Biology subjects

Knopf, F.

Publications and source records attributed to Knopf, F..

6 recordsLinked to original sources

bioRxiv2023

Fine-tuning of Fgf8 morphogen gradient by heparan sulfate proteoglycans in the extracellular matrix

Embryonic development is orchestrated by the action of morphogens, which spread out from a local source and activate, in a field of target cells, different cellular programs based on their concentration gradient. Fibroblast growth factor 8 (Fgf8) is a morphogen with important functions in embryonic organizing centers. It forms a gradient in the extracellular space by free diffusion, interaction with the extracellular matrix (ECM) and receptor-mediated endocytosis. However, morphogen gradient regulation by ECM is still poorly understood. Here we show that specific Heparan Sulfate Proteoglycans (HSPGs) bind Fgf8 directly in the ECM of living zebrafish embryos, thus affecting its diffusion and signaling. Using single-molecule Fluorescence Correlation Spectroscopy, we quantify this binding in vivo, and find two different modes of interaction. First, reducing or increasing the concentration of specific HSPGs in the extracellular space alters Fgf8 diffusion, and thus, its gradient shape. Second, ternary complex formation of Fgf8 ligand with Fgf-receptors and HSPGs at the cell surface requires HSPG attachment to the cell membrane. Together, our results show that graded Fgf8 morphogen distribution is achieved by constraining free Fgf8 diffusion through successive interactions with HSPGs at the cell surface and in ECM space. Statement of significanceFgf8 is a secreted morphogen signaling molecule that instructs neighboring arrays of undifferentiated cells about their position and cellular identity in tissue. Fgf8 and other morphogens are often distributed in a graded fashion, and can typically work at very low concentrations. To reproducibly generate information in developing tissue, mechanisms have evolved to carefully control distribution and concentration of Fgf8 morphogen. We show that freely diffusing Fgf8 morphogen moves through interstitial cell spaces on its way to target cells, and while doing so, interacts with ECM molecules in these spaces and at cell surfaces via low affinity, reversible binding. These interactions are important tuning mechanisms that contribute to forming the Fgf8 morphogen gradient and to cell surface receptor binding, and thus, to controlling cell type identity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/565243v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@b6d073org.highwire.dtl.DTLVardef@2f64c1org.highwire.dtl.DTLVardef@17fbf19org.highwire.dtl.DTLVardef@1db334a_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractTo generate a gradient, the morphogen Fgf8 shuttles between fast free diffusion through extracellular space (i), slow diffusion or immobility (ii, iii) when bound to Heparan Sulfate Proteoglycans (HSPGs), in the extracellular matrix (ECM) and at cell surface receptors (iv), as revealed by single molecule studies in living zebrafish embryos.

developmental biology↗

bioRxiv2023

Preclinical assessment of CAR-NK cell-mediated killing efficacy and pharmacokinetics in a rapid zebrafish xenograft model of metastatic breast cancer

Natural killer (NK) cells are attractive effectors for adoptive immunotherapy of cancer. Results from first-in-human studies using chimeric antigen receptor (CAR)-engineered primary NK cells and NK-92 cells are encouraging in terms of efficacy and safety. In order to further improve treatment strategies and to test the efficacy of CAR-NK cells in a personalized manner, preclinical screening assays using patient-derived tumor samples are needed. Zebrafish (Danio rerio) embryos and larvae represent an attractive xenograft model to study growth and dissemination of patient-derived tumor cells because of their superb live cell imaging properties. Injection into the organisms circulation allows investigation of metastasis, cancer cell-to-immune cell-interactions and studies of the tumor cell response to anti-cancer drugs. Here, we established a zebrafish larval xenograft model to test the efficacy of CAR-NK cells against metastatic breast cancer in vivo by injecting metastatic breast cancer cells followed by CAR-NK cell injection into the Duct of Cuvier (DoC). We validated the functionality of the system with two different CAR-NK cell lines specific for PD-L1 and ErbB2 (PD-L1.CAR NK-92 and ErbB2.CAR NK- 92 cells) against the PD-L1-expressing MDA-MB-231 and ErbB2-expressing MDA-MB-453 breast cancer cell lines. Injected cancer cells were viable and populated peripheral regions of the larvae, including the caudal hematopoietic tissue (CHT), simulating homing of cancer cells to blood forming sites. CAR-NK cells injected 2.5 hours later migrated to the CHT and rapidly eliminated individual cancer cells throughout the organism. Confocal live-cell imaging demonstrated intravascular migration and real-time interaction of CAR-NK cells with MDA-MB-231 cells, explaining the rapid and effective in vivo cytotoxicity. Thus, our data suggest that zebrafish larvae can be used for rapid and cost-effective in vivo assessment of CAR-NK cell potency and to predict patient response to therapy.

cancer biology↗

bioRxiv2023

DanioCTC: Injection of circulating tumor cells from metastatic breast cancer patients in zebrafish xenografts for analysis of metastasis

Circulating tumor cells (CTCs) are considered as metastatic precursor cells, and zebrafish xenografts provide an in vivo model to study cancer cell spread. Currently, the low number of patient-derived CTCs limits their analysis in animal models. We present DanioCTC, a xenograft workflow for injecting CTCs from metastatic breast cancer (MBC) patients into zebrafish embryos to study cell dissemination in vivo. The study successfully adapts existing workflows and combines diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the automated cell micromanipulator CellCelector setup to enrich and isolate MBC-derived CTCs and to finally inject them into Zebrafish embryos, where their dissemination was tracked up to 3 days post-injection. MDA-MB-231 cells were used as a standard xenotransplantation control, and these cells were frequently found in the head and blood-forming regions of the tail. Using DLA aliquots spiked with MBA-MB-231 cells, the newly established DanioCTC workflow confirmed the dissemination of MDA-MB-231 cells into these regions. CTCs from an MBC patient were then enriched by DLA, Parsortix, and flow cytometry, isolated with the CellCelectorTM and xenografted into zebrafish embryos. CTCs were mainly detected in the head and trunk, unlike MDA-MB-231 cells, which were present in the head and tail. DanioCTC presents a significant breakthrough in the use of zebrafish embryos as a model to study CTC dissemination in vivo, which can be used for patient-derived CTCs instead of cell culture-derived cancer cells as a crucial step towards understanding the biology of metastatic breast cancer. Statement of significanceDanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies on CTC dissemination and personalized treatment in vivo, therefore advancing our toolkit to fight metastatic cancer.

cancer biology↗

bioRxiv2023

Compartmentalization and synergy of osteoblasts drive bone formation in the regenerating fin

Zebrafish faithfully regenerate their fins after amputation which includes restoration of bone tissue and a component of cell plasticity. It is currently unclear how different cell populations of the regenerate divide labor to allow for efficient regenerate growth and proper patterning. Here, we studied lineage relationships of FACS-enriched epidermal, blastemal and bone forming fin regenerate cells by single cell (sc) RNA sequencing, lineage tracing, targeted osteoblast ablation and electron microscopy to show that the majority of osteoblasts in the outgrowing regenerate derive from osterix+ osteoblasts, while mmp9+ cells give rise to a limited cell number at the fin segment joints. A third population of distal blastema cells contributes to distal osteoblast progenitors, suggesting compartmentalization during appendage regeneration. Fin elongation and bone formation are carried out by distinct regenerate cell populations, and these variably depend on Fgf signaling. Ablation of osterix+ osteoblasts irreversibly impairs patterning of segment joints, and prevents bone matrix formation in the proximal regenerate. The resulting reduced regenerate length is partially compensated for by the distal regenerate which shows increased Wnt signaling activity. Surprisingly, ablation of joint cells does not abolish the formation of segment joints. Our study characterizes rare fin regenerate cell populations, indicates intricate osteoblast-blastema lineage relationships, inherent detection and compensation of impaired regeneration, and demonstrates zonation of the elongating regenerate. Furthermore, it sheds light on the variable dependence of bone formation on growth factor signaling.

developmental biology↗

bioRxiv2021

The distinct role of ALDH1A1 and ALDH1A3 in the regulation of prostate cancer metastases

Cancer stem cells (CSC) are characterized by high self-renewal capacity, tumor-initiating potential, and therapy resistance. Aldehyde dehydrogenase (ALDH)+ cell population serves as an indicator of prostate CSCs with increased therapy resistance, enhanced DNA double-strand break repair, and activated epithelial-mesenchymal transition (EMT) and migration. Numerous ALDH genes contribute to ALDH enzymatic activity; however, only some of them showed clinical relevance. We found that ALDH1A1 and ALDH1A3 genes functionally regulate CSC properties and radiation sensitivity of PCa. We revealed a negative correlation between ALDH1A1 and ALDH1A3 expression in publicly available prostate cancer (PCa) datasets and demonstrated that ALDH1A1 and ALDH1A3 have opposing predictive value for biochemical recurrence-free survival. Our data suggest an association of ALDH1A1 with the metastatic burden, elucidating the role of ALDH genes in the metastatic spread and homing to the bone, which can be, at least partially, attributed to regulating the transforming growth factor beta 1 (TGFB1) and matrix metalloproteinases (MMPs). ALDH genes play a diverse role in PCa development under AR and {beta}-catenin-dependent regulation, with ALDH1A1 becoming dominant in later stages of tumor development when PCa cells gain androgen independence. Taken together, our results indicate that ALDH1A1 and ALDH1A3 modulate PCa radiosensitivity, regulate CSCs phenotype, and spread of PCa cells to the bone, therefore having clinical implication for identifying patients at high risk for progression to metastatic disease.

cancer biology↗

bioRxiv2021

Osteoblast cell death triggers a pro-osteogenic inflammatory response regulated by reactive oxygen species and glucocorticoid signaling in zebrafish

In zebrafish, transgenic labeling approaches, robust regenerative responses and excellent in vivo imaging conditions enable precise characterization of immune cell behavior in response to injury. Here, we monitored osteoblast-immune cell interactions in bone, a tissue which is particularly difficult to in vivo image in tetrapod species. Ablation of individual osteoblasts leads to recruitment of neutrophils and macrophages in varying numbers, depending on the extent of the initial insult, and initiates generation of cathepsinK+ osteoclasts from macrophages. Induced osteoblast death triggers the production of pro-inflammatory cytokines and reactive oxygen species, which are needed for successful macrophage recruitment. Excess glucocorticoid signaling as it occurs during the stress response inhibits macrophage recruitment, maximum speed and changes the macrophages phenotype. While osteoblast loss is compensated for within a day by contribution of committed osteoblasts, macrophages continue to populate the region. Their presence is required for osteoblasts to fill the lesion site. Our model enables visualization of homeostatic bone repair after microlesions at single cell resolution and demonstrates a pro-osteogenic function of tissue-resident macrophages in non-mammalian vertebrates. Summary statementLaser-mediated osteoblast ablation induces recruitment of tissue-resident macrophages by a release of reactive oxygen species. The presence of macrophages is required for osteoblasts to repopulate the lesion site and can be modulated by glucocorticoids.

developmental biology↗