Fibroblastic reticular cell–derived HGF orchestrates sympathetic nerves in tumor-induced lymph node remodeling and metastasis

Fibroblastic reticular cell–derived HGF orchestrates sympathetic nerves in tumor-induced lymph node remodeling and metastasis | 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 Fibroblastic reticular cell–derived HGF orchestrates sympathetic nerves in tumor-induced lymph node remodeling and metastasis Zhi-Gang Zhang, Jun Li, Xiang Zhang, Li-Peng Hu, Qing Li, Hui Li, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6607085/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 Lymph nodes (LNs) are critical peripheral immune organs extensively innervated by both sympathetic and sensory nerves. During tumor metastasis, LNs undergo significant structural remodeling and enlargement; however, the role of neural innervation in this process remains unclear. Here, using whole-organ three-dimensional (3D) imaging, we observed pronounced elongation and increased branching specifically in sympathetic nerve fibers, but not sensory nerves, during tumor-induced LN enlargement (TLNE), suggesting adaptive neural remodeling. Single-nucleus RNA sequencing further revealed activation of fibroblastic reticular cells (FRCs) during TLNE, characterized by enriched neuro-related signaling pathways and substantial secretion of hepatocyte growth factor (HGF). Functional validation using targeted HGF inhibitors and adeno-associated virus (AAV)-mediated HGF silencing confirmed that FRC-derived HGF critically drives sympathetic nerve growth. Additionally, both HGF inhibition and sympathetic nerve denervation significantly reduced TLNE and tumor-induced LN metastasis, highlighting the importance of adaptive sympathetic innervation in tumor-associated LN remodeling. These findings identify a previously unrecognized FRC-HGF-sympathetic nerve axis and propose neural regulation as a potential therapeutic strategy for tumor-induced LN metastasis. Biological sciences/Cancer Health sciences/Diseases/Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Lymph nodes (LNs), serving as secondary lymphoid organs, are integral to adaptive immunity and are innervated by both sensory and sympathetic nerves 1 – 3 . Sensory nerves continuously monitor LN status and, upon activation, transmit information to the brain while releasing neuropeptides like calcitonin gene related peptide (CGRP) and Substance P to assist immune responses 1 , 4 . In response to upstream signals, sympathetic nerves regulate LN activities through multiple mechanisms: direct modulation of immune cell receptors via neurotransmitters, control of vascular constriction to alter local oxygen and nutrient availability, and transmission of rhythmic information from the brain to synchronize immune responses across dispersed LNs 5–7 . An essential characteristic feature of LNs during immune responses is expansion, typically accompanied by the activation and proliferation of resident immune cells 8 . This expansion is facilitated by various stromal cells, particularly fibroblastic reticular cells (FRCs) 9–11 . During LN expansion, significant alterations occur in its internal structure and microenvironment, raising questions about how peripheral nerves adapt to and regulate these changes. Research has shown that during virus-induced LN enlargement, sympathetic nerves undergo denervation, leading to reduced neurotransmitter release and decreased viral replication efficiency 12 . Unlike the self-limiting LN enlargement caused by viral infections, tumor-induced lymph node enlargement (TLNE) is persistent and systemic. LNs serve as major conduits for tumor metastasis, especially in cancers such as breast cancer (BC) and colorectal cancer (CRC). Therefore, we are particularly interested in whether peripheral nerves undergo remodeling during TLNE and the underlying mechanisms, aiming to comprehensively understand the interactions between the nervous system, tumors, and the immune system. In this study, we established a tumor-induced popliteal LN (PLN) metastasis model by inoculating CRC cells into the footpads of mice 13 . Utilizing Hydrogel-based Reinforcement of Three-Dimensional Imaging Solvent-Cleared Organs (HYBRiD) technology, we visualized neural changes within the PLNs at an organ-wide level 14 , 15 . Our findings revealed significant elongation and increased branching of sympathetic nerves, which positively correlated with the degree of LN expansion. To investigate the underlying mechanisms, we performed single-cell sequencing (snRNA-seq) on PLNs post-tumor induction, mapping the immune and stromal components during tumor-induced expansion. Further analysis indicated that during LN expansion FRCs were associated with nerve growth signals and secreted hepatocyte growth factor (HGF), a multifunctional protein critical for cell proliferation, differentiation, migration, and tissue repair and regeneration. Based on these observations, we postulated that the activation of FRCs and the release of HGF facilitate sympathetic nerve outgrowth during TLNE. This hypothesis was subsequently validated through both in vivo and in vitro experiments. Our results underscore a novel role for FRCs in nerve remodeling and elucidate the coordinated interaction between immune responses, neural regulation, and the stromal environment in LN functionality. Results 1. TLNE drives adaptive sympathetic nerve outgrowth The murine CRC cell line MC38 was used for footpad injection to establish a LN metastasis mouse model ( Extended Data Fig. 1 a, b). PLNs around the injection site showed gradual expansion, with significant enlargement detectable as early as 3 days post-injection ( Extended Data Fig. 1 c, d). To investigate changes in innervation within the PLNs during TLNE, we examined PLNs at various time points using PGP9.5 staining and the HYBRiD technique to generate complete 3D images of nerve morphology ( Extended Data Fig. 1 e, f). The results revealed a significant increase in innervation within the PLNs as early as day 3. Furthermore, continuous axon elongation and branching of nerve fibers, indicative of nerve fiber outgrowth, were evident at days 5, 7, and 10 (Fig. 1 a-e). LNs are primarily innervated by sympathetic and sensory nerves 3 . Our 3D imaging of tyrosine hydroxylase (TH) staining revealed a significant increase in both the length and branching of sympathetic nerve fibers in the PLNs after 3 days ( Extended Data Fig. 2 a-c). To further confirm the outgrowth of sympathetic fibers, we generated dopamine β-hydroxylase (DBH)-P2A-EGFP or calcitonin related polypeptide alpha (CALCA)-P2A-BFP mice and captured additional 3D images. This analysis clearly demonstrated that the outgrowing fibers were DBH-positive sympathetic fibers (Fig. 1 f-h, Extended Data Video 1, Extended Data Fig. 3 a, b), rather than CALCA-positive sensory fibers, as no significant outgrowth was observed in the PLNs after 3 days ( Extended Data Fig. 4 a-e). Further validation through 3D imaging and immunofluorescence (IF) staining confirmed the co-localization of neurofilament light (NFL) and TH on the newly sprouted nerve fibers, providing strong evidence that these fibers were predominantly sympathetic ( Extended Data Fig. 5 a, b). Correlation analysis revealed a strong relationship between the lengths and branch numbers of sympathetic nerve fibers and PLN expansion at days 0, 5, and 10, suggesting that LN innervation adapts to their expansion during TLNE (Fig. 1 i, j). In summary, our results demonstrate that tumor-induced sympathetic nerve outgrowth plays a key role in TLNE. 2. FRCs coordinate sympathetic nerve remodeling during TLNE To explore the mechanism underlying adaptive nerve fiber outgrowth in LNs during TLNE, we performed single-nucleus RNA sequencing (snRNA-seq) to investigate interactions between different cell subsets. The footpad metastasis mouse model at 2.5 days post-inoculation was analyzed for snRNA-seq (Fig. 2 a). The involved cells were categorized into 12 subsets based on marker expression: fibroblasts (FBs), marginal reticular cells (MRCs), lymphatic endothelial cells (LECs), blood endothelial cells (BECs), T-zone reticular cells (TRCs), myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs), and various immune cells. Previous reports classified FBs, MRCs, and TRCs as fibroblastic reticular cells (FRCs) 16 , 17 . The various cell subsets were displayed using t-distributed stochastic neighbor embedding (tSNE) plots (Fig. 2 b-e, Extended Data Fig. 6a-c ). Especially, gene ontology-biological process (GO-BP) analysis indicated no significantly direct correlation between immune cells and adaptive nerve fiber outgrowth within LNs ( Extended Data Fig. 6d ). Flow cytometry further verified changes in immune cell subsets during TLNE, showing a marked increase in B cells, neutrophils, and macrophages after 3 days ( Extended Data Fig. 7a-h ). According to snRNA-seq data, FBs constituted 30.68% of stromal cells and MRCs constituted 5.92%, which were both vial members of FRCs (Fig. 2 f). Further GO-BP analysis indicated that both these members were closely associated with axon guidance, neuron projection guidance, and other neurobiological processes, suggesting that FRCs contribute to the outgrowth of sympathetic nerve fibers in PLNs (Fig. 2 g). To further elucidate the relationship between FRCs and neural signaling pathways, we conducted GO and gene set enrichment analysis (GSEA). FBs were positively correlated with synapse assembly and organization (Fig. 2 h, Extended Data Fig. 8a, b ), while MRCs were correlated with neurotransmitter regulation and neuron migration (Fig. 2 i, Extended Data Fig. 8c, d ). These findings underscored the pivotal roles of FRCs in nerve fiber outgrowth during TLNE. 3. FRCs promote nerve fiber outgrowth through HGF secretion, enhanced by elevated matrix stiffness To explore the key factors involved in this progression, we thus analyzed axon outgrowth-related genes and pathways filtered out by snRNA-seq data of the PLNs. Several nerve related factors, particularly ones related to axon elongation and growth, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and HGF, were screened (Fig. 2 j) 18–21 . Gene expression analysis revealed that only HGF exhibited significant upregulation in FBs and MRCs, suggesting the important roles of which in TLNE in PLNs (Fig. 2 k-m, Extended Data Fig. 9a, b ). To investigate how tumor cells induce HGF secretion by FRCs thus promote nerve fiber outgrowth, we added MC38 cell culture medium (CM) to mice LNs derived primary FRCs. Primary FRCs were significantly activated by CM ( Extended Data Fig. 10a ). And this phenomenon was further verified by IF staining on α-SMA ( Extended Data Fig. 10b ). The relative mRNA expression levels of Acta2 and Col1a1 were elevated in the CM group (Fig. 3 a). Quantification of HGF levels showed that treatment of tumor cell supernatant significantly enhanced HGF release from FRCs (Fig. 3 b). To examine the effects of FRCs on neural cells, we used PC-12 cells ( Extended Data Fig. 10c ). PC-12 cells treated with CM from FRCs alone (CM1) or FRCs / MC38 cells (CM2) showed significant axonogenesis compared to the vehicle group, with PC-12 cells treated with CM2 exhibiting significantly longer nerve axons than those treated with CM1 ( Extended Data Fig. 10d-f ). To verify HGF's role in sympathetic nerve fiber outgrowth in LNs, we used two inhibitors: Norleual TFA, which competitively inhibits HGF by mimicking its hinge region, and SRI 31215, which blocks the proteolytic activation of pro-HGF by targeting matriptase, hepsin, and HGF activator (HGFA). Both Norleual TFA and SRI 31215 attenuated PC-12 cell elongation induced by CM1 or CM2 in vitro (Fig. 3 c-f). Previous studies indicate that LN expansion is primarily driven by FRCs activation and biomechanical changes 10, 11 . To investigate these changes during TLNE, we performed immunohistochemical (IHC) staining on α-smooth muscle actin (α-SMA) in LNs. Significant α-SMA expression could be observed at as early as 2 days post-inoculation, with elevated levels at 5, 7, and 10 days (Fig. 3 g). Additionally, Sirius red staining also revealed significant collagen deposition in the LNs following the similar trend as α-SMA (Fig. 3 h, upper ). Further analysis using polarized light microscopy showed that collagen fibers were primarily composed of type I and III collagen (Fig. 3 h, lower ). To assess matrix stiffness, we used atomic force microscopy (AFM) to measure the Young’s modulus, revealing a series of continuous significant increases of Young’s modulus in PLNs from day 0 to day 10 (Fig. 3 i). These findings indicate that TLNE induces biomechanical changes and FRC activation, leading to PLN expansion. To investigate the effects of mechanical properties on HGF secretion, primary FRCs were cultured on Matrigel with stiffnesses of 0.5 kPa and 12 kPa (Fig. 3 j). Higher Acta2 and Col1a1 expressions could be observed on the stiffer Matrigel, as well as HGF secretion, indicating that FRCs showed significantly higher activation (Fig. 3 k-m). These data indicated that elevated matrix stiffness enhances FRC activation and increases HGF secretion, thus contributing to nerve fiber outgrowth. 4. HGF inhibition suppresses sympathetic nerve outgrowth in TLNE and LN metastasis To further confirm the in vivo role of HGF, we employed Norleual TFA and adeno-associated virus (AAV)-mediated silencing of HGF expression. Norleual TFA or AAV was administered into the popliteal fat pad of mice 6 hours prior to MC38 tumor cell inoculation, with additional injections every three days thereafter (days 0, 3, and 6). PLNs were harvested at day 7, cleared using HYBRiD, and subsequently analyzed (Fig. 4 a). Three-dimensional imaging of TH-stained sympathetic fibers showed significant suppression of sympathetic nerve outgrowth following treatment with Norleual TFA or AAV-mediated HGF silencing (Fig. 4 b-e). Consistently, 3D imaging of DBH-positive sympathetic nerve fibers produced similar findings (Fig. 4 f-i). Together, these results indicate that silencing HGF expression effectively inhibits sympathetic reinnervation within PLNs, reinforcing HGF's critical role in sympathetic nerve fiber expansion during TLNE. Additionally, we investigated the impact of HGF inhibition on TLNE and LN metastasis. Administration of the HGF inhibitor Norleual TFA and AAV-mediated HGF silencing significantly suppressed PLN expansion induced by tumor cell invasion (Fig. 4 j, k). IHC analysis of pan-cytokeratin (panCK) and α-SMA in PLNs further demonstrated that HGF silencing markedly inhibited tumor cell-induced LN metastasis and FRC activation (Fig. 4 l, m). These findings indicate that targeting HGF effectively attenuates TLNE and LN metastasis driven by tumor cells. 5. Sympathetic nerve denervation inhibits LN metastasis To further elucidate the role of the sympathetic nervous system in TLNE, we performed sympathetic denervation by injecting 6-hydroxydopamine (6-OHDA) into the fat pad surrounding the PLNs of mice five days prior to tumor cell inoculation. PLNs were harvested at days 5 and 7 post-inoculation and analyzed by IF and IHC staining (Fig. 5 a). The results demonstrated that sympathetic denervation markedly inhibited PLN expansion triggered by tumor cell invasion (Fig. 5 b-e). Imaging of tdTomato-labeled tumor cells and panCK staining further indicated that sympathetic denervation effectively suppressed MC38 cell-induced LN metastasis (Fig. 5 f-i). Moreover, the elevated percentages of B cells, Marcrophages, and neutrophils observed during TLNE were significantly reduced following 6-OHDA administration ( Extended Data Fig. 11a, b ). Collectively, these findings underscore the crucial role of sympathetic innervation in promoting LN metastasis during tumor progression. Taken together, our findings demonstrate that during TLNE, LN innervation dynamically adapts to accommodate structural expansion. Activated FRCs secrete HGF, facilitating sympathetic nerve fiber outgrowth, accompanied by biomechanical changes within the LN microenvironment. Increased matrix stiffness further enhances FRC activation, thereby promoting additional sympathetic nerve growth. Importantly, inhibition of HGF signaling or sympathetic nerve denervation effectively suppresses TLNE and tumor-induced LN metastasis (Fig. 5 j). Discussion LNs are peripheral immune organs that enlarge in response to infection or other stimuli. Stromal cells, especially FRCs, play a critical role by secreting extracellular matrix (ECM) and modulating microenvironmental stiffness, thereby supporting LN expansion and maintaining structural integrity 9–11 . Recent studies have shown that cold-induced sympathetic activity activates the β-adrenergic receptor (β-AR) signaling pathway in FRCs, indicating that LN function is regulated by the nervous system 22 . However, the mechanisms underlying LN volume changes and the role of the nervous system in regulating this process remain unclear. Specifically, how the nervous system adapts to and manages the increase in LN volume is not well understood. Additionally, whether the immune system modulates the structure and function of the nervous system remain an intriguing and unanswered question 23–25 . To investigate these questions, we utilized a tumor-induced LN metastasis model to induce LN enlargement 13 . Unexpectedly, as the LNs expanded, we observed significant growth of nerve fiber, including axon elongation and increased nerve branching, which were subsequently verified to be predominantly sympathetic nerves. Remarkably, FRCs played a crucial role, they not only provided structural support during LN expansion but also released neurotrophic factors, promoting nerve growth and facilitating neural adaptation to the organ's enlargement. Notably, the increased stiffness of the ECM during LN enlargement further augmented this nerve growth process. Our results revealed that during the early stages of tumor cell metastasis, FRCs exhibit a highly active state within two days post-metastasis, followed by significant nerve regrowth on the third day. Previous research has primarily focused on the role of FRCs in LN enlargement without linking it to neural remodeling 26,27 . Our observation of this sequential order suggested a potentially inseparable connection between nerve fibers and FRC activation within LNs. Subsequently, we verified two key findings. First, during TLNE, the adaptive growth of nerve fibers within LNs predominantly involves sympathetic nerves, and this growth correlates positively with the extent of LN expansion. Although sensory nerves are abundantly distributed within LNs, their alterations are minimal relative to the expansion of LNs. This underscores the importance of the efferent regulation by the peripheral nervous system in maintaining LN functionality. Second, through comprehensive snRNA-seq data analysis, combined with in vitro and in vivo experiments, we found that activated FRCs secrete substantial amounts of HGF. Previous studies have shown that HGF is a crucial growth factor in cancer development and progression, contributing to the formation of a metastatic microenvironment 28,29 . Additionally, HGF is well-documented to promote axon growth 21,30 . By inhibiting HGF using various methods, we observed the disappearance of sympathetic nerve regrowth associated with LN enlargement. These findings demonstrated that early-stage sympathetic nerve regrowth in TLNE is dependent on FRC activation and subsequent HGF secretion. The regulation of LNs by the nervous system during tumor metastasis is critically important. LNs are dispersed throughout the body, each monitoring specific regions for immune challenges. Sympathetic nerves play a key role in coordinating immune responses across this network. When a single LN detects a threat, sensory nerves transmit signals to the brain, which then commands sympathetic nerves to regulate immune responses across all LNs. This orchestrated "whole-body response" suggests that the collective immune efficacy of the LN network may surpass that of an individual LN. Rapid neural regulation aligns with the swift responses characteristic of adaptive immunity, emphasizing the importance of neural involvement in tumor immune surveillance and response. The physiological significance of sympathetic nerve reinnervation observed during LN metastasis remains to be elucidated. Potential mechanisms may involve altering the LN microenvironment through nerve fiber growth and branching, as well as regulating immune and stromal cells via neurotransmitters such as norepinephrine (NE). Notably, previous studies have shown that sympathetic nerves accompany blood vessels into LNs, suggesting that reinnervating nerves might influence vascular neogenesis and exert regulatory effects on blood vessels. Therefore, future research should focus on elucidating these potential interactions. FRCs, key contributors to LN expansion, not only provide structural support but also direct the adaptive growth of nerve fibers by secreting HGF. During TLNE, FRCs likely serve as critical coordinators of both LN remodeling and nerve fiber regeneration, with HGF functioning as an essential mediator. Understanding this mechanism deepens our comprehension of LN biology and suggests novel pathways for innovative immunotherapeutic and neuroregulatory approaches. HGF, identified as a pivotal mediator of LN-associated nerve growth, emerges as a promising therapeutic target for facilitating neural remodeling. Moreover, directly manipulating neural signals within LNs could enhance immune responses while minimizing unnecessary inflammation. Importantly, inhibiting HGF signaling or performing sympathetic nerve denervation significantly suppresses TLNE and associated LN metastasis, highlighting the potential of targeting both HGF and sympathetic innervation in treating tumor-induced LN metastasis. These findings provide critical insights into potential therapeutic strategies with significant scientific and clinical relevance for improving outcomes in cancer patients. Declarations Data availability All data are available upon request and relevant data are available in the Source Data. The snRNA-seq data from this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GSE280461. ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (ID 82230087, 82350123, 82203228, to Z.-G. Zhang; ID 82073023, 81871923, to J. Li; ID 82372821, 82103357 to L.-P. Hu; ID 82002485, to Q. Li; ID 82203228, to D.-X. Li; ID 82103348, to Y.-Y. Wang), the Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (ID 20181708, to Z.-G. Zhang), Innovative research team of high-level local universities in Shanghai (ID SHSMU-ZDCX20210802, to Z.-G. Zhang), Shanghai Pilot Program for Basic Research - Shanghai Jiao Tong University (ID 21TQ1400225, to Z.-G. Zhang), 111 project (ID B21024, to Z.-G. Zhang), Shanghai Science and Technology Commission Sailing Project (ID 22YF1445600 to D.-X. Li; ID 21YF1445200 to L.-P. Hu), the Natural Science Foundation of Shanghai (ID 21ZR1461300 to L.-P. Hu; ID 22ZR1460000 to X.-L. Zhang), Innovative research team of high-level local universities in Shanghai (ID SHSMU-ZDCX20210802 to X.-L. Zhang), the Shanghai Municipal Health Commission (ID 20214Y0200 to D.-X. Li), Key Areas Research and Development Programs of Guangdong Province (ID 2023B1111050009 to M.-J. Xu). We thank Prof. Zhi-Feng Shao, Dr. Xiao-Mei Yang, Dr. Yan-Li Zhang, Dr. Lei Zhu, Dr. Lin-Li Yao and Dr. Ni Zhang for assistance with our experiments. AUTHOR CONTRIBUTIONS J.L. and Z.-G.Z. designed and supervised the overall study, analyzed data, and drafted the manuscript. J.L., X.Z., L.-P.H. and Q.L. constructed the mouse footpad model and collected the LN tissues. X.Z. and G.-H.S. performed the HYBRiD clearing and whole-mount tissue 3D imaging. J.L., H.L., Y.-Q.Z. and J.-X.X. analyzed the single-nucleus RNA sequencing data. X.Z., G.-H.S. and Y.-K.L. analyzed the data of 3D imaging. Y.-Y.W., T.-S.B., S.Z. and C.-J.X. assisted with analysis of IHC and IF staining. 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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-6607085","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":460103973,"identity":"edfac4c3-ef32-4a13-a0b8-315400409a45","order_by":0,"name":"Zhi-Gang Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYFACHhBhw2AAJA+QoiWNdC2HwVqIA/L9Zw9+Lvh13t6c//DDAwwV9+waCGlhbDiXLD2z73bizhlpBgcYzhQnE9TCzNhjIM3bczvB4AYPwwHGtoRkgg5jY+Yx/s3bc87e4PwZIrXwsPGYSfP8OMC44UAOWIsdQS0SPDxm1rwNyYkbbgD9knAmIYGgFvn+M8a3ef7YAR12+PGHDxUJ9gS1gAFjG5QBtCKxgTg9fxBMIm0ZBaNgFIyCkQQAmks84GFtvo4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8965-223X","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Zhi-Gang","middleName":"","lastName":"Zhang","suffix":""},{"id":460103974,"identity":"bbe8accb-e4a9-4414-a5cb-dc4f9ccbbcbe","order_by":1,"name":"Jun Li","email":"","orcid":"","institution":"State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""},{"id":460103975,"identity":"ed1095d6-b5d1-4801-a701-02b6abcde17d","order_by":2,"name":"Xiang Zhang","email":"","orcid":"","institution":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Zhang","suffix":""},{"id":460103976,"identity":"eb998d63-dbab-4001-8f58-42a556752998","order_by":3,"name":"Li-Peng Hu","email":"","orcid":"","institution":"State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Li-Peng","middleName":"","lastName":"Hu","suffix":""},{"id":460103977,"identity":"c62e39e9-4c95-46c0-bf20-890254ad0965","order_by":4,"name":"Qing Li","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Li","suffix":""},{"id":460103978,"identity":"fc93863e-047d-465e-be3a-77da12a37efc","order_by":5,"name":"Hui Li","email":"","orcid":"","institution":"State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Li","suffix":""},{"id":460103979,"identity":"c1f2fdb7-1549-4a3b-bf8d-7a81bed5f183","order_by":6,"name":"Guang-Hong Su","email":"","orcid":"","institution":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.","correspondingAuthor":false,"prefix":"","firstName":"Guang-Hong","middleName":"","lastName":"Su","suffix":""},{"id":460103980,"identity":"1eca0a82-6f9f-4cfc-b7d8-31577d8c61b8","order_by":7,"name":"Jia-Xuan Xie","email":"","orcid":"","institution":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.","correspondingAuthor":false,"prefix":"","firstName":"Jia-Xuan","middleName":"","lastName":"Xie","suffix":""},{"id":460103981,"identity":"e584189c-e475-49a9-890c-fd7bfa906a78","order_by":8,"name":"Yao-Qi Zhou","email":"","orcid":"","institution":"State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.","correspondingAuthor":false,"prefix":"","firstName":"Yao-Qi","middleName":"","lastName":"Zhou","suffix":""},{"id":460103982,"identity":"52d9fd01-bde2-4784-99bc-612a6154d56d","order_by":9,"name":"Yang-Yang Wang","email":"","orcid":"","institution":"Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Yang-Yang","middleName":"","lastName":"Wang","suffix":""},{"id":460103983,"identity":"fc55348b-0e52-4a97-a6f4-ba2e0416b39d","order_by":10,"name":"Tian-Shang Bao","email":"","orcid":"","institution":"Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Tian-Shang","middleName":"","lastName":"Bao","suffix":""},{"id":460103984,"identity":"f5962c98-d984-44a2-88ca-c1507cf7a01d","order_by":11,"name":"Yan-Kun Li","email":"","orcid":"","institution":"School of Biomedical Engineering, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Yan-Kun","middleName":"","lastName":"Li","suffix":""},{"id":460103985,"identity":"ec87a15a-d298-401d-ae1a-d982404e4d78","order_by":12,"name":"Shan Zhang","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Zhang","suffix":""},{"id":460103986,"identity":"acd3aec1-17a8-44de-acaa-9b2eb9c6bccc","order_by":13,"name":"Chun-Jie Xu","email":"","orcid":"","institution":"Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Chun-Jie","middleName":"","lastName":"Xu","suffix":""},{"id":460103987,"identity":"5b1dd164-add9-46f7-a2b6-6b7db025ca11","order_by":14,"name":"Jian Song","email":"","orcid":"","institution":"Institute of Cardiovascular Sciences, Guangxi Academy of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Song","suffix":""},{"id":460103988,"identity":"fd08abba-402d-43da-9c14-f5cd2bb8e460","order_by":15,"name":"Shuang-Qin Yi","email":"","orcid":"","institution":"Department of Frontier Health Sciences, Graduate School of Human Health Sciences, Tokyo Metropolitan University","correspondingAuthor":false,"prefix":"","firstName":"Shuang-Qin","middleName":"","lastName":"Yi","suffix":""},{"id":460103989,"identity":"ffab878a-86ad-4614-bab0-f22090a7da24","order_by":16,"name":"Min-Juan Xu","email":"","orcid":"","institution":"Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine","correspondingAuthor":false,"prefix":"","firstName":"Min-Juan","middleName":"","lastName":"Xu","suffix":""},{"id":460103990,"identity":"a9a71b8b-61b9-42df-8d0f-451ebaadb8e8","order_by":17,"name":"Xiao-Wei Li","email":"","orcid":"","institution":"School of Biomedical Engineering, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Xiao-Wei","middleName":"","lastName":"Li","suffix":""},{"id":460103991,"identity":"a36971c2-b126-40dc-b8a3-a6173f0853ea","order_by":18,"name":"Jia Xu","email":"","orcid":"","institution":"Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Xu","suffix":""},{"id":460103992,"identity":"bf7c122a-ca4a-47b7-ad0c-418162431043","order_by":19,"name":"Xue-Li Zhang","email":"","orcid":"","institution":"State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Xue-Li","middleName":"","lastName":"Zhang","suffix":""},{"id":460103993,"identity":"c2ba789e-4876-40ef-93b2-2b30943dab6f","order_by":20,"name":"Shu-Heng Jiang","email":"","orcid":"","institution":"State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Shu-Heng","middleName":"","lastName":"Jiang","suffix":""},{"id":460103994,"identity":"745852a6-fab1-44a0-8a14-26c208ea6864","order_by":21,"name":"Dong-Xue Li","email":"","orcid":"","institution":"State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Dong-Xue","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-05-07 02:00:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6607085/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6607085/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83439524,"identity":"64da295d-2691-4222-9221-13efabd8e3bf","added_by":"auto","created_at":"2025-05-26 09:12:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2962786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTumor cells induce the expansion of PLNs and the outgrowth of sympathetic nerve fibers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003e3D images of PGP9.5-positive nerve fibers in PLNs at 0 d, 1 d, 2 d, 3 d, 5 d, 7 d and 10 d. Scale bars: upper - 300 μm; lower - 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb-e. \u003c/strong\u003eThe outgrowth of PGP9.5-positive nerve fibers is represented by axon elongation (\u003cstrong\u003eb\u003c/strong\u003e) and branching (\u003cstrong\u003ed\u003c/strong\u003e) in PLNs at 0 d, 5 d and 10 d, respectively (the original red fluorescence has been replaced with green fluorescence). The statistical results are shown on the right (\u003cstrong\u003ec, e\u003c/strong\u003e). Scale bars: 100 μm. All nerve fibers within the images in each group were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef. \u003c/strong\u003e3D images of DBH-EGFP-positive sympathetic nerve fibers in PLNs at 0 d, 1 d, 2 d, 3 d, 5 d, 7 d and 10 d. Scale bars: upper - 300 μm; lower - 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg, h. \u003c/strong\u003eThe statistical results of the lengths of nerve fibers (\u003cstrong\u003eg\u003c/strong\u003e) and the number of nerve branches (\u003cstrong\u003eh\u003c/strong\u003e) of DBH-P2A-EGFP-positive nerve fibers. All nerve fibers within the images in each group were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei, j. \u003c/strong\u003eThe correlations between the lengths of the sympathetic nerve fibers (\u003cstrong\u003ei\u003c/strong\u003e) or the numbers of sympathetic nerve branches (\u003cstrong\u003ej\u003c/strong\u003e) and the volume of the PLNs.\u003c/p\u003e\n\u003cp\u003e*: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; NS: no significant difference.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/6523a2b577f7e36616afe201.jpg"},{"id":83439504,"identity":"13e01f02-3326-4a3b-8752-d693adf1dc5c","added_by":"auto","created_at":"2025-05-26 09:12:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1935664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFRCs are closely correlated with the outgrowth of nerve fibers in PLNs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eSnRNA-seq analyses were performed to investigate the correlations between stromal cells, immune cells and nerves in PLNs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. \u003c/strong\u003et-SNE image plots of different cell subsets in PLNs (the snRNA-seq data from this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GSE280461).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. \u003c/strong\u003eThe total cells in the PLNs were divided into 12 cell subsets according to the data of the snRNA-seq.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed. \u003c/strong\u003eThe number of FBs, MRCs, LECs and BECs in the data of the snRNA-seq.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee. \u003c/strong\u003eThe markers used for the classification of different cell subsets in PLNs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef. \u003c/strong\u003eThe ratio of FBs, MRCs, LECs, BECs and other cell subsets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg. \u003c/strong\u003eGO-BP analysis of different cell subsets in the single-cell sequencing data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh, i. \u003c/strong\u003eGO analyses of the correlation between FBs (\u003cstrong\u003eh\u003c/strong\u003e)/MRCs (\u003cstrong\u003ei\u003c/strong\u003e) and neural signalling pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej. \u003c/strong\u003eThe screening process for nerve growth factors, neurotrophic factors and other nerve-related factors in the single-cell sequencing data. (2.5 d vs. 0 d, P\u0026lt;0.05, |log\u003csub\u003e2\u003c/sub\u003eFC|\u0026gt;0.25)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ek. \u003c/strong\u003eThe expression of HGF in different cell subsets of PLNs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003el, m. \u003c/strong\u003eThe expression of HGF in different cell subsets is displayed by t-SNE image plots (\u003cstrong\u003el\u003c/strong\u003e). HGF was highly expressed in FBs/MRCs and considered closely correlated with nerve fiber outgrowth (\u003cstrong\u003em\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/c0a3be55516a2ae5e65a881f.jpg"},{"id":83439511,"identity":"6c9a60bf-1cea-466b-8264-39d8927a2f16","added_by":"auto","created_at":"2025-05-26 09:12:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2827397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFRC-derived HGF promotes the outgrowth of nerve fibers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eThe relative mRNA expression levels of \u003cem\u003eActa2\u003c/em\u003e and \u003cem\u003eCol1a1\u003c/em\u003e in FRCs treated with culture medium from MC38 cells or the control at 12 h, 24 h and 48 h. Each group was repeated at least 5 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. \u003c/strong\u003eThe concentration and relative mRNA expression level of HGF in the supernatant of FRCs treated with CM from MC38 cells or the control at 48 h. Each group was repeated at least 5 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec-f. \u003c/strong\u003eImages of PC12 cells treated with CM from primary FRCs/MC38 cells or the control at 48 h. Norleual TFA or SRI 31215 was added into PC12 cells treating with culture medium (\u003cstrong\u003ec, d\u003c/strong\u003e). The statistical results of the lengths of nerve fibers (\u003cstrong\u003ee\u003c/strong\u003e) and the lengths of TH positive nerve fibers are shown on the right (\u003cstrong\u003ef\u003c/strong\u003e). Scale bars: 20 μm (\u003cstrong\u003ec\u003c/strong\u003e), all PC12 cells in each group were counted; Scale bars: 12 μm (\u003cstrong\u003ed\u003c/strong\u003e), at least 5 PC12 cells in each group were counted. Each group was repeated at least 5 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eg. \u003c/strong\u003eα-SMA immunostaining of PLNs at different time points. The statistical results of α-SMA immunostaining of PLNs at different time points are shown below. Scale bars: 50 μm. Each group was repeated at least 4 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh. \u003c/strong\u003eSirius red staining of PLNs at different time points. Polarised light images are shown below. The statistical results of Sirius red staining of PLNs at different time points are shown below. Scale bars: 50 μm. Each group was repeated at least 4 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei. \u003c/strong\u003eStatistical analysis of the Young’s modulus value of the PLNs at different time points. Each group was repeated at least 4 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej. \u003c/strong\u003eThe procedure for culturing primary FRCs in Matrigel with different stiffnesses of 0.5 kPa and 12 kPa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ek, l. \u003c/strong\u003eThe visible images of FRCs cultured in 0.5 kPa and 12 kPa Matrigel (\u003cstrong\u003ek\u003c/strong\u003e). The relative mRNA expression levels of \u003cem\u003eActa2\u003c/em\u003e and \u003cem\u003eCol1a1\u003c/em\u003e in FRCs are shown on the right (\u003cstrong\u003el\u003c/strong\u003e). Scale bars: 30 μm. Each group was repeated at least 5 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003em. \u003c/strong\u003eThe expression level of HGF in FRCs cultured in 0.5 kPa and 12 kPa Matrigel was determined by ELISA and qPCR, respectively. Each group was repeated at least 5 times.\u003c/p\u003e\n\u003cp\u003e*: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; NS: no significant difference.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/97cb47294976d4c73619b760.jpg"},{"id":83439526,"identity":"ddb344b4-8965-48f8-ae04-99ddeba1bd24","added_by":"auto","created_at":"2025-05-26 09:12:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1827760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting HGF expression attenuates the innervation changes in TLNE and LN metastasis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eThe procedure used to establish the C57BL/6 mouse model in which mice were injected with HGF inhibitor or AAV, followed by inoculation of MC38 cells into the footpads.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb, c. \u003c/strong\u003e3D images of DBH-P2A-EGFP-positive sympathetic nerve fibers in PLNs that were treated with HGF inhibitor or vehicle (\u003cstrong\u003eb\u003c/strong\u003e), AAV-sh-HGF or sh-negative control (NC) (\u003cstrong\u003ec\u003c/strong\u003e) and inoculated with MC38 cells after 7 days. Scale bars: 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed, e. \u003c/strong\u003eStatistical analyses of the lengths of nerve fibers (\u003cstrong\u003ed\u003c/strong\u003e) and the number of nerve branches (\u003cstrong\u003ee\u003c/strong\u003e) in the 3D images of DBH-P2A-EGFP-positive sympathetic nerve fibers in PLNs treated with an HGF inhibitor or vehicle, AAV-sh-HGF or sh-NC. All nerve fibers within the images in each group were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef, g. \u003c/strong\u003e3D images of TH-positive sympathetic nerve fibers in PLNs that were treated with HGF inhibitor or vehicle (\u003cstrong\u003ef\u003c/strong\u003e), AAV-sh-HGF or sh-NC (\u003cstrong\u003eg\u003c/strong\u003e) and inoculated with MC38 cells after 7 days. Scale bars: 300 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh, i. \u003c/strong\u003eStatistical analyses of the lengths of nerve fibers (\u003cstrong\u003eh\u003c/strong\u003e) and the number of nerve branches (\u003cstrong\u003ei\u003c/strong\u003e) in the 3D images of TH-positive sympathetic nerve fibers in PLNs treated with an HGF inhibitor or vehicle, AAV-sh-HGF or sh-NC. All nerve fibers within the images in each group were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej, k. \u003c/strong\u003ePhotos of PLNs that were treated with HGF inhibitor or vehicle, AAV-sh-HGF or sh-NC and inoculated with MC38 cells after 7 days(\u003cstrong\u003ej\u003c/strong\u003e). The statistical result is shown below (\u003cstrong\u003ek\u003c/strong\u003e). Scale bars: 3 mm. At least 4 samples in each group were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003el, m. \u003c/strong\u003eIHC staining of panCK (\u003cstrong\u003el\u003c/strong\u003e) and α-SMA (\u003cstrong\u003em\u003c/strong\u003e) in PLNs treated with HGF inhibitor or vehicle, AAV-sh-HGF or sh-NC and inoculated with MC38 cells after 7 days. The statistical analyses are shown on the right. Scale bars: 100 μm.Each group was repeated at least 4 times.\u003c/p\u003e\n\u003cp\u003e*: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/f1113cdcc1ed72cc04d2880d.jpg"},{"id":83439510,"identity":"3c10304a-7ec2-4e42-bcaa-5c3d6bc17176","added_by":"auto","created_at":"2025-05-26 09:12:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2231411,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSympathetic nerve denervationsuppresses LN metastasis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eThe procedure used to establish the C57BL/6 mouse model in which mice were injected with 6-OHDA, followed by inoculation of MC38-tdTomatocells into the footpads.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb-e. \u003c/strong\u003ePhotos of PLNs that were treated with 6-OHDA or Vitamin C solution (Vc) and inoculated with MC38-tdTomato cells after 5 (\u003cstrong\u003eb\u003c/strong\u003e) or 7 (\u003cstrong\u003ed\u003c/strong\u003e) days respectively. The statistical results are shown on the right (\u003cstrong\u003ec, e\u003c/strong\u003e).Scale bars: 3 mm. At least 4 samples in each group were counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef, g. \u003c/strong\u003eIF staining of tdTomato/DAPI in PLNs treated with 6-OHDA or Vc and inoculated with MC38-tdTomato cells after 7 days (\u003cstrong\u003ef\u003c/strong\u003e). The statistical result is shown on the right (\u003cstrong\u003eg\u003c/strong\u003e). Scale bars: 100 μm. Each group was repeated at least 5 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh, i. \u003c/strong\u003eIHC staining of panCK in PLNs treated with 6-OHDA or Vc and inoculated with MC38-tdTomato cells after 7 days (\u003cstrong\u003eh\u003c/strong\u003e). The statistical result is shown on the right (\u003cstrong\u003ei\u003c/strong\u003e). Scale bars: 100 μm. Each group was repeated at least 5 times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ej. \u003c/strong\u003eThe mechanism by which TLNE induces activated FRCs and the outgrowth of sympathetic nerve fibers in LNs. HGF inhibition or sympathetic nerve denervation suppresses tumor cell-induced LN metastasis.\u003c/p\u003e\n\u003cp\u003e**: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; NS: no significant difference.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/7459045786e3bbb0b6e1e29a.jpg"},{"id":85417312,"identity":"3b8af730-b69b-4acf-bce5-ddf2ae944e1d","added_by":"auto","created_at":"2025-06-25 14:59:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12860089,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/67934ced-d52d-4e4b-853e-0fc80c1975df.pdf"},{"id":83439503,"identity":"d9da6e92-f3ed-4152-a2c3-e461993139f7","added_by":"auto","created_at":"2025-05-26 09:12:31","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":101376,"visible":true,"origin":"","legend":"Method","description":"","filename":"Methods.doc","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/985ab1a6001bb4a961843226.doc"},{"id":83440003,"identity":"b0086f97-54ea-4a80-8c92-9a624700aaa8","added_by":"auto","created_at":"2025-05-26 09:20:32","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24727040,"visible":true,"origin":"","legend":"Extended Data Figures and Figure legends","description":"","filename":"ExtendedDataFiguresandFigurelegends.doc","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/706b59a68dad578f2dda0474.doc"},{"id":83440001,"identity":"9c6d3a78-7614-45e3-858b-d6c5d8982075","added_by":"auto","created_at":"2025-05-26 09:20:32","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":46931192,"visible":true,"origin":"","legend":"Extended Data Video 1","description":"","filename":"ExtendedDataVideo1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6607085/v1/28c748701944f78bd83257fa.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Fibroblastic reticular cell–derived HGF orchestrates sympathetic nerves in tumor-induced lymph node remodeling and metastasis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLymph nodes (LNs), serving as secondary lymphoid organs, are integral to adaptive immunity and are innervated by both sensory and sympathetic nerves\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Sensory nerves continuously monitor LN status and, upon activation, transmit information to the brain while releasing neuropeptides like calcitonin gene related peptide (CGRP) and Substance P to assist immune responses\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In response to upstream signals, sympathetic nerves regulate LN activities through multiple mechanisms: direct modulation of immune cell receptors via neurotransmitters, control of vascular constriction to alter local oxygen and nutrient availability, and transmission of rhythmic information from the brain to synchronize immune responses across dispersed LNs\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn essential characteristic feature of LNs during immune responses is expansion, typically accompanied by the activation and proliferation of resident immune cells\u003csup\u003e8\u003c/sup\u003e. This expansion is facilitated by various stromal cells, particularly fibroblastic reticular cells (FRCs)\u003csup\u003e9\u0026ndash;11\u003c/sup\u003e. During LN expansion, significant alterations occur in its internal structure and microenvironment, raising questions about how peripheral nerves adapt to and regulate these changes. Research has shown that during virus-induced LN enlargement, sympathetic nerves undergo denervation, leading to reduced neurotransmitter release and decreased viral replication efficiency\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Unlike the self-limiting LN enlargement caused by viral infections, tumor-induced lymph node enlargement (TLNE) is persistent and systemic. LNs serve as major conduits for tumor metastasis, especially in cancers such as breast cancer (BC) and colorectal cancer (CRC). Therefore, we are particularly interested in whether peripheral nerves undergo remodeling during TLNE and the underlying mechanisms, aiming to comprehensively understand the interactions between the nervous system, tumors, and the immune system.\u003c/p\u003e \u003cp\u003eIn this study, we established a tumor-induced popliteal LN (PLN) metastasis model by inoculating CRC cells into the footpads of mice\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Utilizing Hydrogel-based Reinforcement of Three-Dimensional Imaging Solvent-Cleared Organs (HYBRiD) technology, we visualized neural changes within the PLNs at an organ-wide level\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our findings revealed significant elongation and increased branching of sympathetic nerves, which positively correlated with the degree of LN expansion. To investigate the underlying mechanisms, we performed single-cell sequencing (snRNA-seq) on PLNs post-tumor induction, mapping the immune and stromal components during tumor-induced expansion. Further analysis indicated that during LN expansion FRCs were associated with nerve growth signals and secreted hepatocyte growth factor (HGF), a multifunctional protein critical for cell proliferation, differentiation, migration, and tissue repair and regeneration. Based on these observations, we postulated that the activation of FRCs and the release of HGF facilitate sympathetic nerve outgrowth during TLNE. This hypothesis was subsequently validated through both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments. Our results underscore a novel role for FRCs in nerve remodeling and elucidate the coordinated interaction between immune responses, neural regulation, and the stromal environment in LN functionality.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. TLNE drives adaptive sympathetic nerve outgrowth\u003c/h2\u003e \u003cp\u003eThe murine CRC cell line MC38 was used for footpad injection to establish a LN metastasis mouse model (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). PLNs around the injection site showed gradual expansion, with significant enlargement detectable as early as 3 days post-injection (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate changes in innervation within the PLNs during TLNE, we examined PLNs at various time points using PGP9.5 staining and the HYBRiD technique to generate complete 3D images of nerve morphology (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). The results revealed a significant increase in innervation within the PLNs as early as day 3. Furthermore, continuous axon elongation and branching of nerve fibers, indicative of nerve fiber outgrowth, were evident at days 5, 7, and 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-e).\u003c/p\u003e \u003cp\u003eLNs are primarily innervated by sympathetic and sensory nerves\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Our 3D imaging of tyrosine hydroxylase (TH) staining revealed a significant increase in both the length and branching of sympathetic nerve fibers in the PLNs after 3 days (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). To further confirm the outgrowth of sympathetic fibers, we generated dopamine β-hydroxylase (DBH)-P2A-EGFP or calcitonin related polypeptide alpha (CALCA)-P2A-BFP mice and captured additional 3D images. This analysis clearly demonstrated that the outgrowing fibers were DBH-positive sympathetic fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h, \u003cb\u003eExtended Data Video 1, Extended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b), rather than CALCA-positive sensory fibers, as no significant outgrowth was observed in the PLNs after 3 days (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-e). Further validation through 3D imaging and immunofluorescence (IF) staining confirmed the co-localization of neurofilament light (NFL) and TH on the newly sprouted nerve fibers, providing strong evidence that these fibers were predominantly sympathetic (\u003cb\u003eExtended Data\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCorrelation analysis revealed a strong relationship between the lengths and branch numbers of sympathetic nerve fibers and PLN expansion at days 0, 5, and 10, suggesting that LN innervation adapts to their expansion during TLNE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, j). In summary, our results demonstrate that tumor-induced sympathetic nerve outgrowth plays a key role in TLNE.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. FRCs coordinate sympathetic nerve remodeling during TLNE\u003c/h3\u003e\n\u003cp\u003eTo explore the mechanism underlying adaptive nerve fiber outgrowth in LNs during TLNE, we performed single-nucleus RNA sequencing (snRNA-seq) to investigate interactions between different cell subsets. The footpad metastasis mouse model at 2.5 days post-inoculation was analyzed for snRNA-seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe involved cells were categorized into 12 subsets based on marker expression: fibroblasts (FBs), marginal reticular cells (MRCs), lymphatic endothelial cells (LECs), blood endothelial cells (BECs), T-zone reticular cells (TRCs), myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs), and various immune cells. Previous reports classified FBs, MRCs, and TRCs as fibroblastic reticular cells (FRCs)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The various cell subsets were displayed using t-distributed stochastic neighbor embedding (tSNE) plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-e, \u003cb\u003eExtended Data Fig.\u0026nbsp;6a-c\u003c/b\u003e). Especially, gene ontology-biological process (GO-BP) analysis indicated no significantly direct correlation between immune cells and adaptive nerve fiber outgrowth within LNs (\u003cb\u003eExtended Data Fig.\u0026nbsp;6d\u003c/b\u003e). Flow cytometry further verified changes in immune cell subsets during TLNE, showing a marked increase in B cells, neutrophils, and macrophages after 3 days (\u003cb\u003eExtended Data Fig.\u0026nbsp;7a-h\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eAccording to snRNA-seq data, FBs constituted 30.68% of stromal cells and MRCs constituted 5.92%, which were both vial members of FRCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Further GO-BP analysis indicated that both these members were closely associated with axon guidance, neuron projection guidance, and other neurobiological processes, suggesting that FRCs contribute to the outgrowth of sympathetic nerve fibers in PLNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). To further elucidate the relationship between FRCs and neural signaling pathways, we conducted GO and gene set enrichment analysis (GSEA). FBs were positively correlated with synapse assembly and organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, \u003cb\u003eExtended Data Fig.\u0026nbsp;8a, b\u003c/b\u003e), while MRCs were correlated with neurotransmitter regulation and neuron migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, \u003cb\u003eExtended Data Fig.\u0026nbsp;8c, d\u003c/b\u003e). These findings underscored the pivotal roles of FRCs in nerve fiber outgrowth during TLNE.\u003c/p\u003e\n\u003ch3\u003e3. FRCs promote nerve fiber outgrowth through HGF secretion, enhanced by elevated matrix stiffness\u003c/h3\u003e\n\u003cp\u003eTo explore the key factors involved in this progression, we thus analyzed axon outgrowth-related genes and pathways filtered out by snRNA-seq data of the PLNs. Several nerve related factors, particularly ones related to axon elongation and growth, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and HGF, were screened (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej)\u003csup\u003e18\u0026ndash;21\u003c/sup\u003e. Gene expression analysis revealed that only HGF exhibited significant upregulation in FBs and MRCs, suggesting the important roles of which in TLNE in PLNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek-m, \u003cb\u003eExtended Data Fig.\u0026nbsp;9a, b\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo investigate how tumor cells induce HGF secretion by FRCs thus promote nerve fiber outgrowth, we added MC38 cell culture medium (CM) to mice LNs derived primary FRCs. Primary FRCs were significantly activated by CM (\u003cb\u003eExtended Data Fig.\u0026nbsp;10a\u003c/b\u003e). And this phenomenon was further verified by IF staining on α-SMA (\u003cb\u003eExtended Data Fig.\u0026nbsp;10b\u003c/b\u003e). The relative mRNA expression levels of \u003cem\u003eActa2\u003c/em\u003e and \u003cem\u003eCol1a1\u003c/em\u003e were elevated in the CM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Quantification of HGF levels showed that treatment of tumor cell supernatant significantly enhanced HGF release from FRCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). To examine the effects of FRCs on neural cells, we used PC-12 cells (\u003cb\u003eExtended Data Fig.\u0026nbsp;10c\u003c/b\u003e). PC-12 cells treated with CM from FRCs alone (CM1) or FRCs / MC38 cells (CM2) showed significant axonogenesis compared to the vehicle group, with PC-12 cells treated with CM2 exhibiting significantly longer nerve axons than those treated with CM1 (\u003cb\u003eExtended Data Fig.\u0026nbsp;10d-f\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo verify HGF's role in sympathetic nerve fiber outgrowth in LNs, we used two inhibitors: Norleual TFA, which competitively inhibits HGF by mimicking its hinge region, and SRI 31215, which blocks the proteolytic activation of pro-HGF by targeting matriptase, hepsin, and HGF activator (HGFA). Both Norleual TFA and SRI 31215 attenuated PC-12 cell elongation induced by CM1 or CM2 \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-f).\u003c/p\u003e \u003cp\u003ePrevious studies indicate that LN expansion is primarily driven by FRCs activation and biomechanical changes\u003csup\u003e10,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To investigate these changes during TLNE, we performed immunohistochemical (IHC) staining on α-smooth muscle actin (α-SMA) in LNs. Significant α-SMA expression could be observed at as early as 2 days post-inoculation, with elevated levels at 5, 7, and 10 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Additionally, Sirius red staining also revealed significant collagen deposition in the LNs following the similar trend as α-SMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, \u003cb\u003eupper\u003c/b\u003e). Further analysis using polarized light microscopy showed that collagen fibers were primarily composed of type I and III collagen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, \u003cb\u003elower\u003c/b\u003e). To assess matrix stiffness, we used atomic force microscopy (AFM) to measure the Young\u0026rsquo;s modulus, revealing a series of continuous significant increases of Young\u0026rsquo;s modulus in PLNs from day 0 to day 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). These findings indicate that TLNE induces biomechanical changes and FRC activation, leading to PLN expansion.\u003c/p\u003e \u003cp\u003eTo investigate the effects of mechanical properties on HGF secretion, primary FRCs were cultured on Matrigel with stiffnesses of 0.5 kPa and 12 kPa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Higher \u003cem\u003eActa2\u003c/em\u003e and \u003cem\u003eCol1a1\u003c/em\u003e expressions could be observed on the stiffer Matrigel, as well as HGF secretion, indicating that FRCs showed significantly higher activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek-m). These data indicated that elevated matrix stiffness enhances FRC activation and increases HGF secretion, thus contributing to nerve fiber outgrowth.\u003c/p\u003e\n\u003ch3\u003e4. HGF inhibition suppresses sympathetic nerve outgrowth in TLNE and LN metastasis\u003c/h3\u003e\n\u003cp\u003eTo further confirm the \u003cem\u003ein vivo\u003c/em\u003e role of HGF, we employed Norleual TFA and adeno-associated virus (AAV)-mediated silencing of HGF expression. Norleual TFA or AAV was administered into the popliteal fat pad of mice 6 hours prior to MC38 tumor cell inoculation, with additional injections every three days thereafter (days 0, 3, and 6). PLNs were harvested at day 7, cleared using HYBRiD, and subsequently analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Three-dimensional imaging of TH-stained sympathetic fibers showed significant suppression of sympathetic nerve outgrowth following treatment with Norleual TFA or AAV-mediated HGF silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-e). Consistently, 3D imaging of DBH-positive sympathetic nerve fibers produced similar findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-i). Together, these results indicate that silencing HGF expression effectively inhibits sympathetic reinnervation within PLNs, reinforcing HGF's critical role in sympathetic nerve fiber expansion during TLNE.\u003c/p\u003e \u003cp\u003eAdditionally, we investigated the impact of HGF inhibition on TLNE and LN metastasis. Administration of the HGF inhibitor Norleual TFA and AAV-mediated HGF silencing significantly suppressed PLN expansion induced by tumor cell invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej, k). IHC analysis of pan-cytokeratin (panCK) and α-SMA in PLNs further demonstrated that HGF silencing markedly inhibited tumor cell-induced LN metastasis and FRC activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el, m). These findings indicate that targeting HGF effectively attenuates TLNE and LN metastasis driven by tumor cells.\u003c/p\u003e\n\u003ch3\u003e5. Sympathetic nerve denervation inhibits LN metastasis\u003c/h3\u003e\n\u003cp\u003eTo further elucidate the role of the sympathetic nervous system in TLNE, we performed sympathetic denervation by injecting 6-hydroxydopamine (6-OHDA) into the fat pad surrounding the PLNs of mice five days prior to tumor cell inoculation. PLNs were harvested at days 5 and 7 post-inoculation and analyzed by IF and IHC staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The results demonstrated that sympathetic denervation markedly inhibited PLN expansion triggered by tumor cell invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-e). Imaging of tdTomato-labeled tumor cells and panCK staining further indicated that sympathetic denervation effectively suppressed MC38 cell-induced LN metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-i). Moreover, the elevated percentages of B cells, Marcrophages, and neutrophils observed during TLNE were significantly reduced following 6-OHDA administration (\u003cb\u003eExtended Data Fig.\u0026nbsp;11a, b\u003c/b\u003e). Collectively, these findings underscore the crucial role of sympathetic innervation in promoting LN metastasis during tumor progression.\u003c/p\u003e \u003cp\u003eTaken together, our findings demonstrate that during TLNE, LN innervation dynamically adapts to accommodate structural expansion. Activated FRCs secrete HGF, facilitating sympathetic nerve fiber outgrowth, accompanied by biomechanical changes within the LN microenvironment. Increased matrix stiffness further enhances FRC activation, thereby promoting additional sympathetic nerve growth. Importantly, inhibition of HGF signaling or sympathetic nerve denervation effectively suppresses TLNE and tumor-induced LN metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ej).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLNs are peripheral immune organs that enlarge in response to infection or other stimuli. Stromal cells, especially FRCs, play a critical role by secreting extracellular matrix (ECM) and modulating microenvironmental stiffness, thereby supporting LN expansion and maintaining structural integrity\u003csup\u003e9\u0026ndash;11\u003c/sup\u003e. Recent studies have shown that cold-induced sympathetic activity activates the β-adrenergic receptor (β-AR) signaling pathway in FRCs, indicating that LN function is regulated by the nervous system\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, the mechanisms underlying LN volume changes and the role of the nervous system in regulating this process remain unclear. Specifically, how the nervous system adapts to and manages the increase in LN volume is not well understood. Additionally, whether the immune system modulates the structure and function of the nervous system remain an intriguing and unanswered question\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo investigate these questions, we utilized a tumor-induced LN metastasis model to induce LN enlargement\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Unexpectedly, as the LNs expanded, we observed significant growth of nerve fiber, including axon elongation and increased nerve branching, which were subsequently verified to be predominantly sympathetic nerves. Remarkably, FRCs played a crucial role, they not only provided structural support during LN expansion but also released neurotrophic factors, promoting nerve growth and facilitating neural adaptation to the organ's enlargement. Notably, the increased stiffness of the ECM during LN enlargement further augmented this nerve growth process.\u003c/p\u003e \u003cp\u003eOur results revealed that during the early stages of tumor cell metastasis, FRCs exhibit a highly active state within two days post-metastasis, followed by significant nerve regrowth on the third day. Previous research has primarily focused on the role of FRCs in LN enlargement without linking it to neural remodeling\u003csup\u003e26,27\u003c/sup\u003e. Our observation of this sequential order suggested a potentially inseparable connection between nerve fibers and FRC activation within LNs.\u003c/p\u003e \u003cp\u003eSubsequently, we verified two key findings. First, during TLNE, the adaptive growth of nerve fibers within LNs predominantly involves sympathetic nerves, and this growth correlates positively with the extent of LN expansion. Although sensory nerves are abundantly distributed within LNs, their alterations are minimal relative to the expansion of LNs. This underscores the importance of the efferent regulation by the peripheral nervous system in maintaining LN functionality. Second, through comprehensive snRNA-seq data analysis, combined with \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments, we found that activated FRCs secrete substantial amounts of HGF. Previous studies have shown that HGF is a crucial growth factor in cancer development and progression, contributing to the formation of a metastatic microenvironment\u003csup\u003e28,29\u003c/sup\u003e. Additionally, HGF is well-documented to promote axon growth\u003csup\u003e21,30\u003c/sup\u003e. By inhibiting HGF using various methods, we observed the disappearance of sympathetic nerve regrowth associated with LN enlargement. These findings demonstrated that early-stage sympathetic nerve regrowth in TLNE is dependent on FRC activation and subsequent HGF secretion.\u003c/p\u003e \u003cp\u003eThe regulation of LNs by the nervous system during tumor metastasis is critically important. LNs are dispersed throughout the body, each monitoring specific regions for immune challenges. Sympathetic nerves play a key role in coordinating immune responses across this network. When a single LN detects a threat, sensory nerves transmit signals to the brain, which then commands sympathetic nerves to regulate immune responses across all LNs. This orchestrated \"whole-body response\" suggests that the collective immune efficacy of the LN network may surpass that of an individual LN. Rapid neural regulation aligns with the swift responses characteristic of adaptive immunity, emphasizing the importance of neural involvement in tumor immune surveillance and response.\u003c/p\u003e \u003cp\u003eThe physiological significance of sympathetic nerve reinnervation observed during LN metastasis remains to be elucidated. Potential mechanisms may involve altering the LN microenvironment through nerve fiber growth and branching, as well as regulating immune and stromal cells via neurotransmitters such as norepinephrine (NE). Notably, previous studies have shown that sympathetic nerves accompany blood vessels into LNs, suggesting that reinnervating nerves might influence vascular neogenesis and exert regulatory effects on blood vessels. Therefore, future research should focus on elucidating these potential interactions.\u003c/p\u003e \u003cp\u003eFRCs, key contributors to LN expansion, not only provide structural support but also direct the adaptive growth of nerve fibers by secreting HGF. During TLNE, FRCs likely serve as critical coordinators of both LN remodeling and nerve fiber regeneration, with HGF functioning as an essential mediator. Understanding this mechanism deepens our comprehension of LN biology and suggests novel pathways for innovative immunotherapeutic and neuroregulatory approaches. HGF, identified as a pivotal mediator of LN-associated nerve growth, emerges as a promising therapeutic target for facilitating neural remodeling. Moreover, directly manipulating neural signals within LNs could enhance immune responses while minimizing unnecessary inflammation. Importantly, inhibiting HGF signaling or performing sympathetic nerve denervation significantly suppresses TLNE and associated LN metastasis, highlighting the potential of targeting both HGF and sympathetic innervation in treating tumor-induced LN metastasis. These findings provide critical insights into potential therapeutic strategies with significant scientific and clinical relevance for improving outcomes in cancer patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available upon request and relevant data are available in the Source Data. The snRNA-seq data from this study have been deposited in the Gene Expression Omnibus (GEO) with the accession code GSE280461.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (ID 82230087, 82350123, 82203228, to Z.-G. Zhang; ID 82073023, 81871923, to J. Li; ID 82372821, 82103357 to L.-P. Hu; ID 82002485, to Q. Li; ID 82203228, to D.-X. Li; ID 82103348, to Y.-Y. Wang), the Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (ID 20181708, to Z.-G. Zhang), Innovative research team of high-level local universities in Shanghai (ID SHSMU-ZDCX20210802, to Z.-G. Zhang), Shanghai Pilot Program for Basic Research - Shanghai Jiao Tong University (ID 21TQ1400225, to Z.-G. Zhang), 111 project (ID B21024, to Z.-G. Zhang), Shanghai Science and Technology Commission Sailing Project (ID 22YF1445600 to D.-X. Li; ID 21YF1445200 to L.-P. Hu), the Natural Science Foundation of Shanghai (ID 21ZR1461300 to L.-P. Hu; ID 22ZR1460000 to X.-L. Zhang), Innovative research team of high-level local universities in Shanghai (ID SHSMU-ZDCX20210802 to X.-L. Zhang), the Shanghai Municipal Health Commission (ID 20214Y0200 to D.-X. Li), Key Areas Research and Development Programs of Guangdong Province (ID 2023B1111050009 to M.-J. Xu).\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Zhi-Feng Shao, Dr. Xiao-Mei Yang, Dr. Yan-Li Zhang, Dr. Lei Zhu, Dr. Lin-Li Yao and Dr. Ni Zhang for assistance with our experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.L. and Z.-G.Z. designed and supervised the overall study, analyzed data, and drafted the manuscript. J.L., X.Z., L.-P.H. and Q.L. constructed the mouse footpad model and collected the LN tissues. X.Z. and G.-H.S. performed the HYBRiD clearing and whole-mount tissue 3D imaging. J.L., H.L., Y.-Q.Z. and J.-X.X. analyzed the single-nucleus RNA sequencing data. X.Z., G.-H.S. and Y.-K.L. analyzed the data of 3D imaging. Y.-Y.W., T.-S.B., S.Z. and C.-J.X. assisted with analysis of IHC and IF staining. J.S., S.-Q.Y., X.-W.L. and J.X. technically assisted with experiments and analyzed data. J.X. provided samples. X.-L.Z., S.-H.J. and D.-X.L. supervised this study and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING FINANCIAL INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuang, S. 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During tumor metastasis, LNs undergo significant structural remodeling and enlargement; however, the role of neural innervation in this process remains unclear. Here, using whole-organ three-dimensional (3D) imaging, we observed pronounced elongation and increased branching specifically in sympathetic nerve fibers, but not sensory nerves, during tumor-induced LN enlargement (TLNE), suggesting adaptive neural remodeling. Single-nucleus RNA sequencing further revealed activation of fibroblastic reticular cells (FRCs) during TLNE, characterized by enriched neuro-related signaling pathways and substantial secretion of hepatocyte growth factor (HGF). Functional validation using targeted HGF inhibitors and adeno-associated virus (AAV)-mediated HGF silencing confirmed that FRC-derived HGF critically drives sympathetic nerve growth. Additionally, both HGF inhibition and sympathetic nerve denervation significantly reduced TLNE and tumor-induced LN metastasis, highlighting the importance of adaptive sympathetic innervation in tumor-associated LN remodeling. These findings identify a previously unrecognized FRC-HGF-sympathetic nerve axis and propose neural regulation as a potential therapeutic strategy for tumor-induced LN metastasis.\u003c/p\u003e","manuscriptTitle":"Fibroblastic reticular cell–derived HGF orchestrates sympathetic nerves in tumor-induced lymph node remodeling and metastasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-26 09:12:22","doi":"10.21203/rs.3.rs-6607085/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":"257e9b80-6e77-4719-81ed-a991e5c28ebf","owner":[],"postedDate":"May 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48876733,"name":"Biological sciences/Cancer"},{"id":48876734,"name":"Health sciences/Diseases/Cancer"}],"tags":[],"updatedAt":"2025-06-25T14:51:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-26 09:12:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6607085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6607085","identity":"rs-6607085","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}