Bioinformatic analysis and cellular functional assays uncover the role of NXPH4 in breast cancer.

Introduction
Breast cancer remains one of the leading causes of cancer-related mortality worldwide, with a consistently increasing disease burden [1, 2]. Despite advances in screening, surgical intervention, radiotherapy, chemotherapy, and targeted therapy [3, 4], as well as the identification of numerous tumor diagnostic and prognostic biomarkers through approaches such as multi-omics [5, 6], the overall survival rate remains unsatisfactory. This is primarily due to late diagnosis and high rates of metastasis and recurrence [7]. Therefore, the identification of novel biomarkers is critical to improve early detection and therapeutic strategies for breast cancer.
The neurexophilin (NXPH) family comprises four members (NXPH1−4), which share a conserved protein structure characterized by an N-terminal hydrophobic signal peptide, a variable domain, a glycosylated central region, a linker, and a highly conserved C-terminal sequence containing six cysteine residues [8]. As a synaptic secreted protein, NXPH4 has been implicated in the pathogenesis of several malignancies, including hepatocellular carcinoma [9], colorectal cancer [10], bladder cancer [11], and lung cancer [12]. However, its role in breast cancer has not yet been elucidated.
Based on multi-omics databases including The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) project, and the Gene Expression Omnibus (GEO), this study comprehensively analyzed the expression profile of NXPH4 across pan-cancer and normal tissues. We further systematically investigated its expression patterns and potential functions in BRCA, and evaluated its prognostic value for clinical outcome prediction. These findings suggest that NXPH4 may serve as a novel biomarker to improve diagnosis and personalized treatment strategies for BRCA patients, and may provide a foundation for developing new targeted therapies.

Materials and methods

Analysis of NXPH4 expression levels
Data pertaining to NXPH4 expression across 33 cancer types and matched normal samples were acquired from TCGA and the GTEx project. Statistical analysis was performed using R software (version 4.3.3). For presentation, cancer types demonstrating a statistically significant difference (P < 0.05) were selected. Furthermore, expression data from the GEO datasets GSE22820, GSE25407, and GSE42568 were utilized to independently verify NXPH4 expression levels between tumor and normal tissues.

Single-cell sequencing and spatial transcriptomic analysis of NXPH4 Expression​​
Interrogating the expression patterns of NXPH4 at single-cell resolution, we analyzed datasets BRCA_EMTAB8107, BRCA_GSE150660, and BRCA_GSE148673 from the TISCH database. Cell type distributions were visualized following dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP), which also revealed the expression levels and distribution of NXPH4 across different cell subtypes. To spatially resolve this expression, we obtained spatial transcriptomic data of BRCA tissue sections from the Sparkle database. Using the cotrazmR package, we deconvoluted the cellular composition of the tumor microenvironment (TME) and annotated microregions based on the predominant cell type. A heatmap was used to visualize the expression landscape of NXPH4 across these distinct microregions, and a comparative analysis of its average expression in tumor versus normal tissue was presented in a bar plot.

Analysis of the association between NXPH4 Expression, clinical characteristics, and survival Prognosis​​
The correlation between NXPH4 expression levels and key clinical characteristics—including TNM stage, age, PR status, ER status, HER-2 status, and PAM50 subtype—was assessed. A heatmap was constructed to visualize these associations using R.The diagnostic value of NXPH4 for breast cancer was evaluated by performing Receiver Operating Characteristic (ROC) curve analysis with the pROCpackage in R. The Area Under the Curve (AUC) was calculated to quantify the diagnostic power.To analyze survival outcomes, patients were stratified into high- and low-NXPH4 expression groups based on an optimal cut-off value determined by the surv_cutpointfunction from the survminerpackage. Survival differences between these groups were compared using the log-rank test via the survivalpackage, with a significance threshold set at P < 0.05. The resulting survival curves were visualized using the survminerand ggplot2packages.

Biological characterization and genomic enrichment analysis of NXPH4​​
The biological functions and pathways associated with NXPH4 were investigated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, implemented with the “clusterProfiler” and “circlize” packages in R. To further identify coordinated biological processes differentially enriched between the high- and low-risk groups, we conducted a Gene Set Enrichment Analysis (GSEA). A significance threshold of P < 0.05 was applied to define statistically significant terms and pathways in all analyses.

Analysis of the association between NXPH4 and immune infiltration in BRCA​​
For evaluating the correlation of NXPH4 expression with immune cell infiltration, Spearman correlation coefficients were calculated using multiple algorithms on the TCGA-BRCA cohort, and these findings were validated in independent GEO datasets. A correlation heatmap summarized the results. For a deeper investigation into the immunomodulatory role of NXPH4, we compared the expression levels of immune-related genes across four key dimensions: immunostimulators, immunoinhibitors, chemokines, and human leukocyte antigens (HLA), between the high and low expression groups.

Analysis of the relationship between NXPH4 methylation and prognosis​​
The role of NXPH4 promoter methylation in breast cancer was investigated using the UALCAN database; additionally, the prognostic value of methylation at specific CpG sites was assessed with the MethSurv online tool. Survival prognosis plots were generated based on the selected gene regions and CpG sites to evaluate their correlation with patient outcomes.

Drug sensitivity analysis​​
Drug response data for chemotherapeutic and targeted agents were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC1 and GDSC2) database. The R package “pRRophetic” was employed to predict the chemosensitivity of BRCA patients to these drugs. The half-maximal inhibitory concentration (IC50), defined as the drug concentration required to inhibit 50% of cell growth, served as a key metric for evaluating drug sensitivity in breast cancer. Differences in IC50 values between high-risk and low-risk groups were compared using the Wilcoxon test, with a significance threshold set at P < 0.05.

Cell culture and quantitative RT-qPCR​​
The human breast cancer cell lines MCF-7, SK-BR-3, AU565, MDA-MB-468, and MDA-MB-231 were obtained from commercial suppliers. MCF-7 cells (Laibaiha Biotechnology, Shanghai) were maintained in MEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 0.01 mg/ml insulin. SK-BR-3 cells (Wuhan Servicebio Biotechnology) were cultured in McCoy’s 5 A medium containing 10% FBS and 1% penicillin-streptomycin. AU565, MDA-MB-468, and MDA-MB-231 cells (Procell Life Science, Wuhan) were grown in RPMI-1640 medium (AU565) or DMEM (MDA-MB-468 and MDA-MB-231), both supplemented with 10% FBS and 1% penicillin-streptomycin. The non-tumorigenic epithelial cell line MCF-10 A, preserved in our laboratory, was cultured in a specialized mammary epithelial growth medium.Total RNA was extracted using the E.N.Z.A.® Total RNA Kit I (Omega, R6834-01), and reverse transcription quantitative PCR (RT-qPCR) was performed using a commercial kit (ComWin Biotech, China). Cells were maintained in phosphate-buffered saline (PBS) and incubated in a CO₂ incubator (Thermo Fisher Scientific, USA). Morphological examination was carried out using an inverted optical microscope (Olympus, Japan).The following primer sequences were used for RT-qPCR: NXPH4 – forward: 5′-GCAGCGAAAACTTGAGGGTAT-3′, reverse: 5′-AAGGTCTTCGGACGGCCTA-3′;β-actin (internal control) – forward: 5′-AACCGCGAGAAGATGACCCAG-3′, reverse: 5′-GGATAGCACAGCCTGGATAGCAA-3′. All primers were synthesized by Sangon Biotech (Shanghai).

Cell transfection
When SK-BR-3 and MDA-MB-468 cells reached approximately 90% confluence, they were digested with trypsin and counted. The cells were then seeded into 12-well plates at a density of 3 × 10⁵ cells per well and cultured until they reached approximately 60% confluence for transfection. Transfection was performed using siNC, NXPH4 siRNA#1, and NXPH4 siRNA#2, respectively. Transfection complexes were prepared according to the manufacturer’s instructions for the siRNA reagent: six 1.5 mL EP tubes were filled with basal medium, which was used to dilute the siRNA and Lipofectamine 2000 separately. After incubation at room temperature for 5 min, the two mixtures were combined in equal volumes, incubated for another 20 min, and then added to the wells. After 6 h of transfection, the medium was replaced with complete medium. At 24 and 48 h post-transfection, cell morphology was observed, and cells were harvested for subsequent experiments. The siRNA sequences used are listed in Table 1.

CCK-8 and edu proliferation assays
Cell proliferation was assessed using the CCK-8 assay. SK-BR-3 and MDA-MB-468 cells were seeded in 96-well plates at a density of 2 × 10³ cells per well and cultured for 0, 24, 48, and 72 h. Following each incubation period, 10 µL of CCK-8 reagent was added to each well, and the plates were incubated for an additional 2 h. The optical density (OD) at 450 nm was then measured using a microplate reader (Thermo Fisher Scientific).For the EdU assay, cells were seeded in 12-well plates and allowed to adhere overnight. The assay was subsequently performed according to the manufacturer’s instructions using an EdU imaging kit (Cy3) from APExBIO Technology. After staining, images were captured under a fluorescence microscope.

Analysis of apoptosis via western blot
Cells were lysed on ice using RIPA lysis buffer (Solarbio) to extract total protein. The protein lysates were separated by SDS-PAGE and subsequently transferred onto 0.45 μm PVDF membranes (Millipore Corporation). After blocking, the membranes were incubated overnight at 4 °C with primary antibodies against BAX and BCL-2 (1:1000, Wuhan SanYing Biotechnology, Co., Ltd.). Following incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (Biosharp). Anti-β-actin antibody (1:2500) was used as a loading control.​.

Statistical analysis​​
Bioinformatic analyses were performed using R (version 4.3.3) and relevant R packages. Comparisons between two groups were conducted using the Student’s t-test or the Wilcoxon test, as appropriate. All statistical tests were two-sided, and a P-value of less than 0.05 was considered statistically significant. Significance levels were denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Results

Analysis and validation of NXPH4 expression in pan-cancer and breast cancer cells
To investigate the expression pattern of NXPH4 across human cancers, we integrated normal tissue data from the GTEx database with tumor tissue data from TCGA. Expression analysis revealed that NXPH4 was significantly overexpressed in the majority of cancer types in the combined TCGA and GTEx dataset, although it was downregulated in a few malignancies (Fig. 1A). Consistently, analysis of the TCGA database alone showed significantly increased NXPH4 expression in 16 cancer types, as well as in 14 matched pairs of tumor and adjacent normal tissues (Fig. 1B and C). To further validate the differential expression of NXPH4 in breast cancer, we analyzed its expression in datasets from the Gene Expression Omnibus (GEO). The results confirmed that NXPH4 expression was significantly higher in tumor tissues compared to normal breast tissues across multiple independent cohorts (P < 0.05) (Fig. 1D). Finally, we experimentally measured NXPH4 mRNA levels using RT-qPCR in a normal mammary epithelial cell line and several breast cancer cell lines. NXPH4 expression was significantly upregulated in all tested breast cancer cell lines relative to normal epithelial cells, suggesting a potential role for NXPH4 in breast tumorigenesis (Fig. 1E).

NXPH4 was significantly upregulated in malignant cells
Analysis of breast cancer single-cell RNA sequencing data from the TISCH database, based on marker gene expression, showed that NXPH4 is primarily highly expressed in malignant cells (Fig. 2A). Furthermore, spatial transcriptomics of breast cancer sections demonstrated a significant positive correlation between NXPH4 expression and the local proportion of malignant cells (Fig. 2B). These consistent findings indicate that the dysregulated expression and tumor microenvironmental effects of NXPH4 originate mainly from malignant cells, suggesting an important role in breast cancer development.

Knocking down NXPH4 affects the proliferation and apoptosis levels of breast cancer
The knockdown efficiency of NXPH4 in SK-BR-3 and MDA-MB-468 cell lines was confirmed by quantitative RT-qPCR (Fig. 3A). The proliferative capacity of both breast cancer cell lines was assessed using CCK-8 and EdU assays. The results showed that proliferation was significantly inhibited in the siNXPH4#1 and siNXPH4#2 groups compared to the control group (Fig. 3B-D). Western blot analysis of apoptosis-related proteins revealed a statistically significant increase (P < 0.05) in the apoptosis rate in both MDA-MB-468 and SK-BR-3 cells following transfection with NXPH4-targeting siRNAs, compared to the siCtrl group (Fig. 3E, F).

Association of NXPH4 with clinicopathological features and prognosis in BRCA​
To investigate the relationship between NXPH4 expression and clinicopathological characteristics as well as molecular subtypes in breast cancer, we first visualized gene expression patterns—including that of NXPH4—along with patient age, pathological stage (T, N, M stage), ER, PR, and HER2 status, and PAM50 molecular subtypes using a heatmap. Color gradients reflect differences in gene expression, clearly illustrating the distribution pattern of NXPH4 expression across different clinical subgroups (Fig. 4A). Compared with normal breast tissues, NXPH4 expression was significantly elevated in breast cancer tissues across all age groups and TNM stages. NXPH4 expression was lower in ER- and PR-positive cases than in negative ones, whereas HER2-positive tumors showed higher NXPH4 expression. Among the four major breast cancer subtypes, NXPH4 expression was highest in the basal-like subgroup (Fig. 4B). Using the optimal cut-off value for NXPH4 expression, patients were stratified into high- and low-expression groups. Kaplan–Meier survival analysis revealed that patients with high NXPH4 expression had significantly worse overall survival (OS, P = 0.011), disease-specific survival (DSS, P = 0.006), progression-free interval (PFI, P = 0.003), and disease-free interval (DFI, P = 0.023) compared to those with low expression (Fig. 4C). These results suggest that high NXPH4 expression is associated with unfavorable prognosis and shorter survival in BRCA patients. To further evaluate the diagnostic value of NXPH4 in breast cancer, ROC analysis was performed. NXPH4 demonstrated predictive capability for breast cancer diagnosis, with an area under the curve (AUC) of 0.711, indicating a reasonably good diagnostic performance (Fig. 4D).

Functional enrichment analysis of NXPH4-associated differentially expressed genes
Having established the association between NXPH4 and multiple pathological indicators as well as prognosis in breast cancer, this study further conducted a preliminary exploration into the potential mechanisms by which NXPH4 promotes breast carcinogenesis. Based on median NXPH4 expression, patients were stratified into high- and low-expression groups, identifying a total of 610 differentially expressed genes (DEGs) (Fig. 5A). These DEGs were implicated in various biological processes. Gene Ontology (GO) enrichment analysis for biological processes revealed involvement in pattern specification processes, regulation of hormone levels, axon development, presynapse assembly, collagen-containing extracellular matrix organization, passive transmembrane transporter activity, and channel activity, among others (Fig. 5B–D). Several of these processes are closely associated with tumor initiation and progression. For instance, pattern specification processes may play roles in embryonic development and aberrant differentiation of tumor cells, while regulation of hormone levels is linked to the progression of hormone-related cancers such as breast cancer. Presynaptic components, involved in intercellular signaling, may influence tumor cell proliferation and invasion by modulating communication between tumor cells and their microenvironment, thereby promoting tumor growth or metastasis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted enrichment in pathways including biosynthesis of cofactors, signaling pathways regulating pluripotency of stem cells, and IL-17 signaling pathway (Fig. 5E). To further investigate the biological significance of NXPH4 expression patterns, Gene Set Enrichment Analysis (GSEA) was performed using the Reactome and KEGG pathway databases. The results revealed significant enrichment of six key pathways associated with NXPH4 expression, including Cell Surface Interaction at the Vascular Wall, Collagen Formation, Cell Cycle, Mitotic G1 Phase and G1/S Transition, S Phase, and Extracellular Matrix Organization (Figs. 5F–K)—all of which are recognized features related to breast cancer pathogenesis. These findings suggest that NXPH4 may contribute to breast cancer development and progression by participating in processes such as vascular wall cell interactions, collagen formation, cell cycle regulation, and extracellular matrix organization. This implicates NXPH4 as a potential therapeutic target in breast cancer.

Association between NXPH4 expression and immune cell infiltration across multiple datasets
The association between NXPH4 expression and immune cell infiltration within the tumor microenvironment was evaluated across multiple independent cancer datasets, with results visualized in a heatmap (Fig. 6A). The analysis included several cohorts, such as TCGA_BRCA, GSE88770, and GSE97342, and encompassed a variety of immune cell types. The results indicated that the correlation between NXPH4 and immune infiltration was both dataset-specific and cell type-specific. Notably, in the TCGA_BRCA dataset, NXPH4 showed significant positive correlations (p < 0.05) with certain T-cell subsets, macrophages, and related immune populations.To further investigate the association between NXPH4 and immune regulatory molecules in breast cancer, we analyzed the expression levels of immunomodulatory molecules—including immunostimulators, chemokines, immunoinhibitors, and human leukocyte antigens (HLA)—stratified by NXPH4 expression levels (Fig. 6B). Strong correlations were observed between NXPH4 and various immune-related genes, such as immunostimulators (CD276, CXCR4, TNFRSF4), chemokines (CCL2, CCL3, CCL4), immunoinhibitors (CD160, CTLA4, IDO1), and HLA molecules (HLA-A, HLA-B, HLA-DMA). These results suggest that in breast cancer, NXPH4 may modulate the tumor immune microenvironment by recruiting or promoting the infiltration of specific immune cells and regulating the expression of key immunomodulatory molecules. This potentially facilitates the formation of an immunosuppressive microenvironment conducive to immune evasion, thereby supporting a pro-tumorigenic role for NXPH4 from an immunoregulatory perspective.

Association of NXPH4 methylation with prognosis
Further exploration using the methylation module of the UALCAN online tool revealed that the promoter methylation level of NXPH4 in BRCA was significantly higher than in normal breast tissues (P < 0.05, Fig. 7A). Furthermore, significant differences in NXPH4 promoter methylation were observed across different tumor stages and molecular subtypes, with the notable exception of Stage 4 (P < 0.05, Fig. 7B, C). This study also identified a correlation between NXPH4 methylation and patient prognosis. Survival analysis demonstrated that patients with high methylation levels at relevant CpG sites had a significantly shorter overall survival than those with low methylation levels (Fig. 7D).

Association between NXPH4 expression and chemotherapeutic drug response
The relationship between NXPH4 and drug targets and pathways in breast cancer was analyzed using the GDSC database (GDSC1 and GDSC2). As shown in Fig. 8A, a substantial number of drugs are associated with key pathways relevant to breast cancer biology, such as the WNT signaling pathway, RTK signaling pathway, p53 pathway, cell cycle regulation, and DNA replication. Dysregulation of these pathways constitutes an important molecular basis for breast cancer development and progression, and also influences tumor cell responses to drugs. By modulating these pathways, NXPH4 alters biological behaviors of tumor cells—such as proliferation, apoptosis, and DNA damage repair—thereby affecting the inhibitory efficacy of drugs on tumor cells, ultimately reflected in changes in drug IC50 values. For example, Palbociclib, a known drug targeting cell cycle-related proteins, is associated with the “cell cycle” pathway; Doxorubicin, a drug related to DNA damage, is linked to pathways involving “DNA replication” and “genomic integrity”; Cyclophosphamide, an alkylating chemotherapeutic agent, is also associated with “DNA replication” and “genomic integrity” pathways, exerting its anti-tumor effects through DNA damage. NXPH4-mediated regulation of these pathways may influence tumor cell responses to DNA damage induced by Cyclophosphamide.

In the GDSC1 dataset, NXPH4 showed a positive correlation with the IC50 values of certain drugs (e.g., Bleomycin), indicating that higher NXPH4 expression may be associated with increased tumor cell tolerance to these drugs (Fig. 8B). In contrast, in the GDSC2 dataset, NXPH4 was negatively correlated with the IC50 values of most drugs, suggesting that higher NXPH4 expression renders tumor cells more sensitive to the corresponding drugs (Fig. 8C). These findings imply that NXPH4 expression levels may influence tumor cell drug responses.

Discussion
According to the World Health Organization’s global breast cancer survey, there were 2.3 million new cases of breast cancer and 670,000 deaths worldwide in 2022. By 2050, the number of new cases and deaths from breast cancer are projected to increase by 38% and 68%, respectively [2]. Therefore, further research on BRCA has become increasingly urgent, with particular emphasis on identifying biomarkers that may influence patient prognosis.
Recent studies have increasingly untangled the oncogenic roles of NXPH4. However, the specific mechanisms through which NXPH4 functions in breast cancer remain unclear and warrant further investigation. In our study, NXPH4 was demonstrated to be highly expressed in various cancers, and its elevated expression in breast cancer tissues—particularly in malignant cells—suggests a potential role in promoting tumor progression by regulating the cell cycle and signaling pathways. GO and KEGG enrichment analyses revealed that NXPH4-associated genes were significantly enriched in processes such as pattern specification, regulation of hormone levels, and passive transmembrane transporter activity. These functions may be linked to the Wnt, Hedgehog, and Notch pathways, as well as to the stimulation of cancer cell growth by estrogen, progesterone, and androgen [13–15], indicating that NXPH4 may facilitate tumor invasion by altering intercellular communication [16]. Further exploration via GSEA indicated that NXPH4-associated genes were markedly enriched in pathways related to cell cycle regulation (e.g., G1/S transition, S-phase progression) and extracellular matrix organization. These findings are consistent with previous reports: dysregulation of the cell cycle is a hallmark of cancer [17–19], and remodeling of the extracellular matrix is a critical step in metastasis [20–22]. High NXPH4 expression was positively correlated with levels of T-cell and macrophage infiltration, and was associated with the expression of multiple immunomodulatory molecules (e.g., CD276, CTLA−4, HLA family), suggesting that NXPH4 may contribute to the formation of a tumor microenvironment through immune cell recruitment. Concurrently, its association with immunosuppressive molecules such as IDO1 implies a potential role in fostering a tolerant microenvironment to evade immune attack [23]. While our correlation analyses suggest a link between NXPH4 and the immune microenvironment, this observation requires direct experimental confirmation in future research.
Epigenetics refers to heritable molecular mechanisms that regulate gene expression without altering the DNA sequence itself, playing a critical role in gene regulation, genomic stability, and tumorigenesis [24, 25]. While promoter hypermethylation is generally associated with transcriptional repression, our study observed a concurrent increase in both methylation and expression of NXPH4, with methylation levels at specific sites correlating with patient prognosis. This finding is consistent with recent studies demonstrating that hypermethylation of CORO1A and BYSL enhances the malignant behavior of tumor cells [26, 27]. This apparent paradox may be explained by the combined effects of the following factors: (1) DNA hypermethylation often silences tumor suppressor genes, thereby inhibiting the expression of genes that constrain cancer growth [28]; (2) methylation changes can epigenetically reshape the tumor microenvironment (TME), influencing immune cell function and potentially facilitating immune escape, which in turn affects clinical outcomes [29]; and (3) although global methylation levels may be elevated, the expression of certain pro-tumor genes may remain unaltered [30]. This could occur if methylation does not affect core promoter regions or if stronger regulatory mechanisms—such as transcription factor activity—override the repressive effects of methylation. However, the above conclusions will require further experimental validation.
Drug sensitivity analysis revealed that cells with high NXPH4 expression exhibited resistance to certain chemotherapeutic agents (e.g., bleomycin) but increased sensitivity to some targeted drugs (e.g., palbociclib). For breast cancer patients with high NXPH4 expression, combination therapeutic strategies targeting the cell cycle or immune checkpoints may be worth exploring—a direction consistent with our earlier functional enrichment findings.
The primary objective of this study was to evaluate the potential of NXPH4 as a prognostic indicator for BRCA. We further investigated its potential roles in DNA methylation, clinical relevance, and functional enrichment, and aimed to identify potentially sensitive drugs to address clinical therapeutic challenges. A limitation of this study is that its conclusions are primarily based on bioinformatic analyses and in vitro expression validation in cell lines. The specific oncogenic mechanisms of NXPH4 in breast cancer require further validation through functional experiments in both in vitro and in vivo models.

Conclusion
In summary, our cellular experiments demonstrate that NXPH4 knockdown suppresses the proliferation of breast cancer cells. Further bioinformatic analyses reveal a significant correlation between high NXPH4 expression, promoter methylation at specific sites, and poor patient prognosis, underscoring a critical function of NXPH4 in regulating these processes. Moreover, elevated NXPH4 expression, coupled with its association with immune-related molecules, contributes to an immunosuppressive tumor microenvironment (TME), thereby potentially compromising the efficacy of anti-tumor immunotherapy and fostering treatment resistance. Our findings suggest that co-targeting NXPH4 with cell cycle inhibitors or immune checkpoint blockers may enhance the effectiveness of immunotherapy, offering a novel strategic avenue to suppress tumor progression and improve patient survival.