Characterization, isolation, and in vitro culture of leptomeningeal fibroblasts

Remšík, Ján; Saadeh, Fadi; Tong, Xinran; Li, Min Jun; Snyder, Jenna; Bale, Tejus; Wu, Jean; Derderian, Camille; Guber, David; Chi, Yudan; Murali, Rajmohan; Boire, Adrienne

doi:10.1016/j.jneuroim.2021.577727

Cited by 10 publications

(12 citation statements)

References 33 publications

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“…Cells within cluster 6 expressed high levels of Acta2 (which encodes for α-smooth muscle actin), as well as Tnn, Postn , and Mmp13 , and were thus classified as myofibroblasts 50 . Lastly, cells within cluster 7 expressed Slc38a2 and Slc47a1 , consistent with the phenotype of dura and leptomeningeal fibroblasts 54,55,56 recently reported to be enriched in several transporters, and were thus assigned as Ly6a - dura fibroblasts ( Figure 3C ).…”

Section: Meningeal Rarres2+ Ly6a+ Fibroblasts Acquire Lymphoid Tissue...supporting

confidence: 85%

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The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronicTrypanosoma bruceiinfection

Quintana

Sinton

Chandrasegaran

et al. 2023

Preprint

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The meningeal space is an important structure in the brain borders, which provides immunosurveillance for the central nervous system, but the impact of infections on the meningeal immune landscape is far from being fully understood. The extracellular protozoan parasite Trypanosoma brucei, which causes Human African Trypanosomiasis (HAT) or sleeping sickness, accumulate in the meningeal spaces, ultimately inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, combining single cell transcriptomics and mass cytometry by time of flight (CyTOF), coupled with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges during chronic infection. These infection-induced ectopic structures are defined by the presence of ER-TR7+ fibroblastic reticular cells (FRCs) and follicular dendritic cells (FDCs) that initiate a signalling cascade driving local T cell activation towards a T follicular helper (TFH)-like phenotype, as well as B cell class switching. Furthermore, the GC-like B cells found in the infected meninges produce high-affinity autoantibodies able to recognise mouse brain antigens. We found that systemic lymphotoxin β (LTβ) signalling blockade led to a significant depletion of meningeal FDC-like cells and autoreactive B cells, indicating that LTβ signalling is critical to induce and maintain local responses in the meninges. In humans, we identified the presence of autoreactive IgG antibodies able to recognise human brain lysates in the cerebrospinal fluid of second stage HAT patients compared to first stage HAT patients, consistent with our findings in experimental infections. Taken together, our data provide evidence that the meningeal immune response results in the acquisition of lymphoid tissue-like properties during chronic T. brucei infection, broadening our understanding of meningeal immunity in the context of chronic infections. These findings have wider implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity in mice and humans, as observed in other autoimmune neurodegenerative disorders such as neuropsychiatric lupus and multiple sclerosis.

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Section: Meningeal Rarres2+ Ly6a+ Fibroblasts Acquire Lymphoid Tissue...supporting

confidence: 85%

“…Slc38a2 and Slc47a1, consistent with the phenotype of dura and leptomeningeal fibroblasts 54,55,56 recently reported to be enriched in several transporters, and were thus assigned as Ly6adura fibroblasts (Figure 3C).…”

Section: Cells Within Cluster 4 Expressed High Levels Of the Antioxid...supporting

confidence: 78%

The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronicTrypanosoma bruceiinfection

Quintana

Sinton

Chandrasegaran

et al. 2023

Preprint

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“…The varying results found demonstrate that in-depth characterization of the primary human LMCs is needed for more accurate experimental applications. Particularly as the majority of the studies into the role and function of the LMCs focuses on the use of animal derived LMCs or cells derived from meningioma tumors [31][32][33][34][35][36][37].…”

Section: Introductionmentioning

confidence: 99%

Characterization of Primary Human Leptomeningeal Cells in 2D culture

Abubaker

Greaney

Newport

et al. 2023

Preprint

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Maintaining the integrity of brain barriers is critical for a healthy central nervous system. While extensive research has focused on the blood-brain barrier (BBB) of the brain vasculature and blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus, the barriers formed by the meninges have not received as much attention. These membranes create a barrier between the brain and cerebrospinal fluid (CSF), as well as between CSF and blood. Recent studies, particularly on the arachnoid barrier, have revealed its critical role in facilitating the exchange of substances between the brain and CSF. Moreover, this barrier has been implicated in the development of neurological and immunological disorders. In order to gain a deeper comprehension of the functioning and significance of the meningeal barriers, sophisticated models of these barriers, comparable to those of the BBB and BCSFB, need to be created. The aim of this paper is to investigate the characteristics of leptomeningeal cells (LMCs) that form the meningeal barriers, in a cultured environment, including their morphology, proteomics, and barrier properties, and to determine whether passaging of these cells affects their behavior in comparison to their in vivo state. The study employed techniques such as immunofluorescence, immunoblotting, and TEER to analyze the cells. The results indicate that higher passage numbers significantly alter the morphology and protein localization and expression of the LMCs. Additionally, cultured LMCs showed an increase in Vimentin and Cytokeratin expression and a lack of junctional proteins localization on the cell membrane, which could suggest loss of epithelial properties due to culture, preventing barrier formation. This study shows that the optimal LMC passage range is between passages two and five for experimentation, as past this range showed cell properties consistent with EMT at the higher passages.

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“…Like melanocytes, these specialized meningeal cells arise from mesenchymal tissues in the neural tube 8 . Many studies have examined the roles of the meningeal cells in neural development and inflammation, but their role in cancer has never been explored [9][10][11][12] . The CSF environment is metabolically harsh compared to other sites of disease.…”

Section: Introductionmentioning

confidence: 99%

“…The leptomeninges consist of the pia mater, the arachnoid mater, and the CSF space. The pia mater covers the brain’s surface and consists of ICAM1- and SLC38A2-expressing fibroblast-like meningeal cells held together by tight junctions on top of a basement membrane 7,8 . Like melanocytes, these specialized meningeal cells arise from mesenchymal tissues in the neural tube 8 .…”

Section: Introductionmentioning

confidence: 99%

Spatial transcriptomics analysis identifies a unique tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease

Alhaddad,

Ospina,

Khaled

et al. 2023

Preprint

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Leptomeningeal disease (LMD) remains a rapidly lethal complication for late-stage melanoma patients. The inaccessible nature of the disease site and lack of understanding of the biology of this unique metastatic site are major barriers to developing efficacious therapies for patients with melanoma LMD. Here, we characterize the tumor microenvironment of the leptomeningeal tissues and patient-matched extra-cranial metastatic sites using spatial transcriptomic analyses within vitroandin vivovalidation. We show the spatial landscape of melanoma LMD to be characterized by a lack of immune infiltration and instead exhibit a higher level of stromal involvement. We show that the tumor-stroma interactions at the leptomeninges activate pathways implicated in tumor-promoting signaling, mediated through upregulation of SERPINA3 at the tumor-stroma interface. Our functional experiments establish that the meningeal stroma is required for melanoma cells to survive in the CSF environment and that these interactions lead to a lack of MAPK inhibitor sensitivity in the tumor. We show that knocking down SERPINA3 or inhibiting the downstream IGR1R/PI3K/AKT axis results in re-sensitization of the tumor to MAPK-targeting therapy and tumor cell death in the leptomeningeal environment. Our data provides a spatial atlas of melanoma LMD, identifies the tumor-promoting role of meningeal stroma, and demonstrates a mechanism for overcoming microenvironment-mediated drug resistance unique to this metastatic site.

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Characterization, isolation, and in vitro culture of leptomeningeal fibroblasts

Cited by 10 publications

References 33 publications

The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronicTrypanosoma bruceiinfection

The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronicTrypanosoma bruceiinfection

Characterization of Primary Human Leptomeningeal Cells in 2D culture

Spatial transcriptomics analysis identifies a unique tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease

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