Structural and Functional Characteristics of Leptomeningeal Lymphatic Vessels in Leptomeningeal Metastases from Lung Cancer Patients

Structural and Functional Characteristics of Leptomeningeal Lymphatic Vessels in Leptomeningeal Metastases from Lung Cancer Patients | 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 Structural and Functional Characteristics of Leptomeningeal Lymphatic Vessels in Leptomeningeal Metastases from Lung Cancer Patients Xiaoyu Hua, Minting Ye, Da Liu, Hainan Li, Chongzhu Fan, WenFeng Mai, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5244229/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 Background: The purpose of this study is to explore whether there are lymphatic vessels in the human leptomeninges, and their potential role in the immune response to central nervous system tumors, in order to determine their significance in the field of tumor biology, especially the role they play in the growth, metastasis, and immune response of tumors. Methods: We used immunohistochemistry and immunofluorescence techniques to examine the leptomeninges in 46 patients diagnosed with leptomeningeal metastasis in cerebrospinal fluid cytology (29 positive biopsy specimens, 17 negative) as well as 11 epilepsy patients. We visualized lymphatic vessels in the human leptomeninges using LYVE1 and PDPN antibodies, labeled tumor cells with CK, T cells with CD3, and blood vessels with CD31 and α-smooth muscle actin. By comparing the lymphatic vessel density and T cell count in tumor areas versus non-tumor areas, and observing whether there was infiltration of tumor cells into the lymphatic vessels, we analyzed the presence and function of human leptomeningeal lymphatic vessels. Results: The research results confirmed the existence of lymphatic vessels in the human leptomeninges, with a significant increase in lymphatic vessel density and T cell count around the tumor compared to non-tumor areas (P < 0.05). At the same time, infiltration of tumor cells was observed within the lymphatic vessels. Conclusions: These findings suggest that the lymphatic vessels in the leptomeninges not only structurally resemble extracranial brain lymphatic vessels, but also function similarly in tumor immune response and metastasis pathways. These findings challenge traditional understanding of immune responses to central nervous system tumors and provide important clues for further research on the role of intracranial lymphatic vessels in tumor biology. Lymphatic Podoplanin LYVE1 Leptomeningeal metastases Leptomeninges T cells Lymphatic invasion Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Leptomeningeal metastasis (LM) is an unpleasant and potentially fatal phenomenon in advanced cancer patients. LM involves the spread of malignant cells to leptomeninges, the subarachnoid space, and other compartments of the cerebrospinal fluid (CSF) system [ 1 ]. LM occurs in approximately 3–5% of patients with advanced non-small cell lung cancer. However, because to improved outcomes from novel molecular treatments, the prevalence of LM among particular categories of patients with targetable mutations has increased[ 2 , 3 ]. The diagnosis and treatment of LM have become the focus of recent research because of its difficulty to diagnose and the poor treatment outcomes and short survival time of patients. Previously, the prevailing belief was that the central nervous system (CNS) lacked lymphatic vessels and that LM primarily occurred through direct invasion, CSF dissemination, and blood dissemination. However, recent animal experiments and human studies have substantiated the presence of lymphatic vessels within the cranial cavity that drains into deep cervical lymph nodes[ 4 – 7 ]. Given that the cranial cavity has a functioning glymphatic system, it may be necessary to reevaluate established beliefs about tolerance and immune privilege in the CNS. Many different neurological illnesses may involve meningeal lymphatic vessel dysfunction as their underlying etiology[ 8 , 9 ]. Therefore, we hope to gain a deeper understanding of the mechanism of interaction between leptomeningeal lymphatic vessels and CNS tumors by studying specimens from LM patients to provide strong support for the development of more effective tumor treatment strategies. The characterization of the endocranial lymphatic circulatory system in animals has crucial implications for our comprehension of the physiopathological mechanisms in the CNS [ 4 , 5 , 10 ]. In previous studies, magnetic resonance imaging (MRI) has been used to visualize the meningeal lymphatic network, while immunofluorescence has been used to visualize lymphatic endothelial cells in the meningeal lymphatic system with molecular markers[ 11 ]. In mice, macromolecules and immune cells from CSF are drained via the meningeal lymphatic system. Moreover, meningeal lymphatics play crucial roles in immune surveillance and inflammation in the CNS, making them promising therapeutic targets[ 12 ]. However, the majority of evidence now available about meningeal lymphatic activity comes from research conducted on animals, whereas human data include only observations made from postmortem specimens[ 13 – 15 ]. Moreover, studies on the changes in lymphatic vessels in malignant tumors in the CNS are lacking[ 16 , 17 ]. However, to our knowledge, recent research has only confirmed the existence of lymphatic vessels in leptomeninges[ 13 ]. We hope to further explore the functions of leptomeningeal lymphatic vessels in promoting metastasis, regulating immune responses, and influencing the tumor microenvironment. Our samples were collected from living humans, and to our knowledge, this is the first study of the functional status of lymphatic vessels around human CNS tumors, which will more accurately reflect the true role of lymphatic vessels in the development of diseases. Ultimately, we found that the lymphatic vessels in the leptomeninges not only structurally resemble extracranial lymphatic vessels but also function similarly in the tumor immune response and the metastatic pathway. Results Strategy To determine the presence and localization of lymphangions within human leptomeninges, we used immunofluorescence and immunohistochemistry techniques to analyze leptomeningeal tissue samples collected from LM subjects (n = 46) and control subjects with epilepsy (n = 11) during preventricular puncture and surgical procedures (Supplementary Fig. 1). Table 1 includes demographic information for each individual, including age, sex, surgical location, and EGFR mutation status. Initially, we used LYVE1, podoplanin (PDPN), Prox1, and VEGFR3 to visualize lymphatic endothelial cells. However, in alignment with the literature[ 13 , 18 , 19 ], detecting Prox1 has proven particularly challenging (Supplementary Fig. 2d and 2h). Moreover, given the widespread distribution of VEGFR3 in the brain parenchyma, these markers do not seem to be the most suitable for this purpose (Supplementary Fig. 2c and 2g). Consequently, we ultimately opted to employ LYVE1 and PDPN to visualize the lymphatic vessels. We utilized CD31 and alpha-smooth muscle actin (a-SMA) as markers for blood vessels and smooth muscle tissue, respectively. In contrast to arteries and veins, lymphatic vessels express LYVE1 and/or PDPN but lack expression of CD31 and/or a-SMA. Furthermore, we used CK and TTF-1 to identify tumor cells in the leptomeninges and used CD3 to label T cells. Table 1 Patient material. Patients with leptomeningeal metastases Epileptic Biopsy positive Biopsy negative Sex (f/m) 19/10 11/6 5/6 Age (yrs) 53 ± 10.2 54 ± 8.9 19 ± 12.2 EGFR (n; %) 26/29(89%) 14/17(82%) – Surgical location (leptomeningeal location) Right 8 6 7 Left 21 11 4 Quantitative characteristics of lymphatic vessels LVA (%) 7.18 ± 3.93 3.36 ± 1.42 3.39 ± 1.34 T cell per leptomeningeal (per/µm 2 ) 21.2e‒05 ± 9.6e‒05 8.1e‒05 ± 2.7e‒05 10.2 ± 4.5e‒05 Categorical data presented as n (%), and quantitative data shown as average ± standard deviation. Lymphatic vessels are present in human leptomeninges In the cortex and leptomeninges of human brain samples, we observed that lymphatic vessels were widely distributed throughout the leptomeninges, especially concentrated in areas rich in blood vessels; morphologically, LYVE1 + endothelial cells formed a distorted network extending along arteries and arterioles (Fig. 1 a-e). Additionally, we found that lymphatic vessels could exist adjacent to or independent of blood vessels (Fig. 1 c). The lateral side of the outer walls of most subarachnoid space blood vessels clearly contained green fluorescent cells labeled with LYVE1, and a large artery surrounded by LYVE1-labeled cells with several LYVE1-positive small arteries feeding into this large vessel (Fig. 1 c). We also observed LYVE1-positive cells along the arterial pathway crossing through the cerebral cortex (Fig. 1 c-e). To validate the lymphatic nature of LYVE1-positive immune reaction structures, we confirmed the colabeling of LYVE1 and PDPN in lymphatic vessels (Fig. 1 f-h). Moreover, similar to other peripheral lymphatic vessels, there are two types of lymphatic vessels present in the leptomeninges: one type expresses both LYVE1 and PDPN markers (Supplementary Fig. 3a-d), whereas the other expresses only PDPN markers (Supplementary Fig. 3e-h). Both types of lymphatic smooth muscle markers were negative. The first type is similar to the pre-collected lymphatic vessels found in other parts of the peripheral system[ 20 ]. The quantity of this type of lymphatic vessel shows little difference between epilepsy patients and those with LM from lung cancer. The second type of lymphatic vessels is significantly increased in number in patients with LM from lung cancer, and they are mainly concentrated around the tumor (Supplementary Fig. 4a-c). This type of lymphatic vessel is characterized by an increased PDPN-positive vascular area, forming irregular networks (Supplementary Fig. 4c). Within these networks, endothelial cells are dilated, and there are formations of bridge-like structures, valve-like structures, and small tubes (Supplementary Fig. 4d-e). This type of lymphatic vessel is similar to the initial lymphatic vessels found in other extracranial regions and resembles the immature, newly formed lymphatic vessels around extracranial tumors. Compared with leptomeningeal arteries and veins, both types of lymphatic vessels presented negative staining for the blood endothelial cell marker CD31 and the smooth muscle marker a-SMA (Fig. 1 a-b). This is similar to the description of lymphatic vessels in postmortem human samples presented by Goodman and Louveau[ 5 , 16 ]. LM in lung cancer patients: Leptomeningeal lymphangiogenesis, lymphatic network remodeling, and lymphatic vessel invasion After specific lymphatic vessel staining of leptomeningeal specimens from 46 patients diagnosed with LM via CSF cytology (29 positive on biopsy, 17 negative) and 11 patients with epilepsy, we observed an increased area of PDPN-positive vessels around the tumor and the formation of irregular lymphatic vascular networks (Fig. 2 a-d). Within these networks, endothelial cell expansion accompanied by the formation of bridge-like structures, valve-like structures, and small tubes was observed (Supplementary Fig. 4d-e), which resembled the description of lymphatic vessel remodeling structures around pancreatic cancer presented by Shen et al[ 21 ]. The analysis of lymphatic vessel density in the three groups of patients revealed that the lymphatic vessel density around the leptomeningeal tumor in patients with positive leptomeningeal biopsy for LM was significantly greater than that in the other two groups (Fig. 2 e-h), and this difference was statistically significant ( P < 0.05). The difference between the latter two groups was not significant (Fig. 2 g), with average values of 7.18 ± 3.93%, 3.36 ± 1.42%, and 3.39 ± 1.34%, respectively (Table 1 ). In addition, we detected lymphatic tumor cell infiltration in 18 out of 29 patients with positive leptomeningeal biopsies (Fig. 2 b-d). We observed significant expansion of lumens formed by PDPN-positive cells, which were filled with CK-positive tumor cells (Fig. 2 b-c). These results suggest that lung cancer metastasis may lead to lymphangiogenesis and remodeling of the lymphatic network in the leptomeninges. Compared with nontumor patients, LM patients have more T lymphocytes in the leptomeninges We wanted to determine whether lymphocytes exist inside or near the lymphatic vessels in the leptomeninges. Moreover, we aimed to verify the differences in the number of lymphocytes among three groups: patients with positive CSF cytology for LM and positive or negative biopsy specimens and a control group of epilepsy patients. To answer this question, we stained tissue samples from a total of 29/57 patients, including 11 samples from lung cancer patients with LM, 7 samples of negative biopsy tissue from lung cancer patients with LM, and 11 samples from patients in the control group with epilepsy, with an anti-CD3 antibody (a specific T lymphocyte marker). In all the leptomeninges samples we studied, we found lymphocytes outside the blood vessels. In the leptomeninges, we observed CD3-positive T cells located outside the vascular walls and interspersed among LYVE1/PDPN-positive cells (Fig. 3 a-d and 3 f-g). In the leptomeninges of lung cancer patients with LM and positive biopsy tissue, there were many CD3-positive cells between the vessel wall and LYVE1-positive cells (Fig. 3 d-e and 3 g-h). Similarly, in lung cancer patients with LM with negative biopsy tissue and in epilepsy patients, a moderate number of CD3-positive T lymphocytes were observed within the interstices surrounded by lymphatic endothelial cells (Fig. 3 a-c and 3 f). To determine the differences in T lymphocyte numbers among the three groups of patients, we quantified CD3 expression in the most densely populated areas of each slice and measured the leptomeninges area of this region, comparing the ratio of these two value to assess differences among the three groups. We found that lung cancer patients with LM and positive biopsy tissue had the highest CD3 count (Fig. 3 i), and this difference was statistically significant ( P < 0.05). The difference between the latter two groups was not significant (Fig. 3 i), with average values of 21.2e‒05 ± 9.6e‒05 per/µm 2 , 8.1e‒05 ± 2.7e‒05 per/µm 2 , and 10.2 ± 4.5e‒05 per/µm 2 , respectively (Table 1 ). Discussion The clearance of CNS fluids and molecular waste and the mechanisms underlying tumor immunity have always been popular research topics. The traditional view is that unlike other parts of the body, the CNS lacks lymphatic vasculature; however, recent groundbreaking research has shown that lymphatic vessels do extend to cover the meningeal tissues of the entire CNS in mice, rats, primates, and humans[ 4 – 7 , 10 ]. However, little is known about the existence of lymphatic vessels in leptomeninges and the role of these vessels in CNS tumors. Therefore, in this study, we performed immunohistochemical and fluorescence staining experiments for endothelial-specific markers of lymphatic vessels in the leptomeninges of 46 lung cancer patients with LM and 11 patients with epilepsy. The results confirmed a situation similar to that reported in Mezey's postmortem studies: that there are indeed lymphatic vessels present in the leptomeninges of humans[ 13 ]. We further explored whether these findings imply that the lymphatic vessels in the leptomeninges have functions similar to those of the peripheral tissue lymphatic system. We know that lymphatic vessels play various important roles in tumor biology, including promoting metastasis, regulating immune responses, and influencing the tumor microenvironment[ 22 – 24 ]. Therefore, we verified each of these aspects one by one. Ultimately, we found that the lymphatic vessels in the leptomeninges not only structurally resemble extracranial lymphatic vessels but also function similarly in the tumor immune response and the metastatic pathway (Fig. 4 ). To our knowledge, there has been no relevant research on this topic thus far. This study is highly important for obtaining a deeper understanding of the interaction between lymphatic vessels and CNS tumors. Specific animal experiments are needed in the future to elucidate the exact mechanisms involved, which will also aid in the development of more effective tumor treatment strategies. In the past decade, the CNS lymphatic system has once again become a focus of attention. In 1987, Andres et al.[ 25 ] described lymphatic vessels in the human dura mater, which was recently confirmed by Aspelund et al.[ 4 ] and Louveau et al.[ 5 ] Moreover, Mezey et al.'s study on human cadaver samples revealed that positive lymphatic marker staining was detected in the pia mater, in the arachnoid, in venous sinuses, and among the layers of the dura mater[ 13 ]. However, existing evidence concerning meningeal lymphatic function has been derived mainly from animal studies, and human data rely entirely on observations from autopsy specimens. Our samples were collected from living humans, and to our knowledge, this is the first study of the functional status of lymphatic vessels around human meningeal tumors, which will more accurately reflect the true role of lymphatic vessels in the development of diseases. We observed that lymphatic vessels are widely distributed throughout the entire leptomeninges, especially in areas rich in blood vessels. This finding is consistent with previous studies on zebrafish and human cadaveric samples[ 13 , 14 ]. We also found LYVE1-positive cells along the arterial path crossing through the cerebral cortex (Fig. 4 ), similar to the findings reported by Mezey et al., suggesting that the 'perivascular' space described by many researchers in the early stages of this research appears to be composed of endothelial cells 13 . In addition, we also observed two types of lymphatic vessels in the subarachnoid space: PDPN + LYVE1 + CD31 − type lymphatic vessels, which are consistent with peripheral pre-collected lymphatic vessels and those described in the meningeal sinuses of mice[ 5 ]. There is no significant difference in the quantity of this type of lymphatic vessel between epilepsy patients and those with LM from lung cancer. The second type is PDPN + LYVE1 − CD31 − lymphatic vessels, which significantly increases in number in patients with lung cancer LM, primarily surrounding the tumor and forming a network-like structure. Within this network, endothelial cells expand, accompanied by the formation of bridge-like structures, valve-like structures, and small vessels. This type of lymphatic vessel is similar to the initial lymphatic vessels described in the extracranial lymphatic system[ 16 ]. Such structures can also be observed around pancreatic cancer, characterized by newly formed, immature lymphatic vessels. Their inner walls consist of a single layer of endothelial cells, which are loosely connected and lack a complete basement membrane, making it easier for tumor cells to invade (Fig. 4 )[ 20 ]. These are clearly speculative possibilities, and more investigations are needed to assess them. Previous studies have shown that the growth of extracranial tumors not only induces angiogenesis but also promotes lymphangiogenesis in some cases, facilitating tumor cell entry into the surrounding lymphatic system. VEGF-C produced by tumor cells can induce the proliferation and expansion of lymphatic vessels toward tumor cells, leading to increased lymphatic vessel growth within 2–3 weeks after tumor xenograft modeling, accompanied by lymph node metastasis[ 21 , 26 – 28 ]. Therefore, we investigated whether there is a significant increase in the number of lymphatic vessels around lesions in the leptomeninges of lung cancer patients with LM and whether there is evidence of tumor cell invasion in these vessels, similar to other extracranial tumors. We observed that the density of leptomeningeal lymphatic vessels was significantly greater in lung cancer patients with LM than in patients without metastatic lesions and those with epilepsy. In addition, we also observed tumor cell infiltration in the leptomeningeal lymphatic vessels of patients with LM, indicating that leptomeninges lymphatic vessels play a role in tumor-induced lymphangiogenesis and remodeling processes as well as in the metastatic pathway as extracranial lymphatic vessels. An early necessary step in the cascade of lymphatic metastasis is the invasion of clusters or individual tumor cells into the lymphatic system[ 29 ]. Furthermore, CSF enters systemic circulation through deep cervical lymph nodes via dural lymphatic vessels[ 5 ]. Therefore, we speculate that the lymphatic pathway may be one route for LM. Our previous studies also revealed a strong correlation between LM associated with lung cancer and deep cervical lymph node involvement[ 30 ]. A study by Smith et al. confirmed that ligation of the cervical lymphatic vessels leads to brain edema and increased protein concentrations in the CSF of cats and rabbits[ 31 ]. Therefore, we speculate that lymphatic vessel infiltration is an important cause of intracranial hypertension in patients with LM. Moreover, severe intracranial hypertension significantly affects the quality of life and outcomes of patients. Therefore, treatment targeting lymphatic vessel lymphangiogenesis and infiltration may significantly improve the quality of life and outcomes of patients. The process of lymphatic vessel invasion (LVI) involves complex mechanisms. Initial lymphatic vessels have discontinuous cell‒cell connections, allowing entry of white blood cells through small gaps between VE-cadherin-positive "buttons"[ 32 ]. However, tumor cells, which are larger and less susceptible to morphological changes than white blood cells are, face barriers when attempting to invade these vessels [ 33 , 34 ]. Signals from tumor cells or the tumor microenvironment can disrupt lymphatic endothelial cell junctions, creating larger pores known as "chemorepulsion-induced defects" or entry gates[ 35 , 36 ]. Drugs targeting these mechanisms are in development to inhibit LVI and lymphatic spread. One approach is to interfere with the local tissue infiltration of tumor cells [ 37 , 38 ]; another strategy is to inhibit tumor-associated lymphangiogenesis to reduce the interface between tumors and lymphatic vessels [ 39 ]. In the future, we could use some of these methods in combination with current regimens for treating lung cancer patients with LM. After the identification of lymphatic vessels in the leptomeninges, our aim was to establish their correlation with the presence and distribution of T lymphocytes, thereby functionally confirming the resemblance between leptomeningeal lymphatic vessels and peripheral lymphatic vessels. Research has indicated that, similar to other organs, the CNS undergoes constant immune surveillance for abnormal cells and pathogens while also relying on immune cells to maintain normal tissue homeostasis. Peripheral-derived T cells, macrophages, and dendritic cells appear to play a role in this process[ 40 – 42 ]. However, in CNS tumors, T lymphocytes are the primary immune cells involved[ 22 ]; hence, we utilized CD3 as a marker for these immune cells in our study. Previous research has demonstrated the important role of dura mater lymphatic vessels in bridging the CNS and peripheral immune system[ 43 – 45 ]. As such, we postulate that leptomeningeal lymphatic vessels also play a pivotal role in this process. In our research sample, we commonly observed the presence of lymphocytes inside or near the lymphatic vessels in the leptomeninges, which is consistent with previous literature reports[ 13 ]. Furthermore, in patients with positive LM biopsy samples, the number of T cells near leptomeningeal lymphatic vessels was significantly greater than that in LM biopsy-negative patients and was also greater than that in control epilepsy patients. Additionally, animal model studies have validated the drainage of soluble tumor antigens from the CNS into cervical lymph nodes via CSF, thereby eliciting specific T-cell responses[ 46 ]. Consequently, we hypothesize that LM elicits an effective antitumor immune response leading to an increase in T-cell populations in this region. Hu et al. demonstrated the crucial role of meningeal lymphatic vessels (MLVs) in intracranial tumor fluid drainage and immunity, highlighting the importance of MLV remodeling induced by brain tumors for immunotherapy[ 22 ]. However, notably, the MLV on the dorsal side of the dura mater does not directly physically contact the tumor. Therefore, further animal experiments are warranted to verify whether the lymphatic vessels in the leptomeninges play a bridging role and elucidate their involvement in the tumor immune response. Some limitations of the present study should be noted. The limited sample size and restricted anatomical focus of this study present additional drawbacks, although it is the largest living human cohort examined for leptomeningeal lymphatic vessels. In addition, all participants, particularly those with LM, underwent various treatment regimens before sample collection, which prevented this study from fully reflecting the initial state of leptomeningeal lymphatic vessels in the CNS. Moreover, owing to the cross-sectional nature of the sampling, we were unable to capture changes in leptomeningeal lymphatic vessels dynamically during tumor immune responses or explore their specific roles in CSF and waste clearance. These limitations hinder our understanding and research into the dynamic nature of these important biological processes. Therefore, the findings reported may not be able to be extrapolated to a broader context of leptomeningeal lymphatic vessels. Conclusions Our research results confirm the existence of lymphatic vessels in the human leptomeninges, and show a significant increase in lymphatic vessel density and T cell count around tumors compared to non-tumor areas. We also observed infiltration of tumor cells into the lymphatic vessels. These findings suggest that the lymphatic vessels in the leptomeninges not only structurally resemble extracranial brain lymphatic vessels, but also function similarly in tumor immune response and metastasis pathways. The discovery of the leptomeningeal lymphatic vessels expands our understanding of the diversity of "brain" lymphatic vessels, beyond the scope of the dural lymphatic vessels. Although the role of dural lymphatic vessels in brain and CSF clearance and tumor immunity is still controversial, the interaction between leptomeningeal lymphatic vessels and dural lymphatic vessels in draining CSF, clearing macromolecules, and potential tumor immunity deserves further exploration. Methods Human leptomeningeal samples All patients were diagnosed with LM by CSF cytology prior to undergoing meningeal biopsy. Leptomeningeal samples were obtained during the preventricular puncture procedures. The specific procedures included ventriculoperitoneal (VP) + Ommaya reservoir implantation in 36 patients, VP surgery in 3 patients, Ommaya reservoir implantation in 3 patients, and external ventricular drainage in 3 patients. Trepanation for the surgical operation and tissue sampling was conducted at a site located 12 cm caudally from the glabella, with a lateral deviation of approximately 1–2 cm from the midline (Supplementary Fig. 1a-c and Table 1 ). A burr hole with a diameter of less than 1 cm was created to expose the underlying leptomeninges. The leptomeninges were removed in one piece by incising it circumferentially (approximately 5 mm in diameter and 1 mm in thickness). Eleven patients with drug-resistant epilepsy due to nonneoplastic causes underwent surgical resection of the epileptogenic focus. The anterior temporal lobe was removed, with a surgical range of 4.5 cm posterior from the temporal pole on the left side and 5.5 cm posterior from the temporal pole on the right side (Supplementary Fig. 1d-f and Table 1 ). Histopathologically, the entire leptomeningeal region of the anterior temporal lobe was selected for staining. The medical records of the patients were used to gather clinical data. This study was approved by the Sanjiu Brain Hospital Ethics Committee. Immunohistochemistry and immunofluorescence For immunohistochemical staining, the samples were fixed in 10% neutral buffered formalin for 48 hours, dehydrated, and embedded in paraffin. Three-micron paraffin-embedded human meningeal coronal sections (Xiamen Elibot Company) were prepared via the glass slide method. The slides were incubated at 50°C overnight, deparaffinized in xylene, and rehydrated in a gradient of ethanol. The slides were placed in an antigen retrieval instrument (Dako link48), and the antigen retrieval solution (10 mM Tris, 1 mM EDTA, pH 9) was heated to 95°C for 20 minutes and cooled to room temperature. Endogenous peroxidases were blocked with 3% H 2 O 2 for 5 minutes, followed by washing with phosphate-buffered saline. The primary antibodies (1:20000 dilution of LYVE1 rabbit monoclonal antibody Abcam AB219556, 1:1000 dilution of PDPN rabbit monoclonal antibody SIGMA HPA007534, 1:100 dilution of CK mouse monoclonal antibody abcarta PA125, 1:100 dilution of TTF-1 mouse monoclonal antibody AM0225, 1:100 dilution of CD31 mouse monoclonal antibody ZSGB ZM-0044, 1:100 dilution of CD3 rabbit monoclonal antibody abcarta PA004) were incubated overnight at 4°C. After 20 minutes of secondary antibody incubation, the slides were washed with phosphate-buffered saline, developed with DAB, and mounted with neutral mounting medium. For immunofluorescence staining, paraffin-embedded sections were dewaxed in water, followed by antigen retrieval with EDTA (pH 9.0) at high temperature and pressure for 90 seconds. After retrieval, the soft meninges and brain tissue were sequentially washed with TBST (5 min × 3 times), incubated with 3% H 2 O 2 at room temperature for 10 minutes, and washed again with TBST (5 min × 3 times). The sections were subsequently blocked with a mixture of 10% goat serum and 10% donkey serum for 30 minutes at room temperature, followed by overnight incubation at 4°C. The primary antibodies used were LYVE1 (Abcam, UK, 1:5000), CD31 (Abcam, UK, 1:8000), PDPN (Sigma, USA, 1:1000), CK (Bioworld, China, 1:100), CD3 (Abcam, UK, 1:8000), and a-SMA (Abcam, UK, 1:10000). After warming for 15 min, the sections were washed with TBST (5 min × 3 times) and then incubated at room temperature for 45 min with the following secondary antibodies: goat anti-rabbit IgG H&L (HRP) (Abcam, UK, 1:4000), goat anti-mouse IgG H&L (HRP) (Abcam, UK, 1:4000), and donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Thermo Fisher, USA, 1:500). Following incubation, the sections were washed with TBST (5 min × 3 times), stained with TSA for 10 min, washed again with TBST (5 min × 3 times), subjected to 20 min of washout, washed with TBST (5 min × 3 times), stained with DAPI for 5 min to counterstain the nuclei, washed with TBST (5 min × 3 times), and finally mounted for observation under a fluorescence microscope. Microscopy and image analysis Images of the immunostained slides were captured using an Axioscope 5 Bio-TL microscope (Carl Zeiss) to obtain high-resolution images. A 3DHISTECH Pannoramic MIDI digital slide scanner (Budapest, Hungary) was used to analyze the immunofluorescence staining. ImageJ software was used for post image processing, which included the relative lymphatic vessel area (LVA) and T-cell density. T-cell density and LVA are assessed using a double-blind method. First, at 100x magnification, the immunohistochemical slides stained for lymphatic endothelial cells and T cells are thoroughly examined to identify the concentrated areas of lymphatic vessels and T cells. Next, images are captured at 200x magnification, and ImageJ software is used to set appropriate thresholds and segmentation methods to separate the T cells and lymphatic vessels from the background. Then, the total area of lymphatic vessels is divided by the total area of the selected region to calculate the relative lymphatic vessel area. Finally, use ImageJ software to outline the leptomeningeal area in the T-cell antibody-stained slide. Calculate the number of T-cells and the area of the leptomeninges, then divide the number of T-cells by the area of the leptomeninges to determine the T-cell density. Two pathologists, who are unaware of the patient's clinical information, each capture a field of view, and the average of their measurements is taken as the T-cell density and relative lymphatic vessel area for the case. If there is a significant discrepancy between the counts of the two pathologists, they will review the slides together and collaboratively determine the final result. Statistics The statistical studies were carried out using IBM SPSS version 27. Normality was assessed via the Kolmogorov‒Smirnov test, and continuous variables were analyzed using the Kruskal‒Wallis (KW) multiple comparison test. P values < 0.05 were considered statistically significant. Abbreviations LM (Leptomeningeal metastasis), CSF (cerebrospinal fluid), CNS (central nervous system), MRI (magnetic resonance imaging), VP (ventriculoperitoneal), LVA (relative lymphatic vessel area), PDPN (podoplanin), a-SMA (alpha-smooth muscle actin), LVI (lymphatic vessel invasion), MLVs (meningeal lymphatic vessels). Declarations Acknowledgments: We thank Zhenbin Zhang, Lijun Dai, and Gaigai He, the pathological technologists and physicians from Guangdong Sanjiu Brain Hospital, for their assistance with pathological image processing and analysis. Funding: This study was supported by the Guangzhou Municipal Science and Technology Project, China [grant numbers 202201011741], the Guangdong Sanjiu Brain Hospital Project, China [grant numbers A392024002], the Science and Technology Projects in Guangzhou (2023A03J1030), and the Clinical Frontier Technology Program of the First Affiliated Hospital of Jinan University, China (JNU1AF-CFTP-2022-a01210). Authors' contributions: Conceptualization: MYL, CZS, LBC, XNL Methodology: XYH, HNL, CZF, DL Investigation: XYH, WFM, WYJ, MNS, XJY Visualization: HNL, CZF, WFM, MNS, XJY Funding acquisition: MYL, CZS, XNL Project administration: MYL, CZS, LBC, XNL Supervision: MYL, CZS, LBC, XNL Writing – original draft: XYH, MTY, DL Writing – review & editing: MYL, CZS, LBC, XNL Ethics approval and consent to participate: This retrospective study was approved by the Ethics Committee of the Guangdong Sanjiu Brain Hospital (No.2024-01-018), and there was informed consent exemption for all patients. Consent for publication: Not applicable. Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The Fig.4 was created with BioRender.com. Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Grossman SA, Krabak MJ: Leptomeningeal carcinomatosis. Cancer Treatment Reviews 1999, 25: 103-119. Remon J, Le Rhun E, Besse B: Leptomeningeal carcinomatosis in non-small cell lung cancer patients: A continuing challenge in the personalized treatment era. Cancer Treatment Reviews 2017, 53: 128-137. Chamberlain M, Junck L, Brandsma D, Soffietti R, Rudà R, Raizer J, Boogerd W, Taillibert S, Groves MD, Le Rhun E, et al: Leptomeningeal metastases: a RANO proposal for response criteria. Neuro-oncology 2017, 19: 484-492. Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, Wiig H, Alitalo K: A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. The Journal of Experimental Medicine 2015, 212: 991-999. 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Vona G, Sabile A, Louha M, Sitruk V, Romana S, Schütze K, Capron F, Franco D, Pazzagli M, Vekemans M, et al: Isolation by size of epithelial tumor cells : a new method for the immunomorphological and molecular characterization of circulatingtumor cells. The American Journal of Pathology 2000, 156: 57-63. Shaw Bagnall J, Byun S, Begum S, Miyamoto DT, Hecht VC, Maheswaran S, Stott SL, Toner M, Hynes RO, Manalis SR: Deformability of Tumor Cells versus Blood Cells. Scientific Reports 2015, 5: 18542. Kerjaschki D, Bago-Horvath Z, Rudas M, Sexl V, Schneckenleithner C, Wolbank S, Bartel G, Krieger S, Kalt R, Hantusch B, et al: Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse. The Journal of Clinical Investigation 2011, 121: 2000-2012. Kretschy N, Teichmann M, Kopf S, Atanasov AG, Saiko P, Vonach C, Viola K, Giessrigl B, Huttary N, Raab I, et al: In vitro inhibition of breast cancer spheroid-induced lymphendothelial defects resembling intravasation into the lymphatic vasculature by acetohexamide, isoxsuprine, nifedipin and proadifen. British Journal of Cancer 2013, 108: 570-578. Zhang N, Ng AS, Cai S, Li Q, Yang L, Kerr D: Novel therapeutic strategies: targeting epithelial-mesenchymal transition in colorectal cancer. The Lancet Oncology 2021, 22: e358-e368. Mantovani A, Allavena P, Marchesi F, Garlanda C: Macrophages as tools and targets in cancer therapy. Nature Reviews Drug Discovery 2022, 21: 799-820. Dieterich LC, Detmar M: Tumor lymphangiogenesis and new drug development. Advanced Drug Delivery Reviews 2016, 99: 148-160. Galea I, Bechmann I, Perry VH: What is immune privilege (not)? Trends In Immunology 2007, 28: 12-18. Ellwardt E, Walsh JT, Kipnis J, Zipp F: Understanding the Role of T Cells in CNS Homeostasis. Trends In Immunology 2016, 37: 154-165. Oosterhof N, Chang IJ, Karimiani EG, Kuil LE, Jensen DM, Daza R, Young E, Astle L, van der Linde HC, Shivaram GM, et al: Homozygous Mutations in CSF1R Cause a Pediatric-Onset Leukoencephalopathy and Can Result in Congenital Absence of Microglia. American Journal of Human Genetics 2019, 104: 936-947. Prinz M, Priller J: The role of peripheral immune cells in the CNS in steady state and disease. Nature Neuroscience 2017, 20: 136-144. Waisman A, Liblau RS, Becher B: Innate and adaptive immune responses in the CNS. The Lancet Neurology 2015, 14: 945-955. Mundt S, Greter M, Flügel A, Becher B: The CNS Immune Landscape from the Viewpoint of a T Cell. Trends In Neurosciences 2019, 42: 667-679. Calzascia T, Masson F, Di Berardino-Besson W, Contassot E, Wilmotte R, Aurrand-Lions M, Rüegg C, Dietrich P-Y, Walker PR: Homing phenotypes of tumor-specific CD8 T cells are predetermined at the tumor site by crosspresenting APCs. Immunity 2005, 22: 175-184. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-5244229","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":364913821,"identity":"99205d47-37e1-4c89-879e-8b5e7a9ea444","order_by":0,"name":"Xiaoyu Hua","email":"","orcid":"","institution":"Department of Medical Imaging Centre, The First Affiliated Hospital, Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Hua","suffix":""},{"id":364913822,"identity":"d7cd74e4-dfb2-4fbc-b267-50d1ffd78911","order_by":1,"name":"Minting Ye","email":"","orcid":"","institution":"Department of 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Lai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIie2RsUrEQBCGJwzsNWvSbvBQH2HkYLki6IPYzHFw3VXXBokEtBGv9i0UXyCykCvzCusbpDoFRdy9UtgEO4v9YHea/2OYGYBI5D+C7llQUiC+2v6jvMgQjR1VGNQ0nYjl+eN9u8zvxIpGOzFAcZJJfex6JU+dPFNDadrhm+Vy7sJASroyMxIIyuIqpOS1mBG3fhZgUvNpqs1RY6FdrauAkiFoxeKgNEyuaJMyJZUJKgIne8XfXkmqht3/UktSQ0qGUqvF7WHJzvHj44iS13JDiwevuPiNW7Iybsk8MAt1u2fb768vT7fd++eXP+XWGNuXRVAJwH+LRyKRSOQXP6R0TG32NOWcAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Medical Oncology, Guangdong Sanjiu Brain Hospital","correspondingAuthor":true,"prefix":"","firstName":"Mingyao","middleName":"","lastName":"Lai","suffix":""}],"badges":[],"createdAt":"2024-10-11 07:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5244229/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5244229/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67279786,"identity":"679016d2-2192-412c-8a5a-42af1a9ed2d7","added_by":"auto","created_at":"2024-10-23 08:53:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5258994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePresence of lymphatic vessels in the leptomeninges.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) and (\u003cstrong\u003eb\u003c/strong\u003e) were obtained from two epilepsy patients, whereas (\u003cstrong\u003ef\u003c/strong\u003e) was from a lung cancer patient with LM. Leptomeningeal vessels, including arteries and veins (asterisk), were labeled with the smooth muscle cell marker α-smooth muscle actin (α-SMA) and the endothelial cell marker CD31. (\u003cstrong\u003ea\u003c/strong\u003e) and (\u003cstrong\u003eb\u003c/strong\u003e) LYVE1-labeled lymphatic endothelial cells widely distributed throughout the entire leptomeningeal area, particularly concentrated in vascular-rich regions, forming tubular-like structures. LYVE1-positive endothelial cells formed a complex network extending along arteries and arterioles, with LYVE1-positive lumen tissue not colabeled with α-SMA or CD31. (\u003cstrong\u003ec\u003c/strong\u003e) is a magnified portion of (\u003cstrong\u003ea\u003c/strong\u003e), showing LYVE1 labeling around a major artery and adjacent arterioles. (\u003cstrong\u003ed\u003c/strong\u003e) and (\u003cstrong\u003ee\u003c/strong\u003e) are magnified views of (\u003cstrong\u003eb\u003c/strong\u003e). (\u003cstrong\u003ec-e\u003c/strong\u003e) LYVE1-positive cells along the arterial path crossing through the cerebral cortex (arrows). (\u003cstrong\u003ef-h\u003c/strong\u003e) The colocalization of thelymphatic endothelial markers LYVE1 and PDPN. SAS, subarachnoid space. (Scale bars: \u003cstrong\u003ec-h\u003c/strong\u003e, 50 μm; \u003cstrong\u003ea\u003c/strong\u003e, 200 μm; \u003cstrong\u003eb\u003c/strong\u003e, 500 μm).\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5244229/v1/eb5141707eff824f5e0e46de.png"},{"id":67279787,"identity":"29dd8cb2-ff6d-46f4-84e1-b5d68d4c455f","added_by":"auto","created_at":"2024-10-23 08:53:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3946004,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased density of lymphatic vessels and infiltration of cancer cells in a lung cancer patient with LM.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea-e\u003c/strong\u003e) are from three lung cancer patients with LM and with positive biopsy samples. (\u003cstrong\u003ef\u003c/strong\u003e) is from a patient with epilepsy. (\u003cstrong\u003eh\u003c/strong\u003e) is from a lung cancer patient with LM with negative biopsy tissue. (\u003cstrong\u003ea-e\u003c/strong\u003e) There is an increased area of PDPN-positive vessels surrounding the tumor, forming irregular tubes. Within the network of these tubes, endothelial cell expansion accompanies the formation of bridge-like structures or small tubes. (\u003cstrong\u003eb-c\u003c/strong\u003e) Significant expansion of lumens formed by PDPN-positive cells was observed, with these lumens filled with CK-positive tumor cells (arrows). (\u003cstrong\u003ee\u003c/strong\u003e) A significant increase in lymphatic vessel density, marked by PDPN, was observed around the tumor labeled with CK, compared to (f) and (h). (\u003cstrong\u003eg\u003c/strong\u003e) A statistical graph showing the density of lymphatic vessels in the three patient groups. Patientswith lung cancer LM and positive biopsy tissues presented a significant increase in lymphatic vessel density. P, positive biopsy tissue from lung cancer patients with LM; N, negative biopsy tissue from lung cancer patients with LM; E, epilepsy patient. SAS, subarachnoid space. (Scale: \u003cstrong\u003ea, f-h\u003c/strong\u003e, 200 μm; \u003cstrong\u003eb, d-e\u003c/strong\u003e, 100 μm; \u003cstrong\u003ec\u003c/strong\u003e, 50 μm).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5244229/v1/0a8ffa8fa0ec101a2f8203c0.png"},{"id":67278918,"identity":"a20ba5f3-881c-494b-8636-71e28250d35e","added_by":"auto","created_at":"2024-10-23 08:45:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3116580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant increase in the number of leptomeningeal T cells in lung cancer patients with LM.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) and (\u003cstrong\u003ec\u003c/strong\u003e) were obtained from two epilepsy patients; (\u003cstrong\u003eb\u003c/strong\u003e) and (\u003cstrong\u003ef\u003c/strong\u003e) were obtained from two LM patients whose biopsy tissues were negative; (\u003cstrong\u003ed-e\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eg-h\u003c/strong\u003e) were obtained from two LM patients whose biopsy tissues were positive. (\u003cstrong\u003ea-d\u003c/strong\u003e) and (\u003cstrong\u003ef\u003c/strong\u003e) Some CD3-positive T cells (arrows) can be observed surrounding and within lumens enclosed by LYVE1/PDPN-positive cells outside the vascular wall. (\u003cstrong\u003ed-e\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eg-h\u003c/strong\u003e) T cells showed a notable increase in the number of surrounding CK-marked tumor cells compared to nontumor areas a and b (arrows). (\u003cstrong\u003ei\u003c/strong\u003e) Quantitative analysis of T-cell numbers per square micrometer in lymphatic vessels of the leptomeninges revealed significantly greater counts in patients with LM and positive biopsy tissue, indicating statistical significance.P, positive biopsy tissue fromlung cancer patients with LM; N, negative biopsy tissue fromlung cancer patients with LM; E, epilepsy patient. SAS, subarachnoid space. (Scale: \u003cstrong\u003ec\u003c/strong\u003e, 50 μm; \u003cstrong\u003ea-b, d-h\u003c/strong\u003e, 100 μm)\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5244229/v1/32325bbf69db1f905f6db6f9.png"},{"id":67278920,"identity":"5f38d70a-d993-4182-9442-72f03a5266f6","added_by":"auto","created_at":"2024-10-23 08:45:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2943168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of lymphatic characteristics in the leptomeninges of patients with LM from lung cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe leptomeninges include the arachnoid mater, the subarachnoid space, and the pia mater. The figure illustrates an increase in the area of lymphatic vessels and the number of T cells around the tumor, forming an irregular network of lymphatic vessels. These network structures are initial lymphatic vessels, with thin and discontinuous walls that facilitate tumor cell infiltration. The left pull-out box shows how tumor cells infiltrate the initial lymphatic vessels and eventually drain into the collecting lymphatic vessels. Large arteries in the leptomeninges and the adjacent small arteries are surrounded by circumferential lymphatic endothelial markers. Lymphatic endothelial markers are also present along the arterial pathways traversing the cerebral cortex, the right pull-out box displays a cross-sectional view of the structure, clearly showing tumor cell infiltration along the inner wall of the lymphatic vessel. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5244229/v1/03dfc8be17d077d2d3d3e42e.png"},{"id":67283083,"identity":"10a219e2-103d-474f-b916-535ef80a8d8c","added_by":"auto","created_at":"2024-10-23 09:10:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21818980,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5244229/v1/651657d0-ccda-4945-81ec-a0ab54ed91dc.pdf"},{"id":67278916,"identity":"dd6e7017-22ce-4e17-8cb7-1e8e1cfb2955","added_by":"auto","created_at":"2024-10-23 08:45:50","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1059874,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5244229/v1/83bed717dacf17c08a2f84f6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural and Functional Characteristics of Leptomeningeal Lymphatic Vessels in Leptomeningeal Metastases from Lung Cancer Patients","fulltext":[{"header":"Background","content":"\u003cp\u003eLeptomeningeal metastasis (LM) is an unpleasant and potentially fatal phenomenon in advanced cancer patients. LM involves the spread of malignant cells to leptomeninges, the subarachnoid space, and other compartments of the cerebrospinal fluid (CSF) system [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. LM occurs in approximately 3\u0026ndash;5% of patients with advanced non-small cell lung cancer. However, because to improved outcomes from novel molecular treatments, the prevalence of LM among particular categories of patients with targetable mutations has increased[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The diagnosis and treatment of LM have become the focus of recent research because of its difficulty to diagnose and the poor treatment outcomes and short survival time of patients. Previously, the prevailing belief was that the central nervous system (CNS) lacked lymphatic vessels and that LM primarily occurred through direct invasion, CSF dissemination, and blood dissemination. However, recent animal experiments and human studies have substantiated the presence of lymphatic vessels within the cranial cavity that drains into deep cervical lymph nodes[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Given that the cranial cavity has a functioning glymphatic system, it may be necessary to reevaluate established beliefs about tolerance and immune privilege in the CNS. Many different neurological illnesses may involve meningeal lymphatic vessel dysfunction as their underlying etiology[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, we hope to gain a deeper understanding of the mechanism of interaction between leptomeningeal lymphatic vessels and CNS tumors by studying specimens from LM patients to provide strong support for the development of more effective tumor treatment strategies.\u003c/p\u003e \u003cp\u003eThe characterization of the endocranial lymphatic circulatory system in animals has crucial implications for our comprehension of the physiopathological mechanisms in the CNS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In previous studies, magnetic resonance imaging (MRI) has been used to visualize the meningeal lymphatic network, while immunofluorescence has been used to visualize lymphatic endothelial cells in the meningeal lymphatic system with molecular markers[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In mice, macromolecules and immune cells from CSF are drained via the meningeal lymphatic system. Moreover, meningeal lymphatics play crucial roles in immune surveillance and inflammation in the CNS, making them promising therapeutic targets[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, the majority of evidence now available about meningeal lymphatic activity comes from research conducted on animals, whereas human data include only observations made from postmortem specimens[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, studies on the changes in lymphatic vessels in malignant tumors in the CNS are lacking[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, to our knowledge, recent research has only confirmed the existence of lymphatic vessels in leptomeninges[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We hope to further explore the functions of leptomeningeal lymphatic vessels in promoting metastasis, regulating immune responses, and influencing the tumor microenvironment. Our samples were collected from living humans, and to our knowledge, this is the first study of the functional status of lymphatic vessels around human CNS tumors, which will more accurately reflect the true role of lymphatic vessels in the development of diseases. Ultimately, we found that the lymphatic vessels in the leptomeninges not only structurally resemble extracranial lymphatic vessels but also function similarly in the tumor immune response and the metastatic pathway.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrategy\u003c/h2\u003e \u003cp\u003eTo determine the presence and localization of lymphangions within human leptomeninges, we used immunofluorescence and immunohistochemistry techniques to analyze leptomeningeal tissue samples collected from LM subjects (n\u0026thinsp;=\u0026thinsp;46) and control subjects with epilepsy (n\u0026thinsp;=\u0026thinsp;11) during preventricular puncture and surgical procedures (Supplementary Fig.\u0026nbsp;1). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e includes demographic information for each individual, including age, sex, surgical location, and EGFR mutation status. Initially, we used LYVE1, podoplanin (PDPN), Prox1, and VEGFR3 to visualize lymphatic endothelial cells. However, in alignment with the literature[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], detecting Prox1 has proven particularly challenging (Supplementary Fig.\u0026nbsp;2d and 2h). Moreover, given the widespread distribution of VEGFR3 in the brain parenchyma, these markers do not seem to be the most suitable for this purpose (Supplementary Fig.\u0026nbsp;2c and 2g). Consequently, we ultimately opted to employ LYVE1 and PDPN to visualize the lymphatic vessels. We utilized CD31 and alpha-smooth muscle actin (a-SMA) as markers for blood vessels and smooth muscle tissue, respectively. In contrast to arteries and veins, lymphatic vessels express LYVE1 and/or PDPN but lack expression of CD31 and/or a-SMA. Furthermore, we used CK and TTF-1 to identify tumor cells in the leptomeninges and used CD3 to label T cells.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient material.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePatients with leptomeningeal metastases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEpileptic\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiopsy positive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiopsy negative\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (f/m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11/6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5/6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (yrs)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54\u0026thinsp;\u0026plusmn;\u0026thinsp;8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;12.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEGFR (n; %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26/29(89%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14/17(82%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eSurgical location (leptomeningeal location)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRight\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeft\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eQuantitative characteristics of lymphatic vessels\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLVA (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT cell per leptomeningeal (per/\u0026micro;m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.2e‒05\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6e‒05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.1e‒05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7e‒05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5e‒05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eCategorical data presented as n (%), and quantitative data shown as average\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLymphatic vessels are present in human leptomeninges\u003c/h3\u003e\n\u003cp\u003eIn the cortex and leptomeninges of human brain samples, we observed that lymphatic vessels were widely distributed throughout the leptomeninges, especially concentrated in areas rich in blood vessels; morphologically, LYVE1\u0026thinsp;+\u0026thinsp;endothelial cells formed a distorted network extending along arteries and arterioles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-e). Additionally, we found that lymphatic vessels could exist adjacent to or independent of blood vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The lateral side of the outer walls of most subarachnoid space blood vessels clearly contained green fluorescent cells labeled with LYVE1, and a large artery surrounded by LYVE1-labeled cells with several LYVE1-positive small arteries feeding into this large vessel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We also observed LYVE1-positive cells along the arterial pathway crossing through the cerebral cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e). To validate the lymphatic nature of LYVE1-positive immune reaction structures, we confirmed the colabeling of LYVE1 and PDPN in lymphatic vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h). Moreover, similar to other peripheral lymphatic vessels, there are two types of lymphatic vessels present in the leptomeninges: one type expresses both LYVE1 and PDPN markers (Supplementary Fig.\u0026nbsp;3a-d), whereas the other expresses only PDPN markers (Supplementary Fig.\u0026nbsp;3e-h). Both types of lymphatic smooth muscle markers were negative. The first type is similar to the pre-collected lymphatic vessels found in other parts of the peripheral system[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The quantity of this type of lymphatic vessel shows little difference between epilepsy patients and those with LM from lung cancer. The second type of lymphatic vessels is significantly increased in number in patients with LM from lung cancer, and they are mainly concentrated around the tumor (Supplementary Fig.\u0026nbsp;4a-c). This type of lymphatic vessel is characterized by an increased PDPN-positive vascular area, forming irregular networks (Supplementary Fig.\u0026nbsp;4c). Within these networks, endothelial cells are dilated, and there are formations of bridge-like structures, valve-like structures, and small tubes (Supplementary Fig.\u0026nbsp;4d-e). This type of lymphatic vessel is similar to the initial lymphatic vessels found in other extracranial regions and resembles the immature, newly formed lymphatic vessels around extracranial tumors. Compared with leptomeningeal arteries and veins, both types of lymphatic vessels presented negative staining for the blood endothelial cell marker CD31 and the smooth muscle marker a-SMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b). This is similar to the description of lymphatic vessels in postmortem human samples presented by Goodman and Louveau[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eLM in lung cancer patients: Leptomeningeal lymphangiogenesis, lymphatic network remodeling, and lymphatic vessel invasion\u003c/h3\u003e\n\u003cp\u003eAfter specific lymphatic vessel staining of leptomeningeal specimens from 46 patients diagnosed with LM via CSF cytology (29 positive on biopsy, 17 negative) and 11 patients with epilepsy, we observed an increased area of PDPN-positive vessels around the tumor and the formation of irregular lymphatic vascular networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-d). Within these networks, endothelial cell expansion accompanied by the formation of bridge-like structures, valve-like structures, and small tubes was observed (Supplementary Fig.\u0026nbsp;4d-e), which resembled the description of lymphatic vessel remodeling structures around pancreatic cancer presented by Shen et al[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The analysis of lymphatic vessel density in the three groups of patients revealed that the lymphatic vessel density around the leptomeningeal tumor in patients with positive leptomeningeal biopsy for LM was significantly greater than that in the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-h), and this difference was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The difference between the latter two groups was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg), with average values of 7.18\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93%, 3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42%, and 3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, we detected lymphatic tumor cell infiltration in 18 out of 29 patients with positive leptomeningeal biopsies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-d). We observed significant expansion of lumens formed by PDPN-positive cells, which were filled with CK-positive tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c). These results suggest that lung cancer metastasis may lead to lymphangiogenesis and remodeling of the lymphatic network in the leptomeninges.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCompared with nontumor patients, LM patients have more T lymphocytes in the leptomeninges\u003c/h3\u003e\n\u003cp\u003eWe wanted to determine whether lymphocytes exist inside or near the lymphatic vessels in the leptomeninges. Moreover, we aimed to verify the differences in the number of lymphocytes among three groups: patients with positive CSF cytology for LM and positive or negative biopsy specimens and a control group of epilepsy patients. To answer this question, we stained tissue samples from a total of 29/57 patients, including 11 samples from lung cancer patients with LM, 7 samples of negative biopsy tissue from lung cancer patients with LM, and 11 samples from patients in the control group with epilepsy, with an anti-CD3 antibody (a specific T lymphocyte marker). In all the leptomeninges samples we studied, we found lymphocytes outside the blood vessels. In the leptomeninges, we observed CD3-positive T cells located outside the vascular walls and interspersed among LYVE1/PDPN-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef-g). In the leptomeninges of lung cancer patients with LM and positive biopsy tissue, there were many CD3-positive cells between the vessel wall and LYVE1-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-h). Similarly, in lung cancer patients with LM with negative biopsy tissue and in epilepsy patients, a moderate number of CD3-positive T lymphocytes were observed within the interstices surrounded by lymphatic endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). To determine the differences in T lymphocyte numbers among the three groups of patients, we quantified CD3 expression in the most densely populated areas of each slice and measured the leptomeninges area of this region, comparing the ratio of these two value to assess differences among the three groups. We found that lung cancer patients with LM and positive biopsy tissue had the highest CD3 count (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), and this difference was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The difference between the latter two groups was not significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), with average values of 21.2e‒05\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6e‒05 per/\u0026micro;m\u003csup\u003e2\u003c/sup\u003e, 8.1e‒05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7e‒05 per/\u0026micro;m\u003csup\u003e2\u003c/sup\u003e, and 10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5e‒05 per/\u0026micro;m\u003csup\u003e2\u003c/sup\u003e, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe clearance of CNS fluids and molecular waste and the mechanisms underlying tumor immunity have always been popular research topics. The traditional view is that unlike other parts of the body, the CNS lacks lymphatic vasculature; however, recent groundbreaking research has shown that lymphatic vessels do extend to cover the meningeal tissues of the entire CNS in mice, rats, primates, and humans[\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, little is known about the existence of lymphatic vessels in leptomeninges and the role of these vessels in CNS tumors. Therefore, in this study, we performed immunohistochemical and fluorescence staining experiments for endothelial-specific markers of lymphatic vessels in the leptomeninges of 46 lung cancer patients with LM and 11 patients with epilepsy. The results confirmed a situation similar to that reported in Mezey's postmortem studies: that there are indeed lymphatic vessels present in the leptomeninges of humans[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We further explored whether these findings imply that the lymphatic vessels in the leptomeninges have functions similar to those of the peripheral tissue lymphatic system. We know that lymphatic vessels play various important roles in tumor biology, including promoting metastasis, regulating immune responses, and influencing the tumor microenvironment[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, we verified each of these aspects one by one. Ultimately, we found that the lymphatic vessels in the leptomeninges not only structurally resemble extracranial lymphatic vessels but also function similarly in the tumor immune response and the metastatic pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To our knowledge, there has been no relevant research on this topic thus far. This study is highly important for obtaining a deeper understanding of the interaction between lymphatic vessels and CNS tumors. Specific animal experiments are needed in the future to elucidate the exact mechanisms involved, which will also aid in the development of more effective tumor treatment strategies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the past decade, the CNS lymphatic system has once again become a focus of attention. In 1987, Andres et al.[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] described lymphatic vessels in the human dura mater, which was recently confirmed by Aspelund et al.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and Louveau et al.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Moreover, Mezey et al.'s study on human cadaver samples revealed that positive lymphatic marker staining was detected in the pia mater, in the arachnoid, in venous sinuses, and among the layers of the dura mater[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, existing evidence concerning meningeal lymphatic function has been derived mainly from animal studies, and human data rely entirely on observations from autopsy specimens. Our samples were collected from living humans, and to our knowledge, this is the first study of the functional status of lymphatic vessels around human meningeal tumors, which will more accurately reflect the true role of lymphatic vessels in the development of diseases. We observed that lymphatic vessels are widely distributed throughout the entire leptomeninges, especially in areas rich in blood vessels. This finding is consistent with previous studies on zebrafish and human cadaveric samples[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We also found LYVE1-positive cells along the arterial path crossing through the cerebral cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), similar to the findings reported by Mezey et al., suggesting that the 'perivascular' space described by many researchers in the early stages of this research appears to be composed of endothelial cells\u003csup\u003e13\u003c/sup\u003e. In addition, we also observed two types of lymphatic vessels in the subarachnoid space: PDPN\u003csup\u003e+\u003c/sup\u003eLYVE1\u003csup\u003e+\u003c/sup\u003eCD31\u003csup\u003e\u0026minus;\u003c/sup\u003e type lymphatic vessels, which are consistent with peripheral pre-collected lymphatic vessels and those described in the meningeal sinuses of mice[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. There is no significant difference in the quantity of this type of lymphatic vessel between epilepsy patients and those with LM from lung cancer. The second type is PDPN\u003csup\u003e+\u003c/sup\u003eLYVE1\u003csup\u003e\u0026minus;\u003c/sup\u003eCD31\u003csup\u003e\u0026minus;\u003c/sup\u003e lymphatic vessels, which significantly increases in number in patients with lung cancer LM, primarily surrounding the tumor and forming a network-like structure. Within this network, endothelial cells expand, accompanied by the formation of bridge-like structures, valve-like structures, and small vessels. This type of lymphatic vessel is similar to the initial lymphatic vessels described in the extracranial lymphatic system[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Such structures can also be observed around pancreatic cancer, characterized by newly formed, immature lymphatic vessels. Their inner walls consist of a single layer of endothelial cells, which are loosely connected and lack a complete basement membrane, making it easier for tumor cells to invade (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These are clearly speculative possibilities, and more investigations are needed to assess them.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that the growth of extracranial tumors not only induces angiogenesis but also promotes lymphangiogenesis in some cases, facilitating tumor cell entry into the surrounding lymphatic system. VEGF-C produced by tumor cells can induce the proliferation and expansion of lymphatic vessels toward tumor cells, leading to increased lymphatic vessel growth within 2\u0026ndash;3 weeks after tumor xenograft modeling, accompanied by lymph node metastasis[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, we investigated whether there is a significant increase in the number of lymphatic vessels around lesions in the leptomeninges of lung cancer patients with LM and whether there is evidence of tumor cell invasion in these vessels, similar to other extracranial tumors. We observed that the density of leptomeningeal lymphatic vessels was significantly greater in lung cancer patients with LM than in patients without metastatic lesions and those with epilepsy. In addition, we also observed tumor cell infiltration in the leptomeningeal lymphatic vessels of patients with LM, indicating that leptomeninges lymphatic vessels play a role in tumor-induced lymphangiogenesis and remodeling processes as well as in the metastatic pathway as extracranial lymphatic vessels. An early necessary step in the cascade of lymphatic metastasis is the invasion of clusters or individual tumor cells into the lymphatic system[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, CSF enters systemic circulation through deep cervical lymph nodes via dural lymphatic vessels[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, we speculate that the lymphatic pathway may be one route for LM. Our previous studies also revealed a strong correlation between LM associated with lung cancer and deep cervical lymph node involvement[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A study by Smith et al. confirmed that ligation of the cervical lymphatic vessels leads to brain edema and increased protein concentrations in the CSF of cats and rabbits[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, we speculate that lymphatic vessel infiltration is an important cause of intracranial hypertension in patients with LM. Moreover, severe intracranial hypertension significantly affects the quality of life and outcomes of patients. Therefore, treatment targeting lymphatic vessel lymphangiogenesis and infiltration may significantly improve the quality of life and outcomes of patients. The process of lymphatic vessel invasion (LVI) involves complex mechanisms. Initial lymphatic vessels have discontinuous cell‒cell connections, allowing entry of white blood cells through small gaps between VE-cadherin-positive \"buttons\"[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, tumor cells, which are larger and less susceptible to morphological changes than white blood cells are, face barriers when attempting to invade these vessels [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Signals from tumor cells or the tumor microenvironment can disrupt lymphatic endothelial cell junctions, creating larger pores known as \"chemorepulsion-induced defects\" or entry gates[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Drugs targeting these mechanisms are in development to inhibit LVI and lymphatic spread. One approach is to interfere with the local tissue infiltration of tumor cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]; another strategy is to inhibit tumor-associated lymphangiogenesis to reduce the interface between tumors and lymphatic vessels [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the future, we could use some of these methods in combination with current regimens for treating lung cancer patients with LM.\u003c/p\u003e \u003cp\u003eAfter the identification of lymphatic vessels in the leptomeninges, our aim was to establish their correlation with the presence and distribution of T lymphocytes, thereby functionally confirming the resemblance between leptomeningeal lymphatic vessels and peripheral lymphatic vessels. Research has indicated that, similar to other organs, the CNS undergoes constant immune surveillance for abnormal cells and pathogens while also relying on immune cells to maintain normal tissue homeostasis. Peripheral-derived T cells, macrophages, and dendritic cells appear to play a role in this process[\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, in CNS tumors, T lymphocytes are the primary immune cells involved[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; hence, we utilized CD3 as a marker for these immune cells in our study. Previous research has demonstrated the important role of dura mater lymphatic vessels in bridging the CNS and peripheral immune system[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. As such, we postulate that leptomeningeal lymphatic vessels also play a pivotal role in this process. In our research sample, we commonly observed the presence of lymphocytes inside or near the lymphatic vessels in the leptomeninges, which is consistent with previous literature reports[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, in patients with positive LM biopsy samples, the number of T cells near leptomeningeal lymphatic vessels was significantly greater than that in LM biopsy-negative patients and was also greater than that in control epilepsy patients. Additionally, animal model studies have validated the drainage of soluble tumor antigens from the CNS into cervical lymph nodes via CSF, thereby eliciting specific T-cell responses[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Consequently, we hypothesize that LM elicits an effective antitumor immune response leading to an increase in T-cell populations in this region. Hu et al. demonstrated the crucial role of meningeal lymphatic vessels (MLVs) in intracranial tumor fluid drainage and immunity, highlighting the importance of MLV remodeling induced by brain tumors for immunotherapy[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, notably, the MLV on the dorsal side of the dura mater does not directly physically contact the tumor. Therefore, further animal experiments are warranted to verify whether the lymphatic vessels in the leptomeninges play a bridging role and elucidate their involvement in the tumor immune response.\u003c/p\u003e \u003cp\u003eSome limitations of the present study should be noted. The limited sample size and restricted anatomical focus of this study present additional drawbacks, although it is the largest living human cohort examined for leptomeningeal lymphatic vessels. In addition, all participants, particularly those with LM, underwent various treatment regimens before sample collection, which prevented this study from fully reflecting the initial state of leptomeningeal lymphatic vessels in the CNS. Moreover, owing to the cross-sectional nature of the sampling, we were unable to capture changes in leptomeningeal lymphatic vessels dynamically during tumor immune responses or explore their specific roles in CSF and waste clearance. These limitations hinder our understanding and research into the dynamic nature of these important biological processes. Therefore, the findings reported may not be able to be extrapolated to a broader context of leptomeningeal lymphatic vessels.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur research results confirm the existence of lymphatic vessels in the human leptomeninges, and show a significant increase in lymphatic vessel density and T cell count around tumors compared to non-tumor areas. We also observed infiltration of tumor cells into the lymphatic vessels. These findings suggest that the lymphatic vessels in the leptomeninges not only structurally resemble extracranial brain lymphatic vessels, but also function similarly in tumor immune response and metastasis pathways. The discovery of the leptomeningeal lymphatic vessels expands our understanding of the diversity of \"brain\" lymphatic vessels, beyond the scope of the dural lymphatic vessels. Although the role of dural lymphatic vessels in brain and CSF clearance and tumor immunity is still controversial, the interaction between leptomeningeal lymphatic vessels and dural lymphatic vessels in draining CSF, clearing macromolecules, and potential tumor immunity deserves further exploration.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHuman leptomeningeal samples\u003c/h2\u003e \u003cp\u003eAll patients were diagnosed with LM by CSF cytology prior to undergoing meningeal biopsy. Leptomeningeal samples were obtained during the preventricular puncture procedures. The specific procedures included ventriculoperitoneal (VP)\u0026thinsp;+\u0026thinsp;Ommaya reservoir implantation in 36 patients, VP surgery in 3 patients, Ommaya reservoir implantation in 3 patients, and external ventricular drainage in 3 patients. Trepanation for the surgical operation and tissue sampling was conducted at a site located 12 cm caudally from the glabella, with a lateral deviation of approximately 1\u0026ndash;2 cm from the midline (Supplementary Fig.\u0026nbsp;1a-c and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A burr hole with a diameter of less than 1 cm was created to expose the underlying leptomeninges. The leptomeninges were removed in one piece by incising it circumferentially (approximately 5 mm in diameter and 1 mm in thickness). Eleven patients with drug-resistant epilepsy due to nonneoplastic causes underwent surgical resection of the epileptogenic focus. The anterior temporal lobe was removed, with a surgical range of 4.5 cm posterior from the temporal pole on the left side and 5.5 cm posterior from the temporal pole on the right side (Supplementary Fig.\u0026nbsp;1d-f and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Histopathologically, the entire leptomeningeal region of the anterior temporal lobe was selected for staining. The medical records of the patients were used to gather clinical data. This study was approved by the Sanjiu Brain Hospital Ethics Committee.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and immunofluorescence\u003c/h2\u003e \u003cp\u003eFor immunohistochemical staining, the samples were fixed in 10% neutral buffered formalin for 48 hours, dehydrated, and embedded in paraffin. Three-micron paraffin-embedded human meningeal coronal sections (Xiamen Elibot Company) were prepared via the glass slide method. The slides were incubated at 50\u0026deg;C overnight, deparaffinized in xylene, and rehydrated in a gradient of ethanol. The slides were placed in an antigen retrieval instrument (Dako link48), and the antigen retrieval solution (10 mM Tris, 1 mM EDTA, pH 9) was heated to 95\u0026deg;C for 20 minutes and cooled to room temperature. Endogenous peroxidases were blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 5 minutes, followed by washing with phosphate-buffered saline. The primary antibodies (1:20000 dilution of LYVE1 rabbit monoclonal antibody Abcam AB219556, 1:1000 dilution of PDPN rabbit monoclonal antibody SIGMA HPA007534, 1:100 dilution of CK mouse monoclonal antibody abcarta PA125, 1:100 dilution of TTF-1 mouse monoclonal antibody AM0225, 1:100 dilution of CD31 mouse monoclonal antibody ZSGB ZM-0044, 1:100 dilution of CD3 rabbit monoclonal antibody abcarta PA004) were incubated overnight at 4\u0026deg;C. After 20 minutes of secondary antibody incubation, the slides were washed with phosphate-buffered saline, developed with DAB, and mounted with neutral mounting medium.\u003c/p\u003e \u003cp\u003eFor immunofluorescence staining, paraffin-embedded sections were dewaxed in water, followed by antigen retrieval with EDTA (pH 9.0) at high temperature and pressure for 90 seconds. After retrieval, the soft meninges and brain tissue were sequentially washed with TBST (5 min \u0026times; 3 times), incubated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at room temperature for 10 minutes, and washed again with TBST (5 min \u0026times; 3 times). The sections were subsequently blocked with a mixture of 10% goat serum and 10% donkey serum for 30 minutes at room temperature, followed by overnight incubation at 4\u0026deg;C. The primary antibodies used were LYVE1 (Abcam, UK, 1:5000), CD31 (Abcam, UK, 1:8000), PDPN (Sigma, USA, 1:1000), CK (Bioworld, China, 1:100), CD3 (Abcam, UK, 1:8000), and a-SMA (Abcam, UK, 1:10000). After warming for 15 min, the sections were washed with TBST (5 min \u0026times; 3 times) and then incubated at room temperature for 45 min with the following secondary antibodies: goat anti-rabbit IgG H\u0026amp;L (HRP) (Abcam, UK, 1:4000), goat anti-mouse IgG H\u0026amp;L (HRP) (Abcam, UK, 1:4000), and donkey anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) highly cross-adsorbed secondary antibody (Thermo Fisher, USA, 1:500). Following incubation, the sections were washed with TBST (5 min \u0026times; 3 times), stained with TSA for 10 min, washed again with TBST (5 min \u0026times; 3 times), subjected to 20 min of washout, washed with TBST (5 min \u0026times; 3 times), stained with DAPI for 5 min to counterstain the nuclei, washed with TBST (5 min \u0026times; 3 times), and finally mounted for observation under a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopy and image analysis\u003c/h2\u003e \u003cp\u003eImages of the immunostained slides were captured using an Axioscope 5 Bio-TL microscope (Carl Zeiss) to obtain high-resolution images. A 3DHISTECH Pannoramic MIDI digital slide scanner (Budapest, Hungary) was used to analyze the immunofluorescence staining. ImageJ software was used for post image processing, which included the relative lymphatic vessel area (LVA) and T-cell density. T-cell density and LVA are assessed using a double-blind method. First, at 100x magnification, the immunohistochemical slides stained for lymphatic endothelial cells and T cells are thoroughly examined to identify the concentrated areas of lymphatic vessels and T cells. Next, images are captured at 200x magnification, and ImageJ software is used to set appropriate thresholds and segmentation methods to separate the T cells and lymphatic vessels from the background. Then, the total area of lymphatic vessels is divided by the total area of the selected region to calculate the relative lymphatic vessel area. Finally, use ImageJ software to outline the leptomeningeal area in the T-cell antibody-stained slide. Calculate the number of T-cells and the area of the leptomeninges, then divide the number of T-cells by the area of the leptomeninges to determine the T-cell density. Two pathologists, who are unaware of the patient's clinical information, each capture a field of view, and the average of their measurements is taken as the T-cell density and relative lymphatic vessel area for the case. If there is a significant discrepancy between the counts of the two pathologists, they will review the slides together and collaboratively determine the final result.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eThe statistical studies were carried out using IBM SPSS version 27. Normality was assessed via the Kolmogorov‒Smirnov test, and continuous variables were analyzed using the Kruskal‒Wallis (KW) multiple comparison test. \u003cem\u003eP\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLM (Leptomeningeal metastasis), CSF (cerebrospinal fluid), CNS (central nervous system), MRI (magnetic resonance imaging), VP (ventriculoperitoneal), LVA (relative lymphatic vessel area), PDPN (podoplanin), a-SMA (alpha-smooth muscle actin), LVI (lymphatic vessel invasion), MLVs (meningeal lymphatic vessels).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Zhenbin Zhang, Lijun Dai, and Gaigai He, the pathological technologists and physicians from Guangdong Sanjiu Brain Hospital, for their assistance with pathological image processing and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Guangzhou Municipal Science and Technology Project, China [grant numbers 202201011741], the Guangdong Sanjiu Brain Hospital Project, China [grant numbers A392024002], the Science and Technology Projects in Guangzhou (2023A03J1030), and the Clinical Frontier Technology Program of the First Affiliated Hospital of Jinan University, China (JNU1AF-CFTP-2022-a01210).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization:\u0026nbsp;MYL,\u0026nbsp;CZS,\u0026nbsp;LBC,\u0026nbsp;XNL\u003c/p\u003e\n\u003cp\u003eMethodology:\u0026nbsp;XYH,\u0026nbsp;HNL,\u0026nbsp;CZF,\u0026nbsp;DL\u003c/p\u003e\n\u003cp\u003eInvestigation:\u0026nbsp;XYH,\u0026nbsp;WFM,\u0026nbsp;WYJ, MNS, XJY\u003c/p\u003e\n\u003cp\u003eVisualization:\u0026nbsp;HNL,\u0026nbsp;CZF,\u0026nbsp;WFM,\u0026nbsp;MNS, XJY\u003c/p\u003e\n\u003cp\u003eFunding acquisition:\u0026nbsp;MYL,\u0026nbsp;CZS,\u0026nbsp;XNL\u003c/p\u003e\n\u003cp\u003eProject administration:\u0026nbsp;MYL,\u0026nbsp;CZS,\u0026nbsp;LBC,\u0026nbsp;XNL\u003c/p\u003e\n\u003cp\u003eSupervision:\u0026nbsp;MYL,\u0026nbsp;CZS,\u0026nbsp;LBC,\u0026nbsp;XNL\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft:\u0026nbsp;XYH,\u0026nbsp;MTY,\u0026nbsp;DL\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing:\u0026nbsp;MYL,\u0026nbsp;CZS,\u0026nbsp;LBC,\u0026nbsp;XNL\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective study was approved by the Ethics Committee of the Guangdong Sanjiu Brain Hospital (No.2024-01-018), and there was informed consent exemption for all patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;The Fig.4 was created with BioRender.com.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGrossman SA, Krabak MJ: \u003cstrong\u003eLeptomeningeal carcinomatosis.\u003c/strong\u003e \u003cem\u003eCancer Treatment Reviews \u003c/em\u003e1999, 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Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lymphatic, Podoplanin, LYVE1, Leptomeningeal metastases, Leptomeninges, T cells, Lymphatic invasion","lastPublishedDoi":"10.21203/rs.3.rs-5244229/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5244229/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The purpose of this study is to explore whether there are lymphatic vessels in the human leptomeninges, and their potential role in the immune response to central nervous system tumors, in order to determine their significance in the field of tumor biology, especially the role they play in the growth, metastasis, and immune response of tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe used immunohistochemistry and immunofluorescence techniques to examine the leptomeninges in 46 patients diagnosed with leptomeningeal metastasis in cerebrospinal fluid cytology (29 positive biopsy specimens, 17 negative) as well as 11 epilepsy patients. We visualized lymphatic vessels in the human leptomeninges using LYVE1 and PDPN antibodies, labeled tumor cells with CK, T cells with CD3, and blood vessels with CD31 and α-smooth muscle actin. By comparing the lymphatic vessel density and T cell count in tumor areas versus non-tumor areas, and observing whether there was infiltration of tumor cells into the lymphatic vessels, we analyzed the presence and function of human leptomeningeal lymphatic vessels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The research results confirmed the existence of lymphatic vessels in the human leptomeninges, with a significant increase in lymphatic vessel density and T cell count around the tumor compared to non-tumor areas (P \u0026lt; 0.05). At the same time, infiltration of tumor cells was observed within the lymphatic vessels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e These findings suggest that the lymphatic vessels in the leptomeninges not only structurally resemble extracranial brain lymphatic vessels, but also function similarly in tumor immune response and metastasis pathways. These findings challenge traditional understanding of immune responses to central nervous system tumors and provide important clues for further research on the role of intracranial lymphatic vessels in tumor biology.\u003c/p\u003e","manuscriptTitle":"Structural and Functional Characteristics of Leptomeningeal Lymphatic Vessels in Leptomeningeal Metastases from Lung Cancer Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-23 08:45:45","doi":"10.21203/rs.3.rs-5244229/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":"b134625c-0eb1-4876-8a53-558239f2df12","owner":[],"postedDate":"October 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-21T16:38:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-23 08:45:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5244229","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5244229","identity":"rs-5244229","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}