Biology subjects

de Winde, C. M.

Publications and source records attributed to de Winde, C. M..

6 recordsLinked to original sources

bioRxiv2025

Integration of lymphatic vasculature to a human lymph node-on-chip enhances physiological immune properties

To study systemic human innate and adaptive immune responses in detail, competent in vitro lymph node (LN) models with LN stromal cells (LNSCs) are required to recapitulate the physiological microenvironment. The multicellular organisation of LNs possesses a challenge for designing such microphysiological systems (MPS), particularly with the structural complexity of LNs and the lymphatic vasculature. Here, we established an organotypic LN model with integrated lymphatics in an organ-on-chip (OoC) platform containing a printed sacrificial structure, and studied the influence of a perfused lymphatic endothelial cell (LEC)-lined channel on the LN-on-chip microenvironment. Upon one-week of culture under lymphatic flow, LECs lined the tubular structure forming a lymphatic vessel through the LN model, and stable metabolic conditions within the LN-on-chip were confirmed. Interestingly, LECs in the LN-on-chip displayed the phenotype found in human LNs with upregulation of LEC-specific LN markers, such as atypical chemokine receptor 4 (ACKR4). The presence of the LEC-lined perfused vessel in the LN-on-chip resulted in the increase of native immune cells, most notably B cells, and the secretion of survival and migratory signals, namely interleukin-7 (IL-7) and CC motif chemokine ligand 21 (CCL21). Likewise, LECs promoted the abundance of immune cell clusters closer to the vessel. As such, these features represent an enhanced physiological microenvironment to allow for immune cell migration and interactions for efficient LN functioning. This approach paves the way for LN integration into multi-OoC (MOC) platforms to investigate immunological crosstalk between tissue-derived factors, immune cell trafficking and organ-specific adaptive immune responses.

bioengineering↗

bioRxiv2023

Unbiased method for spectral analysis of cells with great diversity of autofluorescence spectra

Autofluorescence is an intrinsic feature of cells, caused by the natural emission of light by its cellular content, that can complicate analysis of flow cytometry data. Different cell types have different autofluorescence spectra and even within one cell type heterogeneity of autofluorescence spectra can be present, for example as a consequence of activation status or metabolic changes. By using full spectrum flow cytometry, the emission spectrum of a fluorochrome is captured by a set of detectors across a range of wavelengths, creating an unique spectrum for this fluorochrome, that is used to unmix the signal of a full stained sample into the signals of the different fluorochromes. Importantly, this technology can also be used to identify the aut-ofluorescence signal of an unstained sample, which can be used for unmixing purposes and to separate the autofluorescence signal from the fluorophore signals. However, this only works if the sample has one homogeneous autofluorescence spectrum. To analyze samples with a heterogeneous autofluorescence spectral profile, we here setup an unbiased workflow to detect all different autofluorescence spectra present in a sample to take them along as autofluorescence signatures during the unmixing of the full stained samples. First, clusters of cells with similar autofluorescence spectra are identified by unbiased dimensional reduction and clustering. Then, unique autofluorescence clusters are determined and are used to improve the unmixing accuracy of the full stained sample. This unbiased method allows for the identification of all autofluorescence spectra present in a sample, independent of cell types and intensity of the autofluorescence spectra. Furthermore, this method is equally useful for spectral analysis of different biological samples, including tissue cell suspensions, peripheral blood mononuclear cells and in vitro cultures of (primary) cells.

cell biology↗

bioRxiv2022

Podoplanin expression in fibroblasts determines lymph node architecture and adaptive immune function

Lymph nodes are uniquely organised niches for immune interactions. Fibroblastic reticular cells (FRCs) facilitate immune cell communication and regulate immune function by producing growth factors, chemokines and inflammatory cues. Stromal expression of the glycoprotein Podoplanin (PDPN) is required for lymph node development, but the requirement for PDPN signaling in FRCs in adult lymph nodes has not been directly tested. Using a conditional in vivo deletion model PDGFRmGFP{Delta}PDPN, scRNA-seq revealed that PDPN deletion increased inflammatory signalling in lymph nodes and disrupted stromal-immune cells crosstalk in steady state and during adaptive immune responses. We confirmed that PDPN/CLEC-2 signaling in FRCs switches transcriptional states and alters expression of immune related genes. We conclude that Podoplanin expression impacts the immunoregulatory properties of fibroblastic stroma in lymph nodes and is a key transcriptional regulator of fibroblast function in lymph nodes. Summary: Podoplanin expression regulates the immunoregulatory properties of fibroblastic stroma in lymph nodes

immunology↗

bioRxiv2020

Podoplanin drives dedifferentiation and amoeboid invasion of melanoma

Melanoma is an aggressive skin cancer developing from melanocytes, frequently resulting in metastatic disease. Melanoma cells utilise amoeboid migration as mode of local invasion. Amoeboid invasion is characterized by rounded cell morphology and high actomyosin contractility driven by the RhoA signalling pathway. Migrastatic drugs targeting actin polymerization and contractility to inhibit invasion and metastasis are therefore a promising treatment option. To predict amoeboid invasion and metastatic potential, there is a need for biomarkers functionally linked to contractility pathways. The glycoprotein podoplanin drives actomyosin contractility in lymphoid fibroblasts, and is overexpressed in several cancer types. Here, we show that podoplanin enhances amoeboid invasion in melanoma. Expression of podoplanin in murine melanoma models drives rounded cell morphology, increasing motility and invasion in vivo. Podoplanin expression is upregulated in a subset of dedifferentiated human melanoma, and in vitro is sufficient to suppress melanogenesis and upregulate melanoma-associated markers Mitf and Pou3f2. Together, our data indicates that podoplanin is both a potential biomarker for dedifferentiated invasive melanoma and a promising migrastatic therapeutic target.

cancer biology↗

bioRxiv2019

Podoplanin function is switched by partner proteins on fibroblastic reticular cells

Lymph node expansion is pivotal for adaptive immunity. CLEC-2+ migratory dendritic cells (DCs) interact with fibroblastic reticular cells (FRCs) to inhibit podoplanin-dependent actomyosin contractility, permitting FRC spreading and lymph node expansion. However, the molecular mechanisms controlling lymph node remodelling are not fully understood. We asked how podoplanin is regulated on FRCs in the early phase of lymph node expansion in vivo, and further, which other FRC markers are required for FRCs to respond to CLEC-2+ DCs. We find that expression of podoplanin and its partner proteins CD44 and CD9 in FRCs is coregulated by CLEC-2, and is differentially expressed by specific lymph node stromal populations in vivo. We find that beyond contractility, podoplanin is required for polarity and alignment of FRCs. Both CD44 and CD9 act to dampen podoplanin-dependent contractility, and colocalize with podoplanin in different areas of the cell membrane. Independently of podoplanin, CD44 and CD9 affect the degree of cell-cell contact and overlap between neighbouring FRCs. Further, we show that both CD44 and CD9 are required for FRCs to spread and form protrusions in response to DCs. Our data show that remodelling of the FRC cytoskeleton is a two-step process requiring podoplanin partner proteins CD44 and CD9. Firstly, CLEC-2/podoplanin-binding drives relaxation of actomyosin contractility, and secondly FRCs form protrusions and spread which requires both CD44 and CD9. Together, we show a multi-faceted response of FRCs to DCs, which requires CD44 and CD9 in addition to podoplanin.

cell biology↗

bioRxiv2017

C-type lectin-like receptor 2 (CLEC-2)-dependent DC migration is controlled by tetraspanin CD37

Cell migration is central to evoke a potent immune response. Dendritic cell (DC) migration to lymph nodes is dependent on the interaction of C-type lectin-like receptor 2 (CLEC-2) expressed by DCs, with podoplanin expressed by lymph node stromal cells (LNSCs). However, the underlying molecular mechanisms by which CLEC-2 influences DC migration remain elusive. Here, we show that CLEC-2-dependent DC migration is tightly controlled by tetraspanin CD37, a membrane-organizing protein. Our findings demonstrate a specific molecular interaction between CLEC-2 and CD37. Myeloid cells lacking CD37 (Cd37-/-) expressed less CLEC-2 on their surface compared to wild-type cells, indicating that CD37 is required to stabilize membrane expression of CLEC-2. In addition, CLEC-2-expressing DCs lacking CD37 showed impaired adhesion, migration velocity and displacement on LNSCs. Moreover, Cd37-/- DCs failed to form actin protrusions in a 3D collagen matrix upon podoplanin-induced CLEC-2 stimulation, phenocopying CLEC-2-deficient DCs (CD11c{Delta}CLEC-2). Microcontact printing experiments revealed that CD37 is required for CLEC-2 recruitment in the membrane to its ligand podoplanin. This study demonstrates that tetraspanin CD37 controls CLEC-2 membrane organization and provides new molecular insights underlying CLEC-2-dependent DC migration.

cell biology↗