Selective Targeting uPAR-driven Neutrophil Extracellular Traps Functional Clusters to Attenuate Tumor Progression and Enhance the Response to Immunotherapy in Intrahepatic Cholangiocarcinoma

Selective Targeting uPAR-driven Neutrophil Extracellular Traps Functional Clusters to Attenuate Tumor Progression and Enhance the Response to Immunotherapy in Intrahepatic Cholangiocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Selective Targeting uPAR-driven Neutrophil Extracellular Traps Functional Clusters to Attenuate Tumor Progression and Enhance the Response to Immunotherapy in Intrahepatic Cholangiocarcinoma baobin yin, xiaotian shen, xin zheng, Sun-Zhe Xie, Chen Zhang, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9281659/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cholangiocarcinoma (CCA) is an aggressive malignancy with dismal prognosis; PD-1/PD-L1 blockade benefits few patients, as the tumor microenvironment (TME) is immunologically “cold”. A key CCA-TME feature is massive tumor-associated neutrophil (TAN) infiltration releasing neutrophil extracellular traps (NETs). Conventionally deemed pro-tumorigenic, NETs’ anti-tumor potential is overlooked, leaving their dual roles and regulatory circuits in CCA undefined. Our study finds prominent NETs enrichment in CCA clinical specimens and preclinical models, correlating with poor outcomes. Functional studies show NETs exert dual effects: a dominant pro-tumor arm accelerating growth/metastasis, and a latent immunostimulatory arm rendering the TME “hot”. Integrated multi-omics and bioinformatics analyses dissect these functions into distinct molecular clusters with divergent prognostic value. Pro-tumor clusters are selectively activated by MAPK signaling, present in ~25% of CCA with KRAS mutations. Mechanistically, uPA-loaded NETs engage uPAR on CCA cells; TLR co-reception licenses downstream MAPK activation, tipping toward tumor promotion. We devised a cluster-directed combination: DNase I dismantling NETs scaffolds plus uPAR blockade neutralizing residual pro-tumor fragments. This strategy abolishes oncogenic signaling while sparing—even boosting—STING-dependent anti-tumor immunity, sensitizing KRAS-mutant and wild-type CCA to anti-PD-L1 therapy. Human transcriptomic datasets link low pro-tumor/high immunostimulatory NETs signatures with durable immunotherapy responses. We establish a functional-cluster framework for CCA NETs biology and provide a precision co-targeting regimen turning pro-tumor function into immunotherapeutic opportunity. Biological sciences/Cancer/Cancer microenvironment Biological sciences/Immunology/Tumour immunology Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Cholangiocarcinoma Neutrophil extracellular traps (NETs) Functional clusters KRAS mutation uPA-uPAR axis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cholangiocarcinoma (CCA) is a highly aggressive malignant tumor with a dismal prognosis, and its incidence has increased significantly in recent years. Most patients are diagnosed at an advanced stage, where surgical treatment offters limited efficacy, and postoperative tumor metastasis and rapid progression severely impair patient survival. Currently, CCA treatment has shifted towad a surgery-centered multimodal comprehensive therapy, including immunotherapy with programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors , which has demonstrated substantial potential in various solid tumors [1, 2] . However, improving therapeutic efficacy remains a core focus of current research. A prominent hallmark of the CCA tumor microenvironment (TME) is abundant neutrophil infiltration [3] , making targeting neutrophils and their derivatives a promising therapeutic strategy for CCA. Neutrophils can release neutrophil extracellular traps (NETs), web-like structures composed of extracellular chromatin as a scaffold decorated with various functional proteins [4] . In tumor progression, NETs exert prominent pro-tumor effects: they trap circulating tumor cells to promote metastatic colonization [5] , remodel the extracellular matrix to facilitate invasion [6] , and form physical barriers that block contact between immune cells and tumor cells [7] , thereby attenuating anti-tumor immune responses. Accumulating studies have confirmed that NETs act as a universal driver of tumor progression under diverse pathological conditions, including tumor-derived factors [8, 9] , oncogenic mutations [10] , chemotherapy-induced stress [11] , and chronic inflammation [12, 13] . On the other hand, beyond their well-documented pro-tumor properties, NETs also possess inherent and extensive immunostimulatory potential, which is reflected in their ability to mediate robust pro-inflammatory responses, regulate T cell activation, promote dendritic cell (DC) maturation, and induce the secretion of immune-stimulatory chemokines [14-16] . These functions hold the potential to remodel the CCA TME into an immunologically "hot" state that would be favorable for immunotherapy. This inherent duality of NETs, serving as both tumor promoters and immune activators, remains highly controversial, and its precise manifestation and regulatory mechanisms in CCA or other solid tumors have not been elucidated, making it a critical focus of our investigation. Current NET-targeting interventions mainly fall into two categories [17] : disrupting pre-formed NETs structures and inhibiting NETs formation. Both strategies have significant limitations: the former fails to eliminate residual NET fragments that retain biological activity and pro-tumor effects; the latter globally suppresses NETs production, sacrificing their potential immunostimulatory effects that support immunotherapy. As an underdeveloped alternative, precise targeting of the interaction between NETs and tumor cells, rather than non-specifically inhibiting all NET functions, represents an alternative NETs-targeted strategy in malignancy. In this study, through multi-omics analysis of preclinical models and clinical cohorts, we systematically elucidated the distinct dual effects of NETs in CCA. Specifically, NETs not only directly promote tumor growth and progression, but also sustain an immunostimulatory hot TME with potential anti-tumor activity. We further dissected these effects into independent NETs functional clusters, each with distinct biological functions and prognostic significance. Mechanistically, we identified urokinase-type plasminogen activator receptor (uPAR) as a key NETs receptor that, upon binding to NETs-bound uPA, selectively activates pro-tumor NETs functional clusters associated with MAPK signaling amplification—particularly in CCA with KRAS mutations, which accounts for ~25% of CCA cases and is closely linked to enhanced neutrophil infiltration, aberrant NETs formation, poor response to conventional therapies and unfavorable prognosis [3, 18] . Notably, uPAR selectively mediates the pro-tumor MAPK signaling in response to NETs, whereas its blockade modulates the immunostimulatory NETs functional clusters. Accordingly, we developed a combinatorial strategy targeting NETs functional clusters, disrupting the physical barrier of NETs using Deoxyribonuclease (DNase) in combination with blocking the uPA-uPAR signaling in residual NETs fragments. This strategy selectively inhibits CCA growth while preserving the immunostimulatory TME, and enhances the sensitivity of CCA- especially the KRAS-mutant subtype to immunotherapy. Methods Methods in detail are included in supplementary materials. Results Increased NETs Formation Correlates with Tumor Progression and Poor Progression of CCA To dissect the clinical relevance of neutrophil extracellular traps (NETs) in cholangiocarcinoma (CCA), we first characterized tumor-associated neutrophil (TAN) heterogeneity through single-cell RNA sequencing (scRNA-seq) of CD66b⁺ cells isolated from human CCA specimens. This analysis identified seven distinct transcriptional subsets, with a particular focus on the terminally differentiated PADI4⁺ subset (Figure 1A). Given that peptidylarginine deiminase 4 (PADI4) catalyzes histone citrullination—an obligate step for triggering NETs formation [19] , we posited that this subset possesses intrinsic NET-forming propensity. Employing a customized NETs-scoring algorithm e [20] , we confirmed that the PADI4⁺ TANs indeed exhibited the strongest NET-forming capacity among all subsets (Figure 1B) . Extending this analysis to The Cancer Genome Atlas (TCGA) CCA cohort, we observed elevated expression of pro-NETs signaling molecules in PADI4⁺TAN—including C5a (C5aR1), CXCR4, and oxidized low-density lipoprotein receptor 1 (OLR1)—which w strongly correlated with the pro-NETs PADI4⁺ TAN signatures. These findings collectively indicate the existence of a pro-NETotic signaling microenvironment in CCA (Figure 1C) . Systematic evaluation confirmed universal elevation of NETs-scores in TCGA CCA samples (Figure S1A) , consistent with previously reported TAN enrichment in this malignancy. To consolidate these bioinformatic findings in clinical specimens, we performed multiplex immunofluorescence staining in CCA samples from Huashan cohort, and revealed pronounced enrichment of NETs meshworks—characterized by the co-expression of H3cit (a hallmark of PADI4-citrullinated histones) and myeloperoxidase (MPO)—within tumor nests compared to adjacent stroma (Figures 1D-E) . To recapitulate these clinical observations experimentally, we established three complementary murine CCA models: subcutaneous (s.c.) and orthotopic hepatic (i.hep.) implantation models of the murine CCA cell line SB1 , as well as the YA model (a hydrodynamic injection-induced spontaneous CCA model driven by YAP/AKT plasmid delivery). All models consistently demonstrated prominent NETs accumulation within tumor tissues (Figure 1F and S1B) . Mechanistically, conditioned medium (CM) derived from human and murine CCA single-cell suspensions potently induced NET formation upon incubation with homologous neutrophils, as evidenced by immunofluorescence microscopy and extracellular DNA quantification (Figure 1G and S1C) . To delineate the functional contribution of NETs to CCA progression, we generated padi4 knockout (ko) mice, which are deficient in NETs-forming capacity. In the SB1 s.c., SB1 i.hep., and YA models which were established in the padi4-ko and wild-type (wt) mice, genetic ablation of Padi4 significantly attenuated tumor growth compared to wild-type (wt) controls, whereas NET-competent wt mice exhibited accelerated disease progression (Figure 1H-I and 1P) . Clinically, transcriptomic analysis of two independent public cohorts confirmed that elevated NETs-scores significantly correlated with poorer overall survival in CCA patients (Figures S1D) . Collectively, these findings establish that NETs formation is enriched in the CCA microenvironment and functionally drives malignant progression. NETs Concomitantly Exhibit Pro-Tumor Activity and Potential Immune-Stimulatory Properties in CCA The functional output of NETs in cancer is context-dependent and remains contentious. In vitro, purified NETs robustly accelerated proliferation, migration, and anchorage-independent growth of human-derived CCLP1 and RBE cells, as well as murine-derived SB1 cells (Figure 1J and S1E) , echoing prior observations in CCA and other solid tumors [17, 21] . To dissect the net biological impact of NETs in an intact host, we compared transcriptomes of YA tumors harvested from Padi4-wt (NETs-proficient) versus Padi4-ko (NETs-deficient) mice. Unexpectedly, NETs preservation was accompanied by a pronounced immune-inflammatory signature. We documented elevated expression of T-cell-attracting chemokines (Cxcl9, Ccl5), antigen-presentation machinery (H2-Aa, H2-Eb1), cytolytic mediators (Gzmb), and interferon-γ (Ifng), together with compensatory upregulation of the immune checkpoint molecule CD274 (PD-L1) (Figure 1K) . qPCR validated these findings in both YA and SB1 i.hep. models with intact NETs formation (Figure S1F) . High-dimensional mass cytometry (CyTOF) resolved 13 discrete leukocyte clusters and revealed that NETs-rich tumors harbored heightened global immune infiltration, most notable within CD4⁺ T-cell and effector-memory T-cell compartments (Figure 1L) . Concordant increases in TCRβ and CD11b signal intensity were observed (Figure S1G) , and immunofluorescence confirmed greater abundance of GZMB⁺ cytotoxic lymphocytes juxtaposed to tumor islets (Figure 1M) . Spatial transcriptomics further showed that T-cell–dense neighborhoods expanded in the presence of NETs and were topographically intercalated with malignant cell niches (Figure 1N) , implying facilitated immune–cancer crosstalk. These observations were validated in two independent CCA transcriptomic cohorts. CCA patients with a higher NETs-score displayed elevated expression of interferon-γ (IFN-γ), T cell inflammation-related genes, tertiary lymphoid structure (TLS) markers, inflammatory response factors, and complement system components compared with their low NETs-score counterparts (Figure S1H) . Nevertheless, this immune activation did not confer survival benefit, underscoring the paradox of NETs biology in CCA. We next asked whether tumor-intrinsic signals contribute to NETs-elicited immunogenicity. RNA-seq of NETs-treated CCLP1 and RBE cells, and of malignant cells FACS-purified from YA tumors, consistently revealed up-regulation of immune-activating ligands, exemplified by CCL5 ( Figure S1I-J) . In trans-well chemotaxis assays, conditioned medium from NETs-challenged CCA cells recruited significantly more human PBMCs or murine splenocytes than control medium, confirming that NETs-educated cancer cells actively amplify leukocyte trafficking ( Figure 1O) . To test whether immune stimulation tempers the net tumor-promoting effect of NETs, we blunted adaptive immunity with dexamethasone in the SB1 s.c. model. Under immunosuppression, the growth differential between Padi4-wt and Padi4-ko tumors widened from 1.47-fold to 2.04-fold, indicating that immune-mediated restraint partially offsets—but does not override—NETs-driven oncogenesis (Figure 1P) . Collectively, these findings uncover an inherent duality of NETs in CCA: while they intrinsically foster an immunologically “hot” microenvironment, their direct pro-tumor actions on malignant cells dominate, culminating in accelerated disease progression. NETs Operate through Functionally Distinct Clusters that Differentially Dictate CCA Prognosis To resolve the apparent paradox of NETs biology, we built a multi-step bioinformatics pipeline (Figure 2A). RNA-seq of RBE and CCLP1 cells exposed to purified NETs yielded 812 consistently up-regulated genes (NRGs; fold-change > 1.5; Fig. S2A). Weighted gene co-expression network analysis (WGCNA) of these NRGs across 858 CCA samples (four public cohorts) partitioned NETs-driven transcriptional output into five quasi-independent functional clusters (C1–C5; Fig. S2B). Functional annotation revealed discrete biological themes: C3 and C5 reflect the immune-stimulatory and regulatory effects of NETs via interleukin and receptor-ligand-related signaling, involving the upregulation of T cell immune-stimulatory factors such as CCL5 and CXCL10—consistent with the NETs-induced immune stimulation observed in our clinical cohorts and murine models; C2 is associated with NETs-mediated regulation of TGFβ signaling and extracellular matrix remodeling; C1 corresponds to our prior finding that cancer cells respond to NETs stimulation via Toll-like recptors (TLR) and downstream NF-κB signaling; and C4 is linked to MAPK signaling, primarily reflecting malignant tumor phenotypes closely associated with disease progression (Figure 2B and S2C) . We next established signature scores for each Functional Clusters and the whole set using Gene Set Enrichment Analysis (GSEA) and analyzed their correlation with NETs-score. Overall, NETs-score was positively correlated with NRGs expression (Figure S2D) , confirming that NETs drive the upregulation of these 5 Functional Custers in clinical CCA samples. Specifically, NETs-score showed strong positive correlations with the signatures of C1, C4, and C5, while correlations with C2 and C3 were weaker—suggesting that these two clusters may be co-regulated by other factors in the CCA TME besides NETs (Figure 2C) . Prognostic analysis of each Functional Cluster signature score revealed significant differences in patient outcomes: patients with higher scores for C1 and C5 (reflecting inflammatory and immune activation) had favorable prognosis, whereas those with higher scores for C2, C3, and C4 conferred poor prognosis, with C4 showing the most significant association with adverse outcomes (Figure 2D) . To translate cluster signatures into patient-level taxonomy, we performed principal-component analysis on Fu-iCCA transcriptomes and assigned each case to its dominant response pattern (C1–C5). The resulting five subgroups formed non-overlapping clouds in PCA space, validating their transcriptional distinctiveness (Figure 2E-F) . High-NETs tumours were enriched almost exclusively in C1, C4 and C5; they also displayed advanced TNM stage and a higher KRAS-mutation rate (Figure 2F) . Among these three dominant subgroups, the C4 pattern was the most malignant—characterized by larger tumour volume, vascular invasion, and distant metastasis—and carried the shortest survival (Figure 2G) . Conversely, the C5 pattern was associated with the best outcome. When each subgroup was further stratified by NETs-score, NETs remained adverse in C1 and C4 but became protective in C5 (Figure 2H) . The poor prognostic power of C4 was independently confirmed in Jusakul cohort (Figure S2E) . Finally, to circumvent the need for transcriptomics in routine practice, we trained six machine-learning classifiers to identify C4 tumours using only standard clinical variables. A random-forest model achieved the highest AUC in internal and external validation sets (Figure S2F-H) . Across the Fu-iCCA and Huashan-iCCA cohorts, model-assigned C4 patients exhibited higher levels of tumor-related markers (CA19-9), larger tumor size, and markedly shorter overall survival than other subgroups (Figure S2I-J) . Taken together, in consistent with the murine model findings, systematic bioinformatics analysis reveals NETs exert complex opposing effects on CCA via distinct pro-tumor (poor prognosis) and anti-tumor (favorable prognosis) functional clusters, and the net clinical outcome reflects the balance between these competing functional clusters rather than the mere quantity of NETs. NETs Selectively Activates MAPK Signaling by Amplifying KRAS Mutation (mut) Effects to Drive CCA Progression Therapeutically uncoupling the pro-tumorigenic from the immunostimulatory arm of NETs demands precise targeting of the malignant circuit without disabling global NET functions. Because the C4 cluster—dominated by MAPK-pathway genes—is the strongest predictor of poor outcome and is uniquely linked to NETs exposure, we asked whether KRAS mutation acts as a molecular gatekeeper that licences NETs to engage this programme. Across FU-iCCA Cohort transcriptomes, KRAS-mutant (KRASmut) tumours were the only genotype significantly associated with elevated NET-score (Figure S3A) , a finding corroborated immunohistochemically in the Huashan cohort (Figure 3A) . Among the five functional clusters, only C4 showed a strong correlation with KRASmut status (Figure S3B) . Despite the established correlation between KRASmut and NETs, how KRASmut regulates CCA cells’ sensing of NETs remains elusive. Although KRASmut is a well-established MAPK amplifier, its oncogenic output usually requires additional extracellular cues [22, 23] . We therefore hypothesized that NETs supply the requisite stimulus, electively switching on the C4 programme in KRASmut cells (Figure 3B) . To test this, we established CCLP1-KRASmut cells (by overexpressing KRAS-G12D mutation in KRASwt CCLP1 cells) and RBE-KRASwt cells (by overexpressing KRASwt in KRASmut RBE cells). RNA-seq after NETs stimulation revealed that C4 enrichment changed most dramatically with KRAS genotype; without NETs, KRAS manipulation alone left C4 quiescent (Figure 3C) . MAPK gene-set activity was moderately elevated by KRASmut alone but reached maximal intensity only when NETs and KRASmut coincided (Figure 3D) . Western blotting confirmed a striking synergistic increase in p-ERK in CCLP1-KRAS G12D upon NETs exposure, an effect largely absent in RBE-KRASwt cells (Figure 3E) . In vitro functional assays mirrored this signalling output: NETs enhanced proliferation, invasion, and sphere formation only in the KRAS mutation background, and these gains were fully reversed by the MEK inhibitor trametinib (Figure 3F) . In vivo, NETs-treated CCLP1-KRASmut cells formed rapidly growing subcutaneous tumours; neutralising NETs with the PAD-inhibitor GSK484 equalised growth rates between genotypes (Figure 3G) . We extended these observations to autochthonous models. YA (YAP/AKT) and YAK (YAP/AKT + KRASG12D) tumours were established in Padi4-wt or Padi4-ko livers (Figure 3H) . RNA-seq analyses demonstrated that all functional clusters were generally upregulated in NETs-rich wt CCA mice, with the showing a markedly prominent upregulation of C4 cluster in the YAK model (Figure 3I) . In NETs-proficient (wt) animals, YAK tumours exhibited exaggerated C4 activation, higher tumour burden, and elevated Ki-67 and p-ERK relative compared with the YA controls. In contrast, NETs ablation (Padi4-ko) uniformly suppressed tumour growth and abolished both Ki-67 and p-ERK signals, erasing the genotype-specific advantage of KRAS mutation ( Figure 3I-J) . Collectively, these data establish a two-step model: KRAS mutation licenses CCA cells to interpret NETs as a MAPK-amplifying signal, while NETs provide the extracellular cue that selectively ignites the C4 pro-tumour cluster. Interfering with this KRAS–NETs feed-forward loop offers a precision strategy to neutralise the malignant arm of NET biology without compromising its anti-tumour immunostimulatory capacity. uPA-Enriched NETs Activate Membrane-Bound uPAR to Selectively Initiate Pro-Tumor Effects in CCA Cellular responses to NETs rely on multiple sensors, with potential differences in their downstream functional outputs. We further deciphered the specific mechanisms by which CCA cells sense NETs stimulation and selectively activate the pro-tumor C4 functional cluster characterized by KRASmut-MAPK signaling. We constructed a potential C4 regulatory network via STRING protein-protein interaction analysis and identified PLAUR—which encodes the glycosylphosphatidylinositol-anchored receptor uPAR—emerged as the top hub (Figure S4A) . NETs stimulation rapidly up-regulated uPAR mRNA and protein, and this increment was amplified in KRAS-mutant (KRASmut) cells; no such induction was observed for other reported NET sensors (TLR2/4/9 or CCDC25) (Figure S4B) . High-resolution immunofluorescence revealed uPAR enrichment at the cell surface in intimate apposition to adherent NETs fibres, implying direct engagement (Figure 4A) . As a well-characterized cell membrane protein, uPAR exerts pro-tumor effects by enhancing integrin signaling, thereby facilitating tumor cell proliferation, invasion, and distant metastasis [26] . Functional validation via siRNA-mediated uPAR knockdown in RBE and CCLP1-KRASmut cells demonstrated that uPAR deficiency downregulated MAPK pathway activation under NETs stimulation (Figure 4B-C) and attenuated the NETs-induced upregulation of cancer cell proliferation (Figure 4D) . To better understand the mechanism by which uPAR responds to NETs and subsequently selectively activates the pro-tumor MAPK pathway, we focused on urokinase-type plasminogen activator (uPA)—the ligand of uPAR which is abundant in TANs [27] . Single-cell profiling of human CCA TANs showed that PLAU (encoding uPA) transcripts were selectively enriched in the PADI4⁺ TAN subset with an enhanced NETs-forming capacity (Figure 4E and S4C) . We therefore hypothesized that uPA released along with the NETs scaffold stimulates the KRAS mutation (KRASmut)-amplified MAPK signaling via uPAR, thereby mediates the selective activation of the pro-tumor functional clusters induced by NETs. Co-culture with KRASmut CCA-conditioned medium further elevated PLAU expression in neutrophils detected by qPCR assays (Figure 4F) . Moreover, typical uPA-enriched NETs structures were induced under phorbol 12-myristate 13-acetate (PMA) stimulation (Figure 4G) . PMA-induced NETs displayed reticular uPA–H3cit co-localization were observed in clinical KRASmut CCA tissues of both clinical specimens and murine YAK models (Figure 4H and S4D) . Across Fu-iCCA Cohort, NETs-scores showed a strong positive correlation with both uPA and uPAR transcript levels (Figure 4I) . To test whether uPA cargo is functionally required, we generated uPA-deficient NETs by siRNA knock-down in dHL-60 cells (a neutrophil-like cell line) prior to PMA-induced NETosis and exposed these cells to CCLP1-KRASmut CM. . Compared with the negative control (siNC)-derived NETs, the uPA-deficient NETs elicited markedly weaker p-ERK induction in CCLP1-KRASmut cells (Figure 4J) . Collectively, these results indicate that uPA-enriched NETs, via uPAR, mediate the selective pro-tumor effects on CCA through the KRASmut-amplified MAPK signaling pathway. Intercepting uPA–uPAR engagement therefore offers a precision node to disable the pro-tumour arm of NETs while sparing their immunostimulatory capacity. uPAR and TLRs Synergistically Mediate Pro-Tumorigenic NETs Stimulation in KRASmut CCA NETs consist of a chromatin scaffold decorated with various proteins, including uPA. Since uPA can be secreted by multiple components of the tumor microenvironment (TME) beyond neutrophils, we further explored the specific mechanisms by which KRASmut CCA cells respond to uPA-enriched NETs stimulation and activate pro-tumorigenic signaling cascades. Notably, stimulation of CCLP1-KRASmut and RBE with uPA alone failed to significantly upregulate pERK levels or the mRNA expression of key MAPK pathway components. In contrast, combined stimulation with uPA and NETs DNA scaffold induced MAPK pathway activation comparable to that induced by uPA-enriched NETs extracts. DNase-mediated disruption of the DNA scaffold effectively abrogated this MAPK activation (Figures 4K-L) , demonstrating that the physical association between uPA and the NET DNA scaffold is essential for pro-tumor signaling. TLRs are well-recognized sensors for NET-derived DNA, and consistently, functional cluster C1 exhibited significant enrichment in TLRs-related pathways. Immunofluorescence analysis revealed that NET stimulation induced pronounced co-localization of TLRs and uPAR in KRASmut CCLP1 and RBE cells, an interaction absent under basal conditions (Figure 4M) . Inhibition of TLRs using chloroquine (CQ) significantly attenuated CCA cell recognition of NETs, resulting in significantly reduced upregulation of key MAPK pathway molecules—an effect comparable to DNase-mediated DNA scaffold disruption. Importantly, this inhibitory effect could not be restored by exogenous uPA supplementation (Figure 4N) . Collectively, these findings demonstrate that the selective pro-tumorigenic effects of NETs in CCA require two sequential and synergistic steps: TLRs-mediated recognition of the NETs chromatin scaffold, followed by uPAR-mediated sensing of scaffold- -bound uPA, ultimately activating KRASmut-amplified pro-tumor MAPK signaling. Therapeutic Targeting of the uPA-uPAR Axis Selectively Abrogates Pro-Tumorigenic NET Effects While Preserving Immunostimulatory Activity We next evaluated the therapeutic potential of targeting the uPA-uPAR axis to attenuate the selective pro-tumor effects of uPA-enriched NETs in CCA. In KRASmut CCA cells, UPARANT [28] —a specific uPA-uPAR interaction inhibitor—significantly reduced NETs-induced MAPK/ERK pathway activation (Figure 5A) to an extent comparable to trametinib (direct MAPK inhibition), chloroquine (TLR inhibition), or DNase (scaffold disruption) (Figure 5A; Figure S5A). Concordantly, UPARANT markedly attenuated NET-driven malignant proliferation in vitro (Figure 5B) . Notably, beyond suppressing the selective activation of the C4-mediated pro-tumor MAPK pathway, UPARANT preserved or even enhanced the expression of immune activation-related genes (including CCL5, CXCL9, and ISG15) across the other functional clusters. This immune-sparing profile mirrored that of direct MAPK pathway inhibition (Figure 5C and S5A) , consistent with previous observations in related models. In contrast, direct intervention of the NETs scaffold using CQ or DNase non-selectively suppressed both pro-tumorigenic signaling pathways and immune activation genes (Figure S5A) . Consistently, UPARANT treatment did not impair but rather enhanced the in vitro chemotaxis of PBMCs toward NETs-stimulated CCA-CM (Figure 5D) . Since NETs-rich inflammatory stimuli can also activate MAPK in KRASwt contexts,, UPARANT similarly exerted inhibitory effects on NETs-induced pro-tumor phenotypes while maintaining immunostimulatory capacity in KRASwt cells (Figure S5B-D) . We next investigated the mechanistic basis for the reciprocal relationship between NET-induced MAPK signaling and immunostimulatory functional clusters. We found that the NET-driven immunostimulation was associated with tumor-intrinsic IFN responses (marked by ISG15), which correlated with activation of Stimulator of Interferon Genes (STING) and its downstream JAK-STAT signaling (Figure S5E) . Mechanistically, the UPARANT-enhanced expression of immune-stimulatory factors and PBMC chemotaxis in the presence of NETs were completely abrogated by direct inhibition of STING or STING-related JAK-STAT signaling (Figures 5E-F), indicating that uPA-uPAR blockade indirectly potentiates STING-JAK/STAT signaling. This aligns with previous reports that direct inhibition of MAPK/ERK signaling relieves the suppression of STING-JAK/STAT, thereby enhancing antitumor immunty [29-31] . In vivo validation was performed by establishing the SB1 i.hep. model padi4-ko or wt mice. UPARANT significantly reduced tumor burden in padi4-wt mice, with immunofluorescence analysis confirming decreased KI67 and pERK levels. Importantly, UPARANT preserved and even enhanced NETs-recruited anti-tumor T cells and dendritic cells (DCs)cells, whereas, it exerted minimal effects on KI67, pERK, and immune cell infiltration in padi4-ko mice (Figure 5G) . qPCR analysis of tumor tissues further confirmed that UPARANT did not affect the NETs-induced expression of immune effector molecules such as Ccl5 and Cxcl9 (Figure S5F) . Collectively, these results demonstrate that NETs-bound uPA selectively activates pro-tumorigenic functional clusters (specifically C4) via uPAR engagement while sparing immunostimulatory programs, establishing the uPA-uPAR axis as a promising therapeutic target that can uncouple tumor-promoting from immune-activating effects of NETs. uPA-uPAR axis Blockade Selectively Abrogates the Pro-Tumor Effects and Preserves Immunostimulatory Capacity of NETs in CCA Current therapeutic strategies targeting NETs primarily include disrupting formed NETs structures (DNase) and pharmacological blockade of NETosis (disulfiram or PADI4 inhibitors) (disulfiram or PADI4 inhibitors). While DNase effectively degrades NETs in vitro, in vivo application disrupts large-scale NETs meshworks to relieve tumor encapsulation and protection from immune cell attack, however, residual NET fragments and their associated components retain biological activity. We therefore proposed a combinatorial strategy to modulate NETs functional clusters—employing DNase to dismantle large-scale NETs architectures while utilizing UPARANT to selectively neutralize selectively pro-tumorigenic functional clusters (represented by C4) within residual NETs fragments while preserving their immune-stimulatory properties while abrogating tumor-promoting effects. Given the prominent activation of C4 cluster in KRASmut contexts, we evaluated this strategy in the YAP/AKT/KRAS (YAK) model, comparing DNase monotherapy, UPARANT monotherapy, DNase plus UPARANT combination, and the PADI4 inhibitor GSK484. GSK484 and the combination regimen significantly reduced tumor burden, with the latter showing superior efficacy. Both GSK484 and DNase plus UPARANT comparably suppressed tumor cell proliferation (Ki67) and MAPK activity (pERK). Notably, while DNase alone enhanced T cell infiltration, this immunostimulatory effect was further amplified by combination with UPARANT (Figures 6A-B) . Transcriptomic profiling revealed that DNase plus UPARANT collectively orchestrated an inflamed tumor microenvironment, characterized by significant upregulation of immune-promoting factors, antigen-presenting machinery, and interferon-responsive genes—an effect surpassing DNase monotherapy. In contrast, GSK484 monotherapy exhibited minimal immunostimulatory activity (Figure 6C) . Immunofluorescence further confirmed that DNase plus UPARANT treatment facilitated T cell penetration into cancer cell-enriched regions following NET barrier disruption, suggesting enhanced anti-tumor immune surveillance (Figure 6D) . Interestingly, CD274 (PD-L1) expression paralleled immune-stimulatory molecule upregulation across NET functional clusters, Padi4-wt versus ko comparisons, and pre/post-treatment analyses.. This observation prompted investigation of whether DNase plus UPARANT-induced TME remodeling could synergize with anti-PD-L1 blockade. In the YAK model, combination with DNase plus UPARANT markedly enhanced the limited efficacy of anti-PD-L1 monotherapy, significantly reducing tumor burden and prolonging survival compared to either treatment alone. This regimen also promoted abundant infiltration of granzyme B-positive (GZMB⁺) immune effector cells (Figure 6E and S6) . These findings were reproducible in the SB1 intrahepatic model, where DNase plus UPARANT similarly potentiated anti-PD-L1 efficacy and immune cell infiltration (Figure 6F and S6) . Finally, we interrogated the relationship between NETs, NETs functional clusters, and immunotherapy response using transcriptomic data from an independent published Ni cohort, in which patient response to immunotherapy was predicted based on CTLA4 and CXCL9 levels as reported by the original researchers [32] . Intriguingly, patients predicted to have a favorable response had relatively higher NET-score (Figure 6G) —a finding that appears contradictory to the established pro-tumorigenic role of NETs but is reconciled by our identification of distinct functional clusters. Indeed, responder-enriched tumors displayed higher representation of the C5 immune-activation cluster, whereas the C4 MAPK-associated cluster showed no correlation with immunotherapeutic benefit (Figure 6H) . Collectively, these findings demonstrate that the DNase plus UPARANT strategy outperforms existing NET-targeting monotherapies by selectively abrogating pro-tumorigenic NET signaling while preserving and enhancing immunostimulatory activity, thereby reshaping the TME toward an inflamed phenotype and synergizing with checkpoint blockade in KRASmut CCA (Figure 7) . Discussion TANs regulate tumor progression via NETs. Blocking NETs inhibits tumor progression, consistent with this and other studies, while NETs also exhibit protential antitumor effects—though relevant evidence in CCA remains scarce [4, 33, 34] . Our previous pan-cancer studies have shown that NETs may reflect pro-tumor or anti-tumor effects depending on cancer type [20] . Unlike TANs subsets with stage-specific and spatial-specific biological characteristics [35, 36] , the functional diversity of NETs likely stems from the distinct roles of their components [37, 38] . In this study, we observed the dual effects of NETs on CCA: they directly promote the malignant phenotypes of CCA cells, while their mediated pro-inflammatory effects may elicit potential antitumor responses. Bioinformatics analysis dissected NETs-CCA interactions, leading to the concept of C1–C5 functional clusters informed by the pro-/anti-inflammatory duality of NETs [39] . These clusters are independent, with individual clusters exerting pro-/anti-tumor effects, and their dynamic balance drives the overall outcomes in mouse models and patients. The differential responses of CCA to NETs reflect the selective activation of these clusters, with cluster-specific responses correlating with prognosis—highlighting the complexity of NETs biology in CCA. Notably, referring to the dual pro-/anti-tumor effects of NETs-CCA crosstalk under the novel functional clusters framework, pro-tumor clusters (e.g., C4) directly enhance CCA malignancy, aligning with the reported pro-tumor roles of NETs in other cancers through various context-dependent mechanisms [40, 41] . Our study identifies the C4 cluster as a key driver of pro-tumor effects by activating the MAPK pathway—signaling for enhancing malignant phenotypes under external stimuli, including NETs [42] . MAPK activation strongly correlates with KRAS mutations, which are present in ~25% of CCA cases and associated with the most aggressive subtype. Notably, KRAS mutations alone are often insufficient to sustain robust MAPK activation; additional extracellular stimuli are typically required, either from autocrine signals in KRAS-mutant cancer cells or paracrine signals from the TME [22, 23, 43] . Combined with our data, TME NETs stimulation and CCA KRASmut cooperate to promote malignant phenotypes: KRASmut boosts TANs recruitment/NETs formation and enhances CCA responsiveness to NETs, leading to more robust pro-tumor MAPK activation of C4 in CCA—a pattern similar to KRASmut-amplified inflammation in lung/pancreatic cancer [22, 44] . In contrast, the anti-tumor functions of NETs have long been overshadowed by the extensive reports on their pro-tumor roles in various malignancies. Among the limited reports, direct cytotoxicity of NETs against tumor cells has been documented as one of the mechanisms [45-47] . Our novel perspective extends this understanding by highlighting that anti-tumor clusters maintain a potential hot TME and promote immune infiltration in CCA. In autoimmune diseases, NETs are highly pro-inflammatory, promote dendritic cell maturation and reduce T cell activation thresholds [13-15] . As a highly inflammatory solid tumor, CCA exhibits similar NETs-mediated pro-inflammatory characteristics, which was confirmed using NETs-deficient models and multiple detection approaches. Specifically, NETs induce CCA cells to secrete chemokines such as CCL5 and CXCL9, which recruit T cells and DCs to the tumor site. Such triggered immunogenicity is reported to rely on signaling pathways activated by DNA damage or inflammatory signals, which function as tumor-suppressive mechanisms aiding cancer treatment [48, 49] . Recruited immune cells localize around tumor areas with elevated immune checkpoint expression, consistent with prior reports that the reticular structure of NETs acts as a physical barrier blocking immune-tumor contact and upregulates immune checkpoint expression [7, 50] . NETs consist of a DNA scaffold and functionally distinct proteins attached to it. Under pathological conditions, NETs show abnormal production (excessive/insufficient) and altered component composition [51-53] . The selective activation of NETs functional clusters implies that distinct NETs components bind to specific cell surface receptors on CCA cells. We therefore focused on two key receptor families: uPAR and TLRs. Core gene analysis identified uPAR as the key receptor mediating the selective activation of the pro-tumor C4 cluster by NETs. uPAR, a membrane glycoprotein implicated in tumor invasion and metastasis, is overexpressed in various cancers and linked to poor prognosis—though its role in NETs-regulated CCA progression remains unclear. Notably, it is well-documented that uPAR promotes tumor growth via downstream integrin-related signaling and the MAPK cascade, a mechanism that underpins its pro-tumor effects, thus making uPAR a promising cancer target [54-56] . Moreover, uPAR is reported to be closely correlated with KRASmut [57, 58] . Consistently, our study reveals that uPAR is indispensable for NETs-induced C4 activation and subsequent KRASmut-amplified MAPK signaling in CCA, linking uPAR’s intrinsic pro-malignancy pathway to NETs-driven CCA progression. Additionally, targeting uPAR reduces the selective pro-tumor effects of NETs without impairing—even partially enhancing—the immunostimulatory effects of other NETs clusters. We also demonstrated that CCA cells recognize NETs via uPAR by binding to its ligand uPA enriched in CCA-derived NETs. Similar to high uPA expression in pancreatic cancer TANs [27] , uPA and the NETs-forming enzyme PADI4 are highly expressed in CCA-derived TANs and released extracellularly by binding to the NETs chromatin scaffold. A key finding is that uPA alone weakly activates the KRASmut-MAPK-mediated C4 cluster via uPAR; and the NETs chromatin scaffold is essential for robust activation of the uPA-uPAR signaling pathway. Among NETs components, the chromatin scaffold serves as both the physical basis for NETs structure and a platform for NETs-bound proteins, facilitating their retention on the cell surface or mediating the hydrolysis of functional fragments [59, 60] . Recognition of the NETs chromatin scaffold involves TLRs, another key receptor family [61] . In CCA, our study confirms that uPAR and TLRs act synergistically to enable CCA cells to fully respond to uPA-bound NETs and selectively activate the downstream C4 (KRAS-MAPK) cluster, the interaction between TLRs and uPAR is also reported in macrophage inflammatory responses and endotoxin-driven colorectal cancer cell invasion [62, 63] . TLRs broadly mediate NETs pro-inflammation (enriched in the C2 cluster) but are universally activated in CCA patients, suggesting they act as common receptors for pan-inflammatory signals, including NETs. In the context of NETs stimulation in CCA, TLRs may also serve as shared receptors for multiple functional clusters. Critical supporting evidence comes from antagonizing TLRs-mediated NETs scaffold recognition: this treatment reduced both KRASmut-MAPK signaling in the pro-tumor C4 cluster and upregulation of immunostimulatory molecules in anti-tumor clusters. An important finding is that targeting uPAR enhances the selective activation of immunostimulatory NETs functional clusters, mediated by STING pathway. Cancerous STING senses chromatin fragments and initiates an intrinsic interferon (IFN) response via the JAK-STAT pathway, thereby upregulating molecules such as CCL5 to boost tumor immunogenicity [64-66] . Moreover, the anti-tumor immunity activated by STING signaling is reported to be enhanced by targeting MAPK pathway, including in CCA [29, 30] . STING is also closely associated with NETs: in stromal cells, NETs signals are internalized via membrane receptors (e.g., TLRs) to trigger STING-mediated inflammatory and immune responses [67, 68] . In our study, NETs-induced pro-tumor MAPK activation in CCA is uPAR-dependent; thus, targeting uPAR mimics MAPK pathway inhibition—activating STING signaling—consistent with prior reports. Collectively, TLRs, uPAR, and STING form a NETs cluster signaling axis where TLRs recognize the NETs structure, with subsequent activation of the uPAR-MAPK pro-tumor cluster via NETs-bound uPA or initiation of STING-mediated intrinsic immune responses via NETs-DNA fragments sensing. Current NETs-targeting strategies include two approaches [17] : disrupting existing NETs using DNase and reducing NETs formation with agents such as disulfiram or PADI4 inhibitors. Though these approaches show similar NETs-targeting efficacy in various tumor and disease models, their mechanisms and limitations differ, and such differences have barely been comparatively investigated. DNase fully abrogates NETs effects in vitro but only hydrolyzes the reticular structure of NETs into smaller fragments in vivo; these residual fragments and their bound proteins retain biological stimulatory effects on tumors [69] . In contrast, strategies reducing NETs formation—targeting either the intracellular NETs formation cascade in TANs or TME signals promoting NETs—inherently sacrifice the potential positive immunomodulatory effects of NETs. Targeting NETs-CCA interactions represents a novel direction; for example, targeting CCDC25 in breast cancer reduces NETs-induced metastasis [25, 70] . Our study proposes a novel strategy for selective NETs cluster targeting by combining DNase with uPAR abrogation. Three mechanisms underpin this rationale: (1) DNase disrupts the "shields" structure of NETs, eliminating the physical barrier blocking immune cell attack on CCA; (2) targeting the uPA-uPAR pathway directly antagonizes the pro-tumor effects of residual NETs fragments (specifically KRASmut-amplified, MAPK-mediated C4 cluster effects); (3) importantly, the immunostimulatory effects of NETs fragments are preserved—even compensatorily enhanced—allowing infiltrating immune cells to penetrate the tumor parenchyma through the disrupted NETs barrier by DNase. Notably, MAPK-MEK-ERK inhibitor has also been reported to induce compensatory enhancement of immunogenicity [29, 30] , possibly via activating compensatory inflammatory pathways—this finding shares mechanistic parallels with our observations. In gastric cancer, targeting uPAR also promotes immune cell infiltration—providing additional support for this strategy [71] . Notably, DNase + uPAR abrogation synergizes with anti-PD-L1 inhibitors (which have limited monotherapy efficacy in CCA): DNase weakens myeloid physical immunosuppression by disrupting the NETs physical barrier, and the subsequent NETs fragments induce PD-L1 upregulation that provides a natural target for PD-1/PD-L1 inhibitors, further enhancing the anti-tumor cytotoxicity of immune cells infiltrating CCA under the NETs cluster-targeting strategy. Compared with DNase monotherapy (which retains bioactive NETs fragments) or PADI4 inhibitors (which abrogate both pro-tumor and immune-stimulatory NETs functions), our DNase+uPAR abrogation combination achieves “precision modulation” of CCA-NETs interaction. Certain limitations of this study require further investigation. First, the NETs functional cluster concept was proposed based on NETs-CCA interactions, so its applicability needs verification and extension at the pan-cancer level. Second, the C1-C5 clusters are defined based on transcriptomic data; future studies need to validate cluster-specific protein biomarkers for clinical translation. Third, larger prospective cohorts are required to confirm the correlation between NETs-score/immunostimulatory cluster and immunotherapy efficacy. In summary, our study demonstrates that NETs regulate CCA progression via dual pro-/anti-tumor C1-C5 functional clusters, with KRASmut-amplified MAPK signaling driving direct pro-tumor effects while the immunostimulatory properties of other clusters are preserved. The DNase+uPAR abrogation combination targets this balance by disrupting the NETs barrier and inhibiting the pro-tumor cluster while preserving immune function—synergizing with anti-PD-L1 therapy. This work provides a novel functional cluster-based framework for understanding NETs biology in CCA and a precision therapeutic strategy for KRASmut CCA patients with limited treatment options. Abbreviations CCA: Cholangiocarcinoma DNase: Deoxyribonuclease KRASmut: KRAS-mutant KRASwt: KRAS-wildtype MAPK: Mitogen-activated protein kinase NETs: Neutrophil extracellular traps PD-1: Programmed death-1 PD-L1: Programmed death-ligand 1 PADI4: Peptidylarginine deiminase 4 STING: Stimulator of interferon genes CCL5: Chemokine (C-C motif) ligand 5 CXCL9/10: Chemokine (C-X-C motif) ligand 9/10 GZMB: Granzyme B TANs: Tumor-associated neutrophils TLRs: Toll-like receptors TME: Tumor microenvironment uPAR: Urokinase-type plasminogen activator receptor uPA: Urokinase-type plasminogen activator CyTOF: Mass cytometry WGCNA: Weighted Gene Co-expression Network Analysis Declarations Acknowledgement Consent for publication: Not applicable Competing interests: The authors claim no conflict of interest Availability of data and materials: The data and materials supporting the findings of this study are available in the published article and its supplementary information, or on reasonable request from the corresponding authors. Author Contributions: Luyu Yang, Xiaotian Shen, Lu Lu, Baobin Ying, Lun-Xiu Qin designed the study, Lu-Yu Yang, Xiaotian Shen, Xin Zheng, Sunzhe Xie, Chen Zhang performed the experiments, Haoting Sun, Xiangyu Wang, Hao Wang, Shule Li, Yiran Chen, Zhenchao Chen, Jixuan Chen, Jinyu Wang, Wenwei Zhu, Jinhong Chen joined in the discussion, Lu-Yu Yang, Xiaotian Shen wrote the manuscript. Acknowledgements: We thank all the healthy donors and CCA patients for the source of tissue and blood samples. We sincerely thank Dr. Hu from Ruijin Hospital for generously providing the padi4 knockout mice that supported key experiments in this study. Funding list: This work was jointly supported by the National Natural Science Foundation of China (Key Program, 82430089; General Program, 82373017), the Shanghai Science and Technology Committee (General Program of Natural Science, 24ZR1408800), and the Project of Flexible introduction of High-level Medical and Health Professionals in Fujian Province (No. YJRCTD-2021QLX). Ethics statement: The studies involving humans were approved by the Ethics Committee of Huashan Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Supplementary Materials Supplementary materials include detailed methods, additional figures (Figures S1-S6), and additional table. References [1] Greten T F, Schwabe R, Bardeesy N, et al. Immunology and immunotherapy of cholangiocarcinoma[J]. Nature reviews. Gastroenterology & hepatology. 2023, 20(6): 349-365. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9281659","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":616524511,"identity":"71285dae-02c1-4322-bcd2-2752d875e157","order_by":0,"name":"baobin yin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACZgY2IHmAB8g6cOBDBWla2BIPzjhDnD1gLUDMY3yYt4UI9QbHeY89+FFxR8acf82HA7wNDPL8YgcIaDnMl27Yc+YZj+WMtxsOSO5gMJw5O4GQFh4zCd62wzwGN85uOGB4hiHB4DYRWiT/grWceXAgsY1ILdJgW873MBw4SIwWycM85sYyQL8Y3GAzONhwRoKwX/jOnzF7+Kbijr3B+cOPP/+psJHnlyagReEAjCUBVimBXzkIyDfAWPwHcKsaBaNgFIyCkQ0A1PNNWoTwQMAAAAAASUVORK5CYII=","orcid":"","institution":"Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China","correspondingAuthor":true,"prefix":"","firstName":"baobin","middleName":"","lastName":"yin","suffix":""},{"id":616524512,"identity":"496a2ece-cd3b-450c-9d5e-79113d32e985","order_by":1,"name":"xiaotian shen","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"xiaotian","middleName":"","lastName":"shen","suffix":""},{"id":616524513,"identity":"2aaa12ba-b673-43a0-a17e-df90ac3fc666","order_by":2,"name":"xin zheng","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"xin","middleName":"","lastName":"zheng","suffix":""},{"id":616524514,"identity":"3f2afab9-8e75-4fff-b548-b6abf38f541d","order_by":3,"name":"Sun-Zhe Xie","email":"","orcid":"","institution":"Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Sun-Zhe","middleName":"","lastName":"Xie","suffix":""},{"id":616524515,"identity":"bd651d43-1bdc-4b77-9beb-36904c74f4fa","order_by":4,"name":"Chen Zhang","email":"","orcid":"","institution":"Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Zhang","suffix":""},{"id":616524516,"identity":"6deeaa4b-08a0-491b-877d-92b9232e9cc6","order_by":5,"name":"tianlun wang","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"tianlun","middleName":"","lastName":"wang","suffix":""},{"id":616524517,"identity":"63c492c3-b4e9-418d-a82c-07e10b19e0c2","order_by":6,"name":"haoting sun","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"haoting","middleName":"","lastName":"sun","suffix":""},{"id":616524518,"identity":"319f6bb5-c162-48af-b300-feac89f008e8","order_by":7,"name":"Da Xu","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Da","middleName":"","lastName":"Xu","suffix":""},{"id":616524519,"identity":"c728db7e-94d5-4ca4-954e-0d5dd1570109","order_by":8,"name":"Xiang-Yu Wang","email":"","orcid":"https://orcid.org/0000-0001-8605-0727","institution":"Huashan Hospital. Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xiang-Yu","middleName":"","lastName":"Wang","suffix":""},{"id":616524520,"identity":"ccd530a5-a500-4b8a-a042-0348dedc9fe2","order_by":9,"name":"Hao Wang","email":"","orcid":"","institution":"Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Wang","suffix":""},{"id":616524521,"identity":"a518d708-421b-4df2-aa60-8bb3300546e3","order_by":10,"name":"shule li","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"shule","middleName":"","lastName":"li","suffix":""},{"id":616524522,"identity":"ba3bf482-e766-4144-920a-d2c6fa36f369","order_by":11,"name":"yiran chen","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"yiran","middleName":"","lastName":"chen","suffix":""},{"id":616524523,"identity":"4699c4f6-cca9-4427-9ed8-c79715761778","order_by":12,"name":"zhenchao chen","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"zhenchao","middleName":"","lastName":"chen","suffix":""},{"id":616524524,"identity":"da7ef07e-3805-459b-8d74-47ab12901eb4","order_by":13,"name":"jixuan chen","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"jixuan","middleName":"","lastName":"chen","suffix":""},{"id":616524525,"identity":"719b8cb8-e897-44c3-84de-c267df00251a","order_by":14,"name":"jinyu wang","email":"","orcid":"","institution":"Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"jinyu","middleName":"","lastName":"wang","suffix":""},{"id":616524526,"identity":"d7c1564d-18ad-45f8-8252-07bbaa0a9fe5","order_by":15,"name":"wenwei zhu","email":"","orcid":"","institution":"Department of Infectious Diseases, Huashan Hospital, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"wenwei","middleName":"","lastName":"zhu","suffix":""},{"id":616524527,"identity":"361da723-6e38-4a05-b088-fa6059d0a3c8","order_by":16,"name":"Lu Lu","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Lu","suffix":""},{"id":616524528,"identity":"42aeef7e-c170-48fb-b81f-44d7f7c41f26","order_by":17,"name":"luyu yang","email":"","orcid":"","institution":"Hepatobiliary Surgery, Department of General Surgery, Huashan Hospital \u0026 Cancer Metastasis Institute, Fudan University, Shanghai, China","correspondingAuthor":false,"prefix":"","firstName":"luyu","middleName":"","lastName":"yang","suffix":""},{"id":616524529,"identity":"bbe88bc8-4121-4976-89d4-4857fa061974","order_by":18,"name":"Lun-Xiu Qin","email":"","orcid":"","institution":"Huashan Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Lun-Xiu","middleName":"","lastName":"Qin","suffix":""}],"badges":[],"createdAt":"2026-03-31 15:05:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9281659/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9281659/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106533403,"identity":"a97c1438-6c7c-4568-9103-0bf28f169170","added_by":"auto","created_at":"2026-04-09 14:57:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11391911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePADI4+ TANs drived NETs exert dual effects on CCA progression and TME remodeling in CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eUMAP plot of single-cell RNA sequencing (scRNA-seq) of CD66b⁺ TANs from human CCA tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e NETs-score of TAN subpopulations; the PADI4⁺ TAN shows the highest score.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Expression of pro-NET signaling molecules (C5a, CXCR4, OLR1) in CCA from The Cancer Genome Atlas (TCGA) cohort, comparing tumor vs. adjacent non-tumor tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD-E\u003c/strong\u003e Representative IF images\u003cstrong\u003e (D)\u003c/strong\u003e of NETs in CCA nests vs. adjacent non-tumor tissues and quantification\u003cstrong\u003e (E)\u003c/strong\u003ein Huashan cohort. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF \u003c/strong\u003eQuantification of NETs staining in mouse CCA models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e In vitro validation of human CCA CM-induced NET formation. Left: represntative IF images; Right: quantification of cell-free DNA (cfDNA) in supernatants. Scale bar: 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH-I\u003c/strong\u003e CCA tumor progression In SB1 i.hep xenografts\u003cstrong\u003e (H) \u003c/strong\u003eand YA spontaneous CCA model\u003cstrong\u003e (I) \u003c/strong\u003ein padi4-wt vs. ko mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ\u003c/strong\u003e Effect of NETs on in vitro proliferation of human CCA cell lines CCLP1 and RBE detected by colony formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e RNA sequencing (RNA-seq) of YA tumors: heat map of selected inmmuno-activative genes in padi4 wt vs. ko tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL\u003c/strong\u003e CyTOF analysis of CCA TME of YA model in padi4-wt vs. ko mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM\u003c/strong\u003e IF staining of GZMB⁺ immune cells in YA and SB1 i.hep tumors from padi4-wt vs. ko mice. Scale bar: 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e Spatial omics of immune cells distribution of YA tumors in padi4-wt vs. ko mice: left, spatial maps of stromal and malignet cells; right, quantification of neutrophils and T cell-enriched area.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eO\u003c/strong\u003e Chemotaxis assay: number of human PBMCs migrating to CM from NET-treated RBE and CCLP1 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e Change of SB1 s.c. tumor progression in padi4-wt vs. ko mice with/without dexamethasone.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"FIG1.png","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/7959e5209c72d5ce7d868e4a.png"},{"id":106533409,"identity":"53925ffa-bb49-4755-847c-61106734433d","added_by":"auto","created_at":"2026-04-09 14:57:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5552715,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiverse functional clusters of NETs exert distinct impacts on CCA progression and prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Schematic of the bioinformatics analysis workflow. RNA-seq was performed on RBE/CCLP1 cells stimulated with NETs to identify NETs-regulated genes (NRGs). WGCNA was applied in pooled cohorts to cluster NRGs into 5 independent functional clusters (C1–C5) based on enriched functional modules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Functional annotation of the 5 NETs functional clusters (C1–C5). Core enriched pathways and featured genes of each clusters were shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Correlation analysis between NETs-score and signature scores of each functional cluster in Fu-iCCA cohort by GSEA analysis. NETs-score showed strong positive correlation with C1, C4, and C5 signatures, and weak correlation with C2 and C3 signatures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Kaplan–Meier survival analysis of each NETs functional cluster in Fu-iCCA cohort. Patients were stratified into high-score and low-score groups based on the signature score of each functional cluster (C1–C5).\u003c/p\u003e\n\u003cp\u003eE Dimensionality reduction analysis (UMAP) of CCA patients in Fu-iCCA cohort based on functional cluster enrichment scores. Patients aggregated into distinct subgroups corresponding to C1–C5, effectively distinguishing NET response patterns.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF \u003c/strong\u003eHeatmap of NETs-high/low patient distribution across C1–C5 subgroups (Fu-iCCA cohort). Subgroups were determined via dimensionality reduction analysis as\u003cstrong\u003e in Figure 2E.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e Comparison of clinical characteristics among C1, C4, and C5 subgroups in Fu-iCCA cohort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e Prognostic stratification by NETs-score within C1, C4, and C5 subgroups in Fu-iCCA cohort.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"FIG2.png","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/365297ac2408b9670afd2b61.png"},{"id":106533318,"identity":"4b9beb1d-74a5-42a3-bf24-ce5ac1d0d5a0","added_by":"auto","created_at":"2026-04-09 14:57:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12421753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKRAS mutation amplifies MAPK signaling to mediate NETs-selective activation of pro-tumor function cluster C4 in CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Comparison of NETs levels between KRASmut and non-KRASmut CCA patients in Huashan cohort. NETs were detected by IF staining of H3cit/MPO as in\u003cstrong\u003e Figure 1D-E\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB \u003c/strong\u003eSchematic of the mechanism: NETs act as a stimulus with pro-tumor effects amplified by KRASmut to activate MAPK signaling, thereby selectively promoting pro-tumor function cluster C4 enrichment in CCA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC \u003c/strong\u003eFunctional cluster enrichment scores (C1–C5) by GSEA analysis of RNA-seq data in CCLP1-KRASmut/vector and RBE-KRASwt/vector cells with or without NETs stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Heatmap showing fold change of MAPK signaling-related genes in RNA-seq data of CCLP1 and RBE cells, with grouping consistent with Figure 3B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e Western blot analysis of MAPK signal pERK/ERK level in CCLP1-KRASmut/vector and RBE-KRASwt/vector cells with or without NETs stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e In vitro colony formation and quantification of CCLP1-KRASmut/vector and RBE-KRASwt/vector cells treated with NETs ± MEK inhibitor (trametinib).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e In vivo subcutaneous xenograft model: Tumor size of CCLP1-KRASmut/vector cells in null mice treated with saline or GSK484 (NETs formation inhibitor).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e Tumor of murine CCA models: Yap/AKT (YA) and Yap/AKT + KRAS (YAK) models established in padi4-wildtype (padi4-wt) or padi4-knockout (Padi4-ko) mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e Enrichment scores of C1–C5 functional clusters of RNA-seq data in tumor tissues from YA and YAK models (padi4-wt/ko) by GSEA analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ\u003c/strong\u003e Comparison of tumor burden, KI-67 (proliferation marker) expression, and pERK expression between YA and YAK models (padi4-wt/ko) in \u003cstrong\u003eFigure 3H\u003c/strong\u003e. Scare Bar: 100μm\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns = not significant.\u003c/p\u003e","description":"","filename":"FIG3.png","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/3ec9e09a33b504ddb7805128.png"},{"id":106533407,"identity":"b83c0baf-ea34-4f0c-84f4-a6edaf0569e9","added_by":"auto","created_at":"2026-04-09 14:57:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8762402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003euPA-enriched neutrophil extracellular traps (NETs) activate membrane uPAR to initiate MAPK signaling in KRAS-mutant (KRASmut) cholangiocarcinoma (CCA), with synergistic sensing by TLRs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Immunofluorescence staining showing colocalization of uPAR (red) and NETs (SytoxGreen, green) on the surface of RBE and CCLP1-KRASmut cells stimulated with NETs. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB-C\u003c/strong\u003e Analysis of MAPK pathway activation in RBE and CCLP1-KRASmut cells transfected with siuPAR/siNC followed by NETs stimulation by qPCR detection of key MAPK pathway genes fold change \u003cstrong\u003e(B)\u003c/strong\u003eor WB analysis of pERK/ERK protein levels \u003cstrong\u003e(C)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e In vitro colony formation fold change of RBE and CCLP1-KRASmut cells with siuPAR/siNC transfection and NETs stimulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e T-SNE plot showing PLAU (encoding uPA) high expression in PADI4⁺ TAN in scRNA-seq data, related to\u003cstrong\u003e Figure 1A\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e qPCR analysis of uPA mRNA levels in human peripheral neutrophils from health control treated with CM from CCLP1-KRASmut and RBE cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e Immunofluorescence staining of uPA in NETs structure from PMA-stimulated human peripheral neutrophils from CCA patients. Scale bar: 10 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e Immunofluorescence staining showing colocalization of uPA and NETs marker H3cit close to cancerous region in human KRASmut CCA tissues. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e Correlation analysis between NETs-score and expression of uPA/uPAR in FU-iCCA cohort.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ\u003c/strong\u003e Experimental design and WB analysis of MAPK signal pERK/ERK level in RBE and CCLP1-KRASmut cells treated with NETs derived from dHL60 cells transfected with siuPAR/siNC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK-L\u003c/strong\u003e Analysis of MAPK pathway activation in RBE and CCLP1-KRASmut cells treated with uPA alone, uPA + NETs-DNA backbone, or uPA + NETs-DNA backbone + DNase, by WB analysis of pERK/ERK protein levels \u003cstrong\u003e(K)\u003c/strong\u003eand qPCR detection of key MAPK pathway genes fold change \u003cstrong\u003e(L)\u003c/strong\u003e .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM\u003c/strong\u003e Immunofluorescence staining showing colocalization of TLR4/9 and uPAR in RBE and CCLP1-KRASmut cells stimulated with NETs. Scale bar: 20 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e qPCR analysis of MAPK pathway genes fold change in CCLP1-KRASmut cells treated with NETs, NETs + CQ, NETs (DNase-digested), or NETs + CQ + uPA.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns = not significant.\u003c/p\u003e","description":"","filename":"FIG4.png","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/6790936f895a5bb7fbcb9010.png"},{"id":106533391,"identity":"d37e54d4-e16b-4cb5-8b5c-e9f722b16666","added_by":"auto","created_at":"2026-04-09 14:57:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9318213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking uPA-uPAR axis attenuates NETs-selective activation of pro-tumor function clusters and preserves immune-stimulatory effects in CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e qPCR analysis of MAPK pathway-related genes fold change in RBE and CCLP1-KRASmut cells treated with NETs ± UPARANT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e In vitro colony formation change of RBE and CCLP1-KRASmut cells treated with NETs ± UPARANT..\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e qPCR analysis of immune activation-related genes fold change in RBE and CCLP1-KRASmut cells treated with NETs ± UPARANT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Chemotaxis assay change of PBMCs migrating to CM from NETs-treated RBE and CCLP1-KRASmut cells with/without UPARANT pretreatment, related to co-culture system in Figure 1O.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e qPCR analysis of immune-stimulatory genes fold change in RBE and CCLP1-KRASmut cells treated with NETs ± UPARANT ± STING inhibitor H-151 or JAK-STAT inhibitor ruxolitinib.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e Chemotaxis assay change of PBMCs migrating to CM from RBE and CCLP1-KRASmut cells treated with NETs ± UPARANT ± H-151 or ruxolitinib.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e In vivo validation in SB1 i.hep models established in padi4-wt and ko mice. representative image of gross tumor and Immunofluorescence staining (left) of Ki67, pERK, CD3+ T cells, and CD11c+ DCs in tumors treated with saline or UPARANT, and quantification (right). Scare bar: 50 μm\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns = not significant.\u003c/p\u003e","description":"","filename":"FIG5.png","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/e89cbd2dc890b344dda974a5.png"},{"id":106533408,"identity":"6a41b725-ba88-4f7c-be5c-e53d52211923","added_by":"auto","created_at":"2026-04-09 14:57:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10832303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelective intervention of NETs pro-tumor functional cluster via anti-uPAR and DNase preserves immune stimulation and synergizes with immunotherapy in CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B\u003c/strong\u003e Images (A) and quantification (B) of tumor burden, and immunofluorescence staining of Ki67,pERK and T cells in YAK model mice with indicated treatment targeting NETs. Scale bar: 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e RNAseq analysis of immune-promoting genes in YAK model tumors with indicated treatment targeting NETs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e Immunofluorescence staining showing T cell infiltration through tumor edge regions of YAK model treated with DNase + UPARANT. Scale bar: 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e Tumor burden and overall survival of YAK model mice treated with anti-PD-L1 alone or combined with DNase + UPARANT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e Tumor burden and overall survival of SB1 i.hep model mice treated with anti-PD-L1 alone or combined with DNase + UPARANT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u003c/strong\u003e Comparison of NETs-score between Ni cohort patients with predicted good response (CXCL9/CTLA4-high) and poor response (CXCL9/CTLA4-low) to immunotherapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e Distribution of NETs functional clusters C1-C5 in Cohort 3 patients stratified by predicted immunotherapy response.\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± SD; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ns = not significant.\u003c/p\u003e","description":"","filename":"FIG6.png","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/87a12e20864ce1b2e9f1b8ec.png"},{"id":106533381,"identity":"057f77cd-2461-41fa-883d-5f66c0d093aa","added_by":"auto","created_at":"2026-04-09 14:57:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5006931,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic of Selective Targeting of uPAR-driven Neutrophil Extracellular Traps Functional Clusters Attenuates Tumor Progression and Preserves Immunostimulatory Potential to Boost Cholangiocarcinoma Immunotherapy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn CCA PADI4⁺ TANs secrete uPA-enriched NETs, and then binds tumor-cell uPAR to activate pro-tumor MAPK signaling, which is further amplified by KRASmut. DNase I degrades the NETs DNA barrier and anti-uPAR blocks uPA-uPAR/MAPK signaling, preserving anti-tumor NETs functions to restore STING-mediated immunity, induce CCL5/CXCL9, and generate a hot TME (enriched DC/T cells), with anti-PD-L1 combination controlling CCA and improving prognosis.\u003c/p\u003e","description":"","filename":"FIG7.png","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/7f25327c236e999decb94f60.png"},{"id":108491075,"identity":"353a5989-21fa-4c95-8a53-94ef893a03ee","added_by":"auto","created_at":"2026-05-05 09:52:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":58404164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/7ef80910-3f59-4595-85cc-dc162cc93b76.pdf"},{"id":106533402,"identity":"a7145a57-812b-441c-bfd1-9ac0ffedeffa","added_by":"auto","created_at":"2026-04-09 14:57:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4702118,"visible":true,"origin":"","legend":"supplementary material","description":"","filename":"supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/a55a7f1788145e9360457e22.docx"},{"id":106533317,"identity":"04236f3b-0c95-41ee-a15d-5364c0402784","added_by":"auto","created_at":"2026-04-09 14:57:04","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2962748,"visible":true,"origin":"","legend":"unprocessed IF images","description":"","filename":"unprocessedIFimages.tif","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/2ac8ecf060ce10503c844806.tif"},{"id":106533400,"identity":"d4d5a2ab-63e0-47e3-91ba-4e80a918eeac","added_by":"auto","created_at":"2026-04-09 14:57:19","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":39633760,"visible":true,"origin":"","legend":"Unprocessed original images of western blots","description":"","filename":"Unprocessedoriginalimagesofwesternblots.tif","url":"https://assets-eu.researchsquare.com/files/rs-9281659/v1/60867083bc08049c59294317.tif"}],"financialInterests":"(Not answered)","formattedTitle":"Selective Targeting uPAR-driven Neutrophil Extracellular Traps Functional Clusters to Attenuate Tumor Progression and Enhance the Response to Immunotherapy in Intrahepatic Cholangiocarcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCholangiocarcinoma (CCA) is a highly aggressive malignant tumor with a dismal prognosis, and its incidence has increased significantly in recent years. Most patients are diagnosed at an advanced stage, where surgical treatment offters limited efficacy, and postoperative tumor metastasis and rapid progression severely impair patient survival. Currently, CCA treatment has shifted towad a surgery-centered multimodal comprehensive therapy, including immunotherapy with programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors , which has demonstrated substantial potential in various solid tumors\u003csup\u003e[1, 2]\u003c/sup\u003e. However, improving therapeutic efficacy remains a core focus of current research. A prominent hallmark of the CCA tumor microenvironment (TME) is abundant neutrophil infiltration\u003csup\u003e[3]\u003c/sup\u003e, making targeting neutrophils and their derivatives a promising therapeutic strategy for CCA.\u003c/p\u003e\n\u003cp\u003eNeutrophils can release neutrophil extracellular traps (NETs), web-like structures composed of extracellular chromatin as a scaffold decorated with various functional proteins\u003csup\u003e[4]\u003c/sup\u003e. In tumor progression, NETs exert prominent pro-tumor effects: they trap circulating tumor cells to promote metastatic colonization\u003csup\u003e[5]\u003c/sup\u003e, remodel the extracellular matrix to facilitate invasion\u003csup\u003e[6]\u003c/sup\u003e, and form physical barriers that block contact between immune cells and tumor cells\u003csup\u003e[7]\u003c/sup\u003e, thereby attenuating anti-tumor immune responses. Accumulating studies have confirmed that NETs act as a universal driver of tumor progression under diverse pathological conditions, including tumor-derived factors\u003csup\u003e[8, 9]\u003c/sup\u003e, oncogenic mutations \u003csup\u003e[10]\u003c/sup\u003e, chemotherapy-induced stress\u003csup\u003e[11]\u003c/sup\u003e , and chronic inflammation\u003csup\u003e[12, 13]\u003c/sup\u003e. On the other hand, beyond their well-documented pro-tumor properties, NETs also possess inherent and extensive immunostimulatory potential, which is reflected in their ability to mediate robust pro-inflammatory responses, regulate T cell activation, promote dendritic cell (DC) maturation, and induce the secretion of immune-stimulatory chemokines\u003csup\u003e[14-16]\u003c/sup\u003e. These functions hold the potential to remodel the CCA TME into an immunologically \u0026quot;hot\u0026quot; state that would be favorable for immunotherapy. This inherent duality of NETs, serving as both tumor promoters and immune activators, remains highly controversial, and its precise manifestation and regulatory mechanisms in CCA or other solid tumors have not been elucidated, making it a critical focus of our investigation.\u003c/p\u003e\n\u003cp\u003eCurrent NET-targeting interventions mainly fall into two categories\u003csup\u003e[17]\u003c/sup\u003e: disrupting pre-formed NETs structures and inhibiting NETs formation. Both strategies have significant limitations: the former fails to eliminate residual NET fragments that retain biological activity and pro-tumor effects; the latter globally suppresses NETs production, sacrificing their potential immunostimulatory effects that support immunotherapy. As an underdeveloped alternative, precise targeting of the interaction between NETs and tumor cells, rather than non-specifically inhibiting all NET functions, represents an alternative NETs-targeted strategy in malignancy.\u003c/p\u003e\n\u003cp\u003eIn this study, through multi-omics analysis of preclinical models and clinical cohorts, we systematically elucidated the distinct dual effects of NETs in CCA. Specifically, NETs not only directly promote tumor growth and progression, but also sustain an immunostimulatory hot TME with potential anti-tumor activity. We further dissected these effects into independent\u0026nbsp;NETs functional clusters, each with distinct biological functions and prognostic significance. Mechanistically, we identified urokinase-type plasminogen activator receptor (uPAR) as a key NETs receptor that, upon binding to NETs-bound uPA, selectively activates pro-tumor\u0026nbsp;NETs functional clusters\u0026nbsp;associated with MAPK signaling amplification\u0026mdash;particularly in CCA with KRAS mutations, which accounts for ~25% of CCA cases and is closely linked to enhanced neutrophil infiltration, aberrant NETs formation, poor response to conventional therapies and unfavorable prognosis\u003csup\u003e[3, 18]\u003c/sup\u003e. Notably, uPAR selectively mediates the pro-tumor MAPK signaling in response to NETs, whereas its blockade modulates the immunostimulatory NETs functional clusters. Accordingly, we developed a combinatorial strategy targeting NETs functional clusters, disrupting the physical barrier of NETs using Deoxyribonuclease (DNase) in combination with blocking the uPA-uPAR signaling in residual NETs fragments. This strategy selectively inhibits CCA growth while preserving the immunostimulatory TME, and enhances the sensitivity of CCA- especially the KRAS-mutant subtype to immunotherapy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eMethods in detail are included in supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIncreased NETs Formation Correlates with Tumor Progression and Poor Progression of CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo dissect the clinical relevance of neutrophil extracellular traps (NETs) in cholangiocarcinoma (CCA), we first characterized tumor-associated neutrophil (TAN) heterogeneity through single-cell RNA sequencing (scRNA-seq) of CD66b⁺ cells isolated from human CCA specimens. This analysis identified seven distinct transcriptional subsets, with a particular focus on the terminally differentiated PADI4⁺ subset \u003cstrong\u003e(Figure 1A).\u003c/strong\u003e Given that peptidylarginine deiminase 4 (PADI4) catalyzes histone citrullination\u0026mdash;an obligate step for triggering NETs formation\u003csup\u003e[19]\u003c/sup\u003e, we posited that this subset possesses intrinsic NET-forming propensity. Employing a customized NETs-scoring algorithm e\u003csup\u003e[20]\u003c/sup\u003e, we confirmed that the PADI4⁺ TANs indeed exhibited the strongest NET-forming capacity among all subsets\u003cstrong\u003e\u0026nbsp;(Figure 1B)\u003c/strong\u003e. Extending this analysis to The Cancer Genome Atlas (TCGA) CCA cohort, we observed elevated expression of pro-NETs signaling molecules in PADI4⁺TAN\u0026mdash;including C5a (C5aR1), CXCR4, and oxidized low-density lipoprotein receptor 1 (OLR1)\u0026mdash;which w strongly correlated with the pro-NETs PADI4⁺ TAN signatures. These findings collectively indicate the existence of a pro-NETotic signaling microenvironment in CCA\u003cstrong\u003e\u0026nbsp;(Figure 1C)\u003c/strong\u003e. Systematic evaluation confirmed universal elevation of NETs-scores \u0026nbsp;in TCGA CCA samples \u003cstrong\u003e(Figure S1A)\u003c/strong\u003e, consistent with previously reported TAN enrichment in this malignancy. To consolidate these bioinformatic findings in clinical specimens, we performed multiplex immunofluorescence staining in CCA samples from Huashan cohort, and revealed pronounced enrichment of NETs meshworks\u0026mdash;characterized by the co-expression of H3cit (a hallmark of PADI4-citrullinated histones) and myeloperoxidase (MPO)\u0026mdash;within tumor nests compared to adjacent stroma \u003cstrong\u003e(Figures 1D-E)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo recapitulate these clinical observations experimentally, we established\u0026nbsp;three complementary murine CCA models: subcutaneous (s.c.) and orthotopic hepatic (i.hep.) implantation models of the murine CCA cell line SB1 , as well as the YA model (a hydrodynamic injection-induced spontaneous CCA model driven by YAP/AKT plasmid delivery). All models consistently demonstrated prominent NETs accumulation within tumor tissues\u003cstrong\u003e\u0026nbsp;(Figure 1F and S1B)\u003c/strong\u003e. Mechanistically, conditioned medium (CM) derived from human and murine CCA single-cell suspensions potently induced NET formation upon incubation with homologous neutrophils, as evidenced by immunofluorescence microscopy and extracellular DNA quantification\u003cstrong\u003e\u0026nbsp;(Figure 1G and S1C)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo delineate the functional contribution of NETs to CCA progression, we generated padi4 knockout (ko) mice, which are deficient in NETs-forming capacity. In the SB1 s.c., SB1 i.hep., and YA models which were established in the padi4-ko and wild-type (wt) mice, genetic ablation of Padi4 significantly attenuated tumor growth compared to wild-type (wt) controls, whereas NET-competent wt mice exhibited accelerated disease progression \u003cstrong\u003e(Figure 1H-I and 1P)\u003c/strong\u003e. Clinically, transcriptomic analysis of two independent public cohorts confirmed that elevated NETs-scores significantly correlated with poorer overall survival in CCA patients\u003cstrong\u003e\u0026nbsp;(Figures S1D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings establish that NETs formation is enriched in the CCA microenvironment and functionally drives malignant progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNETs Concomitantly Exhibit Pro-Tumor Activity and Potential Immune-Stimulatory Properties in CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe functional output of NETs in cancer is context-dependent and remains contentious. In vitro, purified NETs robustly accelerated proliferation, migration, and anchorage-independent growth of human-derived CCLP1 and RBE cells, as well as murine-derived SB1 cells \u003cstrong\u003e(Figure 1J and S1E)\u003c/strong\u003e , echoing prior observations in CCA and other solid tumors \u003csup\u003e[17, 21]\u003c/sup\u003e. To dissect the net biological impact of NETs in an intact host, we compared transcriptomes of YA tumors harvested from Padi4-wt (NETs-proficient) versus Padi4-ko (NETs-deficient) mice. Unexpectedly, NETs preservation was accompanied by a pronounced immune-inflammatory signature. We documented elevated expression of T-cell-attracting chemokines (Cxcl9, Ccl5), antigen-presentation machinery (H2-Aa, H2-Eb1), cytolytic mediators (Gzmb), and interferon-\u0026gamma; (Ifng), together with compensatory upregulation of the immune checkpoint molecule \u003cem\u003eCD274 (PD-L1)\u003c/em\u003e \u003cstrong\u003e(Figure 1K)\u003c/strong\u003e. qPCR validated these findings in both YA and SB1 i.hep. models with intact NETs formation \u003cstrong\u003e(Figure S1F)\u003c/strong\u003e. High-dimensional mass cytometry (CyTOF) resolved 13 discrete leukocyte clusters and revealed that NETs-rich tumors harbored heightened global immune infiltration, most notable within CD4⁺ T-cell and effector-memory T-cell compartments \u003cstrong\u003e(Figure 1L)\u003c/strong\u003e. Concordant increases in TCR\u0026beta; and CD11b signal intensity were observed \u003cstrong\u003e(Figure S1G)\u003c/strong\u003e , and immunofluorescence confirmed greater abundance of GZMB⁺ cytotoxic lymphocytes juxtaposed to tumor islets \u003cstrong\u003e(Figure 1M)\u003c/strong\u003e. Spatial transcriptomics further showed that T-cell\u0026ndash;dense neighborhoods expanded in the presence of NETs and were topographically intercalated with malignant cell niches \u003cstrong\u003e(Figure 1N)\u003c/strong\u003e, implying facilitated immune\u0026ndash;cancer crosstalk.\u003c/p\u003e\n\u003cp\u003eThese observations were validated in two independent CCA transcriptomic cohorts. CCA patients with a higher NETs-score displayed elevated expression of interferon-\u0026gamma; (IFN-\u0026gamma;), T cell inflammation-related genes, tertiary lymphoid structure (TLS) markers, inflammatory response factors, and complement system components compared with their low NETs-score counterparts \u003cstrong\u003e(Figure S1H)\u003c/strong\u003e. Nevertheless, this immune activation did not confer survival benefit, underscoring the paradox of NETs biology in CCA.\u003c/p\u003e\n\u003cp\u003eWe next asked whether tumor-intrinsic signals contribute to NETs-elicited immunogenicity. RNA-seq of NETs-treated CCLP1 and RBE cells, and of malignant cells FACS-purified from YA tumors, consistently revealed up-regulation of immune-activating ligands, exemplified by CCL5 \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;S1I-J)\u003c/strong\u003e. In trans-well chemotaxis assays, conditioned medium from NETs-challenged CCA cells recruited significantly more human PBMCs or murine splenocytes than control medium, confirming that NETs-educated cancer cells actively amplify leukocyte trafficking \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;1O)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo test whether immune stimulation tempers the net tumor-promoting effect of NETs, we blunted adaptive immunity with dexamethasone in the SB1 s.c. model. Under immunosuppression, the growth differential between Padi4-wt and Padi4-ko tumors widened from 1.47-fold to 2.04-fold, indicating that immune-mediated restraint partially offsets\u0026mdash;but does not override\u0026mdash;NETs-driven oncogenesis\u003cstrong\u003e\u0026nbsp;(Figure 1P)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings \u0026nbsp;uncover an inherent duality of NETs in CCA: while they intrinsically foster an immunologically \u0026ldquo;hot\u0026rdquo; microenvironment, their direct pro-tumor actions on malignant cells dominate, culminating in accelerated disease progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNETs Operate through Functionally Distinct Clusters that Differentially Dictate CCA Prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo resolve the apparent paradox of NETs biology, we built a multi-step bioinformatics pipeline (Figure 2A). RNA-seq of RBE and CCLP1 cells exposed to purified NETs yielded 812 consistently up-regulated genes (NRGs; fold-change \u0026gt; 1.5; Fig. S2A). Weighted gene co-expression network analysis (WGCNA) of these NRGs across 858 CCA samples (four public cohorts) partitioned NETs-driven transcriptional output into five quasi-independent functional clusters (C1\u0026ndash;C5; Fig. S2B).\u003c/p\u003e\n\u003cp\u003eFunctional annotation revealed discrete biological themes: C3 and C5 reflect the immune-stimulatory and regulatory effects of NETs via interleukin and receptor-ligand-related signaling, involving the upregulation of T cell immune-stimulatory factors such as CCL5 and CXCL10\u0026mdash;consistent with the NETs-induced immune stimulation observed in our clinical cohorts and murine models; C2 is associated with NETs-mediated regulation of TGF\u0026beta; signaling and extracellular matrix remodeling; C1 corresponds to our prior finding that cancer cells respond to NETs stimulation via Toll-like recptors (TLR) and downstream NF-\u0026kappa;B signaling; and C4 is linked to MAPK signaling, primarily reflecting malignant tumor phenotypes closely associated with disease progression \u003cstrong\u003e(Figure 2B and S2C)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWe next established signature scores for each Functional Clusters and the whole set using Gene Set Enrichment Analysis (GSEA) and analyzed their correlation with NETs-score. Overall, NETs-score was positively correlated with NRGs expression\u003cstrong\u003e\u0026nbsp;(Figure S2D)\u003c/strong\u003e, confirming that NETs drive the upregulation of these 5 Functional Custers in clinical CCA samples. Specifically, NETs-score showed strong positive correlations with the signatures of C1, C4, and C5, while correlations with C2 and C3 were weaker\u0026mdash;suggesting that these two clusters may be co-regulated by other factors in the CCA TME besides NETs \u003cstrong\u003e(Figure 2C)\u003c/strong\u003e. Prognostic analysis of each Functional Cluster signature score revealed significant differences in patient outcomes: patients with higher scores for C1 and C5 (reflecting inflammatory and immune activation) had favorable prognosis, whereas those with higher scores for C2, C3, and C4 conferred poor prognosis, with C4 showing the most significant association with adverse outcomes \u003cstrong\u003e(Figure 2D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo translate cluster signatures into patient-level taxonomy, we performed principal-component analysis on Fu-iCCA transcriptomes and assigned each case to its dominant response pattern (C1\u0026ndash;C5). The resulting five subgroups formed non-overlapping clouds in PCA space, validating their transcriptional distinctiveness\u003cstrong\u003e\u0026nbsp;(Figure 2E-F)\u003c/strong\u003e. High-NETs tumours were enriched almost exclusively in C1, C4 and C5; they also displayed advanced TNM stage and a higher KRAS-mutation rate \u003cstrong\u003e(Figure 2F)\u003c/strong\u003e. Among these three dominant subgroups, the C4 pattern was the most malignant\u0026mdash;characterized by larger tumour volume, vascular invasion, and distant metastasis\u0026mdash;and carried the shortest survival\u0026nbsp;\u003cstrong\u003e(Figure 2G)\u003c/strong\u003e. Conversely, the C5 pattern was associated with the best outcome. When each subgroup was further stratified by NETs-score, NETs remained adverse in C1 and C4 but became protective in C5\u003cstrong\u003e\u0026nbsp;(Figure 2H)\u003c/strong\u003e. The poor prognostic\u0026nbsp;power of C4 was independently confirmed\u0026nbsp;in\u0026nbsp;Jusakul cohort\u0026nbsp;\u003cstrong\u003e(Figure S2E)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFinally, to circumvent the need for transcriptomics in routine practice, we trained six machine-learning classifiers to identify C4 tumours using only standard clinical variables. A random-forest model achieved the highest AUC in internal and external validation sets \u003cstrong\u003e(Figure S2F-H)\u003c/strong\u003e. Across the Fu-iCCA and Huashan-iCCA cohorts, model-assigned C4 patients exhibited higher levels of tumor-related markers (CA19-9), larger tumor size, and markedly shorter overall survival than other subgroups \u003cstrong\u003e(Figure S2I-J)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTaken together, in consistent with the murine model findings, systematic bioinformatics analysis reveals NETs exert complex opposing effects on CCA via distinct pro-tumor (poor prognosis) and anti-tumor (favorable prognosis) functional clusters, and the net clinical outcome reflects the balance between these competing functional clusters rather than the mere quantity of NETs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNETs Selectively Activates MAPK Signaling by Amplifying KRAS Mutation (mut) Effects to Drive CCA Progression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTherapeutically uncoupling the pro-tumorigenic from the immunostimulatory arm of NETs demands precise targeting of the malignant circuit without disabling global NET functions. Because the C4 cluster\u0026mdash;dominated by MAPK-pathway genes\u0026mdash;is the strongest predictor of poor outcome and is uniquely linked to NETs exposure, we asked whether KRAS mutation acts as a molecular gatekeeper that licences NETs to engage this programme. Across FU-iCCA Cohort transcriptomes, KRAS-mutant (KRASmut) tumours were the only genotype significantly associated with elevated NET-score\u003cstrong\u003e\u0026nbsp;(Figure S3A)\u003c/strong\u003e, a finding corroborated immunohistochemically in the Huashan cohort\u0026nbsp;\u003cstrong\u003e(Figure 3A)\u003c/strong\u003e. Among the five functional clusters, only C4 showed a strong correlation with\u0026nbsp;KRASmut status\u003cstrong\u003e\u0026nbsp;(Figure S3B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eDespite the established correlation between KRASmut and NETs, how KRASmut regulates CCA cells\u0026rsquo; sensing of NETs remains elusive. Although KRASmut is a well-established MAPK amplifier, its oncogenic output usually requires additional extracellular cues\u003csup\u003e[22, 23]\u003c/sup\u003e. We therefore hypothesized that NETs supply the requisite stimulus, electively switching on the C4 programme in KRASmut cells\u0026nbsp;\u003cstrong\u003e(Figure 3B)\u003c/strong\u003e. To test this, we established CCLP1-KRASmut cells (by overexpressing KRAS-G12D mutation in KRASwt CCLP1 cells) and RBE-KRASwt cells (by overexpressing KRASwt in KRASmut RBE cells).\u0026nbsp;RNA-seq after NETs stimulation revealed that C4 enrichment changed most dramatically with KRAS genotype; without NETs, KRAS manipulation alone left C4 quiescent\u003cstrong\u003e\u0026nbsp;(Figure 3C)\u003c/strong\u003e.\u0026nbsp;MAPK gene-set activity was moderately elevated by KRASmut alone but reached maximal intensity only when NETs and KRASmut coincided\u003cstrong\u003e\u0026nbsp;(Figure 3D)\u003c/strong\u003e.\u0026nbsp;Western blotting confirmed a striking synergistic increase in p-ERK in CCLP1-KRAS G12D upon NETs exposure, an effect largely absent in RBE-KRASwt cells\u0026nbsp;\u003cstrong\u003e(Figure 3E)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eIn vitro functional assays mirrored this signalling output: NETs enhanced proliferation, invasion, and sphere formation only in the KRAS mutation background, and these gains were fully reversed by the MEK inhibitor trametinib\u003cstrong\u003e\u0026nbsp;(Figure 3F)\u003c/strong\u003e. In vivo, NETs-treated CCLP1-KRASmut cells\u0026nbsp;formed rapidly growing subcutaneous tumours; neutralising NETs with the PAD-inhibitor GSK484 equalised growth rates between genotypes\u0026nbsp;\u003cstrong\u003e(Figure 3G)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWe extended these observations to autochthonous models. YA (YAP/AKT) and YAK (YAP/AKT + KRASG12D) tumours were established in Padi4-wt or Padi4-ko livers \u003cstrong\u003e(Figure 3H)\u003c/strong\u003e. RNA-seq analyses demonstrated that all functional clusters were generally upregulated in NETs-rich wt CCA mice, with the showing a markedly prominent upregulation of C4 cluster in the YAK model \u003cstrong\u003e(Figure 3I)\u003c/strong\u003e. In NETs-proficient (wt) animals, YAK tumours exhibited exaggerated C4 activation, higher tumour burden, and elevated Ki-67 and p-ERK relative compared with the YA controls. In contrast, NETs ablation (Padi4-ko) uniformly suppressed tumour growth and abolished both Ki-67 and p-ERK signals, erasing the genotype-specific advantage of KRAS mutation\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003e\u003cstrong\u003eFigure\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;3I-J)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCollectively, these data establish a two-step model: KRAS mutation licenses CCA cells to interpret NETs as a MAPK-amplifying signal, while NETs provide the extracellular cue that selectively ignites the C4 pro-tumour cluster. Interfering with this KRAS\u0026ndash;NETs feed-forward loop offers a precision strategy to neutralise the malignant arm of NET biology without compromising its anti-tumour immunostimulatory capacity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003euPA-Enriched NETs Activate Membrane-Bound uPAR to Selectively Initiate Pro-Tumor Effects in CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCellular responses to NETs rely on multiple sensors, with potential differences in their downstream functional outputs. We further deciphered the specific mechanisms by which CCA cells sense NETs stimulation and selectively activate the pro-tumor C4 functional cluster characterized by KRASmut-MAPK signaling. We constructed a potential C4 regulatory network via STRING protein-protein interaction analysis and identified PLAUR\u0026mdash;which encodes the glycosylphosphatidylinositol-anchored receptor uPAR\u0026mdash;emerged as the top hub \u003cstrong\u003e(Figure S4A)\u003c/strong\u003e. NETs stimulation rapidly up-regulated uPAR mRNA and protein, and this increment was amplified in KRAS-mutant (KRASmut) cells; no such induction was observed for other reported NET sensors (TLR2/4/9 or CCDC25) \u003cstrong\u003e(Figure S4B)\u003c/strong\u003e. High-resolution immunofluorescence revealed uPAR enrichment at the cell surface in intimate apposition to adherent NETs fibres, implying direct engagement \u003cstrong\u003e(Figure 4A)\u003c/strong\u003e. As a well-characterized cell membrane protein, uPAR exerts pro-tumor effects by enhancing integrin signaling, thereby facilitating tumor cell proliferation, invasion, and distant metastasis\u003csup\u003e[26]\u003c/sup\u003e. Functional validation via siRNA-mediated uPAR knockdown in RBE and CCLP1-KRASmut cells demonstrated that uPAR deficiency downregulated MAPK pathway activation under NETs stimulation \u003cstrong\u003e(Figure 4B-C)\u003c/strong\u003e and attenuated the NETs-induced upregulation of cancer cell proliferation\u003cstrong\u003e\u0026nbsp;(Figure 4D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo better understand the mechanism by which uPAR responds to NETs and subsequently selectively activates the pro-tumor MAPK pathway, we focused on urokinase-type plasminogen activator (uPA)\u0026mdash;the ligand of uPAR which is abundant in TANs \u003csup\u003e[27]\u003c/sup\u003e. Single-cell profiling of human CCA TANs showed that\u0026nbsp;PLAU\u0026nbsp;(encoding uPA) transcripts were selectively enriched in the PADI4⁺ TAN subset with an enhanced NETs-forming capacity\u003cstrong\u003e\u0026nbsp;(Figure 4E and S4C)\u003c/strong\u003e. We therefore hypothesized that uPA \u0026nbsp;released along with the NETs scaffold stimulates the KRAS mutation (KRASmut)-amplified MAPK signaling via uPAR, thereby mediates the selective activation of the pro-tumor functional clusters induced by NETs. Co-culture with KRASmut CCA-conditioned medium further elevated PLAU expression in neutrophils detected by qPCR assays \u003cstrong\u003e(Figure 4F)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMoreover, typical uPA-enriched NETs structures were induced under phorbol 12-myristate 13-acetate (PMA) stimulation\u003cstrong\u003e\u0026nbsp;(Figure 4G)\u003c/strong\u003e. PMA-induced NETs displayed reticular uPA\u0026ndash;H3cit co-localization were observed in clinical KRASmut CCA tissues of both clinical specimens and murine YAK models \u003cstrong\u003e(Figure 4H and S4D)\u003c/strong\u003e. Across Fu-iCCA Cohort, NETs-scores showed a strong positive correlation with both uPA and uPAR transcript levels \u003cstrong\u003e(Figure 4I)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo test whether uPA cargo is functionally required, we generated uPA-deficient NETs by siRNA knock-down in dHL-60 cells (a neutrophil-like cell line) prior to PMA-induced NETosis and exposed these cells to CCLP1-KRASmut CM. . Compared with the negative control (siNC)-derived NETs, the uPA-deficient NETs elicited markedly weaker p-ERK induction in CCLP1-KRASmut cells \u003cstrong\u003e(Figure 4J)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCollectively, these results indicate that uPA-enriched NETs, via uPAR, mediate the selective pro-tumor effects on CCA through the KRASmut-amplified MAPK signaling pathway. Intercepting uPA\u0026ndash;uPAR engagement therefore offers a precision node to disable the pro-tumour arm of NETs while sparing their immunostimulatory capacity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003euPAR and TLRs Synergistically Mediate Pro-Tumorigenic NETs Stimulation in KRASmut CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNETs consist of a chromatin scaffold decorated with various proteins, including uPA. Since uPA can be secreted by multiple components of the tumor microenvironment (TME) beyond neutrophils, we further explored the specific mechanisms by which KRASmut CCA cells respond to uPA-enriched NETs stimulation and activate pro-tumorigenic signaling cascades. Notably, stimulation of CCLP1-KRASmut and RBE with uPA alone failed to significantly upregulate pERK levels or the mRNA expression of key MAPK pathway components. In contrast, combined stimulation with uPA and NETs DNA scaffold induced MAPK pathway activation comparable to that induced by uPA-enriched NETs extracts. DNase-mediated disruption of the DNA scaffold effectively abrogated this MAPK activation \u003cstrong\u003e(Figures 4K-L)\u003c/strong\u003e, demonstrating that the physical association between uPA and the NET DNA scaffold is essential for pro-tumor signaling.\u003c/p\u003e\n\u003cp\u003eTLRs are well-recognized sensors for NET-derived DNA, and consistently, functional cluster C1 exhibited significant enrichment in TLRs-related pathways. Immunofluorescence analysis revealed that NET stimulation induced pronounced co-localization of TLRs and uPAR in KRASmut CCLP1 and RBE cells, an interaction absent under basal conditions \u003cstrong\u003e(Figure 4M)\u003c/strong\u003e. Inhibition of TLRs using chloroquine (CQ) significantly attenuated CCA cell recognition of NETs, resulting in significantly reduced upregulation of key MAPK pathway molecules\u0026mdash;an effect comparable to DNase-mediated DNA scaffold disruption. Importantly, this inhibitory effect could not be restored by exogenous uPA supplementation\u003cstrong\u003e\u0026nbsp;(Figure 4N)\u003c/strong\u003e. Collectively, these findings demonstrate that the selective pro-tumorigenic effects of NETs in CCA require two sequential and synergistic steps: TLRs-mediated recognition of the NETs chromatin scaffold, followed by uPAR-mediated sensing of scaffold- -bound uPA, ultimately activating KRASmut-amplified pro-tumor MAPK signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTherapeutic Targeting of the uPA-uPAR Axis Selectively Abrogates Pro-Tumorigenic NET Effects While Preserving Immunostimulatory Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next evaluated the therapeutic potential of targeting the uPA-uPAR axis to attenuate the selective pro-tumor effects of uPA-enriched NETs in CCA. In KRASmut CCA cells, UPARANT\u003csup\u003e[28]\u003c/sup\u003e\u0026mdash;a specific uPA-uPAR interaction inhibitor\u0026mdash;significantly reduced NETs-induced MAPK/ERK pathway activation\u003cstrong\u003e\u0026nbsp;(Figure 5A)\u003c/strong\u003e to an extent comparable to trametinib (direct MAPK inhibition), chloroquine (TLR inhibition), or DNase (scaffold disruption) (Figure 5A; Figure S5A). Concordantly, UPARANT markedly attenuated NET-driven malignant proliferation in vitro \u0026nbsp; \u003cstrong\u003e(Figure 5B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eNotably, beyond suppressing the selective activation of the C4-mediated pro-tumor MAPK pathway, UPARANT preserved or even enhanced the expression of immune activation-related genes (including CCL5, CXCL9, and ISG15) across the other functional clusters. This immune-sparing profile mirrored that of direct MAPK pathway inhibition \u003cstrong\u003e(Figure 5C and S5A)\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003econsistent with previous observations in related models. In contrast, direct intervention of the NETs scaffold using CQ or DNase non-selectively \u0026nbsp;suppressed both pro-tumorigenic signaling pathways and immune activation genes \u003cstrong\u003e(Figure S5A)\u003c/strong\u003e. Consistently, UPARANT treatment did not impair but rather enhanced the in vitro chemotaxis of PBMCs toward NETs-stimulated CCA-CM \u003cstrong\u003e(Figure 5D)\u003c/strong\u003e. Since NETs-rich inflammatory stimuli can also activate MAPK in KRASwt contexts,, UPARANT similarly exerted inhibitory effects on NETs-induced pro-tumor phenotypes while maintaining immunostimulatory capacity in KRASwt cells\u003cstrong\u003e\u0026nbsp;(Figure S5B-D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eWe next investigated the mechanistic basis for the reciprocal relationship between NET-induced MAPK signaling and immunostimulatory functional clusters. We found that the NET-driven immunostimulation was associated with tumor-intrinsic IFN responses (marked by ISG15), which correlated with \u0026nbsp;activation of Stimulator of Interferon Genes (STING) and its downstream JAK-STAT signaling\u003cstrong\u003e\u0026nbsp;(Figure S5E)\u003c/strong\u003e. Mechanistically, the UPARANT-enhanced expression of immune-stimulatory factors and PBMC chemotaxis in the presence of NETs were completely abrogated by direct inhibition of STING or STING-related JAK-STAT signaling \u003cstrong\u003e(Figures 5E-F),\u003c/strong\u003e indicating that uPA-uPAR blockade indirectly potentiates STING-JAK/STAT signaling. This aligns with previous reports that direct inhibition of MAPK/ERK signaling relieves the suppression of STING-JAK/STAT, thereby enhancing antitumor immunty \u003csup\u003e[29-31]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn vivo validation was performed by establishing the SB1 i.hep. model padi4-ko or wt mice. UPARANT significantly reduced tumor burden in padi4-wt mice, with immunofluorescence analysis confirming decreased KI67 and pERK levels. Importantly, UPARANT preserved and even enhanced NETs-recruited anti-tumor T cells and dendritic cells (DCs)cells, whereas, it exerted minimal effects on KI67, pERK, and immune cell infiltration in padi4-ko mice\u003cstrong\u003e\u0026nbsp;(Figure 5G)\u003c/strong\u003e. qPCR analysis of tumor tissues further confirmed that UPARANT did not affect the NETs-induced expression of immune effector molecules such as\u003cem\u003e\u0026nbsp;Ccl5\u003c/em\u003e and \u003cem\u003eCxcl9\u003c/em\u003e \u003cstrong\u003e(Figure S5F)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that NETs-bound uPA selectively activates pro-tumorigenic functional clusters (specifically C4) via uPAR engagement while sparing immunostimulatory programs, establishing the uPA-uPAR axis as a promising therapeutic target that can uncouple tumor-promoting from immune-activating effects of NETs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003euPA-uPAR axis Blockade Selectively Abrogates the Pro-Tumor Effects and Preserves Immunostimulatory Capacity of NETs in CCA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCurrent therapeutic strategies targeting NETs primarily include disrupting formed NETs structures (DNase) and pharmacological blockade of NETosis (disulfiram or PADI4 inhibitors) (disulfiram or PADI4 inhibitors). While DNase effectively degrades NETs in vitro, in vivo application disrupts large-scale NETs meshworks to relieve tumor encapsulation and protection from immune cell attack, however, residual NET fragments and their associated components retain biological activity. We therefore proposed a combinatorial strategy to modulate NETs functional clusters\u0026mdash;employing DNase to dismantle large-scale NETs architectures while utilizing UPARANT to selectively neutralize \u0026nbsp;selectively pro-tumorigenic functional clusters (represented by C4) within residual NETs fragments while preserving their immune-stimulatory \u0026nbsp;properties while abrogating tumor-promoting effects.\u003c/p\u003e\n\u003cp\u003eGiven the prominent activation of C4 cluster in KRASmut contexts, we evaluated this strategy in the YAP/AKT/KRAS (YAK) model, comparing DNase monotherapy, UPARANT monotherapy, DNase plus UPARANT combination, and the PADI4 inhibitor GSK484. GSK484 and the combination regimen significantly reduced tumor burden, with the latter showing superior efficacy. Both GSK484 and DNase plus UPARANT comparably suppressed tumor cell proliferation (Ki67) and MAPK activity (pERK). Notably, while DNase alone enhanced T cell infiltration, this immunostimulatory effect was further amplified by combination with UPARANT\u003cstrong\u003e\u0026nbsp;(Figures 6A-B)\u003c/strong\u003e. Transcriptomic profiling revealed that DNase plus UPARANT collectively orchestrated an inflamed tumor microenvironment, characterized by significant upregulation of immune-promoting factors, antigen-presenting machinery, and interferon-responsive genes\u0026mdash;an effect surpassing DNase monotherapy. In contrast, GSK484 monotherapy exhibited minimal immunostimulatory activity \u003cstrong\u003e(Figure 6C)\u003c/strong\u003e. Immunofluorescence further confirmed that DNase plus UPARANT treatment facilitated T cell penetration into cancer cell-enriched regions following NET barrier disruption, suggesting enhanced anti-tumor immune surveillance \u003cstrong\u003e(Figure 6D)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInterestingly, CD274 (PD-L1) expression paralleled immune-stimulatory molecule upregulation across NET functional clusters, Padi4-wt versus ko comparisons, and pre/post-treatment analyses.. This observation prompted investigation of whether DNase plus UPARANT-induced TME remodeling could synergize with anti-PD-L1 blockade. In the YAK model, combination with DNase plus UPARANT markedly enhanced the limited efficacy of anti-PD-L1 monotherapy, significantly reducing tumor burden and prolonging survival compared to either treatment alone. This regimen also promoted abundant infiltration of granzyme B-positive (GZMB⁺) immune effector cells \u003cstrong\u003e(Figure 6E and S6)\u003c/strong\u003e. These findings were reproducible in the SB1 intrahepatic model, where DNase plus UPARANT similarly potentiated anti-PD-L1 efficacy and immune cell infiltration\u003cstrong\u003e\u0026nbsp;(Figure 6F and S6)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFinally, we interrogated the relationship between NETs, NETs functional clusters, and immunotherapy response using transcriptomic data from an independent published Ni cohort, in which patient response to immunotherapy was predicted based on CTLA4 and CXCL9 levels as reported by the original researchers\u003csup\u003e[32]\u003c/sup\u003e. Intriguingly, patients predicted to have a favorable response had relatively higher NET-score \u003cstrong\u003e(Figure 6G)\u003c/strong\u003e\u0026mdash;a finding that appears contradictory to the established pro-tumorigenic role of NETs but is reconciled by our identification of distinct functional clusters. Indeed, responder-enriched tumors displayed higher representation of the C5 immune-activation cluster, whereas the C4 MAPK-associated cluster showed no correlation with immunotherapeutic benefit\u003cstrong\u003e\u0026nbsp;(Figure 6H)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eCollectively, these findings demonstrate that the DNase plus UPARANT strategy outperforms existing NET-targeting monotherapies by selectively abrogating pro-tumorigenic NET signaling while preserving and enhancing immunostimulatory activity, thereby reshaping the TME toward an inflamed phenotype and synergizing with checkpoint blockade in KRASmut CCA \u003cstrong\u003e(Figure 7)\u003c/strong\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTANs regulate tumor progression via NETs. Blocking NETs inhibits tumor progression, consistent with this and other studies, while NETs also exhibit protential antitumor effects\u0026mdash;though relevant evidence in CCA remains scarce\u003csup\u003e[4, 33, 34]\u003c/sup\u003e . Our previous pan-cancer studies have shown that NETs may reflect pro-tumor or anti-tumor effects depending on cancer type\u003csup\u003e[20]\u003c/sup\u003e. Unlike TANs subsets with stage-specific and spatial-specific biological characteristics\u003csup\u003e[35, 36]\u003c/sup\u003e, the functional diversity of NETs likely stems from the distinct roles of their components\u003csup\u003e[37, 38]\u003c/sup\u003e. In this study, we observed the dual effects of NETs on CCA: they directly promote the malignant phenotypes of CCA cells, while their mediated pro-inflammatory effects may elicit potential antitumor responses. Bioinformatics analysis dissected NETs-CCA interactions, leading to the concept of C1\u0026ndash;C5 functional clusters informed by the pro-/anti-inflammatory duality of NETs\u003csup\u003e[39]\u003c/sup\u003e. These clusters are independent, with individual clusters exerting pro-/anti-tumor effects, and their dynamic balance drives the overall outcomes in mouse models and patients. The differential responses of CCA to NETs reflect the selective activation of these clusters, with cluster-specific responses correlating with prognosis\u0026mdash;highlighting the complexity of NETs biology in CCA.\u003c/p\u003e\n\u003cp\u003eNotably, referring to the dual pro-/anti-tumor effects of NETs-CCA crosstalk under the novel functional clusters framework, pro-tumor clusters (e.g., C4) directly enhance CCA malignancy, aligning with the reported pro-tumor roles of NETs in other cancers through various context-dependent mechanisms\u003csup\u003e[40, 41]\u003c/sup\u003e. Our study identifies the C4 cluster as a key driver of pro-tumor effects by activating the MAPK pathway\u0026mdash;signaling for enhancing malignant phenotypes under external stimuli, including NETs \u003csup\u003e[42]\u003c/sup\u003e. MAPK activation strongly correlates with KRAS mutations, which are present in ~25% of CCA cases and associated with the most aggressive subtype. Notably, KRAS mutations alone are often insufficient to sustain robust MAPK activation; additional extracellular stimuli are typically required, either from autocrine signals in KRAS-mutant cancer cells or paracrine signals from the TME\u003csup\u003e[22, 23, 43]\u003c/sup\u003e. Combined with our data, TME NETs stimulation and CCA KRASmut cooperate to promote malignant phenotypes: KRASmut boosts TANs recruitment/NETs formation and enhances CCA responsiveness to NETs, leading to more robust pro-tumor MAPK activation of C4 in CCA\u0026mdash;a pattern similar to KRASmut-amplified inflammation in lung/pancreatic cancer\u003csup\u003e[22, 44]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn contrast, the anti-tumor functions of NETs have long been overshadowed by the extensive reports on their pro-tumor roles in various malignancies. Among the limited reports, direct cytotoxicity of NETs against tumor cells has been documented as one of the mechanisms\u003csup\u003e[45-47]\u003c/sup\u003e. Our novel perspective extends this understanding by highlighting that anti-tumor clusters maintain a potential hot TME and promote immune infiltration in CCA. In autoimmune diseases, NETs are highly pro-inflammatory, promote dendritic cell maturation and reduce T cell activation thresholds\u003csup\u003e[13-15]\u003c/sup\u003e. As a highly inflammatory solid tumor, CCA exhibits similar NETs-mediated pro-inflammatory characteristics, which was confirmed using NETs-deficient models and multiple detection approaches. Specifically, NETs induce CCA cells to secrete chemokines such as CCL5 and CXCL9, which recruit T cells and DCs to the tumor site. Such triggered immunogenicity is reported to rely on signaling pathways activated by DNA damage or inflammatory signals, which function as tumor-suppressive mechanisms aiding cancer treatment\u003csup\u003e[48, 49]\u003c/sup\u003e . Recruited immune cells localize around tumor areas with elevated immune checkpoint expression, consistent with prior reports that the reticular structure of NETs acts as a physical barrier blocking immune-tumor contact and upregulates immune checkpoint expression \u003csup\u003e[7, 50]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNETs consist of a DNA scaffold and functionally distinct proteins attached to it. Under pathological conditions, NETs show abnormal production (excessive/insufficient) and altered component composition\u003csup\u003e[51-53]\u003c/sup\u003e. The selective activation of NETs functional clusters implies that distinct NETs components bind to specific cell surface receptors on CCA cells. We therefore focused on two key receptor families: uPAR and TLRs. Core gene analysis identified uPAR as the key receptor mediating the selective activation of the pro-tumor C4 cluster by NETs. uPAR, a membrane glycoprotein implicated in tumor invasion and metastasis, is overexpressed in various cancers and linked to poor prognosis\u0026mdash;though its role in NETs-regulated CCA progression remains unclear. Notably, it is well-documented that uPAR promotes tumor growth via downstream integrin-related signaling and the MAPK cascade, a mechanism that underpins its pro-tumor effects, thus making uPAR a promising cancer target\u003csup\u003e[54-56]\u003c/sup\u003e. Moreover, uPAR is reported to be closely correlated with KRASmut\u003csup\u003e[57, 58]\u003c/sup\u003e. Consistently, our study reveals that uPAR is indispensable for NETs-induced C4 activation and subsequent KRASmut-amplified MAPK signaling in CCA, linking uPAR\u0026rsquo;s intrinsic pro-malignancy pathway to NETs-driven CCA progression. Additionally, targeting uPAR reduces the selective pro-tumor effects of NETs without impairing\u0026mdash;even partially enhancing\u0026mdash;the immunostimulatory effects of other NETs clusters.\u003c/p\u003e\n\u003cp\u003eWe also demonstrated that CCA cells recognize NETs via uPAR by binding to its ligand uPA enriched in CCA-derived NETs. Similar to high uPA expression in pancreatic cancer TANs\u003csup\u003e[27]\u003c/sup\u003e, uPA and the NETs-forming enzyme PADI4 are highly expressed in CCA-derived TANs and released extracellularly by binding to the NETs chromatin scaffold. A key finding is that uPA alone weakly activates the KRASmut-MAPK-mediated C4 cluster via uPAR; and the NETs chromatin scaffold is essential for robust activation of the uPA-uPAR signaling pathway. Among NETs components, the chromatin scaffold serves as both the physical basis for NETs structure and a platform for NETs-bound proteins, facilitating their retention on the cell surface or mediating the hydrolysis of functional fragments\u003csup\u003e[59, 60]\u003c/sup\u003e. Recognition of the NETs chromatin scaffold involves TLRs, another key receptor family\u003csup\u003e[61]\u003c/sup\u003e. In CCA, our study confirms that uPAR and TLRs act synergistically to enable CCA cells to fully respond to uPA-bound NETs and selectively activate the downstream C4 (KRAS-MAPK) cluster, the interaction between TLRs and uPAR is also reported in macrophage inflammatory responses and endotoxin-driven colorectal cancer cell invasion\u003csup\u003e[62, 63]\u003c/sup\u003e. TLRs broadly mediate NETs pro-inflammation (enriched in the C2 cluster) but are universally activated in CCA patients, suggesting they act as common receptors for pan-inflammatory signals, including NETs. In the context of NETs stimulation in CCA, TLRs may also serve as shared receptors for multiple functional clusters. Critical supporting evidence comes from antagonizing TLRs-mediated NETs scaffold recognition: this treatment reduced both KRASmut-MAPK signaling in the pro-tumor C4 cluster and upregulation of immunostimulatory molecules in anti-tumor clusters.\u003c/p\u003e\n\u003cp\u003eAn important finding is that targeting uPAR enhances the selective activation of immunostimulatory NETs functional clusters, mediated by STING pathway. Cancerous STING senses chromatin fragments and initiates an intrinsic interferon (IFN) response via the JAK-STAT pathway, thereby upregulating molecules such as CCL5 to boost tumor immunogenicity\u003csup\u003e[64-66]\u003c/sup\u003e. Moreover, the anti-tumor immunity activated by STING signaling is reported to be enhanced by targeting MAPK pathway, including in CCA\u003csup\u003e[29, 30]\u003c/sup\u003e. STING is also closely associated with NETs: in stromal cells, NETs signals are internalized via membrane receptors (e.g., TLRs) to trigger STING-mediated inflammatory and immune responses\u003csup\u003e[67, 68]\u003c/sup\u003e. In our study, NETs-induced pro-tumor MAPK activation in CCA is uPAR-dependent; thus, targeting uPAR mimics MAPK pathway inhibition\u0026mdash;activating STING signaling\u0026mdash;consistent with prior reports. Collectively, TLRs, uPAR, and STING form a NETs cluster signaling axis where TLRs recognize the NETs structure, with subsequent activation of the uPAR-MAPK pro-tumor cluster via NETs-bound uPA or initiation of STING-mediated intrinsic immune responses via NETs-DNA fragments sensing.\u003c/p\u003e\n\u003cp\u003eCurrent NETs-targeting strategies include two approaches\u003csup\u003e[17]\u003c/sup\u003e: disrupting existing NETs using DNase and reducing NETs formation with agents such as disulfiram or PADI4 inhibitors. Though these approaches show similar NETs-targeting efficacy in various tumor and disease models, their mechanisms and limitations differ, and such differences have barely been comparatively investigated. DNase fully abrogates NETs effects in vitro but only hydrolyzes the reticular structure of NETs into smaller fragments in vivo; these residual fragments and their bound proteins retain biological stimulatory effects on tumors\u003csup\u003e[69]\u003c/sup\u003e. In contrast, strategies reducing NETs formation\u0026mdash;targeting either the intracellular NETs formation cascade in TANs or TME signals promoting NETs\u0026mdash;inherently sacrifice the potential positive immunomodulatory effects of NETs. Targeting NETs-CCA interactions represents a novel direction; for example, targeting CCDC25 in breast cancer reduces NETs-induced metastasis\u003csup\u003e[25, 70]\u003c/sup\u003e. Our study proposes a novel strategy for selective NETs cluster targeting by combining DNase with uPAR abrogation. Three mechanisms underpin this rationale: (1) DNase disrupts the \u0026quot;shields\u0026quot; structure of NETs, eliminating the physical barrier blocking immune cell attack on CCA; (2) targeting the uPA-uPAR pathway directly antagonizes the pro-tumor effects of residual NETs fragments (specifically KRASmut-amplified, MAPK-mediated C4 cluster effects); (3) importantly, the immunostimulatory effects of NETs fragments are preserved\u0026mdash;even compensatorily enhanced\u0026mdash;allowing infiltrating immune cells to penetrate the tumor parenchyma through the disrupted NETs barrier by DNase. Notably, MAPK-MEK-ERK inhibitor has also been reported to induce compensatory enhancement of immunogenicity\u003csup\u003e[29, 30]\u003c/sup\u003e, possibly via activating compensatory inflammatory pathways\u0026mdash;this finding shares mechanistic parallels with our observations. In gastric cancer, targeting uPAR also promotes immune cell infiltration\u0026mdash;providing additional support for this strategy\u003csup\u003e[71]\u003c/sup\u003e. Notably, DNase + uPAR abrogation synergizes with anti-PD-L1 inhibitors (which have limited monotherapy efficacy in CCA): DNase weakens myeloid physical immunosuppression by disrupting the NETs physical barrier, and the subsequent NETs fragments induce PD-L1 upregulation that provides a natural target for PD-1/PD-L1 inhibitors, further enhancing the anti-tumor cytotoxicity of immune cells infiltrating CCA under the NETs cluster-targeting strategy. Compared with DNase monotherapy (which retains bioactive NETs fragments) or PADI4 inhibitors (which abrogate both pro-tumor and immune-stimulatory NETs functions), our DNase+uPAR abrogation combination achieves \u0026ldquo;precision modulation\u0026rdquo; of CCA-NETs interaction.\u003c/p\u003e\n\u003cp\u003eCertain limitations of this study require further investigation. First, the NETs functional cluster concept was proposed based on NETs-CCA interactions, so its applicability needs verification and extension at the pan-cancer level. Second, the C1-C5 clusters are defined based on transcriptomic data; future studies need to validate cluster-specific protein biomarkers for clinical translation. Third, larger prospective cohorts are required to confirm the correlation between NETs-score/immunostimulatory cluster and immunotherapy efficacy.\u003c/p\u003e\n\u003cp\u003eIn summary, our study demonstrates that NETs regulate CCA progression via dual pro-/anti-tumor C1-C5 functional clusters, with KRASmut-amplified MAPK signaling driving direct pro-tumor effects while the immunostimulatory properties of other clusters are preserved. The DNase+uPAR abrogation combination targets this balance by disrupting the NETs barrier and inhibiting the pro-tumor cluster while preserving immune function\u0026mdash;synergizing with anti-PD-L1 therapy. This work provides a novel functional cluster-based framework for understanding NETs biology in CCA and a precision therapeutic strategy for KRASmut CCA patients with limited treatment options.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCA: Cholangiocarcinoma\u003c/p\u003e\n\u003cp\u003eDNase: Deoxyribonuclease\u003c/p\u003e\n\u003cp\u003eKRASmut: KRAS-mutant\u003c/p\u003e\n\u003cp\u003eKRASwt: KRAS-wildtype\u003c/p\u003e\n\u003cp\u003eMAPK: Mitogen-activated protein kinase\u003c/p\u003e\n\u003cp\u003eNETs: Neutrophil extracellular traps\u003c/p\u003e\n\u003cp\u003ePD-1: Programmed death-1\u003c/p\u003e\n\u003cp\u003ePD-L1: Programmed death-ligand 1\u003c/p\u003e\n\u003cp\u003ePADI4: Peptidylarginine deiminase 4\u003c/p\u003e\n\u003cp\u003eSTING: Stimulator of interferon genes\u003c/p\u003e\n\u003cp\u003eCCL5: Chemokine (C-C motif) ligand 5\u003c/p\u003e\n\u003cp\u003eCXCL9/10: Chemokine (C-X-C motif) ligand 9/10\u003c/p\u003e\n\u003cp\u003eGZMB: Granzyme B\u003c/p\u003e\n\u003cp\u003eTANs: Tumor-associated neutrophils\u003c/p\u003e\n\u003cp\u003eTLRs: Toll-like receptors\u003c/p\u003e\n\u003cp\u003eTME: Tumor microenvironment\u003c/p\u003e\n\u003cp\u003euPAR: Urokinase-type plasminogen activator receptor\u003c/p\u003e\n\u003cp\u003euPA: Urokinase-type plasminogen activator\u003c/p\u003e\n\u003cp\u003eCyTOF: Mass cytometry\u003c/p\u003e\n\u003cp\u003eWGCNA: Weighted Gene Co-expression Network Analysis\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors claim no conflict of interest\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: The data and materials supporting the findings of this study are available in the published article and its supplementary information, or on reasonable request from the corresponding authors.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions: Luyu Yang, Xiaotian Shen, Lu Lu, Baobin Ying, Lun-Xiu Qin designed the study, Lu-Yu Yang, Xiaotian Shen, Xin Zheng, Sunzhe Xie, Chen Zhang performed the experiments, Haoting Sun, Xiangyu Wang, Hao Wang, Shule Li, Yiran Chen, Zhenchao Chen, Jixuan Chen, Jinyu Wang, Wenwei Zhu, Jinhong Chen joined in the discussion, Lu-Yu Yang, Xiaotian Shen wrote the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements: We thank all the healthy donors and CCA patients for the source of tissue and blood samples. We sincerely thank Dr. Hu from Ruijin Hospital for generously providing the padi4 knockout mice that supported key experiments in this study.\u003c/p\u003e\n\u003cp\u003eFunding list: This work was jointly supported by the National Natural Science Foundation of China (Key Program, 82430089; General Program, 82373017), the Shanghai Science and Technology Committee (General Program of Natural Science, 24ZR1408800), and the Project of Flexible introduction of High-level Medical and Health Professionals in Fujian Province (No. YJRCTD-2021QLX).\u003c/p\u003e\n\u003cp\u003eEthics statement:\u003c/p\u003e\n\u003cp\u003eThe studies involving humans were approved by the Ethics Committee of Huashan Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.\u003c/p\u003e\n\u003cp\u003eSupplementary\u0026nbsp;Materials\u003c/p\u003e\n\u003cp\u003eSupplementary materials include detailed methods, additional figures (Figures S1-S6), and additional table.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e[1] Greten T F, Schwabe R, Bardeesy N, et al. Immunology and immunotherapy of cholangiocarcinoma[J]. Nature reviews. Gastroenterology \u0026amp; hepatology. 2023, 20(6): 349-365.\u003c/li\u003e\n\u003cli\u003e[2] Ilyas S I, Affo S, Goyal L, et al. Cholangiocarcinoma - novel biological insights and therapeutic strategies[J]. Nature reviews. Clinical oncology. 2023, 20(7): 470-486.\u003c/li\u003e\n\u003cli\u003e[3] Dong L, Lu D, Chen R, et al. Proteogenomic characterization identifies clinically relevant subgroups of intrahepatic cholangiocarcinoma[J]. 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Science advances. 2022, 8(21): eabn3774. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cholangiocarcinoma, Neutrophil extracellular traps (NETs), Functional clusters, KRAS mutation, uPA-uPAR axis","lastPublishedDoi":"10.21203/rs.3.rs-9281659/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9281659/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cholangiocarcinoma (CCA) is an aggressive malignancy with dismal prognosis; PD-1/PD-L1 blockade benefits few patients, as the tumor microenvironment (TME) is immunologically “cold”. A key CCA-TME feature is massive tumor-associated neutrophil (TAN) infiltration releasing neutrophil extracellular traps (NETs). Conventionally deemed pro-tumorigenic, NETs’ anti-tumor potential is overlooked, leaving their dual roles and regulatory circuits in CCA undefined. Our study finds prominent NETs enrichment in CCA clinical specimens and preclinical models, correlating with poor outcomes. Functional studies show NETs exert dual effects: a dominant pro-tumor arm accelerating growth/metastasis, and a latent immunostimulatory arm rendering the TME “hot”. Integrated multi-omics and bioinformatics analyses dissect these functions into distinct molecular clusters with divergent prognostic value. Pro-tumor clusters are selectively activated by MAPK signaling, present in ~25% of CCA with KRAS mutations. Mechanistically, uPA-loaded NETs engage uPAR on CCA cells; TLR co-reception licenses downstream MAPK activation, tipping toward tumor promotion. We devised a cluster-directed combination: DNase I dismantling NETs scaffolds plus uPAR blockade neutralizing residual pro-tumor fragments. This strategy abolishes oncogenic signaling while sparing—even boosting—STING-dependent anti-tumor immunity, sensitizing KRAS-mutant and wild-type CCA to anti-PD-L1 therapy. Human transcriptomic datasets link low pro-tumor/high immunostimulatory NETs signatures with durable immunotherapy responses. We establish a functional-cluster framework for CCA NETs biology and provide a precision co-targeting regimen turning pro-tumor function into immunotherapeutic opportunity.","manuscriptTitle":"Selective Targeting uPAR-driven Neutrophil Extracellular Traps Functional Clusters to Attenuate Tumor Progression and Enhance the Response to Immunotherapy in Intrahepatic Cholangiocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 14:56:08","doi":"10.21203/rs.3.rs-9281659/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"39515eef-36dd-4d41-856e-0cf63456be58","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Reject after peer review","date":"2026-04-30T11:24:24+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65607532,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":65607533,"name":"Biological sciences/Immunology/Tumour immunology"},{"id":65607534,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"}],"tags":[],"updatedAt":"2026-04-30T11:27:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 14:56:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9281659","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9281659","identity":"rs-9281659","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}