BIRC5 expression correlates with immunosuppressive phenotype and predicts inferior response to immunotherapy in lung adenocarcinoma

Introduction

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung malignancies and is the leading cause of cancer-related deaths worldwide (1). Among the histological subtypes, lung adenocarcinoma (LUAD) is most prevalent (2), leading to the majority of deaths attributable to lung cancers (3). Despite huge treatment advancements in LUAD, including radiotherapy, molecular targeted therapy, and immunotherapy, the overall prognosis still remains unsatisfactory (4). Immunotherapies, especially immune checkpoint blockades (ICBs) targeting programmed cell death protein 1 (PD-1), its ligand (PD-L1), or cytotoxic T lymphocyte antigen-4 (CTLA-4) significantly extend overall survival (OS) in patients with diverse types of cancer, including LUAD (5-7). Nonetheless, a significant proportion of LUAD patients treated with ICB failed to achieve an objective response, highlighting the need for the identification of predictive biomarkers for selection of patients who may benefit from this treatment (8). While ICB has shown to be an effective oncologic treatment for LUAD (9), the response to ICB can be affected by unique genomic and immunological landscapes (10). Therefore, understanding correlates between specific gene expression and tumor immune microenvironmental features could help the development of robust biomarkers for immunotherapy and enhance the clinical response and expand the benefit population (11-13).

BIRC5, also known as survivin, is the smallest but functionally most complex member of the inhibitor of apoptosis protein (IAP) family (14,15). BIRC5 plays a pivotal role in shielding cells from both intrinsic and extrinsic apoptotic pathways, primarily by indirectly inhibiting caspase-9 activation and physically preventing direct interactions with apoptosis-promoting molecules (16). Functionally, BIRC5 promotes malignant cell development by stabilizing the mitotic apparatus, maintaining proper chromosome segregation, and preserving microtubule integrity, facilitating safe and efficient cell division (17). Hypoxia induces BIRC5 expression, promoting angiogenesis and tightly linked to cell proliferation. BIRC5 is highly expressed in most human cancers and strongly correlated with tumor growth, recurrence, chemotherapy resistance, and poor prognosis (17). However, a comprehensive analysis of BIRC5 expression in LUAD and its prognostic and predictive value for ICB treatment outcomes has not yet been investigated.

Therefore, we conducted this research to analyze the expression of BIRC5 in LUAD and its correlation with clinicopathological features, as well as its prognostic values, using data from The Cancer Genome Atlas (TCGA: https://www.cancer.gov/ccg/research/genome-sequencing/tcga), and protein expression data from the Human Protein Atlas (HPA: https://www.proteinatlas.org/) online database. To elucidate the relationship between tumor microenvironmental features and BIRC5 expression, we analyzed the bulk-RNA and single-cell sequencing data to investigate the connections of BIRC5 expression level and the immune cells’ infiltrations. We present this article in accordance with the TRIPOD reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-216/rc).

Methods

Patients and databases

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Gene expression data of 535 LUAD and 59 normal lung tissues was downloaded from the TCGA database, with an additional 57 LUAD and matched normal lung tissues categorized for analysis of clinicopathological features and prognostic values. Transcriptomic data in transcripts per million (TPM) formats from TCGA and GTEx (https://gtexportal.org/home/) was processed uniformly to examine BIRC5 expression through both two databases. In addition, BIRC5 expression data from pan-cancer tissues and surrounding normal tissues was retrieved and evaluated using the Oncomine database (https://www.oncomine.org). The protein level of BIRC5 in LUAD and normal lung tissues was evaluated by using the HPA database.

BIRC5-related functional enrichment analysis

To evaluate BIRC5-binding proteins, we utilized the STRING website (https://string-db.org/) v11.0 (archived version) and established specific thresholds to identify the binding proteins, including a network type of complete network, active interaction sources from studies, and a minimum necessary interaction score of low confidence (0.150). We adjusted the significance of network edges to evidence and limited the maximum number of interactors displayed to 20, resulting in the identification of 20 experimentally confirmed proteins that bind to BIRC5. Additionally, we utilized GEPIA2 (http://gepia2.cancer-pku.cn/#index) to evaluate genes with a similar expression pattern to BIRC5 in LUAD and selected the top 100 candidate genes. Subsequently, on the DAVID website (https://david.ncifcrf.gov), we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of BIRC5, 20 BIRC5-binding proteins, and 100 candidate genes.

Single-cell sequencing data analysis

We utilized the CancerSEA database (http://biocc.hrbmu.edu.cn/CancerSEA/home.jsp), a specialized platform for single-cell sequencing data analysis, to investigate the functional status of cancer cells in relation to BIRC5 expression. Correlation analysis was conducted to explore the association between BIRC5 expression and various tumor functional characteristics, and a heatmap was generated based on the results. Moreover, we generated t-distributed stochastic neighbor embedding (t-SNE) diagrams for each single-cell from the CancerSEA website to further evaluate the relationship between BIRC5 and tumor cell functionality.

Differential gene expression analysis

R package “edgeR” was utilized to determine differentially expressed genes (DEGs) between high and low BIRC5 expression groups. A cutoff gene expression fold change of >1.5 or <−1.5 and false discovery rate (FDR) q-value <0.05 was applied to select the most significant DEGs. We listed all of the DEGs by using volcano plot and the top 100 up-regulated genes in high versus low BIRC5 expression group by using heatmap.

Pathway enrichment analysis

We applied three methods [GO, KEGG, gene set enrichment analysis (GSEA)] to perform the pathway enrichment analysis. The curated gene sets of reported signaling pathways (from the KEGG, Hallmark, PID, Reactome databases) were downloaded from the Molecular Signature Database (http://software.broadinstitute.org/gsea/msigdb/index.jsp). R package “clusterProfiler” was used for GO term analysis, and GSEA software V.4.1.0 was utilized to study the relevant pathways between high and low BIRC5 expression groups.

Infiltration of immune cells

To quantify the immune cell infiltration in tumor tissues, we employed single-sample GSEA (ssGSEA) method. The LUAD expression profile data was subjected to immunological datasets encompassing 24 categories of immunocytes, and the infiltration levels were quantified using the R package “GSVA” (18). Furthermore, Spearman correlation and Wilcoxon rank-sum test were implemented to evaluate the association between BIRC5 and various immune cells as well as the relationship of immune cells with the two groups.

Public datasets of anti-PD-1/PD-L1 treated cohort

The clinical and bulk-RNA-seq data of 54 NSCLC patients received PD-1/PD-L1 blockade monotherapy, along with response data available, were downloaded from previous publications (19-21). The clinical responses were assessed by the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 guideline, including complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD). Responders were defined as patients received PD-1/PD-L1 blockade with CR or PR or SD. Non-responders were defined as patients received PD-1/PD-L1 blockade with PD. The definition of progression-free survival (PFS) and OS was consistent with their corresponding published studies. Kaplan-Meier curves with two-sided log-rank tests and Cox proportional hazards model with calculated hazard ratios (HRs) and 95% confidence intervals (CIs) were used to determine the survival difference.

Statistical analysis

BIRC5 expression in normal lung and LUAD groups was analyzed using the Wilcoxon rank-sum test. Patients with LUAD were divided into two groups based on the median expression of BIRC5. The clinicopathological characteristics comparison between high and low BIRC5 expression groups were analyzed using Wilcoxon rank-sum or Kruskal-Wallis tests. Pearson correlation analysis were calculated to evaluate the relatedness of BIRC5 expression and immune cells abundance, immune-related markers expression. Survival outcomes were measured with OS, disease-free survival (DFS), or PFS according to the accessibility for each cohort. Prognostic and predictive values of BIRC5 expression were analyzed by Kaplan-Meier method, log-rank test, and Cox proportional hazards regression analysis. To study the predictive significance of DEGs, a receiver operating characteristic (ROC) curve was developed using the “plotROC” package. The predictive value nomogram for LUAD patients was plotted using the R tool “rms”. All statistical significance testing was two-sided and P values or FDR q-values <0.05 were considered statistically significant. All tests were performed with the R environment v4.0 or GraphPad Prism version 6.0.

Results

Characterization of BIRC5 expression in LUAD

Firstly, we analyzed the distinct expression levels of BIRC5 between normal lung and LUAD tissues using transcriptomic datasets from the TCGA program. The detailed comparison of characteristics between high and low BIRC5 expression in LUAD were listed in Table 1. The expression level of BIRC5 was significantly higher in LUAD tissues compared to normal lung samples (P<0.001; Figure 1A). In the paired tissue samples, LUAD also showed a significantly higher expression level of BIRC5 than normal lung tissues (P<0.001; Figure 1B). To analyze the expression level of BIRC5 in various cancers, we carried out a systemic analysis using transcriptomic datasets from the TCGA program and found that BIRC5 was upregulated in most cancer types, including bladder urothelial carcinoma, bone cancer, cholangiocarcinoma, and glioblastoma multiforme (Figure 1C). Using HPA database, we evaluated the expression levels of BIRC5 in LUAD and normal lung tissues. We observed that LUAD had higher protein levels of BIRC5 than the matched normal lung tissues (Figure 1D). Moreover, tumor with pathological stage II–IV had significantly higher expression of BIRC5 than those with pathological stage I (P<0.001; Figure 2A). We also found that increased tumor (T) stage (P<0.001), node (N) stage (P<0.001), smoking history (P<0.05), and young age (P<0.05) were associated with elevated expression of BIRC5 (Figure 2B-2F).

Figure 1 The expression level of BIRC5. (A) Comparison of BIRC5 expression level between LUAD and normal lung tissues. (B) BIRC5 expression level in 57 matched LUAD and normal lung tissues. (C) Pan-cancer analysis of BIRC5 expression in various cancers and adjacent normal tissues (**, P<0.01; ***, P<0.001). (D) Representative immunohistochemistry images of BIRC5 in both normal and LUAD tissues sourced from the Human Protein Atlas database (https://www.proteinatlas.org/). Image credit goes to the Human Protein Atlas. The links to the normal and tumor tissues of BIRC5 are provided (https://www.proteinatlas.org/ENSG00000089685-BIRC5/tissue/lung#img; https://www.proteinatlas.org/ENSG00000089685-BIRC5/cancer/lung+cancer). HPA, Human Protein Atlas; LUAD, lung adenocarcinoma; TPM, transcripts per million.

Figure 2 Correlation between BIRC5 expression and clinicopathological features. (A) Clinical stage; (B) T stage; (C) N stage; (D) M stage; (E) smoking history; (F) age (years). *, P<0.05; ***, P<0.001; ns, not significant. M, metastasis; N, node; T, tumor; TPM, transcripts per million.

To determine the prognostic value of BIRC5 in LUAD, we examined the correlation between BIRC5 expression and survival outcomes of LUAD patients. The high expression of BIRC5 was correlated with markedly inferior OS (HR =1.79, P<0.001; Figure 3A), DFS (HR =1.98, P<0.001; Figure 3B), and PFS (HR =1.58, P=0.001; Figure 3C). In addition, the ROC analysis of LUAD to determine the predictive significance of BIRC5 revealed an area under the curve (AUC) value of 0.968 (Figure 3D).

Figure 3 Survival analysis of clinical outcomes between patients with high and low BIRC5 expression. (A) Kaplan-Meier plot of overall survival. (B) Kaplan-Meier plot of disease-specific survival. (C) Kaplan-Meier plot of progression-free survival. (D) ROC analysis of LUAD to determine the predictive significance of BIRC5. AUC, area under the curve; CI, confidence interval; FPR, false positive rate; HR, hazard ratio; LUAD, lung adenocarcinoma; ROC, receiver operating characteristic; TPR, true positive rate.

Analysis of BIRC5 co-expression network and enriched pathway

We identified and experimentally validated the binding proteins of BIRC5 using the STRING website to investigate the co-expression network and pathways enrichment of BIRC5 in LUAD. The results showed the presence of 20 proteins, including MED1, MED6, MED18, MED30, MED31, LAMTOR5, H3F3A, H3F3B, AURKB, AURKC, HFE, INCENP, CDCA8, HIST3H3, CASP3, CASP7, CASP9, DIABLO, XIAP and BIRC2 binding to BIRC5 (Figure 4A). Then, we extracted the 100 most closely linked genes to BIRC5 from GEPIA2 database. GO and KEGG enrichment analyses were conducted on the 448 genes. GO enrichment analysis revealed that these genes were significantly associated with chromosome segregation, mitotic nuclear division, nuclear division, mitotic sister chromatid segregation, ATPase activity, microtubule motor activity, tubulin binding, microtubule binding, and other processes (Figure 4B-4D). In addition, we observed that BIRC5 was implicated in tumorigenesis via cell cycle, pyrimidine metabolism, cellular senescence, DNA replication, p53 signaling pathway, and progesterone-mediated oocyte maturation (Figure 4E).

Figure 4 Co-expression network of BIRC5 and enrichment pathway analysis. (A) The binding proteins of BIRC5 by STRING website. (B-D) GO enrichment analysis of BP, CC, MF. (E) KEGG pathways analysis based on BIRC5 binding proteins and interactive genes. ATP, adenosine triphosphate; BP, biological process; CC, cellular component; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function.

Expression level of BIRC5 in single-cells and its effect on functional status

Single-cell transcriptomic sequencing is a critical tool for analyzing the complex landscape of tumor, immune, endothelial, and stromal cells in cancers. In our investigation of BIRC5 expression and its association with tumor function in LUAD at the single-cell level, we utilized the CancerSEA website and found that BIRC5 expression was significantly correlated with the critical cellular processes such as cell cycle, DNA damage, DNA repair, hypoxia, inflammation, and proliferation (Figure 5A). Our analysis further revealed that BIRC5 expression was associated with cell cycle progression, DNA damage response, proliferation, invasion, epithelial-mesenchymal transition (EMT), and inflammation in LUAD (Figure 5B). We also visualized BIRC5 expression profiles in single-cells of LUAD using t-SNE diagrams (Figure 5C). These findings suggest that the high expression of BIRC5 is associated with DNA damage/repair, hypoxia, inflammation, and proliferation. Whether the expression of BIRC5 plays a role in the development of LUAD needs to be confirmed by more experiments.

Figure 5 Expression pattern of BIRC5 in single-cells and its correlation with tumor functional status. (A) Heatmap showed the correlation between BIRC5 expression and different tumor functional status based on CancerSEA database. (B) Correlation between BIRC5 expression and seven significantly different functional states based on CancerSEA database. (C) t-SNE diagram of BIRC5 expression profiles in single-cells. **, P<0.01; ***, P<0.001. EMT, epithelial-mesenchymal transition; t-SNE, t-distributed stochastic neighbor embedding.

Univariate and multivariate analyses

Having noticed the negative association between high BIRC5 expression and clinical outcomes of LUAD patients, we then conducted the univariate and multivariate analyses by adjusting the common clinicopathological parameters. The results showed that pathological stage, residual tumor, and treatment outcome were significantly associated with OS in univariate analyses (Table S1). High BIRC5 expression was also markedly associated with poor OS. In multivariate analyses, high BIRC5 expression was found to be independently associated with shorter OS in multivariate analysis. We performed a subgroup analysis to investigate the impact of BIRC5 expression on OS based on age, sex, and anatomic neoplasm subdivision risk variables. In these subgroups stratified by age, gender, and anatomic neoplasm classification, we observed that elevated BIRC5 expression was significantly associated with worse survival outcomes (Figure S1).

Enriched pathways in tumor with high BIRC5 expression

To identify the different pathways enrichment between high and low BIRC5 expression groups, we performed GSEA using bulk-RNA sequencing data from 535 LUAD patients. The results revealed that tumors with high BIRC5 expression was associated with several enriched pathways including messenger RNA (mRNA) splicing, DNA repair, and translation (Figure 6). Single-cell sequencing data analysis also uncovered a potential mechanistic link between BIRC5 expression and cell cycle, DNA damage/repair, hypoxia, inflammation, and cell proliferation.

Figure 6 The different pathways enrichment between high and low BIRC5 expression groups. FDR, false discovery rate; NES, normalized enrichment score.

Immune infiltration analysis

To determine the immune profiles of tumors with distinct BIRC5 expression levels, we deconvoluted the bulk-RNA sequencing data of 535 LUAD patients to depict immune infiltration landscape. The immune infiltration analysis indicated a negative correlation between BIRC5 expression and infiltration levels of several immunosupportive cells including CD8⁺ T cells, T helper 17 cells, B cells, macrophages, dendritic cells (DCs), and natural killer (NK) cells in LUAD, but a positive correlation was observed between BIRC5 expression and regulatory T cells (Tregs) infiltration levels (Figure 7). These results suggest that tumors with high BIRC5 expression would possess a cold tumor immune microenvironment.

Figure 7 The correlation between immune cell infiltrating and BIRC5 expression level in LUAD. DC, dendritic cell; LUAD, lung adenocarcinoma; NK, natural killer; TPM, transcripts per million; Treg, regulatory T cell.

Predictive value of BIRC5 expression in LUAD received ICB

Generally, tumors with high immunosuppressive cells infiltration are unlikely to respond to immunotherapy. Having noticed LUAD with high BIRC5 expression had elevated Tregs infiltration, we next investigated the treatment outcome of NSCLC patients whose pre-treatment tumors with high BIRC5 expression to PD-1/PD-L1 blockade.

To determine the impact of BIRC5 expression on clinical outcomes in patients treated with PD-1/PD-L1 blockade monotherapy, we conducted an integrated analyses of the transcriptomic and treatment outcome data of patients with advanced NSCLC receiving PD-1/PD-L1 blockade monotherapy. Three publications with 54 NSCLC patients had available transcriptomic and response data (19-21). Details were shown in Table S2. Firstly, we observed that responders had significantly lower BIRC5 expression level than non-responders (P=0.03, Figure 8A). Patients with high BIRC5 expression (≥ median level) showed a markedly lower response rate than those with low BIRC5 expression (< median level) [objective response rate (ORR) 19.0% vs. 41.1%; P=0.02, Figure 8B]. Importantly, patients with high BIRC5 expression had dramatically shorter PFS than those with low BIRC5 expression (median PFS 1.2 vs. 4.5 months; P=0.01, Figure 8C). OS was also shorter in high BIRC5 expression group than in low group (median OS 3.1 vs. 12.7 months; P=0.005, Figure 8D). We did not perform the multivariate analysis because the clinicopathological data of these studies were unavailable.

Figure 8 Predictive value of BIRC5 expression in LUAD received ICB. (A) Responders had significantly lower BIRC5 expression level than non-responders. (B) Patients with high BIRC5 expression (≥ median level) showed a markedly decreased response rate than those with low BIRC5 expression (< median level). (C) Survival analysis of BIRC5 expression in NSCLC patients with PD-1 blockade for PFS. (D) Survival analysis of BIRC5 expression in NSCLC patients with PD-1 blockade for OS. CI, confidence interval; HR, hazard ratio; ICB, immune checkpoint blockade; mOS, median overall survival; mPFS, median progression-free survival; LUAD, lung adenocarcinoma; NSCLC, non-small cell lung cancer; OS, overall survival; PD-1, programmed death-1; PFS, progression-free survival.

Discussion

In this study, we integrated the clinical and transcriptomic data of 535 LUAD samples, 59 normal lung, and 54 patients with NSCLC treated with ICB from online database and found that LUAD had a significantly higher expression level of BIRC5 than normal lung tissues. The elevated BIRC5 expression was markedly associated with unfavorable clinical outcomes. Transcriptomic and single-cell sequencing data analysis revealed that tumors with high BIRC5 expression was associated with enrichment of cancer cell invasion and proliferation related pathways including cell cycle, DNA damage/repair, hypoxia, inflammation, and cell proliferation. The immune infiltration analysis indicated a negative correlation between BIRC5 expression and infiltration levels of CD8⁺ T cells, T helper 17 cells, B cells, macrophages, DCs, and NK cells in LUAD, but a positive correlation was observed between BIRC5 expression and Tregs infiltrations. Importantly, the PFS and OS of NSCLC patients who received PD-1/PD-L1 inhibitors and had high BIRC5 expression were significantly shorter than those NSCLC patients with low BIRC5 expression.

Apoptosis is a crucial process that maintains normal cellular function (22). Normally, apoptosis helps eliminate damaged DNA and cells with irregular cell cycles (23). However, in malignant cells, the apoptotic mechanism is disrupted and the body is unable to effectively eliminate these cells, leading to tumorigenesis and treatment resistance (24,25). The IAP protein family is a highly conserved group of anti-apoptotic factors that primarily inhibit apoptosis by suppressing Caspase activity and regulating NF-κB signaling (26). BIRC5, the smallest member of the IAP family and located on chromosome 17q25, was isolated from the human genome library in 1997 (27). BIRC5 has various biological functions, including the inhibition of apoptosis by suppressing caspase-3 and caspase-7 activity in direct or indirect ways, promoting cell mitosis by assisting in chromosome division and microtubule movement, angiogenesis, and serving as a biomarker for tumor diagnosis, treatment, and prognosis prediction (28). Thus, inhibition of BIRC5 activity may help to inhibit the development and spread of tumor cells, making it a promising target for cancer therapy. Numerous studies have provided evidence for the critical role of BIRC5 in regulating cell cycle, proliferation, progression, and angiogenesis in cancer cells (29-31). Cytokines in lymphocytes have been shown to control the expression of BIRC5, which is essential for the growth and survival of hematopoietic cells. In the context of human cancers, BIRC5 has been reported to inhibit cell apoptosis while stimulating cell proliferation (32,33). Previous studies have revealed that BIRC5 exerts its anti-apoptotic effects through the suppression of both caspase-dependent and caspase-independent apoptotic pathways, in addition to promoting cell growth and development (14,31). In this study, we integrated the bulk-cell and single-cell RNA sequencing data and observed that tumors with high BIRC5 expression were positively correlated with increased cell cycle, DNA damage/repair, hypoxia, and inflammation. Moreover, LUAD patients with high BIRC5 expression had unfavorable clinical outcomes. Collectively, these findings revealed that BIRC5 may play a pivotal role in tumorigenesis and prognosis in LUAD.

In recent years, the role of immune cell infiltration in cancer development has received considerable attention (34). The immune cell infiltrate composition and immune gene signatures have been shown to be the robust predictors of clinical prognosis in several cancers. The presence of CD8 and memory T cells, as well as Th1-biased gene signatures, is associated with a better prognosis, while the presence of M2-like macrophages is associated with a worse prognosis (35,36). In our study, we observed an inverse correlation between BIRC5 expression in LUAD and immune cells such as CD8⁺ T cells, macrophages, B cells, T helper 17 cells, DCs, and NK cells. Moreover, we found that BIRC5 expression was positively correlated with Tregs. Past studies found that Treg representation in diverse murine and human cancers is generally much higher than in corresponding normal tissue and blood, and increased tumor infiltration by Treg expressing specific genes was recently correlated with poor prognosis in NSCLC (37,38). This is also consistent with our result. Now several studies on the heterogeneity of Treg are underway (39,40). Our results suggest that BIRC5 may play a significant role in regulating the tumor immune microenvironment. However, the precise mechanism by which BIRC5 influences the tumor immune microenvironment and the tumorigenesis of LUAD remains unknown. To unravel the detailed mechanism of BIRC5 mediating CD8⁺ T cell phenotype and function transition and regulating Tregs infiltration is worthwhile for the development of novel immunotherapeutic targets and combination therapeutic strategies.

Immunotherapy has been emerged as a successful cancer therapy. However, not all patients respond to immunotherapy. Our findings showed that in LUAD patients treated with PD-1/PD-L1 inhibitors, those with low BIRC5 expression had a significantly higher ORR, PFS and OS than those with high BIRC5 expression. These results suggested that BIRC5 expression might represent as a potential biomarker to predict the response to immunotherapy in LUAD. Further investigations with large sample size are needed to validate its predictive value.

Conclusions

In conclusion, our study indicated that high BIRC5 expression was associated with enrichment of DNA damage/repair, cancer cell invasion and proliferation related pathways, increased Tregs infiltration. These results suggest that it may be associated with the poor prognosis of NSCLC patients treated with ICB. However, our work does still have several flaws. First of all, the expression source of BIRC5 needs to be further clarified. It is currently unclear whether its expression mainly comes from tumor cells, immune cells, or a combination of both. Since bulk transcriptome sequencing cannot distinguish cell type-specific signals, expression of BIRC5 by immune cells may confound the interpretation of tumor cell-derived signals. Future research can precisely analyze the contributions of different cell subsets through single-cell RNA sequencing or spatial transcriptome technology. Another limitation is the lack of functional experiments and independent queue verification. More investigations are needed to confirm the results, and eventually enhance patients’ quality of life.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the TRIPOD reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-216/rc

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-216/prf

Funding: This work was supported in part by grants from Shanghai Municipal Education Commission (No. 16SG18), the Science and Technology Commission of Shanghai Municipality (No. 16411964600), the National Natural Science Foundation of China (No. 81772467), the Shanghai Shen Kang Pharmaceutical Development Co. Ltd (No. SHDC 12015314), and the Backbone Program of Shanghai Pulmonary Hospital (No. FKGG1802).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-216/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. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

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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