Chronic Stress Promotes Pancreatic Ductal Adenocarcinoma Progression via Complement C5a-Recruited Myeloid-Derived Suppressor Cells

Chronic Stress Promotes Pancreatic Ductal Adenocarcinoma Progression via Complement C5a-Recruited Myeloid-Derived Suppressor Cells | 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 Research Article Chronic Stress Promotes Pancreatic Ductal Adenocarcinoma Progression via Complement C5a-Recruited Myeloid-Derived Suppressor Cells siyuan yang, Li Xiao, Pingqian Qi, Ziqing Liu, Zhuomin Chen, Ruijia Wei, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7471626/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Dec, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted 13 You are reading this latest preprint version Abstract Chronic stress is recognized as one of the risk factors for pancreatic ductal adenocarcinoma (PDAC). The autonomic nervous system (ANS), a critical component of the tumor microenvironment (TME), has been closely associated with PDAC tumorigenesis and prognosis, though the underlying mechanisms remain elusive. Here, we demonstrate that aberrant activation of sympathetic nerves (SNS) upregulates the hepatic complement system, thereby recruiting myeloid-derived suppressor cells (MDSCs) into pancreatic malignancy to promote tumor progression. Chronic stress induces SNS-derived release of the neurotransmitter norepinephrine (NE), which enters the liver via systemic circulation and binds to upregulated β1 adrenergic receptor (ADRB1) on hepatocytes, leading to increased expression of complement components C3, C5, and CFH. In chronic stress-model mice, elevated serum and intratumoral Complement component 5a (C5a) levels showed a positive correlation with MDSCs proportions in both the spleen and tumor tissues. Administration of the C5aR1 antagonist PMX-50 disrupted this association, indicating C5a as a critical mediator of MDSCs expansion and recruitment. Infiltrating MDSCs within the TME exhibit elevated expression of Programmed death-ligand 1 and Transforming growth factor-beta, fostering the establishment of an immunosuppressive microenvironment. Intraperitoneal injection of the ADRB1 antagonist atenolol in PDAC mice resulted in marked reductions in serum/tumoral C5a levels, splenic/intratumoral MDSCs proportions, and tumor burden. Our findings suggest that targeting SNS-driven MDSCs recruitment and TME remodeling may offer novel therapeutic strategies for PDAC patients. Chronic stress Sympathetic nervous system Complement system Pancreatic ductal adenocarcinoma Myeloid-derived suppressor cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The complex interplay among tumor microenvironment (TME) components, including parenchymal cells, nerves, and immune cells, is a critical determinant of the poor prognosis of pancreatic ductal adenocarcinoma (PDAC) patients [ 1 , 2 ]. Chronic stress, a psychological state triggered by persistent and unresolved stressors, has been implicated in PDAC pathogenesis and progression through autonomic nervous system (ANS) dysregulation [ 3 ]. The ANS, comprising two major efferent branches (the sympathetic nervous system, SNS, and parasympathetic nervous system, PSNS), not only maintains systemic homeostasis but also increasingly emerges as a key modulator of chronic diseases and tumor progression [ 4 , 5 ]. SNS with aberrant density or hyperactivity release high levels of catecholamines, which enhance PDAC cell survival by suppressing antitumor immune responses [ 6 – 9 ]. In contrast, PSNS exerts dual tumor-suppressive mechanisms via cholinergic signaling in pancreatic tumors: directly downregulating MAPK and PI3K/AKT signaling cascades in cancer cells, and potentially mediating indirect antitumor effects through myeloid cell inhibition [ 10 ]. Paradoxically, however, SNS activation has also been reported to inhibit tumor growth by downregulating CCL2 in macrophages [ 11 ]. This apparent contradiction underscores the complexity of SNS functionality, particularly its interactions with key TME components, including immune cells, stromal cells, and vascular endothelial cells, in shaping tumor behavior. Myeloid-derived suppressor cells (MDSCs), first introduced by Gabrilovich et al.[ 12 ], are a heterogeneous population of immunosuppressive cells implicated in the pathological progression from chronic inflammation to cancer metastasis [ 13 ]. In the setting of chronic inflammation, MDSCs undergo expansion in the bone marrow and spleen and recruitment to lesion sites, establishing an immunosuppressive TME [ 14 ]. Emerging evidence supports the hypothesis that stress-related sympathetic activation mobilizes MDSCs via catecholamine release. Restraint stress models exhibited MDSCs expansion and accumulation in the bone marrow, peripheral blood, and spleen [ 15 , 16 ]. Similar findings were observed in tumor-bearing mice subjected to chronic restraint or unpredictable mild stress (CUMS) [ 17 – 19 ]. However, under repetitive restraint stress, splenic MDSCs numbers were paradoxically reduced [ 20 ]. These data hint that sustained stimulation may activate systemic pathways to modulate MDSCs expansion and spatial distribution. The complement system, a pivotal component of the innate immune system, orchestrates host defense against endogenous and exogenous stimuli to maintain physiological homeostasis. Anaphylatoxin C5a, a key bioactive molecule released upon activation of all complement pathways, engages in multiple facets of tumor biology, including immune evasion, progression, and metastasis [ 21 ]. C5a modulates the immune microenvironment to elicit either pro- or anti-tumor responses, contingent upon tumor type and local C5a concentration [ 22 ]. MDSCs, as critical constituents of the immune microenvironment, exhibit a positive correlation between surface Complement 5a receptor 1 (C5aR1) expression levels and tumor infiltration capacity [ 23 ]. This receptor/ligand interaction enables C5a to recruit MDSCs to tumor sites, ultimately driving CD8⁺ T cell functional exhaustion and accelerating tumor progression [ 24 ]. While chronic stress is known to facilitate PDAC progression through MDSCs, the molecular mechanisms governing MDSCs expansion, systemic redistribution, and immunosuppressive TME modeling remain poorly characterized. Here we provide evidence for a previously unknown regulatory pathway, driven by the SNS-induced C5a, which is mainly produced by NE/ADRB1 axis-activated hepatic cells and promotes the expansion of MDSCs to infiltrate PDAC and facilitate the establishment of immunosuppressive TME through programmed death-ligand 1 (PD-L1) and transforming growth factor-beta (TGF-β1). Thus, SNS couples chronic stress to intrinsically primed immature immune cells to accelerate the progression of PDAC. Materials and Methods Experimental Animals and Cell line Male C57BL/6J mice (6 to 8 weeks old) were used for all experiments. Mice were originally purchased from GemPharmatech Co., Ltd. (Nanjing, China) and housed in the Animal Facility of Nanchang University under specific pathogen-free (SPF) conditions. Mice were group-housed (3–5 per cage) with ad libitum access to food and water, and maintained in a temperature-controlled room (21–25°C) with 40–60% humidity under a 12-hour light/dark cycle (lights on: 6:00 AM to 6:00 PM). Murine pancreatic cancer Pan02 cells were obtained from Fuheng Biotechnology (Shanghai, China) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified 5% CO2 incubator. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Rehabilitation Hospital, Jiangxi Medical College, Nanchang University (Approval No. SFYYXLL-PJ-2021-KY005). Orthotopic Transplantation Model Orthotopic PDAC tumors were generated by surgical implantation of 2 × 10 6 Pan02 cells into the pancreas of male C57BL/6 mice as previously described [25]. Briefly, mice were anesthetized via intraperitoneal injection of Zoletil-50 and placed in a sterile surgical field. A small left abdominal incision was made to expose the pancreas, followed by orthotopic injection of 2 × 10 6 Pan02 cells suspended in 100 μL phosphate-buffered saline (PBS) into the pancreatic head region. A transient tissue bulge confirmed successful intrapancreatic delivery. The needle was withdrawn slowly to minimize cell leakage, and the incision was closed with surgical sutures. Establishment of CUMS Model The CUMS protocol, a widely used chronic stress paradigm [26], was initiated 3 days post-tumor cell inoculation. Mice received 2–3 randomized stressors daily, including restraint, cage tilt (45°), tail suspension, water/food deprivation, circadian rhythm disruption, or white noise exposure. Stressor types and timing were randomized using a non-repetitive schedule (minimum 7-day intervals between identical stressors) over a 14-day period. Open Field Test (OFT) Behavioral analysis was conducted in a 40 × 40 × 35 cm open-field arena. Mice were individually placed at the arena center, and locomotion was recorded for 10 min using ANY-maze™ software (Stoelting Co.). The central zone was defined as the innermost 25% of total area. Total distance moved, central zone dwell time, and central zone path length were quantified. Elevated Plus Maze (EPM) Test The EPM apparatus consisted of two open arms (28 × 5 cm) and two enclosed arms (28 × 5 × 15 cm) elevated 80 cm above ground. Mice were positioned in the central quadrant facing an open arm, and exploratory behavior was video-tracked for 10 min. Metrics included total distance traveled, open arm residence time, and open arm path length. Pharmacological Interventions Tumor-bearing mice were randomly assigned to treatment groups. Starting 3 days post-implantation, the following agents were administered daily via intraperitoneal injection: Atenolol (β1-adrenergic receptor antagonist, 10 mg/kg/day; MedChemExpress, USA), PMX-50 (C5aR1 antagonist, 10 mg/kg/day; MCE, USA), and SB-431542 (TGF-β receptor inhibitor, 30 mg/kg/day; MCE, USA); For PD-L1 blockade, anti-PD-L1 antibody (10 mg/kg; MCE, USA) was injected twice weekly for 2 weeks. Hepatocyte Isolation Following anesthesia with Zoletil-50 (Virbac),An indwelling needle was inserted into the portal vein for sequential perfusion with Liver Perfusion Buffer (Thermo Fisher, USA) and Liver Digest Medium (Thermo Fisher, USA). The distended liver was excised, and the Glisson’s capsule was mechanically disrupted. Hepatocytes were released through 70 μm nylon filters and pelleted via low-speed centrifugation (50 × g, 5 min). Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted using TRIzol™ (Thermo Fisher, USA) and reverse-transcribed with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). qPCR was performed on a StepOnePlus™ system (Applied Biosystems) using Hieff UNICON® SYBR Green Master Mix (Yeasen, China). Primer sequences are listed in Supplementary Table S1. Flow Cytometry(FCM) Single-cell suspensions from spleen/tumor tissues were prepared as follows:Spleen: Mechanically dissociated through 70 μm filters, lysed with RBC Lysis Buffer (BioLegend, USA).Tumor: Collagenase IV (1 mg/ml; Beyotime, China)-digested fragments filtered through 70 μm mesh Cells were stained with: CD16/32 (Clone 2.4G2; BD Biosciences, USA), CD45-BV421 (Clone 30-F11; BioLegend, USA), CD11b-FITC (Clone M1/70; BD Biosciences, USA), Ly-6G/Ly-6C-PE (Clone RB6-8C5; BD Biosciences, USA), and 7-AAD viability dye (BD Biosciences, USA). Data acquisition used a CytoFLEX LX (Beckman Coulter), with analysis via FlowJo (v10.6.2). Cell sorting employed a CytoFLEX SRT system. Enzyme-Linked Immunosorbent Assay (ELISA) Serum was isolated via centrifugation (3,000 × g, 15 min) after 1-hour clotting. Tissue lysates were prepared in RIPA buffer (Beyotime, China) with protease inhibitors. C5a (Novus Biologicals, NBP2-67681, USA) and NE (Elabscience, E-EL-0042c, China) levels were quantified according to manufacturer protocols. Immunohistochemistry Tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned (5 μm). After antigen retrieval (pH 6.0 citrate buffer) and peroxidase quenching, sections were blocked with 5% goat serum and incubated with Ki-67 antibody (ZM-0166; ZSGB-BIO) overnight. DAB chromogen (Dako) and hematoxylin counterstaining were applied. Ki-67 + cells were quantified using ImageJ (≥ 3 fields/section). Transcriptome Sequencing MDSCs sorted from PDAC tissues were lysed in TRIzol®. RNA integrity (RIN > 8.0) was confirmed via Agilent 2100 Bioanalyzer. Libraries were prepared with VAHTS Universal V6 RNA-seq Library Kit (Vazyme) and sequenced (150 bp paired-end) on NovaSeq 6000 (Illumina). Raw reads were processed using fastp (v0.23.2) and aligned to mm10 genome. Statistical Analysis Data are expressed as mean ± SEM. Two-group comparisons used Student’s t-test. Multi-group analyses employed one-way ANOVA with Tukey’s post-hoc test (GraphPad Prism 9.5). P < 0.05 defined statistical significance. Results Chronic Stress Promotes PDAC Progression To investigate the role of chronic stress in PDAC progression, we established a CUMS model in orthotopic PDAC-bearing mice. Following a 3-day postoperative recovery period after tumor cell implantation, mice were subjected to intermittent stressors for 2 weeks prior to behavioral assessments and endpoint analyses (Fig. 1 A). Anxiety-like behaviors were evaluated using the open field test (OFT) and elevated plus maze (EPM). In the OFT (Fig. 1 B), no significant difference in total locomotor distance was observed between control and CUMS groups (Fig. 1 C), indicating unimpaired basal motor function. However, CUMS mice exhibited markedly reduced path length (Fig. 1 D) and dwell time (Fig. 1 E) in the central zone compared to controls. Similarly, EPM analysis revealed comparable total movement distances between groups (Fig. 1 F and 1 G), but CUMS mice demonstrated significantly decreased exploratory activity in open arms and central quadrants, as evidenced by reduced path length (Fig. 1 H) and residence time (Fig. 1 I). These data confirm CUMS-induced anxiety-like behaviors in PDAC-bearing mice. Chronic stress is known to suppress appetite in rodents [ 27 , 28 ]. Body weight measurements every 4 days revealed progressive weight loss in CUMS mice relative to controls (Fig. 1 J), validating the stress model. Meanwhile, CUMS-exposed mice harbored significantly heavier pancreatic tumors compared to controls (Fig. 1 K and 1 L). Immunohistochemical analysis further demonstrated elevated proliferation indices in CUMS tumors, with a pronounced increase in Ki-67 + cells (Fig. 1 M and 1 N). Collectively, these findings indicate that chronic stress exacerbates PDAC growth by enhancing tumor cell proliferation. Chronic Stress Drives MDSCs Expansion and Infiltration Chronic stress promotes tumor progression through both direct SNS-mediated modulation of malignant phenotypes and indirect TME remodeling. MDSCs, core immunosuppressive effectors expressing SNS neurotransmitter receptors, orchestrate multi-layered immune suppression by inhibiting effector T cells and facilitating regulatory T cell differentiation [ 29 ]. Given that stress-related sympathetic activation primarily targets β-adrenergic receptor (β-AR)-expressing MDSCs, we quantified tumor-infiltrating CD11b + Gr1 + MDSCs via flow cytometry in CUMS-treated PDAC mice. CUMS significantly increased MDSCs proportions within PDAC (Figs. 2 A and 2 B) and spleens (Figs. 2 C and 2 D), suggesting systemic MDSCs expansion coupled with enhanced tumor trafficking. Complement C5a Mediates MDSCs Recruitment Solid tumors often hijack the complement system to establish immunosuppressive niches favoring tumor survival [ 24 , 30 ]. As the liver produces up to 90% of circulating complement components [ 31 ], we analyzed hepatic complement expression in CUMS mice. qRT-PCR revealed no change in C2 levels (Fig. 3 A) but significant upregulation of pro-tumorigenic complement factors C3, C5, and CFH (Figs. 3 B– 3 D), indicating stress-induced hepatic reprogramming toward complement synthesis. Among these factors, C5-derived C5a critically recruits MDSCs via binding to C5aR1 on their surface [ 24 ]. CUMS elevated C5a levels in both serum and tumor tissues (Figs. 3 E and 3 F). To explore C5a’s role in stress-driven MDSCs dynamics, we administered the C5aR1 antagonist PMX-50, which attenuated CUMS-induced MDSCs accumulation in tumors (Figs. 3 G and 3 H) and spleens (Figs. 3 I and 3 J), confirming C5a/C5aR1 axis dependency. Notably, C5aR1 blockade inhibited PDAC growth (Figs. 3 K and 3 L), demonstrating that disrupting MDSCs recruitment counteracts stress-induced tumor progression. SNS Regulates C5a Production via NE/ADRB1 Signaling Chronic stress activates SNS to release catecholamines into circulation, modulating systemic physiological responses [ 3 , 32 ]. Serum NE levels were significantly elevated in CUMS mice compared to controls (Fig. 4 A). NE exerts its effects via adrenergic receptors, which contain three subtypes, including β (β1,β2,β3), α1(α1a, α1b, α1d), and α2 (α2a, α2b, α2c) receptors [ 33 ]. Among these, CUMS selectively upregulated hepatic ADRB1 mRNA (Fig. 4 B) without altering other AR subtypes (Figs. 4 C– 4 J). To interrogate ADRB1’s role in stress-induced complement synthesis, we administered the ADRB1 antagonist atenolol (10 mg/kg/day). Atenolol attenuated CUMS-driven hepatic C5 mRNA expression (Fig. 4 K) and reduced serum/tumoral C5a levels (Figs. 4 L and 4 M). Mechanistically, chronic stress triggers SNS-derived NE to activate hepatocyte ADRB1, thereby stimulating C5 synthesis. Crucially, atenolol reversed CUMS-induced MDSCs accumulation in tumors (Figs. 4 N and 4 O) and spleens (Figs. 4 P and 4 Q), concomitant with reduced tumor burden (Figs. 4 R and 4 S). These results establish the NE/ADRB1 axis as a hepatic gateway for stress-mediated complement-MDSCs crosstalk. Chronic Stress Potentiates MDSCs Immunosuppression MDSCs orchestrate immunosuppressive TMEs to facilitate immune evasion. While we demonstrated stress-driven MDSCs expansion, their functional reprogramming remained unexplored. Transcriptomic profiling of tumor-infiltrating MDSCs revealed 560 differentially expressed genes in CUMS mice (260 upregulated, 300 downregulated; Figs. 5 A– 5 C). Gene Ontology (GO) enrichment highlighted immune response regulation and complement receptor activity (Fig. 5 D), while KEGG pathway analysis implicated extracellular matrix interactions and PD-L1/PD-1 signaling (Fig. 5 E). Targeted screening identified upregulated immunosuppressive effectors: CD274 (encoding PD-L1) and TGF-β1 (Fig. 5 F). qRT-PCR confirmed elevated PD-L1 and TGF-β1 mRNA in CUMS-derived MDSCs (Figs. 5 G and 5 H). Functional validation using TGF-β1 receptor inhibitor SB-431542 and PD-L1 antibody (Fig. 5 I) significantly attenuated tumor growth (Fig. 5 J). These findings mechanistically link chronic stress to enhanced MDSCs immunosuppression via PD-L1 and TGF-β1 pathways. Discussion The landscape of neuroimmunology research on PDAC has dramatically evolved over the past decade. Here, we elucidate a novel sympathetic/complement axis through which chronic stress drives PDAC progression. Under systemic chronic stress, SNS-derived NE activates hepatic ADRB1 signaling, upregulating the complement protein C5a in both circulation and the PDAC TME. Concurrently, chronic stress elevates systemic NE levels and splenic MDSCs proportions. As previously reported, C5a recruits MDSCs to infiltrate tumors [ 24 ].Our mechanistic investigations reveal that stress-reprogrammed MDSCs exhibit dual immunosuppressive functions: (1) direct suppression of CD8 + T cell cytotoxicity via PD-L1 overexpression and (2) TME remodeling through TGF-β1 secretion. This dual mechanism synergistically enhances immune evasion and accelerates PDAC progression (Fig. 6 ). Previous studies have demonstrated that chronic stress can directly promote PDAC progression by activating the SNS to release NE [ 9 ]. Concurrently, researchers observed substantial infiltration of immature myeloid-derived immune cells adjacent to tumor tissues. MDSCs, critical immunosuppressive components within the TME, have been robustly associated with poor prognosis in PDAC [ 34 ]. These cells accelerate cancer progression and undermine the efficacy of immunotherapies through molecular mechanisms including establishing immunosuppressive niches, facilitating tumor angiogenesis, mediating metastatic dissemination, and inducing therapy resistance [ 35 ]. Although MDSCs express high levels of β-adrenergic receptors, the molecular mechanisms underlying SNS-mediated regulation of MDSCs remain poorly understood. Notably, our CUMS model significantly enhanced MDSCs infiltration in both the spleen and pancreatic TME, indicating that the SNS drives systemic MDSCs expansion and tumor-specific recruitment. These findings suggest that the SNS indirectly promotes PDAC progression by modulating immunosuppressive properties within the TME. However, the number and behaviors of MDSCs obtained from stressed models showed no significantly differences compared with their untreated littermates[ 15 , 17 ].Catecholamine-driven MDSCs mobilization and immunosuppressive potentiation occur exclusively in tumor-bearing or chronic inflammatory contexts [ 36 ]. These observations suggest that SNS-mediated regulation of MDSCs may involve alternative pathways. Although complement activation has been implicated in modulating MDSCs dynamics [ 37 ], the contextual triggers of this regulation remain elusive. Chronic stress provides a physiological stimulus that pathologically activates the SNS, leading to systemic NE overproduction and subsequent physiological perturbations [ 32 , 38 ]. Our findings corroborate this paradigm by demonstrating a temporally synchronized triad: elevated serum NE levels, upregulated hepatic ADRB1 mRNA expression, and increased C5a concentrations in both circulation and tumors. Critically, pharmacological disruption of the NE/ADRB1 axis via atenolol abrogated these correlations, markedly reducing hepatic complement production. This confirms a direct mechanistic link between chronic stress-induced sympathetic hyperactivity and hepatic complement synthesis. C5a, a central complement component, promotes tumor progression through multiple mechanisms, including fostering immunosuppressive TMEs, inducing angiogenesis, and enhancing cancer cell motility [ 37 , 39 ]. Studies demonstrate that C5a recruits MDSCs into the TME, where they impair cytotoxic T cell responses via inhibitory reactive nitrogen/oxygen species, thereby fueling tumor proliferation [ 24 , 40 ]. In our chronic stress model, serum and intratumoral C5a levels exhibited a positive correlation with MDSCs infiltration. Pharmacological blockade of C5aR1 with PMX-50 significantly attenuated MDSCs recruitment and suppressed tumor growth [ 41 , 42 ]. Although broader immune cell interactions require systematic evaluation, these data establish the C5a/C5aR1 axis as a pivotal pathway through which chronic stress reprograms the immunological landscape of PDAC TME. SNS modulates systemic distribution and local infiltration of MDSCs via neurotransmitter-receptor interactions, yet its functional impact on MDSCs remains largely unknown. After establishing that chronic stress recruits MDSCs into PDAC through SNS-driven complement activation, we focused on functional reprogramming of TME-resident MDSCs. High-throughput comparative profiling revealed that MDSCs isolated from CUMS tumors exhibited upregulated expression of the immunosuppressive mediators PD-L1 and TGF-β1 . The PD-1/PD-L1 axis, a master regulator of immune homeostasis, enables tumor immune evasion by suppressing T cell activation [ 43 ]. MDSCs-derived PD-L1 engages PD-1 on T cells, inducing anergy and apoptosis [ 44 , 45 ]. TGF-β1 further reinforces the immunosuppressive TME by promoting metastatic progression, stromal remodeling, and epithelial-mesenchymal transition [ 46 , 47 ]. Therapeutic blockade of PD-L1 or TGF-β1 signaling significantly impeded tumor progression, suggesting that dual inhibition of these pathways could enhance antitumor immunity and elicit durable clinical responses under chronic stress [ 48 , 49 ]. These findings implicate the SNS in directly potentiating MDSCs immunosuppressive functions to reshape the TME. Future studies must validate this paradigm in human PDAC specimens to inform diagnostic and therapeutic innovations. In summary, our findings demonstrate that chronic stress exacerbates PDAC progression by augmenting TME immunosuppression. Hyperactivated SNS elevate circulating NE levels, which bind to upregulated β1-adrenergic receptors on hepatocytes, triggering activation of the complement system, a critical component of the innate immune response. The resultant surge in C5a within both circulation and PDAC tumors drives marked expansion of MDSCs in the spleen and TME, accelerating tumor growth, a phenomenon reversible by β1-AR or C5aR1 inhibitors. Beyond numerical expansion, chronic stress functionally reprograms infiltrating MDSCs to overexpress PD-L1 and TGF-β1 , thereby amplifying immune suppression and tumor cell survival. These results systematically delineate the neuro-immune-TME axis underlying stress-accelerated PDAC pathogenesis. Therapeutic targeting of NE/C5a-recruited MDSCs may represent a novel strategy to enhance PDAC treatment efficacy. Declarations Authors’contributions S.Y.Y., Z.F., and H.D. conceived and designed the study, performed experiments, interpreted data, and wrote the manuscript. L.X. assisted with flow cytometry analysis and cell culture. Q.W.Y., S.Y.X., M.M.W., Y.J.D., J.P.H., and L.Z. conducted acquisition and quantitative analysis of immunohistochemical images. L.X. and Z.M.C. participated in RNA-Seq experiments. P.Q.Q., R.S.H., R.J.W., and Z.Z.Y. were involved in drug treatment procedures. P.Q.Q., L.X., and Z.Q.L. contributed to manuscript preparation and revision. Z.F. and H.D. supervised the project, including research design, data interpretation, and manuscript composition. Funding This work was supported by the National Science Foundation of China, No.82160546 and 82460116; the Science Foundation of Jiangxi Province, No. 20202BBG73027 and 20242BAB26116; the Foundation of Jiangxi Province for Distinguished Scholars No. JXSQ2023201020. Data availability Data are available upon reasonable request, contact [email protected] . Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Rehabilitation Hospital, Jiangxi Medical College, Nanchang University (Approval No. SFYYXLL-PJ-2021-KY005). The present research adheres to the ARRIVE guidelines. Consent for publication Not applicable. Contributor Information Huan Deng, M. D, Ph. D, [email protected] Zhen Feng, M. D, Ph. D, [email protected] References Siegel, R.L., A.N. Giaquinto, and A. Jemal, Cancer statistics, 2024. CA Cancer J Clin, 2024. 74 (1): p. 12-49. Falcomatà, C., et al., Context-Specific Determinants of the Immunosuppressive Tumor Microenvironment in Pancreatic Cancer. Cancer Discov, 2023. 13 (2): p. 278-297. Kim-Fuchs, C., et al., Chronic stress accelerates pancreatic cancer growth and invasion: a critical role for beta-adrenergic signaling in the pancreatic microenvironment. Brain Behav Immun, 2014. 40 : p. 40-7. 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Faulkner, S., et al., Tumor Neurobiology and the War of Nerves in Cancer. Cancer Discov, 2019. 9 (6): p. 702-710. Corrales, L., et al., Anaphylatoxin C5a creates a favorable microenvironment for lung cancer progression. J Immunol, 2012. 189 (9): p. 4674-83. Ostrand-Rosenberg, S., Cancer and complement. Nat Biotechnol, 2008. 26 (12): p. 1348-9. Han, X., et al., Tumor-Derived Tissue Factor Aberrantly Activates Complement and Facilitates Lung Tumor Progression via Recruitment of Myeloid-Derived Suppressor Cells. Int J Mol Sci, 2017. 18 (1). Ajona, D., et al., A Combined PD-1/C5a Blockade Synergistically Protects against Lung Cancer Growth and Metastasis. Cancer Discov, 2017. 7 (7): p. 694-703. Sun, C., R. Mezzadra, and T.N. Schumacher, Regulation and Function of the PD-L1 Checkpoint. Immunity, 2018. 48 (3): p. 434-452. Noman, M.Z., et al., PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med, 2014. 211 (5): p. 781-90. Lu, C., et al., The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells. Oncoimmunology, 2016. 5 (12): p. e1247135. Derynck, R., S.J. Turley, and R.J. Akhurst, TGFβ biology in cancer progression and immunotherapy. Nat Rev Clin Oncol, 2021. 18 (1): p. 9-34. Xue, V.W., et al., Transforming Growth Factor-β: A Multifunctional Regulator of Cancer Immunity. Cancers (Basel), 2020. 12 (11). Akinleye, A. and Z. Rasool, Immune checkpoint inhibitors of PD-L1 as cancer therapeutics. J Hematol Oncol, 2019. 12 (1): p. 92. Peng, H., et al., Local Release of TGF-β Inhibitor Modulates Tumor-Associated Neutrophils and Enhances Pancreatic Cancer Response to Combined Irreversible Electroporation and Immunotherapy. Adv Sci (Weinh), 2022. 9 (10): p. e2105240. Additional Declarations No competing interests reported. Supplementary Files TableS1.xls Cite Share Download PDF Status: Published Journal Publication published 19 Dec, 2025 Read the published version in Cancer Immunology, Immunotherapy → Version 1 posted Editorial decision: Revision requested 20 Sep, 2025 Reviews received at journal 20 Sep, 2025 Reviews received at journal 18 Sep, 2025 Reviewers agreed at journal 08 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 07 Sep, 2025 Reviewers agreed at journal 05 Sep, 2025 Reviewers agreed at journal 05 Sep, 2025 Reviewers agreed at journal 05 Sep, 2025 Reviewers invited by journal 03 Sep, 2025 Editor assigned by journal 28 Aug, 2025 Submission checks completed at journal 28 Aug, 2025 First submitted to journal 27 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7471626","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511600412,"identity":"c3a36e0b-5ae4-4c9b-81d5-6e408d4ebff9","order_by":0,"name":"siyuan yang","email":"","orcid":"","institution":"Henan Polytechnic University","correspondingAuthor":false,"prefix":"","firstName":"siyuan","middleName":"","lastName":"yang","suffix":""},{"id":511600413,"identity":"67f42567-9b1c-45e7-b52f-f37182080938","order_by":1,"name":"Li Xiao","email":"","orcid":"","institution":"West China Hospital of Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Xiao","suffix":""},{"id":511600414,"identity":"4d016f65-af4b-4f76-98af-fdb08d5b307f","order_by":2,"name":"Pingqian Qi","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Pingqian","middleName":"","lastName":"Qi","suffix":""},{"id":511600415,"identity":"19ec49f0-4cb5-4bd3-b3a4-859cf7f18118","order_by":3,"name":"Ziqing Liu","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Ziqing","middleName":"","lastName":"Liu","suffix":""},{"id":511600416,"identity":"81408541-ede2-442d-89f3-92e7bc16423e","order_by":4,"name":"Zhuomin Chen","email":"","orcid":"","institution":"Nanchang 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University","correspondingAuthor":false,"prefix":"","firstName":"Mengmeng","middleName":"","lastName":"Wang","suffix":""},{"id":511600423,"identity":"5942af59-5974-4cd0-a616-85a75bd3c53a","order_by":11,"name":"Yanjuan Deng","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Yanjuan","middleName":"","lastName":"Deng","suffix":""},{"id":511600424,"identity":"a54296b3-73a8-40e4-86a2-18c1610920e1","order_by":12,"name":"Jinping Hu","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Jinping","middleName":"","lastName":"Hu","suffix":""},{"id":511600425,"identity":"789442a9-e69b-42d7-9ef7-aff4f16d7cfa","order_by":13,"name":"Lv Zhou","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Lv","middleName":"","lastName":"Zhou","suffix":""},{"id":511600426,"identity":"0673aa2c-660d-4e42-8775-26690a3e1474","order_by":14,"name":"Zhen Feng","email":"","orcid":"","institution":"Nanchang University","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Feng","suffix":""},{"id":511600427,"identity":"9123bb69-1c11-4b4c-b69c-433d76d9acc4","order_by":15,"name":"Huan Deng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYBACAxDBA8T8ED4zCVokG0jWYnCAWC3m7M3PHrypuGO3+XbzMwmGCuvEBvazB/Bqsew5Zm4458yz5G13jplJMJxJT2zgyUvA77AbCWbSvG2Hk81u5LBJMLYdTmyQ4DHAr+X+82/SvP8OJxvPAGn5R4yWGzxAWxoO2xlIgLQ0EKHFsienTHLOscMJEjfSjC0SjqUbt/Hk4Ndizn58m8SbmsP2/DOSH974UGMt289+Br8WGEhsAJEJQMxGlHogsCdW4SgYBaNgFIxAAAB9BkPlK5JlMwAAAABJRU5ErkJggg==","orcid":"","institution":"Nanchang University","correspondingAuthor":true,"prefix":"","firstName":"Huan","middleName":"","lastName":"Deng","suffix":""}],"badges":[],"createdAt":"2025-08-27 12:23:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7471626/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7471626/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00262-025-04249-z","type":"published","date":"2025-12-19T15:57:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90912709,"identity":"41210f83-510c-480d-8a1a-41ec474d0aca","added_by":"auto","created_at":"2025-09-09 13:52:23","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1106723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChronic stress promotes PDAC growth.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic diagram of PDAC cell line inoculation, CUMS exposure, and behavioral testing protocol.\u003c/p\u003e\n\u003cp\u003e(B) Movement trajectories of control group (n=11 per group) and CUMS group (n=11 per group) mice in the open field test.\u003c/p\u003e\n\u003cp\u003e(C) CUMS intervention did not affect baseline locomotor activity in the open field test.\u003c/p\u003e\n\u003cp\u003e(D-E) CUMS significantly reduced the distance traveled and time spent in the central zone of the open field.\u003c/p\u003e\n\u003cp\u003e(F) Movement trajectories of control group (n=11 per group) and CUMS group (n=11 per group) mice in the elevated plus maze.\u003c/p\u003e\n\u003cp\u003e(G) CUMS intervention did not impair baseline locomotor activity in the elevated plus maze.\u003c/p\u003e\n\u003cp\u003e(H-I) CUMS significantly decreased the exploration distance and time in the open arms of the elevated plus maze.\u003c/p\u003e\n\u003cp\u003e(J) CUMS induced a reduction in body weight (n=6 per group).\u003c/p\u003e\n\u003cp\u003e(K-L) CUMS significantly increased tumor weight (n=6 per group).\u003c/p\u003e\n\u003cp\u003e(M-N) CUMS markedly elevated the Ki-67 positive rate in pancreatic tumor tissues (original magnification ×400) (n=5 per group).\u003c/p\u003e\n\u003cp\u003en.s., not significant; *P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001. n indicates biological replicates.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471626/v1/f683701f711b67285fbc9902.jpg"},{"id":90912711,"identity":"c84d46ad-d2d9-47bd-817f-cc9556713d38","added_by":"auto","created_at":"2025-09-09 13:52:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":441896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChronic stress promotes MDSCs expansion and infiltration.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) FCM analysis revealed an increased proportion of MDSCs in PDAC tumor tissues under CUMS conditions (n=5 per group).\u003c/p\u003e\n\u003cp\u003e(C-D) CUMS elevated the percentage of MDSCs in the spleen (n=5 per group).\u003c/p\u003e\n\u003cp\u003en.s., not significant; *P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001. n indicates biological replicates.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471626/v1/005fb8fb661a36f5ca51de47.jpg"},{"id":90911829,"identity":"6d71c35b-f953-43b6-b6d1-40e2cffe6794","added_by":"auto","created_at":"2025-09-09 13:44:23","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":907395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChronic stress promotes tumor MDSCs recruitment via complement C5a.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A-D) CUMS increased mRNA levels of C3, C5, and CFH in hepatocytes, while C2 mRNA levels remained unchanged (n=4 per group).\u003c/p\u003e\n\u003cp\u003e(E-F) CUMS elevated C5a levels in peripheral blood and tumor tissues (n=4 per group).\u003c/p\u003e\n\u003cp\u003e(G-H) Under chronic stress, the C5aR1 antagonist PMX-50 reduced the percentage of MDSCs in PDAC tumors (n=5 per group).\u003c/p\u003e\n\u003cp\u003e(I-J) PMX-50 decreased the percentage of MDSCs in the spleen under chronic stress (n=5 per group).\u003c/p\u003e\n\u003cp\u003e(K-L) Blockade of C5a signaling significantly reduced tumor weight (n=6 per group).\u003c/p\u003e\n\u003cp\u003en.s., not significant; *P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001. n indicates biological replicates.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471626/v1/f098ad4a7a41948b8edd8fb8.jpg"},{"id":90914196,"identity":"dd31d12b-aa16-4e8f-8490-7ad66a1bf5da","added_by":"auto","created_at":"2025-09-09 14:00:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1088200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSympathetic nerves regulate C5a-mediated MDSCs recruitment via the NE/ADRB1 axis.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) CUMS increased peripheral blood norepinephrine (NE) levels (n=4 per group).\u003c/p\u003e\n\u003cp\u003e(B-G) CUMS significantly upregulated mRNA levels of ADRB1 (one of the nine NE receptor subtypes) in hepatocytes (n=3-4 per group).\u003c/p\u003e\n\u003cp\u003e(K) Under chronic stress, the ADRB1 inhibitor atenolol markedly reduced hepatic C5 mRNA levels (n=6 per group).\u003c/p\u003e\n\u003cp\u003e(L-M) CUMS combined with atenolol attenuated C5a levels in peripheral blood and tumor tissues of CUMS-exposed mice (n=4 per group).\u003c/p\u003e\n\u003cp\u003e(N-Q) Atenolol significantly decreased the percentage of MDSCs in PDAC tumors and the spleen (n=5 per group).\u003c/p\u003e\n\u003cp\u003e(R-S) Chronic stress-induced tumor weight was significantly reduced by atenolol (n=5 per group).\u003c/p\u003e\n\u003cp\u003en.s., not significant; *P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001. n indicates biological replicates.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471626/v1/e4f3d3631ef3a43ec6bcf93b.jpg"},{"id":90911828,"identity":"704392ee-2c8e-4918-ac02-dd7b10d71711","added_by":"auto","created_at":"2025-09-09 13:44:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":926108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChronic stress enhances the immunosuppressive function of MDSCs in the tumor microenvironment.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Bar plot showing upregulated and downregulated differentially expressed genes (DEGs) between control and CUMS groups (n=4 per group).\u003c/p\u003e\n\u003cp\u003e(B) Volcano plot illustrating DEGs (upregulated and downregulated) between control and CUMS groups.\u003c/p\u003e\n\u003cp\u003e(C) Heatmap displaying hierarchical clustering patterns of DEGs between groups.\u003c/p\u003e\n\u003cp\u003e(D) Top 30 Gene Ontology (GO) enrichment terms of DEGs between control and CUMS groups.\u003c/p\u003e\n\u003cp\u003e(E) Top 30 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (bubble plot) of DEGs.\u003c/p\u003e\n\u003cp\u003e(F) Enrichment analysis of \u003cem\u003eCD274\u003c/em\u003e(PD-L1) and \u003cem\u003eTGF-β1\u003c/em\u003e expression between control and CUMS groups.\u003c/p\u003e\n\u003cp\u003e(G-H) CUMS significantly increased PD-L1 and TGF-β1 mRNA levels in tumor-infiltrating MDSCs (n=4 per group).\u003c/p\u003e\n\u003cp\u003e(I-J) The TGF-β1 inhibitor SB-431542 and anti-PD-L1 antibody attenuated chronic stress-induced tumor growth (n=6 per group).\u003c/p\u003e\n\u003cp\u003en.s., not significant; *P \u0026lt; 0.05; ** P \u0026lt; 0.01; *** P \u0026lt; 0.001. n indicates biological replicates.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471626/v1/ba8763439f1bd42155be593e.jpg"},{"id":90911831,"identity":"6b54c9d5-a31b-4630-af37-40cbe2be1a7a","added_by":"auto","created_at":"2025-09-09 13:44:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1343419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eChronic stress promotes PDAC progression via NE/C5a axis-mediated recruitment of MDSCs.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7471626/v1/6eb7dbc919bc4a2d08af6a88.jpg"},{"id":98813843,"identity":"f3c34bf4-6c11-4a93-b4be-0bf4aff845dd","added_by":"auto","created_at":"2025-12-22 16:04:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6937869,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7471626/v1/1e79cda0-a772-4790-b5dc-763952c450f3.pdf"},{"id":90911823,"identity":"a6d7f306-3cf1-4971-b596-d70b7c0d3830","added_by":"auto","created_at":"2025-09-09 13:44:23","extension":"xls","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":21504,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xls","url":"https://assets-eu.researchsquare.com/files/rs-7471626/v1/7db6d2aa6ed93195a5fa65ad.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chronic Stress Promotes Pancreatic Ductal Adenocarcinoma Progression via Complement C5a-Recruited Myeloid-Derived Suppressor Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe complex interplay among tumor microenvironment (TME) components, including parenchymal cells, nerves, and immune cells, is a critical determinant of the poor prognosis of pancreatic ductal adenocarcinoma (PDAC) patients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Chronic stress, a psychological state triggered by persistent and unresolved stressors, has been implicated in PDAC pathogenesis and progression through autonomic nervous system (ANS) dysregulation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The ANS, comprising two major efferent branches (the sympathetic nervous system, SNS, and parasympathetic nervous system, PSNS), not only maintains systemic homeostasis but also increasingly emerges as a key modulator of chronic diseases and tumor progression [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSNS with aberrant density or hyperactivity release high levels of catecholamines, which enhance PDAC cell survival by suppressing antitumor immune responses [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, PSNS exerts dual tumor-suppressive mechanisms via cholinergic signaling in pancreatic tumors: directly downregulating MAPK and PI3K/AKT signaling cascades in cancer cells, and potentially mediating indirect antitumor effects through myeloid cell inhibition [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Paradoxically, however, SNS activation has also been reported to inhibit tumor growth by downregulating CCL2 in macrophages [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This apparent contradiction underscores the complexity of SNS functionality, particularly its interactions with key TME components, including immune cells, stromal cells, and vascular endothelial cells, in shaping tumor behavior.\u003c/p\u003e\u003cp\u003eMyeloid-derived suppressor cells (MDSCs), first introduced by Gabrilovich et al.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], are a heterogeneous population of immunosuppressive cells implicated in the pathological progression from chronic inflammation to cancer metastasis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In the setting of chronic inflammation, MDSCs undergo expansion in the bone marrow and spleen and recruitment to lesion sites, establishing an immunosuppressive TME [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Emerging evidence supports the hypothesis that stress-related sympathetic activation mobilizes MDSCs via catecholamine release. Restraint stress models exhibited MDSCs expansion and accumulation in the bone marrow, peripheral blood, and spleen [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similar findings were observed in tumor-bearing mice subjected to chronic restraint or unpredictable mild stress (CUMS) [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, under repetitive restraint stress, splenic MDSCs numbers were paradoxically reduced [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These data hint that sustained stimulation may activate systemic pathways to modulate MDSCs expansion and spatial distribution.\u003c/p\u003e\u003cp\u003eThe complement system, a pivotal component of the innate immune system, orchestrates host defense against endogenous and exogenous stimuli to maintain physiological homeostasis. Anaphylatoxin C5a, a key bioactive molecule released upon activation of all complement pathways, engages in multiple facets of tumor biology, including immune evasion, progression, and metastasis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. C5a modulates the immune microenvironment to elicit either pro- or anti-tumor responses, contingent upon tumor type and local C5a concentration [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. MDSCs, as critical constituents of the immune microenvironment, exhibit a positive correlation between surface Complement 5a receptor 1 (C5aR1) expression levels and tumor infiltration capacity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This receptor/ligand interaction enables C5a to recruit MDSCs to tumor sites, ultimately driving CD8⁺ T cell functional exhaustion and accelerating tumor progression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile chronic stress is known to facilitate PDAC progression through MDSCs, the molecular mechanisms governing MDSCs expansion, systemic redistribution, and immunosuppressive TME modeling remain poorly characterized. Here we provide evidence for a previously unknown regulatory pathway, driven by the SNS-induced C5a, which is mainly produced by NE/ADRB1 axis-activated hepatic cells and promotes the expansion of MDSCs to infiltrate PDAC and facilitate the establishment of immunosuppressive TME through programmed death-ligand 1 (PD-L1) and transforming growth factor-beta (TGF-β1). Thus, SNS couples chronic stress to intrinsically primed immature immune cells to accelerate the progression of PDAC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eExperimental Animals and Cell line\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale C57BL/6J mice (6 to 8 weeks old) were used for all experiments. Mice were originally purchased from GemPharmatech Co., Ltd. (Nanjing, China) and housed in the Animal Facility of Nanchang University under specific pathogen-free (SPF) conditions. Mice were group-housed (3\u0026ndash;5 per cage) with ad libitum access to food and water, and maintained in a temperature-controlled room (21\u0026ndash;25\u0026deg;C) with 40\u0026ndash;60% humidity under a 12-hour light/dark cycle (lights on: 6:00 AM to 6:00 PM). Murine pancreatic cancer Pan02 cells were obtained from Fuheng Biotechnology (Shanghai, China) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco) at 37\u0026deg;C in a humidified 5% CO2 incubator. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Rehabilitation Hospital, Jiangxi Medical College, Nanchang University (Approval No. SFYYXLL-PJ-2021-KY005).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOrthotopic Transplantation Model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOrthotopic PDAC tumors were generated by surgical implantation of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e Pan02 cells into the pancreas of male C57BL/6 mice as previously described [25]. Briefly, mice were anesthetized via intraperitoneal injection of Zoletil-50 and placed in a sterile surgical field. A small left abdominal incision was made to expose the pancreas, followed by orthotopic injection of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e Pan02 cells suspended in 100 \u0026mu;L phosphate-buffered saline (PBS) into the pancreatic head region. A transient tissue bulge confirmed successful intrapancreatic delivery. The needle was withdrawn slowly to minimize cell leakage, and the incision was closed with surgical sutures.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEstablishment of CUMS Model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CUMS protocol, a widely used chronic stress paradigm [26], was initiated 3 days post-tumor cell inoculation. Mice received 2\u0026ndash;3 randomized stressors daily, including restraint, cage tilt (45\u0026deg;), tail suspension, water/food deprivation, circadian rhythm disruption, or white noise exposure. Stressor types and timing were randomized using a non-repetitive schedule (minimum 7-day intervals between identical stressors) over a 14-day period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOpen Field Test (OFT)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBehavioral analysis was conducted in a 40 \u0026times; 40 \u0026times; 35 cm open-field arena. Mice were individually placed at the arena center, and locomotion was recorded for 10 min using ANY-maze\u0026trade; software (Stoelting Co.). The central zone was defined as the innermost 25% of total area. Total distance moved, central zone dwell time, and central zone path length were quantified.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eElevated Plus Maze (EPM) Test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EPM apparatus consisted of two open arms (28 \u0026times; 5 cm) and two enclosed arms (28 \u0026times; 5 \u0026times; 15 cm) elevated 80 cm above ground. Mice were positioned in the central quadrant facing an open arm, and exploratory behavior was video-tracked for 10 min. Metrics included total distance traveled, open arm residence time, and open arm path length.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePharmacological Interventions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor-bearing mice were randomly assigned to treatment groups. Starting 3 days post-implantation, the following agents were administered daily via intraperitoneal injection: Atenolol (\u0026beta;1-adrenergic receptor antagonist, 10 mg/kg/day; MedChemExpress, USA), PMX-50 (C5aR1 antagonist, 10 mg/kg/day; MCE, USA), and SB-431542 (TGF-\u0026beta; receptor inhibitor, 30 mg/kg/day; MCE, USA); For PD-L1 blockade, anti-PD-L1 antibody (10 mg/kg; MCE, USA) was injected twice weekly for 2 weeks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHepatocyte Isolation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing anesthesia with Zoletil-50 (Virbac),An indwelling needle\u0026nbsp;was inserted into the portal vein for sequential perfusion with Liver Perfusion Buffer (Thermo Fisher, USA) and Liver Digest Medium (Thermo Fisher, USA). The distended liver was excised, and the Glisson\u0026rsquo;s capsule was mechanically disrupted. Hepatocytes were released through 70 \u0026mu;m nylon filters and pelleted via low-speed centrifugation (50 \u0026times; g, 5 min).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol\u0026trade; \u0026nbsp;(Thermo Fisher, USA) and reverse-transcribed with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). qPCR was performed on a StepOnePlus\u0026trade; system (Applied Biosystems) using Hieff UNICON\u0026reg; SYBR Green Master Mix (Yeasen, China). Primer sequences are listed in Supplementary Table S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFlow Cytometry(FCM)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSingle-cell suspensions from spleen/tumor tissues were prepared as follows:Spleen: Mechanically dissociated through 70 \u0026mu;m filters, lysed with RBC Lysis Buffer (BioLegend, USA).Tumor: Collagenase IV (1 mg/ml; Beyotime, China)-digested fragments filtered through 70 \u0026mu;m mesh Cells were stained with: CD16/32 (Clone 2.4G2; BD Biosciences, USA), CD45-BV421 (Clone 30-F11; BioLegend, USA), CD11b-FITC (Clone M1/70; BD Biosciences, USA), Ly-6G/Ly-6C-PE (Clone RB6-8C5; BD Biosciences, USA), and 7-AAD viability dye (BD Biosciences, USA). Data acquisition used a CytoFLEX LX (Beckman Coulter), with analysis via FlowJo (v10.6.2). Cell sorting employed a CytoFLEX SRT system.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum was isolated via centrifugation (3,000 \u0026times; g, 15 min) after 1-hour clotting. Tissue lysates were prepared in RIPA buffer (Beyotime, China) with protease inhibitors. C5a (Novus Biologicals, NBP2-67681, USA) and NE (Elabscience, E-EL-0042c, China) levels were quantified according to manufacturer protocols.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunohistochemistry\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned (5 \u0026mu;m). After antigen retrieval (pH 6.0 citrate buffer) and peroxidase quenching, sections were blocked with 5% goat serum and incubated with Ki-67 antibody (ZM-0166; ZSGB-BIO) overnight. DAB chromogen (Dako) and hematoxylin counterstaining were applied. Ki-67\u003csup\u003e+\u003c/sup\u003e cells were quantified using ImageJ (\u0026ge; 3 fields/section).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTranscriptome Sequencing\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDSCs sorted from PDAC tissues were lysed in TRIzol\u0026reg;. RNA integrity (RIN \u0026gt; 8.0) was confirmed via Agilent 2100 Bioanalyzer. Libraries were prepared with VAHTS Universal V6 RNA-seq Library Kit (Vazyme) and sequenced (150 bp paired-end) on NovaSeq 6000 (Illumina). Raw reads were processed using fastp (v0.23.2) and aligned to mm10 genome.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as mean \u0026plusmn; SEM. Two-group comparisons used Student\u0026rsquo;s t-test. Multi-group analyses employed one-way ANOVA with Tukey\u0026rsquo;s post-hoc test (GraphPad Prism 9.5). P \u0026lt; 0.05 defined statistical significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eChronic Stress Promotes PDAC Progression\u003c/h2\u003e\u003cp\u003eTo investigate the role of chronic stress in PDAC progression, we established a CUMS model in orthotopic PDAC-bearing mice. Following a 3-day postoperative recovery period after tumor cell implantation, mice were subjected to intermittent stressors for 2 weeks prior to behavioral assessments and endpoint analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Anxiety-like behaviors were evaluated using the open field test (OFT) and elevated plus maze (EPM).\u003c/p\u003e\u003cp\u003eIn the OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), no significant difference in total locomotor distance was observed between control and CUMS groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating unimpaired basal motor function. However, CUMS mice exhibited markedly reduced path length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) and dwell time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) in the central zone compared to controls. Similarly, EPM analysis revealed comparable total movement distances between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), but CUMS mice demonstrated significantly decreased exploratory activity in open arms and central quadrants, as evidenced by reduced path length (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and residence time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These data confirm CUMS-induced anxiety-like behaviors in PDAC-bearing mice.\u003c/p\u003e\u003cp\u003eChronic stress is known to suppress appetite in rodents [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Body weight measurements every 4 days revealed progressive weight loss in CUMS mice relative to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ), validating the stress model. Meanwhile, CUMS-exposed mice harbored significantly heavier pancreatic tumors compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). Immunohistochemical analysis further demonstrated elevated proliferation indices in CUMS tumors, with a pronounced increase in Ki-67\u0026thinsp;+\u0026thinsp;cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN). Collectively, these findings indicate that chronic stress exacerbates PDAC growth by enhancing tumor cell proliferation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eChronic Stress Drives MDSCs Expansion and Infiltration\u003c/h2\u003e\u003cp\u003eChronic stress promotes tumor progression through both direct SNS-mediated modulation of malignant phenotypes and indirect TME remodeling. MDSCs, core immunosuppressive effectors expressing SNS neurotransmitter receptors, orchestrate multi-layered immune suppression by inhibiting effector T cells and facilitating regulatory T cell differentiation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Given that stress-related sympathetic activation primarily targets β-adrenergic receptor (β-AR)-expressing MDSCs, we quantified tumor-infiltrating CD11b\u0026thinsp;+\u0026thinsp;Gr1\u0026thinsp;+\u0026thinsp;MDSCs via flow cytometry in CUMS-treated PDAC mice. CUMS significantly increased MDSCs proportions within PDAC (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and spleens (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting systemic MDSCs expansion coupled with enhanced tumor trafficking.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eComplement C5a Mediates MDSCs Recruitment\u003c/h2\u003e\u003cp\u003eSolid tumors often hijack the complement system to establish immunosuppressive niches favoring tumor survival [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. As the liver produces up to 90% of circulating complement components [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], we analyzed hepatic complement expression in CUMS mice. qRT-PCR revealed no change in C2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) but significant upregulation of pro-tumorigenic complement factors C3, C5, and CFH (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), indicating stress-induced hepatic reprogramming toward complement synthesis.\u003c/p\u003e\u003cp\u003eAmong these factors, C5-derived C5a critically recruits MDSCs via binding to C5aR1 on their surface [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. CUMS elevated C5a levels in both serum and tumor tissues (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). To explore C5a\u0026rsquo;s role in stress-driven MDSCs dynamics, we administered the C5aR1 antagonist PMX-50, which attenuated CUMS-induced MDSCs accumulation in tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) and spleens (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ), confirming C5a/C5aR1 axis dependency. Notably, C5aR1 blockade inhibited PDAC growth (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL), demonstrating that disrupting MDSCs recruitment counteracts stress-induced tumor progression.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSNS Regulates C5a Production via NE/ADRB1 Signaling\u003c/h2\u003e\u003cp\u003eChronic stress activates SNS to release catecholamines into circulation, modulating systemic physiological responses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Serum NE levels were significantly elevated in CUMS mice compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). NE exerts its effects via adrenergic receptors, which contain three subtypes, including β (β1,β2,β3), α1(α1a, α1b, α1d), and α2 (α2a, α2b, α2c) receptors [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Among these, CUMS selectively upregulated hepatic ADRB1 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) without altering other AR subtypes (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ).\u003c/p\u003e\u003cp\u003eTo interrogate ADRB1\u0026rsquo;s role in stress-induced complement synthesis, we administered the ADRB1 antagonist atenolol (10 mg/kg/day). Atenolol attenuated CUMS-driven hepatic C5 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK) and reduced serum/tumoral C5a levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). Mechanistically, chronic stress triggers SNS-derived NE to activate hepatocyte ADRB1, thereby stimulating C5 synthesis. Crucially, atenolol reversed CUMS-induced MDSCs accumulation in tumors (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eN and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eO) and spleens (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eP and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eQ), concomitant with reduced tumor burden (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eR and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eS). These results establish the NE/ADRB1 axis as a hepatic gateway for stress-mediated complement-MDSCs crosstalk.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eChronic Stress Potentiates MDSCs Immunosuppression\u003c/h2\u003e\u003cp\u003eMDSCs orchestrate immunosuppressive TMEs to facilitate immune evasion. While we demonstrated stress-driven MDSCs expansion, their functional reprogramming remained unexplored. Transcriptomic profiling of tumor-infiltrating MDSCs revealed 560 differentially expressed genes in CUMS mice (260 upregulated, 300 downregulated; Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Gene Ontology (GO) enrichment highlighted immune response regulation and complement receptor activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), while KEGG pathway analysis implicated extracellular matrix interactions and PD-L1/PD-1 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eTargeted screening identified upregulated immunosuppressive effectors: CD274 (encoding PD-L1) and TGF-β1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). qRT-PCR confirmed elevated PD-L1 and TGF-β1 mRNA in CUMS-derived MDSCs (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Functional validation using TGF-β1 receptor inhibitor SB-431542 and PD-L1 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI) significantly attenuated tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). These findings mechanistically link chronic stress to enhanced MDSCs immunosuppression via PD-L1 and TGF-β1 pathways.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe landscape of neuroimmunology research on PDAC has dramatically evolved over the past decade. Here, we elucidate a novel sympathetic/complement axis through which chronic stress drives PDAC progression. Under systemic chronic stress, SNS-derived NE activates hepatic ADRB1 signaling, upregulating the complement protein C5a in both circulation and the PDAC TME. Concurrently, chronic stress elevates systemic NE levels and splenic MDSCs proportions. As previously reported, C5a recruits MDSCs to infiltrate tumors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].Our mechanistic investigations reveal that stress-reprogrammed MDSCs exhibit dual immunosuppressive functions: (1) direct suppression of CD8\u003csup\u003e+\u003c/sup\u003e T cell cytotoxicity via PD-L1 overexpression and (2) TME remodeling through TGF-β1 secretion. This dual mechanism synergistically enhances immune evasion and accelerates PDAC progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated that chronic stress can directly promote PDAC progression by activating the SNS to release NE [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Concurrently, researchers observed substantial infiltration of immature myeloid-derived immune cells adjacent to tumor tissues. MDSCs, critical immunosuppressive components within the TME, have been robustly associated with poor prognosis in PDAC [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These cells accelerate cancer progression and undermine the efficacy of immunotherapies through molecular mechanisms including establishing immunosuppressive niches, facilitating tumor angiogenesis, mediating metastatic dissemination, and inducing therapy resistance [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Although MDSCs express high levels of β-adrenergic receptors, the molecular mechanisms underlying SNS-mediated regulation of MDSCs remain poorly understood. Notably, our CUMS model significantly enhanced MDSCs infiltration in both the spleen and pancreatic TME, indicating that the SNS drives systemic MDSCs expansion and tumor-specific recruitment. These findings suggest that the SNS indirectly promotes PDAC progression by modulating immunosuppressive properties within the TME.\u003c/p\u003e\u003cp\u003eHowever, the number and behaviors of MDSCs obtained from stressed models showed no significantly differences compared with their untreated littermates[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].Catecholamine-driven MDSCs mobilization and immunosuppressive potentiation occur exclusively in tumor-bearing or chronic inflammatory contexts [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These observations suggest that SNS-mediated regulation of MDSCs may involve alternative pathways. Although complement activation has been implicated in modulating MDSCs dynamics [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], the contextual triggers of this regulation remain elusive. Chronic stress provides a physiological stimulus that pathologically activates the SNS, leading to systemic NE overproduction and subsequent physiological perturbations [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our findings corroborate this paradigm by demonstrating a temporally synchronized triad: elevated serum NE levels, upregulated hepatic ADRB1 mRNA expression, and increased C5a concentrations in both circulation and tumors. Critically, pharmacological disruption of the NE/ADRB1 axis via atenolol abrogated these correlations, markedly reducing hepatic complement production. This confirms a direct mechanistic link between chronic stress-induced sympathetic hyperactivity and hepatic complement synthesis.\u003c/p\u003e\u003cp\u003eC5a, a central complement component, promotes tumor progression through multiple mechanisms, including fostering immunosuppressive TMEs, inducing angiogenesis, and enhancing cancer cell motility [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Studies demonstrate that C5a recruits MDSCs into the TME, where they impair cytotoxic T cell responses via inhibitory reactive nitrogen/oxygen species, thereby fueling tumor proliferation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In our chronic stress model, serum and intratumoral C5a levels exhibited a positive correlation with MDSCs infiltration. Pharmacological blockade of C5aR1 with PMX-50 significantly attenuated MDSCs recruitment and suppressed tumor growth [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Although broader immune cell interactions require systematic evaluation, these data establish the C5a/C5aR1 axis as a pivotal pathway through which chronic stress reprograms the immunological landscape of PDAC TME.\u003c/p\u003e\u003cp\u003eSNS modulates systemic distribution and local infiltration of MDSCs via neurotransmitter-receptor interactions, yet its functional impact on MDSCs remains largely unknown. After establishing that chronic stress recruits MDSCs into PDAC through SNS-driven complement activation, we focused on functional reprogramming of TME-resident MDSCs. High-throughput comparative profiling revealed that MDSCs isolated from CUMS tumors exhibited upregulated expression of the immunosuppressive mediators \u003cem\u003ePD-L1\u003c/em\u003e and \u003cem\u003eTGF-β1\u003c/em\u003e. The PD-1/PD-L1 axis, a master regulator of immune homeostasis, enables tumor immune evasion by suppressing T cell activation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. MDSCs-derived PD-L1 engages PD-1 on T cells, inducing anergy and apoptosis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. TGF-β1 further reinforces the immunosuppressive TME by promoting metastatic progression, stromal remodeling, and epithelial-mesenchymal transition [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therapeutic blockade of PD-L1 or TGF-β1 signaling significantly impeded tumor progression, suggesting that dual inhibition of these pathways could enhance antitumor immunity and elicit durable clinical responses under chronic stress [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. These findings implicate the SNS in directly potentiating MDSCs immunosuppressive functions to reshape the TME. Future studies must validate this paradigm in human PDAC specimens to inform diagnostic and therapeutic innovations.\u003c/p\u003e\u003cp\u003eIn summary, our findings demonstrate that chronic stress exacerbates PDAC progression by augmenting TME immunosuppression. Hyperactivated SNS elevate circulating NE levels, which bind to upregulated β1-adrenergic receptors on hepatocytes, triggering activation of the complement system, a critical component of the innate immune response. The resultant surge in C5a within both circulation and PDAC tumors drives marked expansion of MDSCs in the spleen and TME, accelerating tumor growth, a phenomenon reversible by β1-AR or C5aR1 inhibitors. Beyond numerical expansion, chronic stress functionally reprograms infiltrating MDSCs to overexpress \u003cem\u003ePD-L1\u003c/em\u003e and \u003cem\u003eTGF-β1\u003c/em\u003e, thereby amplifying immune suppression and tumor cell survival. These results systematically delineate the neuro-immune-TME axis underlying stress-accelerated PDAC pathogenesis. Therapeutic targeting of NE/C5a-recruited MDSCs may represent a novel strategy to enhance PDAC treatment efficacy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026rsquo;contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.Y.Y., Z.F., and H.D. conceived and designed the study, performed experiments, interpreted data, and wrote the manuscript. L.X. assisted with flow cytometry analysis and cell culture. Q.W.Y., S.Y.X., M.M.W., Y.J.D., J.P.H., and L.Z. conducted acquisition and quantitative analysis of immunohistochemical images. L.X. and Z.M.C. participated in RNA-Seq experiments. P.Q.Q., R.S.H., R.J.W., and Z.Z.Y. were involved in drug treatment procedures. P.Q.Q., L.X., and Z.Q.L. contributed to manuscript preparation and revision. Z.F. and H.D. supervised the project, including research design, data interpretation, and manuscript composition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science Foundation of China, No.82160546 and 82460116; the Science Foundation of Jiangxi Province, No. 20202BBG73027 and 20242BAB26116; the Foundation of Jiangxi Province for Distinguished Scholars No. JXSQ2023201020.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available upon reasonable request, contact [email protected].\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Rehabilitation Hospital, Jiangxi Medical College, Nanchang University (Approval No. SFYYXLL-PJ-2021-KY005). The present research adheres to the ARRIVE guidelines.\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eContributor Information\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuan Deng, M. D, Ph. D, [email protected]\u003c/p\u003e\n\u003cp\u003eZhen Feng, M. D, Ph. D, [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel, R.L., A.N. Giaquinto, and A. 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Rasool, \u003cem\u003eImmune checkpoint inhibitors of PD-L1 as cancer therapeutics.\u003c/em\u003e J Hematol Oncol, 2019. \u003cstrong\u003e12\u003c/strong\u003e(1): p. 92.\u003c/li\u003e\n\u003cli\u003ePeng, H., et al., \u003cem\u003eLocal Release of TGF-\u0026beta; Inhibitor Modulates Tumor-Associated Neutrophils and Enhances Pancreatic Cancer Response to Combined Irreversible Electroporation and Immunotherapy.\u003c/em\u003e Adv Sci (Weinh), 2022. \u003cstrong\u003e9\u003c/strong\u003e(10): p. e2105240.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chronic stress, Sympathetic nervous system, Complement system, Pancreatic ductal adenocarcinoma, Myeloid-derived suppressor cells","lastPublishedDoi":"10.21203/rs.3.rs-7471626/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7471626/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChronic stress is recognized as one of the risk factors for pancreatic ductal adenocarcinoma (PDAC). The autonomic nervous system (ANS), a critical component of the tumor microenvironment (TME), has been closely associated with PDAC tumorigenesis and prognosis, though the underlying mechanisms remain elusive. Here, we demonstrate that aberrant activation of sympathetic nerves (SNS) upregulates the hepatic complement system, thereby recruiting myeloid-derived suppressor cells (MDSCs) into pancreatic malignancy to promote tumor progression. Chronic stress induces SNS-derived release of the neurotransmitter norepinephrine (NE), which enters the liver via systemic circulation and binds to upregulated β1 adrenergic receptor (ADRB1) on hepatocytes, leading to increased expression of complement components C3, C5, and CFH. In chronic stress-model mice, elevated serum and intratumoral Complement component 5a (C5a) levels showed a positive correlation with MDSCs proportions in both the spleen and tumor tissues. Administration of the C5aR1 antagonist PMX-50 disrupted this association, indicating C5a as a critical mediator of MDSCs expansion and recruitment. Infiltrating MDSCs within the TME exhibit elevated expression of Programmed death-ligand 1 and Transforming growth factor-beta, fostering the establishment of an immunosuppressive microenvironment. Intraperitoneal injection of the ADRB1 antagonist atenolol in PDAC mice resulted in marked reductions in serum/tumoral C5a levels, splenic/intratumoral MDSCs proportions, and tumor burden. Our findings suggest that targeting SNS-driven MDSCs recruitment and TME remodeling may offer novel therapeutic strategies for PDAC patients.\u003c/p\u003e","manuscriptTitle":"Chronic Stress Promotes Pancreatic Ductal Adenocarcinoma Progression via Complement C5a-Recruited Myeloid-Derived Suppressor Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 13:44:18","doi":"10.21203/rs.3.rs-7471626/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-21T01:39:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-20T16:46:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T12:29:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62021853980797820369629512167112714315","date":"2025-09-09T01:47:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106347650538856409587152225134987826220","date":"2025-09-08T03:58:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299429388303667797550633807835740261044","date":"2025-09-07T17:38:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114147934236651040749089351154983121292","date":"2025-09-05T13:18:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296018710989267494882325670483936495111","date":"2025-09-05T08:32:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158308926509035138989803467261323515535","date":"2025-09-05T06:34:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-03T04:29:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-28T06:43:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-28T06:42:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Immunology, Immunotherapy","date":"2025-08-27T12:20:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-immunology-immunotherapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ciim","sideBox":"Learn more about [Cancer Immunology, Immunotherapy](http://link.springer.com/journal/262)","snPcode":"262","submissionUrl":"https://submission.nature.com/new-submission/262/3","title":"Cancer Immunology, Immunotherapy","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c99ed4a0-6556-45f9-8837-df0b34a9e1fc","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T15:59:35+00:00","versionOfRecord":{"articleIdentity":"rs-7471626","link":"https://doi.org/10.1007/s00262-025-04249-z","journal":{"identity":"cancer-immunology-immunotherapy","isVorOnly":false,"title":"Cancer Immunology, Immunotherapy"},"publishedOn":"2025-12-19 15:57:13","publishedOnDateReadable":"December 19th, 2025"},"versionCreatedAt":"2025-09-09 13:44:18","video":"","vorDoi":"10.1007/s00262-025-04249-z","vorDoiUrl":"https://doi.org/10.1007/s00262-025-04249-z","workflowStages":[]},"version":"v1","identity":"rs-7471626","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7471626","identity":"rs-7471626","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}