Cancer metastasis is the most important prognostic factor determining patient survival, but currently there are very few drugs or therapies that specifically inhibit the invasion and metastasis of ...cancer cells. Currently, human cancer metastasis is largely studied using transgenic and immunocompromised mouse xenograft models, which are useful for analysing end-point tumour growth but are unable to accurately and reliably monitor
in vivo invasion, intravasation, extravasation or secondary tumour formation of human cancer cells. Furthermore, limits in our ability to accurately monitor early stages of tumour growth and detect micro-metastases likely results in pain and suffering to the mice used for cancer xenograft experiments. Zebrafish (
Danio rerio) embryos, however, offer many advantages as a model system for studying the complex, multi-step processes involved during cancer metastasis. This article describes a detailed method for the analysis of human cancer cell invasion and metastasis in zebrafish embryos before they reach protected status at 5 days post fertilisation. Results demonstrate that human cancer cells actively invade within a zebrafish microenvironment, and form metastatic tumours at secondary tissue sites, suggesting that the mechanisms involved during the different stages of metastasis are conserved between humans and zebrafish, supporting the use of zebrafish embryos as a viable model of human cancer metastasis. We suggest that the embryonic zebrafish xenograft model of human cancer is a tractable laboratory model that can be used to understand cancer biology, and as a direct replacement of mice for the analysis of drugs that target cancer invasion and metastasis.
Metastasis is a main cause of death in prostate cancer (PCa). To dissect the molecular cues from cancer cell-microenvironment interaction that drive metastatic cascade, bone metastatic PCa cells were ...intravenously implanted into zebrafish embryos and mice tibia forming metastatic lesions. Transcriptomic analysis showed an elevated expression of stemness genes, pro-inflammatory cytokines and TGF-β family member Activin A in the cancer cells at metastatic onset in both animal models. Consistently, analysis of clinical datasets revealed that the expression of Activin A is specifically elevated in metastases and correlates with poor prognosis in stratified high-risk PCa patients. It is further unveiled that the microenvironment induced Activin A expression by NF-κB activation. The elevated level of Activin A enhanced the invasive ALDH
CSC-like phenotypes and PCa proliferation by activation of Smad and ERK1/2 signaling driving metastasis. Suppression of Activin A or Activin receptor significantly reduced the CSC-like subpopulation, invasion, metastatic growth, and bone lesion formation in zebrafish and mice xenografts, suggesting a functional role of NF-κB-dependent Activin A in PCa metastasis. Overall, our study demonstrates that human PCa cells can display a comparable response with the microenvironment in zebrafish and mice xenografts. Combining both animal models, we uncovered the microenvironment-dependent activin signaling as an essential driver in PCa metastasis with therapeutic potential.
Despite latest advances in prostate cancer (PCa) therapy, PCa remains the third-leading cause of cancer-related death in European men. Dysregulation of microRNAs (miRNAs), small non-coding RNA ...molecules with gene expression regulatory function, has been reported in all types of epithelial and haematological cancers. In particular, miR-221-5p alterations have been reported in PCa.
miRNA expression data was retrieved from a comprehensive publicly available dataset of 218 PCa patients (GSE21036) and miR-221-5p expression levels were analysed. The functional role of miR-221-5p was characterised in androgen- dependent and androgen- independent PCa cell line models (C4-2 and PC-3M-Pro4 cells) by miR-221-5p overexpression and knock-down experiments. The metastatic potential of highly aggressive PC-3M-Pro4 cells overexpressing miR-221-5p was determined by studying extravasation in a zebrafish model. Finally, the effect of miR-221-5p overexpression on the growth of PC-3M-Pro4luc2 cells in vivo was studied by orthotopic implantation in male Balb/cByJ nude mice and assessment of tumor growth.
Analysis of microRNA expression dataset for human primary and metastatic PCa samples and control normal adjacent benign prostate revealed miR-221-5p to be significantly downregulated in PCa compared to normal prostate tissue and in metastasis compared to primary PCa. Our in vitro data suggest that miR-221-5p overexpression reduced PCa cell proliferation and colony formation. Furthermore, miR-221-5p overexpression dramatically reduced migration of PCa cells, which was associated with differential expression of selected EMT markers. The functional changes of miR-221-5p overexpression were reversible by the loss of miR-221-5p levels, indicating that the tumor suppressive effects were specific to miR-221-5p. Additionally, miR-221-5p overexpression significantly reduced PC-3M-Pro4 cell extravasation and metastasis formation in a zebrafish model and decreased tumor burden in an orthotopic mouse model of PCa.
Together these data strongly support a tumor suppressive role of miR-221-5p in the context of PCa and its potential as therapeutic target.
Innate and adaptive immune cells participate in the homeostatic regulation of hematopoietic stem cells (HSCs). Here, we interrogate the contribution of myeloid cells, the most abundant cell type in ...the mammalian bone marrow, in a clinically relevant mouse model of neutropenia. Long-term genetic depletion of neutrophils and eosinophils results in activation of multipotent progenitors but preservation of HSCs. Depletion of myeloid cells abrogates HSC expansion, loss of serial repopulation and lymphoid reconstitution capacity and remodeling of HSC niches, features previously associated with hematopoietic aging. This is associated with mitigation of interferon signaling in both HSCs and their niches via reduction of NK cell number and activation. These data implicate myeloid cells in the functional decline of hematopoiesis, associated with activation of interferon signaling via a putative neutrophil-NK cell axis. Innate immunity may thus come at the cost of system deterioration through enhanced chronic inflammatory signaling to stem cells and their niches.
Cancer metastasis is the most important prognostic factor determining patient survival, but currently there are very few drugs or therapies that specifically inhibit the invasion and metastasis of ...cancer cells. Currently, human cancer metastasis is largely studied using transgenic and immunocompromised mouse xenograft models, which are useful for analysing end-point tumour growth but are unable to accurately and reliably monitor
in vivo invasion, intravasation, extravasation or secondary tumour formation of human cancer cells. Furthermore, limits in our ability to accurately monitor early stages of tumour growth and detect micro-metastases likely results in pain and suffering to the mice used for cancer xenograft experiments. Zebrafish (
Danio rerio) embryos, however, offer many advantages as a model system for studying the complex, multi-step processes involved during cancer metastasis. This article describes a detailed method for the analysis of human cancer cell invasion and metastasis in zebrafish embryos before they reach protected status at 5 days post fertilisation. Results demonstrate that human cancer cells actively invade within a zebrafish microenvironment, and form metastatic tumours at secondary tissue sites, suggesting that the mechanisms involved during the different stages of metastasis are conserved between humans and zebrafish, supporting the use of zebrafish embryos as a viable model of human cancer metastasis. We suggest that the embryonic zebrafish xenograft model of human cancer is a tractable laboratory model that can be used to understand cancer biology, and as a direct replacement of mice for the analysis of drugs that target cancer invasion and metastasis.
To visually and genetically trace single-cell dynamics of human prostate cancer (PCa) cells at the early stage of metastasis, a zebrafish (ZF) xenograft model was employed. The phenotypes of ...intravenously transplanted fluorescent cells were monitored by high-resolution, single-cell intravital confocal and light-sheet imaging. Engrafted osteotropic, androgen independent PCa cells were extravasated from caudle vein, invaded the neighboring tissue, proliferated and formed experimental metastases around caudal hematopoietic tissue (CHT) in four days. Gene expression comparison between cells in culture and in CHT revealed that engrafted PCa cells responded to the ZF microenvironment by elevating expression of epithelial-mesenchymal transition (EMT) and stemness markers. Next, metastatic potentials of ALDH
cancer stem-like cells (CSCs) and ALDH
non-CSCs were analyzed in ZF. Engraftment of CSCs induced faster metastatic onset, however after six days both cell subpopulations equally responded to the ZF microenvironment, resulting in the same increase of stemness genes expression including Nanog, Oct-4 and Cripto. Knockdown of Cripto significantly reduced the vimentin/E-cadherin ratio in engrafted cells, indicating that Cripto is required for transduction of the microenvironment signals from the ZF niche to increase mesenchymal potential of cells. Targeting of either Cripto or EMT transcriptional factors Snail 1 and Zeb1 significantly suppressed metastatic growth. These data indicated that zebrafish microenvironment governed the CSC/EMT plasticity of human PCa cells promoting metastasis initiation.
Mesenchymal stem cells (MSCs) play pivotal roles in tissue (re)generation. In the murine bone marrow, they are thought to reside within the Sca‐1+ CD51+ bone marrow stromal cell population. Here, ...using scRNAseq, we aimed to delineate the cellularheterogeneity of this MSC‐enriched population throughout development. At the fetal stage, the MSC population is relatively homogeneous with subsets predicted to contain stem/progenitor cells, based on transcriptional modeling and marker expression. These subsets decline in relative size throughout life, with postnatal emergence of specialized clusters, including hematopoietic stem/progenitor cell (HSPC) niches. In fetal development, these stromal HSPC niches are lacking, but subsets of endothelial cells express HSPC factors, suggesting that they may provide initial niches for emerging hematopoiesis. This cellular taxonomy of the MSC population upon development is anticipated to provide a resource aiding the prospective identification of cellular subsets and molecular mechanisms driving bone marrow (re)generation.
Mesenchymal stem cells (MSCs) in the bone marrow (BM) are thought to play pivotal roles in tissue (re)generation and provide a supportive microenvironment for hematopoietic stem cells (HSCs). MSCs ...have been well characterized in murine models and isolated using different combinations of markers. Yet, these markers define a broader and more heterogeneous population, of which MSCs are only a minority. Although several studies indicate that bona fide MSCs reside in the BM, further investigations are needed to define their identity and allow their prospective and effective isolation. Here, using a single cell RNA sequencing approach, we aimed to delineate the cellular heterogeneity of the MSC-enriched Sca-1+ CD51+ population throughout development. At the fetal stage (E17), the BMSC population appeared relatively homogeneous with subsets predicted to contain candidate MSCs based on marker expression and computational modeling. The frequency of putative MSC subsets declined upon aging. After birth, the BMSC population was more heterogeneous, and characterized by the emergence of clusters showing transcriptional wiring consistent with specialization towards differentiated (progenitor) subsets. This included the postnatal emergence of a Lepr+ population, expressing HSC regulatory genes and previously established as HSC niches. In the fetal BM, subsets of endothelial cells expressed HSC factors and may thus represent an initial supportive microenvironment for emerging hematopoiesis. Our results shed light on the cellular diversity of BMSCs from different developmental phases, providing a cellular taxonomy of this population at single cell resolution. The exploration of our dataset, and the comparison of transcriptional landscapes from different developmental stages, may provide a resource to identify and further validate factors with a predicted role in BM regeneration.
Metastatic colonization by circulating cancer cells is a highly inefficient process. To colonize distant organs, disseminating cancer cells must overcome many obstacles in foreign microenvironments, ...and only a small fraction of them survives this process. How these disseminating cancer cells cope with stress and initiate metastatic process is not fully understood. In this study, we report that the metastatic onset of prostate cancer cells is associated with the dynamic conversion of metabolism signaling pathways governed by the energy sensors AMPK and mTOR. While in circulation in blood flow, the disseminating cancer cells display decreased mTOR and increased AMPK activities that protect them from stress-induced death. However, after metastatic onset, the mTOR-AMPK activities are reversed, enabling mTOR-dependent tumor growth. Suppression of this dynamic conversion by co-targeting of AMPK and mTOR signaling significantly suppresses prostate cancer cell and tumor organoid growth in vitro and experimental metastasis in vivo, suggesting that this can be a therapeutic approach against metastasizing prostate cancer.
•Disseminating PCa cells display decreased mTOR and increased AMPK activities.•Active AMPK protects PCa cells from stress-induced death in circulation.•After metastatic onset, mTOR-AMPK pathways are reversed for mTOR-dependent growth.•Co-targeting AMPK and mTOR suppresses metastatic onset in vivo and patient organoids.
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