilled water. Dehydration and mounting were completed after the application of hematoxylin counterstaining. The intensity of staining and the proportion of positive cells in the sections were scored. Proportions of less than 10%, 10–25%, 26–50%, 51–75%, and >75% were scored from 0 to 4, respectively. The staining intensity was categorized as no color, light yellow, brown-yellow, and brown, corresponding to scores of 0 to 3. The percentage of positive cells and staining intensity scores were multiplied to determine the final score. A low-NrCAM expression group was formed consisting of patients with SCLC with mildly positive (score =1–3) or negative (score =0) expression, while a high-NrCAM expression group consisted of patients with positive (score =4–6) or strongly positive (score =8) expression.

Quantitative real-time polymerase chain reaction (qRT-PCR)

TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was used to extract total RNA from SCLC tissues and cells, which was subsequently reverse-transcribed into complementary DNA (cDNA). SYBR green reagent was used for qRT-PCR, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was the standard reference. All reactions were carried out in triplicate to ensure reproducibility, and the relative gene expression levels were calculated using the 2^−ΔΔCt method. The following primer sequences were used:

Forward NrCAM, 5'-GAGGTGTCTAGCCCAGTGGA-3';
Reverse NrCAM, 5'-ATGCGGGAAACTTTGAAGAA-3';
Forward GAPDH, 5'-GGEGGCACTGTGATCCTAC-3';
Reverse GAPDH, 5'-TCGGEGAGTGTGGATGAGG-3'.

Western blotting

SCLC tissues and cells were lysed on ice for 20 minutes using a lysis buffer containing protease inhibitors (Roche, Basel, Switzerland). Total proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% bovine serum albumin was applied to prevent nonspecific binding, membranes were incubated with primary antibodies against NrCAM (AB243) and β-actin (AB8226). Horse radish peroxidase-conjugated secondary antibodies corresponding to the species of the primary antibodies were then applied. Protein signals were visualized with a chemiluminescence imaging system (16). All experiments were independently repeated at least three times to ensure reproducibility and reliability of the results.

Vector construction and cell transfection

A lentiviral vector targeting NrCAM knockdown was constructed and transfected into H1688 cells, resulting in three groups: a blank control group (shRNA-NC group), a group targeting the NrCAM-knockdown sequence 1 (shRNA-1 group), and a group targeting NrCAM-knockdown sequence 2 (shRNA-2 group). A flag-tagged lentiviral vector for NrCAM overexpression was established and transfected into H446 cells in two groups: one that overexpressed NrCAM (Flag-NrCAM group) and another that was a blank vector control group (Flag-NC group). NrCAM overexpression or knockdown expression levels were confirmed via qRT-PCR and Western blot tests. All experiments, including transfections and subsequent validations, were independently repeated at least three times to ensure reproducibility.

Cell Counting Kit-8 (CCK-8) assay

Following transfection, H1688 and H446 cells were gathered and placed at 3,000 cells per well in a 96-well plate density. CCK-8 (cat. No. C0038; Beyotime, Nantong, China) reagent was added to the culture media following the designated time for culturing. A microplate reader was used to measure the absorbance at 450 nm after the cells were cultured for 2 hours at room temperature (17). Procedures in each experimental group were independently repeated three times to ensure reproducibility.

Colony formation assay

Transfected H1688 and H446 cells were counted and digested before being planted in 6- or 12-well plates at a density of 1,000 cells per well. The cells were cultured at 37 ℃ in a 5% CO₂ incubator for 7–14 days, with the medium being replaced every 2–3 days to ensure cell growth. In order to observe the colonies, the cells were fixed with 4% paraformaldehyde after incubation and stained with either methyl blue or crystal violet. Under a microscope, the number of developed colonies was noted and counted. Procedures in each experimental group were independently repeated three times to ensure reproducibility.

Transwell assay

For 72 hours, transfected H1688 and H446 cells were cultured in the upper chamber of a Transwell insert covered with RPMI-1640 medium and 50% Matrigel (Corning, Corning, NY, USA). Following their migration to the membrane’s bottom surface, the cells were stained with crystal violet and paraformaldehyde. An optical microscope was used to count the number of migrating cells. Procedures in each experimental group were independently repeated three times to ensure reproducibility (18).

Scratch assay

After transfected H1688 and H446 cells were placed in culture dishes, a sterile pipette tip was used to make a homogeneous scratch over the cell monolayer once the cells had reached 80–90% confluence. The cells were subsequently rinsed with PBS to eliminate dislodged cells, and a fresh culture medium was added. The culture dishes were positioned in a 5% CO₂ incubator at 37 ℃, and the scratched region was intermittently examined under a microscope to monitor and document cell migration. Images were typically taken every 12–24 hours until the wound was fully healed. The speed and extent of cell migration were assessed through the comparison of images obtained at different time points. Procedures in each experimental group were independently repeated three times to ensure reproducibility.

Nude mouse tumorigenicity assay

A total of 21 BALB/c male nude mice (6 weeks old and weighing 18–22 g) were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The mice were housed in a specific pathogen-free facility under controlled environmental conditions, including a temperature of 22±2 ℃, a relative humidity of 50%±10%, and a 12-hour light-dark cycle. All animals had free access to chow and sterile water throughout the experiment. After 1 week of acclimatization, the mice were randomly divided into three groups (n=7 per group). H1688 cells from different transfection groups (including shRNA-NC, shRNA-1, and shRNA-2) were used for tumorigenicity experiments. Subcutaneous injections of approximately 5×10⁶ cells were administered into 6-week-old male nude mice. The mice’s body weight and tumor progression in each group were systematically observed after injection. Tumor volume was assessed beginning on day 12 after injection via measurements of the length and width of the tumor. The experiment concluded at week 5 (30 days), when the maximum tumor volume approximated 2,000 mm³. Tumor tissues were subsequently collected for analysis. NrCAM messenger RNA (mRNA) expression levels in tumor tissues were evaluated using qRT-PCR, while immunohistochemistry staining was conducted to analyze the expression of NrCAM and Ki-67 proteins. A subset of nude mice from the experimental groups was used for tail vein injections to determine the impact of NrCAM on the metastatic capacity of H1688 cells. The animal experiment was performed under a project license (No. 2024-077) granted by the Ethics Board of Nantong Tumor Hospital, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Statistical analysis

SPSS 26.0 software (IBM Corp., Armonk, NY, USA) was used to analyze the data. The mean ± standard deviation (SD) is expressed as continuous variables, and t-tests were used to compare groups. Meanwhile, categorical variables are expressed as numbers and percentages and were compared via the Chi-squared test (χ²). The Kaplan-Meier method was used to analyze survival. In order to determine independent risk factors influencing the prognosis of patients with SCLC, univariate and multivariate Cox regression analyses were employed. Statistical significance was defined as a P value of less than 0.05.

Results

TCGA database analysis

Differential expression analysis was performed on the SCLC and adjacent normal tissues from the GSE6044, GSE111044, and GSE44447 databases, with |log₂FC| >2 and P<0.05 as the screening criteria, and 82, 1,186, and 1,430 significantly differentially expressed genes (DEGs) were identified in these databases, respectively (Figure 1A). Through the intersection of upregulated and downregulated genes from the three databases, 32 genes were significantly upregulated, while five genes were significantly downregulated (Figure 1B). Further ranking of the 32 upregulated genes by their expression FC revealed that INSM1, ASCL1, and NrCAM were the top three genes. A search of the PubMed database for literature related to the upregulation of INSM1, ASCL1, and NrCAM indicated that although INSM1 and ASCL1 have been thoroughly investigated in SCLC, there are no findings regarding the involvement of NrCAM in SCLC. Our study preliminarily examined the expression and functional importance of NrCAM in SCLC.

Figure 1 Results from TCGA database. (A) Changes in transcriptome expression profiles from the GSE6044, GSE111044, and GSE44447 datasets in TCGA database, with DEGs identified based on |log₂FC| >1 and P<0.05. (B) Intersection analysis of DEGs from the GSE6044, GSE111044, and GSE44447 datasets. DEGs, differentially expression genes; FC, fold change; TCGA, The Cancer Genome Atlas.

Immunohistochemical staining of NrCAM in SCLC tumor and adjacent normal tissues

According to immunohistochemical staining outcomes, NrCAM was essentially nonexistent in the surrounding normal tissues (normal #1 and normal #4 in Figure 2), indicating that NrCAM expression was reduced in these tissues. NrCAM showed a brownish-yellow staining pattern in SCLC tissues, with varying expression levels among the tissues from patients with SCLC. NrCAM expression was classified as negative (0 point), weakly positive (1–3 points), positive (4–6 points), and substantially positive (8 points) based on the staining intensity (Figure 2).

Figure 2 Immunohistochemical staining of NrCAM in SCLC and adjacent normal tissues. NrCAM, neuron-related cell adhesion molecule; SCLC, small-cell lung cancer.

Relationship between NrCAM expression and SCLC clinical pathological features

According to immunohistochemical staining results, patients with SCLC with negative and slightly positive NrCAM expression were placed in the low-NrCAM expression group (n=18). On the other hand, the high-NrCAM expression group consisted of individuals with positive and significantly positive expression (n=25). The patient’s TNM stage, tumor size, and differentiation grade correlated with NrCAM expression. The proportion of patients with poorly differentiated tumors and TNM stages III–IV was substantially higher in the high-expression group than in the low-expression group. Moreover, the tumor diameter in the high-expression cohort was significantly lower than that in the low-expression cohort (Table 1).

Survival outcomes of patients with different levels of NrCAM expression

The group with high NrCAM expression had a median survival time of 36.32 months [95% confidence interval (CI): 30.54–42.10], which was shorter than the group with low NrCAM expression, which had a median survival time of 42.38 months (95% CI: 40.88–43.88) (P=0.04, Figure 3).

Figure 3 The relationship between NrCAM expression in SCLC tissues and patient survival. NrCAM, neuron-related cell adhesion molecule; OS, overall survival; SCLC, small-cell lung cancer.

Multivariate and univariate Cox regression analysis of the prognostic factors of patients with SCLC

Univariate Cox regression analysis indicated that TNM stage (III–IV), lymph node metastases, poor differentiation, and high NrCAM expression were the variables potentially linked to a poor prognosis in patients with SCLC (all P values <0.05). Multivariate Cox regression analysis revealed that a poor prognosis in patients with SCLC was independently associated with increased NrCAM expression (P<0.05) (Table 2).

Expression of NrCAM in SCLC cell lines and the development of knockdown vectors

According to qRT-PCR and Western blot analysis, SCLC cell lines (H446, H69, SHP-77, and H1688) had a higher NrCAM expression than did normal HBE cells, with H1688 cells exhibiting the highest expression and H446 cells the lowest (Figure 4A,4B). These results prompted us to choose the H446 cell line for NrCAM overexpression and the H1688 SCLC cell line for NrCAM knockdown in the subsequent studies.

Figure 4 Detection of NrCAM expression in normal HBE and SCLC cell Lines via qRT-PCR and Western blotting. (A) NrCAM expression in normal HBE and SCLC cell lines as detected by qRT-PCR. (B) Western blot detection of NrCAM expression in normal HBE and SCLC cell lines. (C) Western blot detection of NrCAM expression in H1688 cells transfected with NrCAM-knockdown lentivirus vectors. (D) NrCAM expression in H446 cells treated with lentiviral vectors for NrCAM overexpression was assessed via Western blot analysis. *, P<0.05; **, P<0.01. shRNA-NC, blank control group; shRNA-1, group targeting the NrCAM-knockdown sequence 1; shRNA-2, group targeting NrCAM-knockdown sequence 2; Flag-NC, overexpressed NrCAM; Flag-NrCAM, blank vector control group. HBE, human bronchial epithelial; mRNA, messenger RNA; NrCAM, neuron-related cell adhesion molecule; qRT-PCR, quantitative real-time polymerase chain reaction; SCLC, small-cell lung cancer.

The Western blot analysis indicated that, relative to the shRNA-NC group, both shRNA-1 and shRNA-2 groups decreased the NrCAM protein expression in H1688 cells (Figure 4C). In comparison to the Flag-NC group, the Flag-NrCAM group significantly increased NrCAM expression in H446 cells (Figure 4D). These findings confirmed the successful construction of NrCAM-knockdown and NrCAM-overexpression SCLC cell models for further investigation.

Effect of NrCAM knockdown and overexpression on SCLC cell proliferation and colony formation

Results from the CCK-8 assay indicated that relative to the shRNA-NC group, the proliferation of H1688 cells in the shRNA-1 and shRNA-2 groups was inhibited significantly starting on day 3 of culture (P<0.05). By day 4, the inhibition of cell proliferation became even more significant (P<0.01) (Figure 5A). On the other hand, the Flag-NrCAM group considerably increased the proliferation of H446 cells as compared to the Flag-NC group (Figure 5B).

Figure 5 Effect of NrCAM knockdown or overexpression on proliferation and colony formation in SCLC cells. The CCK-8 assay was used to assess the effect of NrCAM knockdown or overexpression on the proliferative capacity of (A) H1688 cells and (B) H446 cells. The effect of NrCAM knockdown or overexpression on the colony formation ability of (C) H1688 cells and (D) H446 cells (crystal violet staining). *, P<0.05; **, P<0.01. shRNA-NC, blank control group; shRNA-1, group targeting the NrCAM-knockdown sequence 1; shRNA-2, group targeting NrCAM-knockdown sequence 2; Flag-NC, overexpressed NrCAM; Flag-NrCAM, blank vector control group. CCK-8, Cell Counting Kit-8; NrCAM, neuron-related cell adhesion molecule; SCLC, small-cell lung cancer.

The findings of the colony formation assay showed that the ability of H1688 cells to form colonies in the shRNA-1 and shRNA-2 groups was considerably reduced (P<0.01) as compared to the shRNA-NC group (Figure 5C). In comparison that in the Flag-NC group, the colony-forming ability of H446 cells in the Flag-NrCAM group was substantially higher (P<0.01) (Figure 5D).

Effect of NrCAM knockdown/overexpression on the invasion and migration ability of SCLC cells

Transwell and scratch experiments demonstrated that compared to that of the shRNA-NC group, the migratory and invasive capacity of H1688 cells in the shRNA-1 and shRNA-2 groups had been substantially suppressed (Figure 6A,6B). On the other hand, the H446 cells in the Flag-NrCAM group exhibited considerably enhanced the migratory and invasion capability as compared to those in the Flag-NC group (P<0.05) (Figure 6C,6D).

Figure 6 The effect of NrCAM knockdown on SCLC cell migration and invasion ability. The influence of NrCAM overexpression or knockdown on the migratory and invasion ability of (A) H1688 cells (100×) and (C) H446 cells (100×) was evaluated via a Transwell experiment (crystal violet staining). The effect of NrCAM overexpression or knockdown on the migratory ability of (B) H1688 cells (40×) and (D) H446 cells (40×) is evaluated using a scratch assay. *, P<0.05; **, P<0.01. shRNA-NC, blank control group; shRNA-1, group targeting the NrCAM-knockdown sequence 1; shRNA-2, group targeting NrCAM-knockdown sequence 2; Flag-NC, overexpressed NrCAM; Flag-NrCAM, blank vector control group. NrCAM, neuron-related cell adhesion molecule; SCLC, small-cell lung cancer.

In vivo subcutaneous tumor formation assay examining the effect of NrCAM knockdown on SCLC cell proliferation

The tumor volumes produced by the shRNA-1 and shRNA-2 groups were noticeably less than those produced by the shRNA-NC group (Figure 7A). According to the tumor development curve, the tumor volume in the shRNA-1 and shRNA-2 groups was less than that of the shRNA-NC group beginning on day 24 after injection, and the difference grew more significant with time (P<0.01) (Figure 7B). Tumor weight in the shRNA-1 and shRNA-2 groups was significantly lower than that in the shRNA-NC group according to measurements of the weight of the removed tumor tissues and the expression level of NrCAM mRNA (Figure 7C). Furthermore, there was a significant reduction (P<0.01) in NrCAM mRNA expression in the tumors of the shRNA-1 and shRNA-2 groups (Figure 7D). Immunohistochemical analysis of the tumors for NrCAM and Ki-67 showed that the expression levels of both NrCAM and Ki-67 were decreased in the tumors of the shRNA-1 and shRNA-2 groups relative to the shRNA-NC group, with Ki-67-positive cell counts reduced by 62.43% (shRNA-1 group) and 63.65% (shRNA-2 group), respectively, indicating a substantial suppression of tumor cell proliferation activity as a result of NrCAM knockdown (Figure 7E).

Figure 7 Effect of NrCAM knockdown on SCLC cell proliferation examined via in vivo subcutaneous tumor formation assay in nude mice. (A) Morphology of the tumors formed after 30 days of inoculation of SCLC cells from different treatment groups into nude mice. (B) Measurement of tumor length and width starting from day 12 after injection and tumor growth curves. (C) Weight analysis of the excised tumors after 30 days of inoculation. (D) qRT-PCR analysis of NrCAM mRNA expression in tumors from different groups. (E) Immunohistochemical staining analysis of NrCAM and Ki-67 expression in tumors. *, P<0.05, **, P<0.01. shRNA-NC, blank control group; shRNA-1, group targeting the NrCAM-knockdown sequence 1; shRNA-2, group targeting NrCAM-knockdown sequence 2; Flag-NC, overexpressed NrCAM; Flag-NrCAM, blank vector control group. mRNA, messenger RNA; NrCAM, neuron-related cell adhesion molecule; qRT-PCR, quantitative real-time polymerase chain reaction; SCLC, small-cell lung cancer.

Discussion

The cell adhesion molecule NrCAM is essential for neurodevelopment and the nervous system’s cell adhesion and migration functions (19,20). Recent research on the tumor microenvironment and cell adhesion molecules indicated that NrCAM is involved in the physiological activities of the nervous system and the etiology and evolution of numerous malignant cancers (13,21,22). Numerous studies have confirmed that NrCAM expression is markedly elevated in various malignancies and is strongly correlated with tumor aggressiveness and prognosis. A study employing TCGA data analysis demonstrated that the expression of NrCAM was enhanced in gastric cancer tissues relative to normal tissues. Moreover, this increased expression was associated with unfavorable prognosis in patients (23). Through downregulating miR-506-3p, the circular RNA of NrCAM increases expression in thyroid cancer, strengthening its function in cancer cell invasion, migration, and proliferation and demonstrating NrCAM’s potential as a therapeutic target (24). Furthermore, NrCAM has been connected to low-grade neuroblastoma in children, suggesting that it may be crucial in the early stages of neuroblastoma development (25). These results indicated that NrCAM might promote tumor cell migration and invasion and be involved in the early development of malignancies. As a result, NrCAM has a promising potential as a diagnostic and therapeutic target, offering novel insights for early cancer screening and targeted treatment.

In this study, through TCGA database screening and immunohistochemical validation, we found that the expression of NrCAM was elevated in SCLC tissues and was significantly associated with the TNM stage, tumor size, and differentiation grade of patients. This indicates that NrCAM may be crucial in the initial phases of SCLC progression. As per the survival analysis, patients with high NrCAM expression had a considerably shorter mean survival time than did those with low expression levels. Further evidence from Cox multivariate analysis supports the idea that elevated NrCAM expression is a distinct risk factor for a poor outcome in patients with SCLC. These clinical correlational studies offer a novel biological foundation for evaluating the prognosis risk of patients with SCLC and further support NrCAM’s value as a prognostic biomarker.

Our study examined the influence of NrCAM on the biological behavior of SCLC cells by developing NrCAM-knockdown and NrCAM-overexpression cell models. In vitro tests indicated that SCLC cell proliferation, colony formation, migration, and invasion capacities were dramatically reduced when NrCAM was knocked down. In contrast, these malignant characteristics were markedly amplified when NrCAM was overexpressed. These data suggest that NrCAM promotes SCLC cell malignancy, possibly driving tumor growth by regulating signaling pathways or the interactions with the extracellular matrix.

Furthermore, in vivo nude mouse tumorigenicity experiments confirmed that promotive effect NrCAM promoted SCLC tumor growth. Knockdown of NrCAM in SCLC cells significantly reduced tumor volume in nude mice, and the downregulation of Ki-67 indicated a reduction in tumor cell proliferation activity. We noted that traditional neuroendocrine markers [synaptophysin (SYP), chromogranin A (CgA), CD56] are widely used for diagnostic purposes but have limited prognostic value and no proven functional roles in tumor progression. In contrast, our study demonstrates that NrCAM is not only overexpressed in SCLC but also plays a functional role in promoting tumor cell proliferation, migration, and invasion, both in vitro and in vivo. Furthermore, high NrCAM expression was independently associated with poor prognosis. These findings suggest that NrCAM may serve as a dual-purpose biomarker with both diagnostic and prognostic potential, offering functional advantages over existing markers.

However, the study involved certain limitations that should be acknowledged. First, the number of participants may be insufficient to draw firm conclusions regarding the relationship between NrCAM expression and clinical pathology and prognosis, and multicenter investigations are required for future validation. Second, we did not thoroughly examine the molecular mechanisms underlying NrCAM’s effect on SCLC cell activity. Future research could further combine transcriptomic, proteomic, and other high-throughput technologies to elucidate the downstream regulatory network of NrCAM and clarify the related mechanisms of action.

Conclusions

NrCAM expression is elevated in SCLC and significantly promotes the malignant behavior of tumors. The results of this study strongly suggest that NrCAM may serve as a valuable biomarker for diagnosis and prognosis, and as a potential therapeutic target in SCLC. Future studies should further examine the molecular mechanisms of NrCAM and evaluate its clinical applicability, offering more possibilities for the precise treatment of SCLC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1273/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1273/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1273/prf

Funding: This work was supported by the Nantong Municipal Health Commission (No. MS2024050), the Jiangsu Provincial Health Commission (No. Z2022036), and the Special Key Project of Clinical Medicine of Nantong University (No. 2022JZ012).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1273/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures in this study involving human participants were performed in accordance with the Declaration of Helsinki and its subsequent amendments, and the study was approved by the Ethics Committee of Nantong Tumor Hospital (No. 2021060). Informed consent was obtained from all the patients. The animal experiment was performed under a project license (No. 2024-077) granted by the Ethics Board of Nantong Tumor Hospital, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)