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Journal of Translational Medicine logoLink to Journal of Translational Medicine
. 2026 Mar 4;24:498. doi: 10.1186/s12967-026-07943-3

Targeting neuropilin-1 and neutralizing interleukin-6 inhibits cancer stem cell formation in bladder cancer cells

Hsiao-Wen Weng 1,2,#, Hui-Kung Ting 3,#, Hsiao-Hsien Wang 4, Ta-Jung Peng 5, Shye-Jye Tang 1,✉, Kuang-Hui Sun 5,6,✉
PMCID: PMC13069746  PMID: 41782148

Abstract

Background

Bladder urothelial carcinoma (BLCA) remains a clinical challenge because of its high recurrence rate and the persistence of cancer stem cells (CSCs) within the tumor microenvironment (TME). Neuropilin-1 (NRP1) is recognized as a key orchestrator of epithelial–mesenchymal transition (EMT) and stemness in several malignancies; however, its specific mechanism in driving BLCA aggressiveness and CSC maintenance has not been fully elucidated. This study aimed to characterize the regulatory role of NRP1 in bladder cancer progression and identify potential therapeutic options.

Methods

The clinical significance of NRP1 was first assessed using TCGA patient datasets and tissue microarrays. The functional roles of NRP1 were determined through CRISPR/Cas9-mediated knockout, shRNA-mediated knockdown, and overexpression of NRP1 in the T24 and 5637 cell lines. Bulk RNA sequencing and GSEA were utilized to identify downstream pathways, and ELISA and Western blotting were used to validate signaling interactions. Finally, a synergistic therapeutic strategy was tested using a subcutaneous xenograft nude mouse model.

Results

High NRP1 expression was significantly associated with advanced tumor stage, chemoresistance, and poor overall survival in patients with BLCA. Mechanistically, NRP1 regulated the IL-6–STAT3 signaling axis; NRP1 depletion significantly reduced IL-6 secretion and phosphorylated STAT3 levels, thereby impairing sphere formation and EMT features. Conversely, exogenous IL-6 treatment rescued the CSC phenotype in NRP1-deficient cells. Clinically, NRP1 expression strongly correlated with IL-6 and STAT3 levels, and patients with high expression of NRP1 and IL-6 had significantly worse survival outcomes, identifying NRP1–IL-6 coexpression as a novel prognostic biomarker in BLCA. Based on these findings, we demonstrated that dual targeting with the NRP1 inhibitor EG00229 and the IL-6-neutralizing antibody siltuximab synergistically suppressed CSC self-renewal and inhibited tumor growth in vivo.

Conclusions

This study provides a comprehensive characterization of the NRP1–IL-6–STAT3 axis as a fundamental driver of bladder cancer stemness. Our findings establish a foundational mechanistic proof-of-concept for a dual-targeting approach. By simultaneously inhibiting NRP1 and neutralizing IL-6, we demonstrated profound suppression of tumor-initiating capacity and CSC self-renewal. This strategy offers significant translational potential for improving clinical outcomes and overcoming therapeutic resistance in patients with aggressive BLCA.

Supplementary information

The online version contains supplementary material available at 10.1186/s12967-026-07943-3.

Keywords: Bladder urothelial carcinoma, Epithelial–mesenchymal transition, Cancer stem cells, Interleukin-6, Neuropilin-1, Tumor microenvironment

Introduction

Globally, bladder urothelial carcinoma (BLCA) places a substantial burden on health care systems, with approximately 614,000 new diagnoses and 220,000 deaths annually [1]. BLCA represents a formidable challenge in oncology, distinguished by its profound heterogeneity and high rates of recurrence following conventional therapies [2, 3]. The inflammatory tumor microenvironment (TME) plays a crucial role in driving BLCA progression. Intricate interactions between cancer cells and various immune cell populations within the TME lead to the secretion of proinflammatory cytokines and growth factors, which collectively foster a permissive environment for tumor growth and metastasis [4, 5]. This inflammatory milieu is a key inducer of the epithelial‒mesenchymal transition (EMT), a process that enables cancer cells to acquire mesenchymal characteristics, such as increased motility and invasiveness, through the activation of master transcription factors (TFs) (e.g., SNAIL, SLUG and ZEB1/2) and the modulation of epithelial and mesenchymal markers [6]. Importantly, EMT is intimately linked to the emergence and maintenance of cancer stem cells (CSCs) [7, 8], which are highly tumorigenic and self-renewing cells that are characterized by their ability to form spheres in vitro and express stemness-associated biomarkers [9]. CSCs are widely recognized as the main drivers of tumor recurrence, metastasis, and therapeutic resistance in BLCA [9, 10].

Among the key inflammatory mediators in the BLCA TME, interleukin-6 (IL-6) has emerged as a master regulator. Elevated IL-6 levels promote chemoresistance through autocrine signaling and sustained STAT3 activation [11, 12], leading to immunosuppression, increased invasiveness, and enhanced metastatic capacity [13]. IL-6 also directly underpins CSC properties by increasing their self-renewal capacity and tumorigenic potential [14, 15]. Consistent with its multifaceted role, IL-6 expression is strongly associated with disease progression across various malignancies, including BLCA [16–18].

Neuropilin-1 (NRP1), a versatile coreceptor for diverse ligands, including TGF-β, vascular endothelial growth factor (VEGF), and semaphorin, is a known modulator of cancer cell proliferation, migration, and metastasis [19–21]. Compelling evidence has implicated NRP1 in regulating EMT and CSC traits, driving phenotypic shifts that confer chemoresistance and metastatic potential [22–25]. The role of NRP1 as a versatile coreceptor is well documented in various malignancies; however, its specific therapeutic relevance in BLCA has only recently gained prominence. In patients with BLCA, NRP1 expression is significantly upregulated in malignant cells compared with adjacent noncancer tissues, and multivariate analyses have established high NRP1 levels as an independent prognostic indicator of reduced overall survival [26]. Furthermore, NRP1 has been implicated in the formation of an immunosuppressive TME in muscle-invasive bladder cancer (MIBC), where it is correlated with dysfunctional effector immune cells and resistance to both adjuvant chemotherapy and immune checkpoint blockade [27]. The druggability of the NRP1 axis is evidenced by the development of clinical-grade agents such as the monoclonal antibody MNRP1685A [28] and the selective small-molecule inhibitor EG00229 [24, 29, 30]. However, the potential for NRP1 inhibition to specifically eradicate bladder CSCs remains largely unexplored. Given the multifaceted role of NRP1 in driving both EMT and the inflammatory milieu, targeting this receptor offers a promising strategy to disrupt the self-renewal capacity of the aggressive CSC populations responsible for the high rates of recurrence in patients with BLCA.

This study investigated the mechanisms through which NRP1 regulates bladder CSC maintenance and BLCA progression. Our findings reveal a pivotal role for NRP1 in controlling IL-6 secretion, whereby NRP1 depletion leads to IL-6 downregulation, impairing CSC traits through the suppression of phospho-STAT3 (Tyr705) activity. Importantly, exogenous IL-6 can rescue the CSC phenotype in NRP1-deficient cells, suggesting that NRP1 inhibition alone may be ineffective in an inflammatory TME due to compensatory IL-6 signaling. Building upon these insights, we explored a novel combinatorial therapeutic strategy and demonstrated that the NRP1 inhibitor EG00229, combined with the anti-IL-6 antibody siltuximab, profoundly affects BLCA stemness. These results reveal a fundamental NRP1–IL-6–STAT3 axis that governs bladder cancer stemness and provide compelling preclinical evidence for a promising therapeutic approach that simultaneously targets NRP1 and IL-6 signaling to overcome BLCA recurrence and progression.

Materials and methods

Cell culture

The human BLCA cell lines T24 and 5637 were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan). The cells were maintained in RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), and 1% GlutaMAX (Gibco, USA). All the cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.

CRISPR/Cas9-mediated gene knockout

NRP1 and IL-6 knockout (KO) cell lines were generated using CRISPR/Cas9 gene editing. Guide RNA (gRNA) target sequences for exon 5 of NRP1 and exon 2 of IL-6 were designed using the “CHOPCHOP” online tool (https://chopchop.cbu.uib.no/). Oligonucleotide pairs for gRNAs were annealed and ligated into BsmBI-digested pAll-Cas9.Ppuro vector (RNAiCore, Taiwan). RNAiCore packaged the resulting Cas9/gRNA vectors into lentiviruses. The cells were transduced at an MOI of 0.5 for 24 h, followed by puromycin selection (2 μg/mL, Sigma‒Aldrich, USA) for 96 h. Single-cell clones were isolated by plating 100 cells onto 100 mm dishes and selecting individual colonies. Gene knockout was validated by Western blotting. Genotyping was performed via PCR amplification of a 400 bp region flanking the gRNA target site, cloning the PCR product into the pGEM-T Easy vector (Promega, USA), and Sanger sequencing to confirm indels. The detailed gRNA sequences and genotyping primer sequences are provided in Table S1.

Lentivirus shRNA-mediated knockdown

For stable gene silencing, two distinct lentiviral shRNAs targeting NRP1 (shNRP1#17, TRCN0000300917; shNRP1#80, TRCN0000322980), one shRNA targeting IL-6 (shIL6#5, TRCN0000059205), and two shRNAs targeting STAT3 (shSTAT3#86, TRCN0000329886; shSTAT3#87, TRCN0000329887) were obtained from RNAiCore. The cells were infected with lentiviruses at an MOI of 2 in 60 mm dishes. Stable cell populations expressing the shRNAs were established by puromycin selection (2 μg/mL). The pLKO_TRC005 empty vectors served as a control, and the pLKO.1 empty vector served as an shIL6 control.

Transient transfection

To overexpress NRP1, pCherry-mNrp1 (Addgene plasmid #21934) or pEGFP-N1-FLAG (Addgene, #60360) plasmids were transiently transfected into T24 or 5637 cells using jetPRIME transfection reagent (Polyplus, France) according to the manufacturer’s instructions. A total of 2 μg of plasmid DNA was used per transfection. The transfection efficiency was monitored by fluorescence microscopy, and protein overexpression was confirmed by Western blot analysis 24 h post transfection.

Clonogenic assay

T24 (500 cells/well) or 5637 (1,000 cells/well) cells were seeded into 60 mm dishes and incubated for 8 days to allow colony formation. Colonies were then fixed with cold methanol for 30 min, followed by three washes with PBS and staining with 0.5% crystal violet for 30 min.

2D horizontal migration assay

In gap closure assays, cells (2 × 104) were seeded onto both sides of two-well culture inserts (Ibidi, Germany) placed in 12-well plates. After overnight incubation at 37 °C, the inserts were removed to create a defined cell-free gap. In the wound healing assays, confluent cell monolayers (90% confluence) in 12-well plates were wounded using a sterile 200 μL pipette tip and then were washed with PBS, after which the medium was replaced. Images were acquired at 0 h (T = 0) and every 6 h thereafter for a total of 18 h.

Transwell migration and invasion assays

Migration assays were performed using 24-well Transwell inserts with 8.0 μm pores (Corning, USA). For invasion assays, inserts were precoated with Matrigel (1:4 dilution, Sigma-Aldrich, USA). T24 and 5637 cells (1 × 105 cells) in 300 μL of 1% FBS medium were added to the upper chambers. The lower chambers contained 700 μL of 20% FBS medium as a chemoattractant. After 24 h (migration) or 48 h (invasion), nonmigrated cells were removed from the upper surface of the membrane with a cotton swab. The membranes were fixed with methanol, stained with 0.5% crystal violet, and imaged using brightfield microscopy.

Sphere/Spheroid formation and invasion assays

For sphere formation, T24 and 5637 cells (4 × 103 cells/well) were cultured in ultralow attachment (ULA) 96-well plates in serum-free DMEM/F12 medium (Gibco, USA) supplemented with B27 supplement (Gibco, USA), 20 ng/mL basic FGF (Gibco, USA), 20 ng/mL epidermal growth factor (Gibco, USA), 0.4% fraction V BSA, and 4 μg/mL insulin (Gibco, USA). Images were captured at the indicated time points using an Olympus I×71 microscope (Olympus, Japan). To ensure objective quantification, sphere numbers and diameters were measured using ImageJ software. Spheres were classified into three size-based cohorts (>25 μm, 50–100 μm, 100–200 μm, and >200 μm) to evaluate changes in sphere-forming efficiency. For spheroid invasion assays, T24 cells (5,000 cells) were initially incubated for 48 h using the hanging drop method in 20 μL of RPMI-1640 medium to form aggregated spheroids. Matrigel (100 μL) or a 1:1 drug dilution was used to coat 48-well plates, which were subsequently incubated for 3 h at 37 °C. The spheroids were then embedded in 20 μL of a Matrigel/Type I collagen mixture (1:1 ratio, Corning, USA) and seeded onto the precoated plates. The matrix was polymerized for 30 min at 37 °C. Medium (200 μL) with or without the specified drugs was added at 0 h. Images were captured every 24 h for 48 h using an Olympus I×71 microscope.

Pharmacological inhibition and combination treatments

T24 and 5637 cells (2.5 × 103 cells/well) were seeded in 96-well plates for cell viability analysis. The cells were treated with varying concentrations of the NRP1 inhibitor EG00229 (25, 50, or 100 μM), the IL-6 neutralizing antibody siltuximab (5 or 10 μg/mL), or their combinations (25 μM EG00229/5 μg/mL siltuximab; 50 μM EG00229/10 μg/mL siltuximab). Viability was assessed at 24, 48, 72, and 96 h using a CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS; Sigma‒Aldrich, USA) or a CCK-8 kit (T-Pro Biotechnology, Taiwan) following the manufacturers’ protocols. DMSO and the IgG isotype control or nonspecific IgG (anti-GFP antibody) served as the respective vehicle controls for EG00229 and siltuximab. To evaluate the impact on self-renewal, T24 or 5637 cells were seeded in ULA 6-well plates and treated with IgG/DMSO control, 50 μM EG00229, 10 μg/mL siltuximab, or the combination (50 μM EG00229/10 μg/mL siltuximab) for 3 days, followed by a 4-day drug withdrawal period prior to sphere quantification.

Western blotting

The cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Roche, Switzerland). The lysates were homogenized through a 21-gauge needle and centrifuged at 12,000 × g for 15 min at 4 °C. The protein concentration was determined by the Bradford assay (Bio-Rad, USA). The samples were denatured at 95 °C for 15 min, separated on 10% polyacrylamide gels, and transferred to nitrocellulose membranes (Cytiva, USA). The membranes were blocked in 3% fraction V BSA/TBST and incubated with primary antibodies (1:1,000) overnight at 4 °C. Following washes, the membranes were incubated with HRP-conjugated secondary antibodies and visualized using a CCD camera. Densitometric analysis was performed using Multi Gauge software (Fujifilm). Table S2 lists all antibodies used.

Quantitative real-time PCR (RT‒qPCR)

Total RNA was extracted using TRIzol (Invitrogen) and reverse-transcribed into cDNA using ImProm-II Reverse Transcriptase (Promega, USA) according to the manufacturer’s guidelines. Quantitative PCR was performed using SYBR Green Master Mix (Ampliqon, Denmark) on a QuantStudio 1 system (Applied Biosystems, USA). Relative gene expression was calculated using the 2-ΔΔCt method. All the qPCR primer sequences used are listed in Table S3.

Enzyme-linked immunosorbent assay (ELISA)

The cells (6 × 105) were seeded in 100 mm dishes and incubated for 24 h. The medium was then replaced with 1% FBS medium and incubated for an additional 24 h. Conditioned medium was collected and centrifuged at 1000 × g for 5 min to remove cellular debris. Specifically, IL-6 levels in the conditioned medium were quantified using a dedicated human IL-6 ELISA kit (R&D Systems, USA) according to the manufacturer’s instructions.

Tissue array and immunohistochemistry

The primary antibodies used for IHC included NRP1 (Abcam, #EPR3113) and IL-6 (Proteintech, #21865–1-AP). Bladder tissue microarray (TMA) slides were obtained from Biomax (#BL804 and #802c). Patient clinicopathologic characteristics are provided in Table S4. TMA slides were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed in Tris-EDTA buffer at 95 °C for 20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The slides were incubated with primary antibodies (5 μg/mL) at room temperature for 1 h using an IHC Prep& Detect Kit (Proteintech, USA). The slides were counterstained with hematoxylin and mounted. Manual scanning was performed using Microvisioneer mvSlide software with an Olympus B×43 microscope (Olympus, Japan). The TMA slides were analyzed in a blinded manner using the IHC Profiler plugin for ImageJ [31]. The Colour Deconvolution 2 plugin was used for DAB and hematoxylin separation. The staining intensity was scored on a scale of 0 (negative), 1 (low positive), 2 (positive), or 3 (high positive).

Patient databases and association studies

Gene expression, survival, and clinical data from the TCGA BLCA cohort were downloaded from UCSC Xena (https://xenabrowser.net/; version 2017–10-13). Patients were stratified into 1st and 4th quartiles on the basis of NRP1 and IL-6 expression levels. Kaplan-Meier survival analysis was performed by the log-rank (Mantel-Cox) test. NRP1 and IL-6 coexpression data were obtained from cBioPortal (https://www.cbioportal.org/), and correlations were analyzed using Spearman’s correlation coefficient. For pancancer analysis, patient survival rates based on NRP1 and IL-6 expression were analyzed across 12 distinct cancer types: BRCA, CESC, COADREAD, GBM, HNSC, KIRC, LIHC, LUNG, OV, PAAD, SARC, and STAD. Data for the pancancer analysis were sourced from UCSC Xena (version 2019–12-06) and the TCGA Pancancer Atlas (for coexpression) [32].

Transcriptome analysis

Total RNA was extracted from pooled T24 cells (empty vector control and NRP1 knockout) using TRIzol reagent. The RNA libraries were prepared and sequenced on the Illumina NovaSeq 6000 platform, generating 150-bp paired-end reads. The raw reads were uploaded to Galaxy (https://usegalaxy.eu/). Adapter sequences were removed using Trimmomatic (v.0.38), and read quality was assessed with FastQC (v.0.73+galaxy0). The reads were aligned to the human reference genome GRCh38 using HISAT2 (v.2.2.1+galaxy1). Exon-overlapping read counts were summarized using Htseq-count (v.0.9.1+galaxy1). Differential gene expression analysis was performed using DESeq2 (v.2.11.40.7+galaxy2). Gene set enrichment analysis was conducted using the GSEA (v.2.2.4) to identify enriched pathways.

Mouse model

Female BALB/c nu/nu mice (CAnN.Cg-Foxn1nu/CrlNarl) were purchased from the National Laboratory Animal Center and bred at the Laboratory Animal Center of the National Yang Ming Chiao Tung University (Yangming Campus). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). The mice were housed under specific pathogen-free conditions in individually ventilated cage (IVC) systems with a 12/12 h light-dark cycle. T24 spheres (5 × 105) in DMEM/F12/Matrigel (1:1) were injected subcutaneously into bilateral flanks of 5–8-week-old mice. When the tumors reached 100–200 mm3, the mice were subjected to intraperitoneal (IP) treatments with either DMSO/IgG, 10 mg/kg EG00229 (Tocris Bioscience, UK), 10 mg/kg siltuximab (Selleckchem, USA), or a combination of EG00229 and siltuximab. Tumor volumes were measured twice weekly using digital calipers and calculated using the following formula: V = 0.5 × length × width2.

Statistical analysis

Statistical comparisons between the control and experimental groups were performed using an unpaired two-tailed Student’s t-test for two groups, one-way ANOVA for more than two groups, or two-way ANOVA for multiple comparisons. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.The notation “ns” denotes not-significant (p > 0.05). All the statistical analyses were conducted using GraphPad Prism software v.9, and the results are presented as the means ± standard deviations.

Results

Clinical relevance of NRP1 expression and its association with stemness and EMT signatures in patients with BLCA

To explore the clinical significance of NRP1 in BLCA progression, we analyzed its expression patterns and correlations with patient outcomes using The Cancer Genome Atlas (TCGA) dataset. In the TCGA BLCA cohort, high NRP1 expression (top quartile) was correlated with poor overall survival (p = 0.0191; Fig. 1A). This association was confirmed in independent datasets (GSE3167, GSE65635, and GSE120736), which linked high NRP1 expression to advanced-stage BLCA [33]. IHC on BLCA TMAs confirmed protein-level elevation, with positive staining (score 2) increasing from 8% in stage I to 26% in stage II and 25% in stage III tumors (Fig. 1B). High NRP1 expression was also associated with upregulated expression of the CSC marker CD44 and drug resistance genes (ABCB1, ABCC1, and ABCG2) (Fig. 1C). Additionally, it promoted EMT, which was characterized by reduced E-cadherin (CDH1) expression and elevated vimentin (VIM) expression (Fig. 1D) and increased expression levels of EMT-related TFs (SNAIL, SLUG, TWIST1/2, and ZEB1/2) (Fig. 1E). Overall, NRP1 promotes BLCA stemness and EMT, promoting progression, resistance, and poor outcomes.

Fig. 1.

Fig. 1

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High expression of NRP1 is correlated with poor survival and promotes stemness and EMT in patients with bladder urothelial carcinoma (BLCA). (A) Kaplan‒Meier survival analysis showing the overall survival of patients with BLCA from the cancer Genome Atlas (TCGA) cohort. Patients were stratified by high (NRP1 mRNA, top quartile, Q4, n = 104) or low (bottom quartile, Q1, n = 106) expression. (B) Distribution of NRP1 IHC intensity scores across 100 human BLCA tumor samples from two independent tissue microarrays (TMAs). (C–E) Analysis of the correlation between NRP1 expression and molecular signatures in the TCGA BLCA cohort, dichotomized into NRP1-high (Q4, n = 102) and NRP1-low (Q1, n = 101) groups. Expression levels of the (C) cancer stem cell marker CD44 and drug resistance-related ABC transporters (ABCB1, ABCC1, and ABCG2), (D) the epithelial marker CDH1 and the mesenchymal marker VIM, and (E) EMT-driving TFs (SNAIL, SLUG, TWIST1, TWIST2, ZEB1, ZEB2). Statistical significance was determined by the log-rank test (A) and a two-tailed Mann‒Whitney U test (C‒E). *p < 0.05, ****p < 0.0001

NRP1 facilitates invasion, migration, and proliferation in BLCA

To investigate the role of NRP1 in bladder cancer progression, we generated NRP1-knockout (KO) T24 and 5637 bladder cancer cells using CRISPR/Cas9. Western blots confirmed that NRP1 was depleted in the KO clones compared with the empty vector (EV) controls (Fig. 2A). Sanger sequencing revealed a 1-bp insertion and an 8-bp deletion in both alleles of T24 KO#1 and a single adenine insertion in one allele of 5637 KO#3 (Supplementary Fig. S1A). For overexpression (OE), we transfected cells with pCherry-mNrp1 or pEGFP-N1 as a control, which was verified by Western blotting and fluorescence microscopy (Supplementary Fig. S1B, C). Compared with the EV controls, NRP1-KO cells exhibited reduced colony formation (Fig. 2B), wound closure (Fig. 2C), migration (Fig. 2D), and invasion (Fig. 2E), whereas OE enhanced these phenotypes. Similarly, shRNA-mediated NRP1 knockdown (Supplementary Fig. S1D, E) suppressed proliferation, migration, and invasion (Supplementary Fig. S1F–H). Collectively, these results demonstrate that NRP1 drives bladder cancer progression.

Fig. 2.

Fig. 2

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NRP1 is essential for bladder cancer cell proliferation, migration, and invasion. (A) Western blot analysis of the NRP1 knockout (KO) efficiencies in T24 and 5637 bladder cancer cells. Whole-cell lysates from empty vector (EV) control and NRP1-KO cells were probed for NRP1; ACTB served as a loading control. (B) Clonogenic growth assay assessing the colony formation capacity of NRP1-overexpressing (OE) cells (vs. pEGFP-N1 empty vector control, ctrl) and NRP1-KO cells (vs. gRNA empty vector, EV). n = 3. (C) Wound healing assay evaluating the migratory capacities of T24 and 5637 cells with NRP1 KO or OE. Representative brightfield images are shown at 0 and 12 h. Scale bars, 100 μm. n = 3. (D, E) Transwell assays quantifying (D) migration through uncoated inserts and (E) invasion through Matrigel-coated inserts of NRP1-modulated T24 and 5637 cells. Scale bars, 100 μm. n = 6 (ctrl and OE). n = 3 (EV and KO). The data are presented as the means ± SDs. Statistical significance was determined by a two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

NRP1 promotes cancer stem cell phenotypes in BLCA

Given the established link between NRP1 expression, EMT, and BLCA progression, we next examined whether NRP1 directly influences the CSC phenotype in BLCA. NRP1 KO in T24 and 5637 cells decreased the number and size of spheres (Fig. 3A and Supplementary Fig. S2A, B); shRNA knockdown similarly reduced the number of spheres (Supplementary Fig. S3A). Conversely, NRP1 OE increased the number of spheres. To confirm the specificity of NRP1 in BLCA, a rescue experiment in which NRP1 was transiently overexpressed in NRP1-KO T24 cells was performed, and successful transfection was verified by Western blotting (Supplementary Fig. S4A). This intervention restored cell proliferation (Supplementary Fig. S4B) and migration in the culture–insert 2-well assay but not in the wound-healing assay (Supplementary Fig. S4C, D) or in the sphere formation assay (Supplementary Fig. S4E) in NRP1-KO cells. RT‒qPCR analysis revealed decreased mRNA levels of CD44 and NANOG in NRP1-KO cells but markedly increased levels in NRP1-OE cells (Fig. 3B–E). The mRNA and protein expression levels of VIM, a mesenchymal marker, were reduced in both T24 and 5637 cells following NRP1 knockdown (Supplementary Fig. S3C–E). In T24 NRP1-KO spheres, FOXC1 and ZEB2 mRNA levels were downregulated (Supplementary Fig. S3D); in 5637-KO cells, TWIST1/2 expression was decreased (Supplementary Fig. S3E). Additional EMT TFs (SLUG, and ZEB1/2) were also downregulated in knockdown-derived spheres (Supplementary Fig. S3F, G). Therefore, NRP1 sustains BLCA CSCs by regulating stemness markers and EMT-related TFs.

Fig. 3.

Fig. 3

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NRP1 is essential for maintaining cancer stem cell characteristics in BLCA. (A) Sphere formation assay (Day 7) assessing the self-renewal capacity of T24 and 5637 BLCA cells upon NRP1 genetic modulation. Representative brightfield images show spheres formed by NRP1-OE and NRP1-KO cells compared with control cells. The number and diameter (>50 μm) of spheres were quantified. Scale bars, 100 μm. n = 3. (B–E) RT–qPCR analysis of stemness-related gene expression in T24 and 5637 spheres following NRP1 OE (B, C) or KO (D, E). Relative mRNA levels (2−ΔΔCt method) were normalized to those of ACTB. n = 3. The data are presented as the means ± SDs. Statistical significance was determined by a two-tailed unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

NRP1 regulates IL-6 secretion and drives cancer stemness via the IL-6/STAT3 axis

To identify the specific mechanisms by which NRP1 sustains the CSC phenotype, we performed bulk RNA-seq on NRP1-KO T24 cells. Unbiased gene set enrichment analysis (GSEA) revealed significant downregulation of pathways related to the inflammatory response and IL-6-JAK-STAT3 signaling (Fig. 4A, B). Although IL-6 is a known driver of bladder cancer aggressiveness [18], its regulation by NRP1 has not been previously characterized in BLCA. However, prior evidence in pancreatic neuroendocrine neoplasms has suggested that NRP1 can be upregulated by IL-6/STAT3 signaling, suggesting a potential reciprocal regulatory circuit [34], and that siRNA-mediated NRP1 inhibition downregulated p-STAT3 (Tyr705) in prostate cancer ARCaPM cells [35]. Given these transcriptomic insights and the established role of IL-6 in CSC maintenance, we hypothesized that NRP1 serves as a master regulator of an IL-6–STAT3 autocrine loop in bladder cancer. Furthermore, the loss of NRP1 consistently decreased IL-6 mRNA levels under both parental (2D) and sphere-forming conditions (Fig. 4C). Quantitative analysis of IL-6 secretion using ELISA confirmed a reduction in the concentration of IL-6, which decreased from approximately 50 pg/mL in EV cells to 30 pg/mL in T24 NRP1-KO cells. Conversely, NRP1-OE cells in sphere cultures strikingly increased IL-6 secretion to approximately 750 pg/mL, whereas NRP1 knockout significantly reduced IL-6 levels (Fig. 4D and Supplementary Fig. S5A). Importantly, a notable decrease in p-STAT3 levels was observed in NRP1-knockout and knockdown CSCs (Fig. 4E, F), and similar reductions in p-STAT3 and VIM levels were observed in NRP1-knockdown cells (Supplementary Fig. S6A).

Fig. 4.

Fig. 4

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NRP1 deficiency downregulates the expression of IL-6 and its downstream signaling pathway. (A) GSEA showing differential pathway enrichment following NRP1 knockout in T24 cells. (B) Enrichment plot of IL6–JAK–STAT3 signaling presented with ES, NES, and FDR. (C) RT‒qPCR analysis of IL-6 expression in NRP1-KO T24 parental cells and spheres. Relative mRNA levels (2−ΔΔCt method) were normalized to those of ACTB. n = 3. (D) Human IL-6 levels in cell culture supernatants from T24-EV- and NRP1-KO parental cells or T24 spheres measured by ELISA. n = 3 (EV and KO parental); n = 4 (ctrl and OE spheres); n = 2 (EV and KO#1 spheres). (E, F) Western blot analysis of NRP1, STAT3, p-STAT3, CD44, SOX2, and VIM protein expression levels in (E) T24 EV and NRP1-KO#1 spheres or (F) 5637 shCTRL and shNRP1 (#17 and #80) knockdown spheres. ACTB served as a loading control. n = 3. The results of the quantification of p-STAT3 are presented as p-STAT3/STAT3 ratios normalized to those of ACTB. The data are presented as the means ± SDs. Statistical significance was determined by a two-tailed unpaired Student’s t test. **p < 0.01, ***p < 0.001

We next explored the reciprocal relationship and downstream effects of IL-6 signaling by generating IL-6-KO T24 cells and confirmed 2- and 4-bp deletions in both alleles (Supplementary Fig. S6B). IL-6 knockout and knockdown impaired sphere formation (Supplementary Fig. S6C, D) and downregulated the expression levels of EMT-related TFs (FOXC1 and ZEB2; Supplementary Fig. S6E) and stemness markers (CD44, OCT4, NANOG, and MYC; Supplementary Fig. S6F). To assess whether NRP1 requires IL-6 to promote stemness, we conducted a rescue experiment through transient transfection of IL-6-KO cells in which NRP1 was successfully overexpressed, as verified by fluorescence microscopy (Supplementary Fig. S6G). NRP1 overexpression partially restored the sphere formation efficiency of IL-6-KO cells (Supplementary Fig. S6H).

Building on these findings, we investigated the potential for a positive feedback loop by treating T24 wild-type cells with recombinant IL-6 protein (rIL-6), which induced an approximate 30-fold upregulation of IL-6 mRNA after 96 hours (Supplementary Fig. S7A) and concurrently elevated NRP1 and p-STAT3 protein levels (Supplementary Fig. S7B). To definitively establish that this increase in IL-6 expression reflects a self-sustaining autocrine circuit rather than an artifact of exogenous exposure, we treated T24 NRP1-OE spheres with an IL-6-neutralizing antibody. This intervention suppressed sphere formation efficiency in NRP1-OE cells (Supplementary Fig. S7C), indicating that blocking secreted IL-6 disrupted the loop even under conditions of elevated NRP1 expression. The essentiality of STAT3 as a downstream effector was further confirmed by overexpressing NRP1 in T24 cells cotransduced with shSTAT3 (#86 and #87) or shCTRL. Western blotting verified that both total STAT3 and phospho-STAT3 were downregulated (Supplementary Fig. S7D), and both STAT3 knockdown constructs effectively abolished NRP1-OE-induced sphere formation (Supplementary Fig. S7E). Collectively, these results indicate that enhanced stemness in NRP1-OE cells requires continuous autocrine IL-6 production and signaling, thereby establishing an NRP1–IL-6–STAT3 positive feedback loop that drives EMT and CSC formation.

Exogenous IL-6 rescues cancer stem cell phenotypes in NRP1-deficient BLCA cells

Because NRP1 KO reduced IL-6 secretion and p-STAT3 levels, we hypothesized that exogenous IL-6 could rescue the CSC and EMT phenotypes of NRP1-deficient cells. In sphere formation assays, rIL-6 treatment increased both the number and size of spheres formed by NRP1-KO cells (Fig. 5A). Moreover, rIL-6 treatment upregulated the expression of stemness markers (CD44, OCT4, SOX2, and NANOG) in both EVs and KO CSCs (Fig. 5B) and restored p-STAT3 levels in NRP1-KO cells to those observed in control cells (Fig. 5C). In NRP1-knockdown T24 and 5637 cells, rIL-6 increased the number and size of spheres (Supplementary Fig. S8A), whereas the IL-6 neutralizing antibody blocked this increase (Supplementary Fig. S8B). Moreover, in NRP1-knockdown T24 CSCs, rIL-6 restored the expression levels of key stemness markers (CD44, OCT4, SOX2, and NANOG) and EMT markers (VIM, SLUG, and ZEB2). Similarly, in 5637 NRP1-knockdown CSCs, rIL-6 rescued the expression of OCT4, FOXC1, and ZEB1/2 (Supplementary Fig. S8C, D). Furthermore, the decrease in IL-6 expression was partial in NRP1-deficient cells, as exogenous IL-6 did not fully restore the expression patterns of EMT-related and stemness-related genes. These results suggest that current NRP1-targeted therapies, which do not address IL-6 signaling concurrently, may have limited efficacy in completely eradicating aggressive CSC populations because of this robust compensatory pathway.

Fig. 5.

Fig. 5

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Exogenous rIL-6 rescues the CSC phenotype in NRP1-deficient cells. (A) Sphere formation (Day 7) of T24 or 5637 EV and NRP1-KO cells (4,000 cells/well) treated with MOCK (0.1% BSA in PBS) or 100 ng/mL rIL-6 in ULA 96-well plates. Images taken at 10× magnification. The number and diameter (>100 μm) of spheres were quantified. Scale bar, 100 μm. n = 3. (B) RT‒qPCR analysis of stemness-related genes in T24 EV and NRP1-KO#1 spheres cultured for seven days and treated with MOCK or 100 ng/mL rIL-6. Relative mRNA expression levels (2−ΔΔCt method) were normalized to those of ACTB. n = 3. (C) Western blot analysis of NRP1, STAT3, p-STAT3, CD44, and SOX2 protein levels in T24 EV and NRP1-KO#1 spheres treated with MOCK or 100 ng/mL rIL-6 for 3 days. ACTB served as a loading control. n = 3. The results of the quantification of p-STAT3 are presented as p-STAT3/STAT3 ratios normalized to those of ACTB. The data are presented as the means ± SDs. Statistical significance was determined by one-way ANOVA with Tukey’s posttest (A, B). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

NRP1 inhibition and IL-6 neutralization reduce tumorigenesis in vitro and in vivo

Building on these insights, we developed a novel therapeutic strategy by combining the NRP1 inhibitor EG00229 with the FDA-approved IL-6-neutralizing antibody, siltuximab, to evaluate their synergistic anticancer efficacy, particularly against CSCs. No significant cytotoxicity was detected in T24 or 5637 cells treated with EG00229 (50 μM), siltuximab (10 μg/mL), or their combination (Supplementary Fig. S9A, B). Moreover, EG00229 specifically inhibited cell proliferation in T24 NRP1-OE cells but not in NRP1-KO cells (Supplementary Fig. S9C). Monotherapies minimally affected primary sphere formation, whereas combination therapy reduced primary sphere size and profoundly inhibited secondary sphere formation by decreasing both the number and size of spheres. Notably, in T24 cells, no spheres larger than 100 μm were detected after combination therapy, indicating a profound impact on self-renewal capacity (Fig. 6A). In spheroid invasion assays, compared with siltuximab monotherapy, the combination therapy decreased the invasion distance to a greater extent, and the efficacy of the combination therapy was comparable to that of EG00229 alone (Fig. 6B). The combination treatment downregulated the expression of the key stemness marker SOX2 and reduced p-STAT3 levels (Fig. 6C). These findings demonstrate that combining NRP1 inhibition with IL-6 neutralization represents a potential strategy for suppressing IL-6/STAT3 axis activation, leading to substantial reductions in CSC self-renewal capacity and stemness marker expression.

Fig. 6.

Fig. 6

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NRP1 and IL-6 dual targeting synergistically inhibits CSCs in vitro and tumorigenesis in vivo. (A) Sphere formation (Day 7) from wild-type T24 primary (2 × 104 cells/well) and secondary (8,000 cells/well) spheres or 5637 primary and secondary spheres (1 × 104 cells/well) treated with IgG/DMSO, 50 μM EG00229, 10 μg/mL siltuximab, or 50 μM EG00229/10 μg/mL siltuximab for 3 days, followed by 4 days withdrawal, in ULA 6-well plates. (B) Spheroid invasion assay. T24 wild-type spheroids (5,000 cells) were treated as indicated. The longest invasion distance was quantified using ImageJ. n = 16 (Dmso/igg); n = 10 (EG00229); n = 9 (siltuximab); n = 9 (EG00229/siltuximab). (C) Western blot analysis of STAT3, p-STAT3, and SOX2 protein levels in T24 and 5637 wild-type spheres treated as indicated. ACTB served as a loading control. n = 3. The results of the quantification of p-STAT3 are presented as p-STAT3/STAT3 ratios normalized to those of ACTB. (D) Images of tumor xenografts resulting from the subcutaneous injection of T24 sphere cells (5 × 105 cells) treated with IgG, IgG/DMSO, 10 mg/kg EG00229, 10 mg/kg siltuximab, or a combination of EG00229/siltuximab for 8 weeks. Five mice per group were used. The data are presented as the means ± SDs. Statistical significance was determined by one-way ANOVA with Tukey’s posttest (A, B) and a two-tailed Mann‒Whitney U test (D). *p < 0.05, **p < 0.01, ****p < 0.0001

To evaluate the effects of EG00229 combined with siltuximab in a xenograft mouse model, mice were injected subcutaneously with T24 CSCs embedded in Matrigel supplemented with IgG/DMSO, EG00229, siltuximab, or EG00229/siltuximab for 8 weeks. Both combination therapy and EG00229 monotherapy significantly reduced tumor volume, but the combination treatment resulted in a greater reduction in tumorigenicity (Fig. 6D), suggesting that combining EG00229 with siltuximab significantly reduces tumor-initiation capacity and tumorigenicity, proposing a potential therapeutic approach for targeting bladder cancer stem cells.

Clinical relevance of the coexpression of IL-6 with NRP1 in BLCA

Our comprehensive analysis of clinical data from the TCGA database further reinforced the clinical relevance of this NRP1–IL-6 axis. Patients with high IL-6 expression in the BLCA cohort had significantly worse overall survival (Fig. 7A). Crucially, the coexpression of high levels of both NRP1 and IL-6 was associated with an even poorer five-year overall survival rate (Fig. 7B), suggesting a synergistic detrimental effect. Patients with high NRP1 expression also presented elevated levels of both STAT3 and IL-6 (Fig. 7C), with phospho-STAT3 (Tyr705) protein expression being significantly greater in these patients (Fig. 7D). Correlation analysis revealed a moderate but significant association between NRP1 and IL-6 mRNA expression in patients with BLCA (Fig. 7E). Although IHC analysis revealed that IL-6 staining intensity in tumors was predominantly low across all stages, higher positive staining (score 2) was detected exclusively in more advanced stage II and III tumors (Fig. 7F). Based on these integrated clinical and mechanistic insights, we propose a model in which NRP1 acts as a central hub, driving cancer stem cell phenotypes through a positive feedback loop involving IL-6 and STAT3. Specifically, NRP1 activity increases IL-6 secretion, which, in turn, increases the phosphorylation of STAT3. This STAT3 activation then further promotes NRP1 expression, creating an autocrine signaling circuit that sustains CSC properties. Consequently, therapeutic strategies that target both NRP1 and IL-6, which are secreted by tumor cells (autocrine) or originate from the tumor microenvironment, may represent a potent approach to effectively reduce CSC formation and overcome therapeutic resistance.

Fig. 7.

Fig. 7

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NRP1 is highly correlated with IL-6 and STAT3 in BLCA patients. (A, B) Kaplan‒Meier survival analysis of overall survival in BLCA patients from the TCGA cohort. (A) Patients stratified by high (Q4, n = 104) or low (Q1, n = 106) IL-6 expression. (B) Comparison between the NRP1highIL-6high (n = 42) and NRP1lowIL-6low (n = 45) groups. (C) Correlation analysis of NRP1 expression with IL-6 and STAT3 mRNA levels in the TCGA BLCA cohort. Patients were stratified into NRP1-high (Q4, n = 102) and NRP1-low (Q1, n = 101) groups. (D) Analysis of phospho-STAT3 (Tyr705) protein levels in the TCGA BLCA cohort, stratified by high (Q4, n = 90) or low (Q1, n = 86) NRP1 expression by reverse phase protein array (RPPA). (E) Spearman correlation coefficients for NRP1 and IL-6 mRNA levels. (F) Distribution of IL-6 IHC intensity scores across 99 tumors from two distinct TMAs. Statistical significance was determined by the log-rank test (A, B) and a two-tailed Mann‒Whitney U test (C, D). ****p < 0.0001

Discussion

Bladder urothelial carcinoma remains a major clinical challenge because of its high recurrence rate, which is intrinsically linked to tumor heterogeneity and the persistence of aggressive CSCs [2, 3]. Conventional therapies often fail to eradicate these resistant cell populations completely, underscoring the need for novel therapeutic strategies specifically aimed at eliminating CSCs. NRP1 is a multifaceted molecule known to participate in diverse biological processes, including growth factor signaling, vascular function, and immune homeostasis [19–21, 36]. The NRP1 antagonist EG00229 was specifically designed to disrupt the binding of VEGF to NRP1 without interfering with VEGFR–ligand interactions [29]. Preclinical studies have demonstrated the ability of EG00229 to reduce the expression of proinflammatory factors (IL-1β, IL-6, and IL-8) and nuclear NF-κB/p65 under hypoxic conditions in human nasal epithelial cells and to suppress the growth of epidermal cancer stem cell xenografts [24, 30, 37]. Extending these observations, our work reveals a novel and crucial mechanism by which NRP1 activates STAT3 to drive IL-6 secretion, thereby establishing a positive-feedback loop that profoundly sustains the bladder CSC phenotype.

IL-6 is a key cytokine linked to tumor drug resistance and therapeutic failure [12, 13], making anti-IL-6/IL-6 R targeted therapies promising options in oncology. However, a significant clinical challenge with anti-IL-6 monotherapy is the potential for compensatory rebound elevation of IL-6 [38, 39], which may limit sustained efficacy. Our findings provide a mechanistic basis for this challenge within the context of BLCA, linking elevated serum IL-6 levels to aggressive disease [18] and identifying NRP1-driven STAT3 activation as a key mediator of invasiveness [40].

NRP1 has been shown to promote cancer stemness by modulating YAP expression [41]. YAP is a well-documented mediator of the production of IL-6 and other proinflammatory cytokines during tumor initiation [15, 42, 43]. Critically, YAP functions as a coactivator of STAT3, contributing to tumorigenesis, angiogenesis, and metastasis [44, 45], although YAP can also exert STAT3-independent effects [46]. This intricate interplay is reinforced by reports that NRP1 expression can be upregulated by exogenous IL-6 [47] and that increased IL-6 levels in tumor tissue are associated with increased phospho-STAT3 and NRP1 expression [34]. These convergent lines of evidence provide a robust explanation for the observed increases in IL-6 and STAT3 expression in BLCA patients with high NRP1 expression and reveal that NRP1 deficiency leads to reduced phospho-STAT3 levels and a subsequent decrease in IL-6 expression in bladder cancer stem cells.

The therapeutic landscape for advanced BLCA remains constrained, with cisplatin-based chemotherapy and immune checkpoint inhibitors serving as the current standards of care [48]. Despite these interventions, treatment resistance and disease recurrence persist as significant clinical hurdles, largely driven by the survival of patients with persistent CSC [8–10]. Our findings demonstrate that targeted NRP1/IL-6 inhibition directly impacts the CSC populations responsible for tumor initiation and recurrence. Notably, this dual-targeting approach yielded significant reductions in secondary sphere formation and tumor-initiating capacity in xenograft models—results that stand in contrast to conventional chemotherapies, which primarily target bulk proliferating cells. Moreover, concurrent NRP1 inhibition disrupts the feedback loop that drives IL-6 re-elevation, potentially overcoming this resistance mechanism that is generated by IL-6 [38, 39]. Therefore, concurrent NRP1 inhibition disrupts the self-sustaining autocrine feedback loop that drives the increase in IL-6 levels. By neutralizing this IL-6-mediated resistance mechanism, we established a robust rationale for the combined use of EG00229 and siltuximab. These agents possess established preclinical tolerability and FDA approval, respectively, which may facilitate a more rapid transition to clinical evaluation. Furthermore, this strategy not only addresses a critical unmet need in BLCA management but also suggests broader pancancer significance, given the established correlation between NRP1/IL-6 coexpression and poor prognosis across multiple tumor types (Supplementary Fig. S10, S11).

Although our findings provide compelling preclinical evidence for the role of the NRP1–IL-6–STAT3 axis in BLCA CSC persistence, the use of established cell lines and subcutaneous xenograft models may not fully recapitulate the complex bladder TME, including stromal interactions and immune infiltration. To enhance clinical relevance, future studies should incorporate more physiologically relevant models, such as patient-derived organoids, primary tumor cultures, patient-derived xenografts (PDXs), or orthotopic implantation models. These approaches would better validate the therapeutic potential of dual NRP1/IL-6 targeting in a setting that mirrors human disease heterogeneity.

Conclusions

This work characterizes the role of NRP1 in sustaining BLCA CSCs via the IL-6/STAT3 axis. Our findings suggest that dual targeting of NRP1 and IL-6 disrupts cancer stemness and tumor progression, offering a potential therapeutic strategy for improving treatment outcomes in patients with BLCA that warrants further clinical evaluation.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (41.8MB, docx)
Supplementary Material 2 (47.5KB, docx)
Supplementary Material 3 (6.2MB, docx)

Acknowledgements

The authors would like to thank the Institute of Biomedical Sciences, Academia Sinica, and Dr. Yuh-Shan Jou for excellently revising the manuscript.

Abbreviations

BLCA

Bladder urothelial carcinoma

CSCs

Cancer stem cells

EMT

Epithelial-mesenchymal transition

EV

Empty vector

IL-6

Interleukin 6

KO

Knockout

NRP1

Neuropilin 1

OE

Overexpression

TCGA

The Cancer Genome Atlas

TME

Tumor microenvironment

Author contributions

HWW, SJT and KHS conceptualized the project and designed all the experiments. HKT, HHW and SJT supervised the project and contributed to revising the manuscript. HWW and TJP conducted most experiments and performed the animal experiments. HWW, SJT and KHS wrote the manuscript. All the authors have read and approved the final manuscript.

Funding

This work was supported by the National Science and Technology Council (NSTC-113-2320-B-019-002, 110-2320-B-A49A-514-MY3, 111-2740-B-A49-001, 112-2740-B-A49-001, 113-2740-B-A49-002, 113-2320-B-A49-025, 114-2314-B-016-003); Tri-Service General Hospital and National Defense Medical Center (TSGH-D-110069, TSGH-D-111076), Taipei City Hospital, Taiwan, and Cheng-Hsin General Hospital, Taipei.

Data availability

All data relevant to the study are shown in the article or supplementary information. RNA-seq data has been deposited in the SRA database (PRJNA1297379 https://www.ncbi.nlm.nih.gov/sra/PRJNA1297379).

Declarations

Ethics approval and consent to participate

All animal experiments in this study were conducted following the NIH Guide for the Care and Use of Laboratory Animals and the ARRIVE Guidelines. The protocol was approved by the IACUC of National Yang Ming Chiao Tung University, Yang Ming Campus.

Consent for publication

All authors give consent for the publication of the manuscript in Journal of Translational Medicine.

Competing interest

The authors declare that they have no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Hsiao-Wen Weng and Hui-Kung Ting contributed equally to this work.

Contributor Information

Shye-Jye Tang, Email: tsj@mail.ntou.edu.tw.

Kuang-Hui Sun, Email: khsun@nycu.edu.tw.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (41.8MB, docx)
Supplementary Material 2 (47.5KB, docx)
Supplementary Material 3 (6.2MB, docx)

Data Availability Statement

All data relevant to the study are shown in the article or supplementary information. RNA-seq data has been deposited in the SRA database (PRJNA1297379 https://www.ncbi.nlm.nih.gov/sra/PRJNA1297379).

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