High expression of SERPINE1 in gastric cancer

Bioinformatic analysis via TIMER revealed elevated the expression of SERPINE1 in GC versus normal tissues (Fig. 1A). UALCAN validation confirmed its upregulation in GC specimens (Fig. 1B). Stage-dependent escalation analysis demonstrated progressive SERPINE1 elevation, particularly showing marked increases from Stage III to IV (Fig. 1C). This suggests that SERPINE1 may be associated with tumor progression or malignancy. With increasing tumor grade, the expression of SERPINE1 showed a gradual upward trend, indicating that SERPINE1 may be related to the malignancy of gastric cancer, i.e., SERPINE1 expression escalates proportionally with tumor grade progression (Fig. 1D). Figure 1E revealed SERPINE1's progressive upregulation in GC lymph node metastasis (normal/N0: low; N1-N3: high; peak in N3). Survival analyses demonstrated significant clinical correlations: elevated SERPINE1 predicted reduced overall survival (OS, Fig. 1F) and shortened disease-free survival (DFS, Fig. 1G) compared to low-expression cohorts (p < 0.05). Differential expression analysis identified upregulated SERPINE1 mRNA in GC versus normal tissues (p < 0.0001, Fig. 1H). Protein-level validation through western blot (Fig. 1I) and immunohistochemistry (Fig. 1J) confirmed concordant overexpression patterns. These results implicate SERPINE1 in gastric carcinogenesis progression, demonstrating clinical correlations with advanced tumor staging, unfavorable survival outcomes, and therapeutic target potential.

Fig. 1

Expression of SERPINE1 in Gastric Cancer and Its Correlation with Clinical Features. (A-B) Bioinformatics via TIMER (A) and UALCAN (B) databases for SERPINE1 mRNA in GC vs. normal tissues. (C-E) Correlation of SERPINE1 with cancer stage (C), tumor grade (D), and lymph node metastasis (E) via one-way ANOVA. (F-G) Kaplan–Meier survival analysis (log-rank test) for SERPINE1 vs. overall survival (F) and DFS (G). (H) qRT-PCR of SERPINE1 mRNA in GC and normal tissues (Student’s t-test). (I) Western blot analysis of SERPINE1 protein expression in GC and normal tissues. (J) IHC staining for SERPINE1 in GC and normal tissues. **p < 0.01

NFATC2 as a potential transcription factor of SERPINE1

To investigate SERPINE1's regulatory mechanisms in gastric carcinogenesis, we used the PROMO database to predict its potential transcription factors, and NFATC2 was identified as a potential transcription factor of SERPINE1. Using the JASPAR database, we obtained the DNA sequence motif bound by NFATC2 (Fig. 2A). The Bioinformatic analyses (GEPIA/TIMER) revealed NFATC2 overexpression in GC with positive SERPINE1 co-expression (Fig. 2B-C). Western blot analysis confirmed elevated NFATC2 protein levels across GC cell lines (AGS/SNU-1/HGC27/MKN45/MKN1), showing maximal expression in AGS and MKN1 (Fig. 2D), which were selected for subsequent experiments. NFATC2 silencing significantly reduced SERPINE1 expression at transcriptional (p < 0.01, Fig. 2E) and translational levels (p < 0.01, Fig. 2F). However, knockdown of SERPINE1 did not result in significant changes in NFATC2 at either the mRNA (Fig. 2G) or protein level (Fig. 2H), indicating that NFATC2 is upstream of SERPINE1. Dual-luciferase reporter assays showed that NFATC2 exerted its biological effects by reducing the activity of the luciferase reporter gene, and this effect was abolished or weakened in the mutant construct (Fig. 2I). ChIP-qPCR experiments further confirmed that NFATC2 protein was significantly enriched in the promoter region of SERPINE1 in AGS and MKN1 cell lines, with higher enrichment in MKN1 cells (Fig. 2J).

Fig. 2

NFATC2 as a Potential Transcription Factor of SERPINE1. (A) Prediction of NFATC2-binding DNA motif in the SERPINE1 promoter via JASPAR database. (B-C) Correlation analysis of NFATC2 and SERPINE1 expression in GC using GEPIA (B) and TIMER (C) databases. (D) Western blot detection of NFATC2 protein levels in human GC cell lines (AGS, SNU-1, HGC27, MKN45, MKN1). (E–F) qRT-PCR (E) and Western blot (F) analysis of SERPINE1 expression after NFATC2 knockdown in AGS cells (shNFATC2 vs. shNC), performed via Student’s t-test. (G-H) qRT-PCR (G) and Western blot (H) analysis of NFATC2 expression after SERPINE1 knockdown in AGS cells (shSERPINE1 vs. shNC), via Student’s t-test. (I) Dual-luciferase reporter assay to validate NFATC2 binding to the SERPINE1 promoter in AGS cells (wild-type vs. mutant constructs). (J) ChIP-qPCR analysis of NFATC2 enrichment in the SERPINE1 promoter region in AGS and MKN1 cells. **p < 0.01, ***p < 0.001

These findings establish NFATC2 as a direct transcriptional regulator of SERPINE1, critically involved in gastric carcinogenesis and clinical progression. Mechanistically, NFATC2 overexpression drives oncogenic advancement and adverse clinical outcomes through SERPINE1 upregulation.

Regulation of gastric cancer malignant phenotype by SERPINE1-mediated NFATC2

Given that NFATC2 is a potential transcription factor of SERPINE1 and SERPINE1 potentially contributes to gastric carcinogenesis, while the role of NFATC2 in GC remains to be explored, we conducted functional rescue experiments to investigate whether SERPINE1 is involved in the regulation of malignant biological behaviors of GC cells by NFATC2. Lentiviral transfection was used to achieve overexpression of SERPINE1 in AGS and MKN1 cells after knocking down NFATC2. Figure 3A demonstrates NFATC2 silencing suppressed SERPINE1 expression, whereas SERPINE1 ectopic expression rescued this regulatory effect. CCK-8 assays revealed NFATC2 knockdown impaired GC cell proliferative capacity, which was counteracted by SERPINE1 overexpression. Additionally, overexpression of SERPINE1 partially restored the decrease in cell viability caused by NFATC2 knockdown, suggesting that SERPINE1 serves as a key effector in NFATC2-mediated regulation of cellular proliferative capacity (Fig. 3B). Clonogenic analysis demonstrated NFATC2 silencing impaired GC cell colony formation, while overexpression of SERPINE1 increased the colony-forming ability. Furthermore, overexpression of SERPINE1 partially restored the decrease in colony-forming ability caused by NFATC2 knockdown, further indicating that SERPINE1 plays a role in the regulation of cell proliferation mediated by NFATC2 (Fig. 3C). Transwell assays revealed NFATC2 knockdown suppressed gastric cancer cell migration/invasion, while SERPINE1 overexpression potentiated these malignant phenotypes. Moreover, SERPINE1 overexpression partially rescued the reduction in migration and invasion abilities induced by NFATC2 silencing, indicating that SERPINE1 is involved in regulating cell migration and invasion mediated by NFATC2 (Fig. 3D). Immunoblotting of EMT markers demonstrated NFATC2 ablation attenuated EMT progression in gastric cancer cells, manifested through E-cadherin elevation with concomitant N-cadherin/Vimentin reduction. Overexpression of SERPINE1 partially promoted this EMT process (Fig. 3E). Overall, our results show that SERPINE1 overexpression enhances GC cell survival, proliferation, migration, and invasion, and promotes the EMT process, while NFATC2 knockdown has opposite effects. Significantly, SERPINE1 overexpression can partially reverse the impacts of NFATC2 knockdown on these biological processes of GC cells.

Fig. 3

Regulation of Gastric Cancer Malignant Phenotype by SERPINE1-mediated NFATC2. (A) Lentiviral transfection for NFATC2 knockdown (shNFATC2) and SERPINE1 overexpression (OE-SERPINE1) in AGS cells, followed by Western blot to detect SERPINE1 protein levels. (B) CCK-8 assay to evaluate cell viability after NFATC2 knockdown and/or SERPINE1 overexpression in AGS cells. (C) Colony formation assay to assess clonogenic potential under NFATC2 knockdown and/or SERPINE1 overexpression conditions in AGS cells. (D) Transwell migration and invasion assays to measure motility of AGS cells with NFATC2 knockdown and/or SERPINE1 overexpression. (E) Western blot analysis of EMT-related proteins (E-cadherin, N-cadherin, Vimentin) in AGS cells after NFATC2 knockdown and/or SERPINE1 overexpression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. NC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. sh-NFATC2#1

SERPINE1 is associated with tumor microenvironment immune infiltration

Following the examination of SERPINE1’s role in mediating NFATC2-regulated malignant biological behaviors of GC cells, we additionally explored SERPINE1’s role in tumor microenvironment immune infiltration. Figure 4A shows the association between SERPINE1 gene expression levels and infiltration levels of different immune cell types in GC. Findings reveal that SERPINE1 expression is significantly linked to infiltration levels of certain immune cells, particularly showing a positive correlation with macrophage, neutrophil, and dendritic cell infiltration. Figure 4B demonstrates that copy number variations (CNVs) of the SERPINE1 gene are closely associated with the infiltration levels of multiple immune cells in GC. Elevated SERPINE1 copy number alterations demonstrated reduced CD8+ T cell and dendritic cell infiltration; contrastingly, neutrophils exhibited mild elevation. This suggests that SERPINE1 amplification may correlate with immune evasion via suppressing cytotoxic T cell recruitment. This suggests that CNVs of the SERPINE1 gene may influence the immune response in the GC microenvironment, thereby affecting disease progression and development. Figure 4C-G reveal associations between SERPINE1 gene expression levels and expression levels of CD2, CD3D, CD3E, CD8 A, and CD8B in GC. Results indicate SERPINE1 expression is positively correlated with these markers, with the strongest correlation noted for CD8 A. This suggests SERPINE1 expression in GC may be linked to T cell activity or presence, particularly CD8+ T cells, with potential implications for the immune microenvironment and disease progression in GC. Figure 4H-I demonstrate the associations between SERPINE1 gene expression and CD206/CD163 expression levels in GC. Results reveal that SERPINE1 expression is positively correlated with both CD206 and CD163, with a stronger association observed for CD163. Both markers are indicative of M2-type macrophages, suggesting that the expression of SERPINE1 in GC may be related to the presence or activity of tumor-associated macrophages (TAMs). Figure 4J illustrates the association between SERPINE1 and CD274 (PD-L1) in GC, showing a modest positive correlation between SERPINE1 and PD-L1 expression levels. This may imply a regulatory link between SERPINE1 and PD-L1 expression in the GC immune microenvironment. Given that PD-L1 is a target for immune checkpoint inhibitors and SERPINE1 is a protein involved in immune regulation and tumor progression, this positive correlation may have important implications for immunotherapy strategies in GC.

Fig. 4

Relationship Between SERPINE1 and Immune Infiltration in the Tumor Microenvironment. A. Correlation between SERPINE1 gene expression levels and infiltration levels of different immune cells. B. Relationship between SERPINE1 copy number variations (CNVs) and infiltration levels of various immune cells. C-G. Correlation between SERPINE1 gene expression levels and CD2, CD3D, CD3E, CD8 A, and CD8B expression levels. H-I. Correlation between SERPINE1 gene expression levels and CD206 and CD163 expression levels. J. Relationship between SERPINE1 and CD274 (PD-L1) in GC, showing a slight positive correlation between their expression levels

NFATC2 induces macrophage polarization via SERPINE1

To further validate the roles of NFATC2 and SERPINE1 on macrophage polarization, complementary experimental validation was performed through in vitro and in vivo mechanistic approaches. GC cells were co-cultured with macrophages (THP1/M0) as depicted in Fig. 5A. Subsequently, flow cytometric profiling and quantitative RT-PCR analysis were implemented to quantify expression dynamics of macrophage surface markers, including CD163, ARG1, and IL10. Quantitative RT-PCR analysis demonstrated NFATC2 silencing downregulated the expression of CD163, ARG1, and IL10, while overexpression of SERPINE1 increased the expression of these genes. Additionally, overexpression of SERPINE1 partially restored the decrease in the expression of these genes caused by NFATC2 knockdown, indicating that SERPINE1 plays a role in NFATC2-mediated M2 polarization of macrophages (Fig. 5B). Flow cytometry analysis revealed that NFATC2 silencing decreased the proportion of CD206+ macrophages (typically associated with M2-type macrophages) induced by GC cells, while overexpression of SERPINE1 increased this percentage. Similarly, SERPINE1 overexpression partially restored the decreased percentage of CD206-positive macrophages caused by NFATC2 knockdown. This indicates that SERPINE1 is involved in NFATC2-mediated regulation of M2 macrophage polarization (Fig. 5C).

Fig. 5

NFATC2 Induces Macrophage Polarization via SERPINE1. (A) Schematic of co-culture system between GC cells (AGS/MKN1) and THP1-derived M0 macrophages. (B) qRT-PCR analysis of M2 macrophage markers (CD163, ARG1, IL10) in THP1 cells co-cultured with AGS cells transfected with shNFATC2 and/or OE-SERPINE1. (C) Flow cytometry to quantify CD206 + M2 macrophages after NFATC2 knockdown and/or SERPINE1 overexpression in GC cells. (D-E) Nude mouse xenograft model: tumor images (D) and weights (E) after subcutaneous injection of AGS cells with shNC, shNFATC2, OE-SERPINE1, or shNFATC2 + OE-SERPINE1 (n = 5/group). (F) Immunohistochemistry staining for CD163 in xenograft tumors to assess M2 macrophage infiltration. (G) ELISA detection of TGF-β and IL-10 secretion in co-culture supernatants or tumor lysates. △p < 0.05, △△△p < 0.001 vs. Blank; *p < 0.05, **p < 0.01, ***p < 0.001 vs. NC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. sh-NFATC2#1

In vivo gastric cancer xenograft studies utilized athymic murine models (n = 5), stratified into four experimental cohorts: NC, shNFATC2#1, OE-SERPINE1, and shNFATC2#1 + OE-SERPINE1. The xenograft tumor images (Fig. 5D) and the changes in tumor weight under different treatment conditions (Fig. 5E) showed that knockdown of NFATC2 significantly inhibited tumor growth, while overexpression of SERPINE1 significantly promoted tumor growth. When both NFATC2 knockdown and SERPINE1 overexpression were performed, the tumor weight increased but did not reach the level seen with SERPINE1 overexpression alone, suggesting that the absence of NFATC2 may partially counteract the promoting effect of SERPINE1 overexpression on tumor growth. Further immunohistochemistry results (Fig. 5F) confirmed these findings. Macrophage polarization is regulated by various cytokines, with TGF-β and IL-10 being two important pro-inflammatory factors that play key roles in M2-type macrophage polarization (Yeh et al. 2018; Li et al. 2022). ELISA results for TGF-β and IL-10 (Fig. 5G) showed that knockdown of NFATC2 reduced the secretion of these cytokines, while overexpression of SERPINE1 increased their secretion. Even under conditions of NFATC2 knockdown, overexpression of SERPINE1 still promoted the secretion of TGF-β and IL-10, although the effect was somewhat diminished. These results provide crucial mechanistic insights into NFATC2/SERPINE1-mediated gastric carcinogenesis and their immunomodulatory functions, identifying novel therapeutic targets for GC intervention. Collectively, our data establish NFATC2/SERPINE1 as critical regulators of gastric oncogenesis via M2 macrophage polarization to influence tumor growth and microenvironment, potentially offering novel therapeutic targets for gastric cancer.

NFATC2/SERPINE1/JAK3/STAT3 signaling forms a positive feedback loop

To explore the mechanisms by which NFATC2 and SERPINE1 regulate GC, we conducted multiple experiments. Western blot results showed that silencing the NFATC2 gene could inhibit the activation of the JAK3/STAT3 pathway, while overexpression of the SERPINE1 gene promoted the activation of this pathway (Fig. 6A). Further analysis using the TIMER and GEPIA databases revealed that STAT3 and NFATC2 expression levels were positively correlated in GC (Fig. 6B-C). STAT3 overexpression induced NFATC2 mRNA upregulation (Fig. 6D). Conversely, when the STAT3 gene was specifically silenced using siRNA interference technology, the expression level of NFATC2 mRNA significantly decreased (Fig. 6E). Our analysis using the JASPAR database identified 93 potential STAT3 binding sites within the NFATC2 promoter (score = 10.21011), with the conserved binding motifs depicted in Fig. 6F. Dual-luciferase reporter systems revealed STAT3 overexpression markedly enhanced WT promoter-driven transcriptional activity (Fig. 6G). This effect was abrogated when mutations were introduced into the promoter to disrupt STAT3 binding sites (Fig. 6G). Chromatin immunoprecipitation (ChIP)-qPCR assays validated the direct interaction between STAT3 and the NFATC2 promoter. Specifically, the anti-STAT3 antibody showed enrichment of promoter DNA fragments relative to control IgG, and this enrichment was further enhanced under conditions of STAT3 overexpression (Fig. 6H). Collectively, these findings confirm that STAT3 functions as a transcription factor for NFATC2, directly activating its promoter to drive gene expression. Our findings further demonstrate SERPINE1 enhances NFATC2 expression via JAK3/STAT3 activation, forming a self-reinforcing positive regulatory circuit that drives gastric carcinogenesis.

Fig. 6

SERPINE1 Promotes NFATC2 Expression via Activation of the JAK3/STAT3 Signaling Pathway, Forming a Positive Feedback Loop. (A) Western blot analysis of JAK3/STAT3 signaling pathway activation (p-JAK3, p-STAT3) in AGS cells with NFATC2 knockdown (shNFATC2) or SERPINE1 overexpression (OE-SERPINE1). (B-C) GEPIA/TIMER database correlation analysis of STAT3 and NFATC2 expression in GC. (D-E) qRT-PCR detection of NFATC2 mRNA levels after STAT3 overexpression (D) or knockdown (E) in AGS cells, via Student’s t-test. (F) JASPAR database prediction of STAT3-binding motifs in the NFATC2 promoter. (G) Dual-luciferase reporter assay of wild-type (WT) and mutant (Mut) NFATC2 promoter constructs in the presence of STAT3 overexpression. (H) ChIP-qPCR assay of STAT3 binding to the NFATC2 promoter in AGS cells. (I) Western blot for p-JAK3/p-STAT3 in AGS cells with OE-NFATC2 and/or sh-JAK3. (J-L) CCK-8 (J), colony formation (K), and Transwell (L) assays to evaluate cell viability, proliferation, and migration/invasion under NFATC2 overexpression and JAK3 inhibition. (M) Western blot analysis of EMT markers (E-cadherin, N-cadherin, Vimentin) in AGS cells with OE-NFATC2 and/or sh-JAK3. *p < 0.05, **p < 0.01, ***p < 0.001 vs. NC; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. OE-NFATC2

To further validate the relationship between NFATC2 and the JAK3/STAT3 signaling pathway, AGS cells were divided into four groups: NC, OE-NFATC2, sh-JAK3, and OE-NFATC2 + sh-JAK3. Western blot analysis (Fig. 6I) demonstrated NFATC2 overexpression upregulated JAK3/STAT3 phosphorylation, whereas JAK3 silencing abrogated this signaling activation. These findings suggest NFATC2 contributes to gastric carcinogenesis via JAK3/STAT3 pathway activation, as evidenced by CCK-8 assays (Fig. 6J) results showed that knocking down JAK3 could reverse the enhancement of GC cell viability caused by overexpression of NFATC2. Colony formation assays (Fig. 6K) indicated that NFATC2 overexpression enhanced cell proliferation and clonogenic ability, while JAK3 knockdown reduced these capabilities. Transwell migration and invasion assays (Fig. 6L) showed that NFATC2 overexpression promoted GC cell migration and invasion, whereas JAK3 depletion attenuated this promoting effect. Western blot analysis of EMT-related proteins (Fig. 6M) revealed that NFATC2 overexpression induced EMT, characterized by E-cadherin suppression with concomitant N-cadherin/Vimentin induction, while JAK3 knockdown reversed this process with opposite protein changes. Collectively, these data demonstrate NFATC2-mediated JAK3/STAT3 activation through SERPINE1, resulting in STAT3 elevation. Simultaneously, STAT3 regulates the upregulation of NFATC2, which further upregulates SERPINE1, forming a positive feedback loop. This oncogenic circuit drives malignant phenotypes (proliferation/clonogenicity), metastatic potential, and EMT progression in gastric cancer cells, fueling disease advancement.

SERPINE1/JAK3/STAT3 increases PD-L1 + M2 macrophages

After exploring the mechanism by which SERPINE1 promotes GC progression by upregulating NFATC2 through the activation of the JAK3/STAT3 signaling pathway, we further investigated the role of this signaling pathway in the tumor immune microenvironment, particularly its effects on PD-L1+ M2 macrophages. GEPIA database interrogation revealed significant STAT3-PD-L1 co-expression in gastric cancer (Fig. 7A). To further explore this relationship, we designed co-culture experiments, as shown in Fig. 7B, AGS GC cells with STAT3 silencing were cocultured with THP1 macrophages. Immunoblotting demonstrated STAT3 knockdown suppressed PD-L1 expression (Fig. 7C). In further co-culture experiments, we examined the proportion of PD-L1+ M2 macrophages after co-culturing GC cells with macrophages. Flow cytometry analysis revealed that NFATC2 overexpression significantly enhances PD-L1 expression in M2 macrophages, while JAK3 knockdownexerted an opposing effect. Notably, JAK3 depletion partially reversed the NFATC2 overexpression-driven upregulation of PD-L1 (Fig. 7D). These findings suggest STAT3 potentially facilitates GC immune evasion through PD-L1 induction, while NFATC2 promotes macrophage PD-L1 expression and M2 polarization via JAK3 to remodel the tumor immune landscape.

Fig. 7

The SERPINE1/JAK3/STAT3 Pathway Increases PD-L1+ M2 Macrophages and Inhibits CD8+ T Cell Activation via the PD-1/PD-L1 Interaction. (A) Pearson correlation analysis via GEPIA database to assess STAT3 and PD-L1 (CD274) expression correlation in GC. (B) Schematic of THP1 macrophage-AGS cell co-culture system for immune phenotype analysis. (C) Western blot detection of PD-L1 protein levels in THP1 cells after STAT3 knockdown (si-STAT3 vs. si-NC), analyzed by Student’s t-test. **p < 0.01, ***p < 0.001 vs. si-NC. (D) Flow cytometry to quantify PD-L1+ CD206+ M2 macrophages and CD8+ T cell activation markers in co-cultures with AGS cells overexpressing NFATC2 and/or knocking down JAK3, performed via one-way ANOVA. △△p < 0.01, △△△p < 0.001 vs. Blank; *p < 0.05, **p < 0.01, ***p < 0.001 vs. NC; #p < 0.05, ##p < 0.01 vs. OE-NFATC2

Knockdown of serpine1 sensitizes gastric cancer xenografts to anti-PD-1 therapy

After gaining a deeper understanding of the regulatory role of the SERPINE1/JAK3/STAT3 signaling pathway in the immune microenvironment of GC, we further explored the impact of Serpine1 knockdown on the sensitivity of GC xenografts to anti-PD-1 therapy. Utilizing a lentiviral-mediated gene silencing approach, we downregulated Serpine1 expression in YTN16 cells, followed by assessing protein levels via Western blot analysis (Fig. 8A). C57BL/6 J mice received subcutaneous injections of YTN16 cells with stable shRNA constructs (sh-NC controls or sh-Serpine1) in the right flank. The mice were then treated with anti-PD-1 antibodies or isotype-matched control IgG as per the experimental protocol (Fig. 8B). Figure 8C displays the size and shape of tumors in different treatment groups, including sh-NC (negative control), sh-Serpine1 (Serpine1 gene knockdown), anti-PD-1 (anti-PD-1 treatment), and sh-Serpine1 + anti-PD-1 (Serpine1 gene knockdown plus anti-PD-1 treatment). The changes in tumor volume over time, showed that compared to the sh-NC group, the sh-Serpine1 and anti-PD-1 groups had slower tumor volume growth, and the sh-Serpine1 + anti-PD-1 group had the slowest tumor volume growth, further confirming the synergistic effect of the combined treatment (Fig. 8D). The tumor weights indicated that Serpine1 knockdown inhibited tumor growth, and anti-PD-1 treatment also showed some inhibitory effect, with the lowest tumor weight in the sh-Serpine1 + anti-PD-1 group, suggesting a synergistic effect in inhibiting tumor growth (Fig. 8E). Serpine1 knockdown combined with PD-1 blockade synergistically enhanced tumor-infiltrating CD4+/CD8+ T cell populations, with the most significant effect observed in the combination treatment (Fig. 8F). Collectively, these findings demonstrate dual targeting synergistically suppresses gastric cancer progression by augmenting antitumor immunity.

Fig. 8

Knockdown of SERPINE1 Sensitizes Gastric Cancer Xenografts to Anti-PD-1 Therapy. (A) Western blot validation of SERPINE1 knockdown in YTN16 cells via lentiviral shRNA (sh-SERPINE1 vs. sh-NC). (B) Experimental design: C57BL/6 J mice inoculated with sh-NC or sh-SERPINE1 YTN16 cells, treated with anti-PD-1 antibody (10 mg/kg) or IgG control (n = 5/group). (C) Representative tumor images from each treatment group (sh-NC, sh-SERPINE1, anti-PD-1, sh-SERPINE1 + anti-PD-1). Tumor weights (D) and growth curves (E) measured over 28 days, analyzed by one-way ANOVA. (F) Flow cytometry analysis of CD8+ T cell infiltration in tumors from each group. **p < 0.01, ***p < 0.001