The tumor microenvironment consists of stromal cells and leukocytes that contribute to cancer progression. Cross-talk between tumor cells and their microenvironment is facilitated by a variety of soluble factors, including growth factors, cytokines such as chemokines. Due to a wide expression of chemokine receptors on cells in the tumor microenvironment, including tumor cells, chemokines affect various processes such as leukocyte recruitment, angiogenesis, tumor cell survival, tumor cell adhesion, proliferation, vascular permeability, immune suppression, invasion and metastasis. Inflammatory chemokines are instrumental players in cancer-related inflammation and significantly contribute to numerous steps during metastasis. Recruitment of myeloid-derived cells to metastatic sites is mainly mediated by the inflammatory chemokines CCL2 and CCL5. Tumor cell homing and extravasation from the circulation in distant organs are also regulated by inflammatory chemokines. Recent experimental evidence demonstrated that besides leukocyte recruitment, tumor cell-derived CCL2 directly activated endothelial cells and together with monocytes facilitated tumor cell extravasation, in a CCL2-and CCL5-dependent manner. Furthermore, CX3CL1 expression in the bone facilitated metastasis of CX3CR1 expressing tumor cells to this site. Current findings in preclinical models strongly suggest that inflammatory chemokines play an important role during metastasis and targeting of the chemokine axis might have a therapeutic potential. 2

Tumor cells interact with blood constituents and these interactions promote metastasis. Selectins are vascular receptors facilitating interactions of tumor cells with platelets, leukocytes, and endothelium, but the role of endothelial E-selectin remains unclear. Here we show that E-selectin is a major receptor for monocyte recruitment to tumor cell-activated endothelium. Experimental and spontaneous lung metastasis using murine tumor cells, without E-selectin ligands, were attenuated in E-selectin-deficient mice. Tumor cellderived CCL2 promoted endothelial activation, resulting in enhanced endothelial E-selectin expression. The recruitment of inflammatory monocytes to metastasizing tumor cells was dependent on the local endothelial activation and the presence of E-selectin. Monocytes promoted transendothelial migration of tumor cells through the induction of E-selectin-dependent endothelial retractions and a subsequent modulation of tight junctions through dephosphorylation of VE-cadherin. Thus, endothelial E-selectin shapes the tumor microenvironment through the recruitment, adhesion, and activation of monocytes that facilitate tumor cell extravasation and thereby metastasis. These findings provide evidence that endothelial E-selectin is a novel factor contributing to endothelial retraction required for efficient lung metastasis.

Drosophila melanogaster plasmatocytes, the phagocytic cells among hemocytes, are essential for immune responses, but also play key roles from early development to death through their interactions with other cell types. They regulate homeostasis and signaling during development, stem cell proliferation, metabolism, cancer, wound responses, and aging, displaying intriguing molecular and functional conservation with vertebrate macrophages. Given the relative ease of genetics in Drosophila compared to vertebrates, tools permitting visualization and genetic manipulation of plasmatocytes and surrounding tissues independently at all stages would greatly aid a fuller understanding of these processes, but are lacking. Here, we describe a comprehensive set of transgenic lines that allow this. These include extremely brightly fluorescing mCherry-based lines that allow GAL4-independent visualization of plasmatocyte nuclei, the cytoplasm, or the actin cytoskeleton from embryonic stage 8 through adulthood in both live and fixed samples even as heterozygotes, greatly facilitating screening. These lines allow live visualization and tracking of embryonic plasmatocytes, as well as larval plasmatocytes residing at the body wall or flowing with the surrounding hemolymph. With confocal imaging, interactions of plasmatocytes and inner tissues can be seen in live or fixed embryos, larvae, and adults. They permit efficient GAL4-independent Fluorescence-Activated Cell Sorting (FACS) analysis/sorting of plasmatocytes throughout life. To facilitate genetic studies of reciprocal signaling, we have also made a plasmatocyte-expressing QF2 line that, in combination with extant GAL4 drivers, allows independent genetic manipulation of both plasmatocytes and surrounding tissues, and GAL80 lines that block GAL4 drivers from affecting plasmatocytes, all of which function from the early embryo to the adult.

Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo.

Heparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and glycocalyx thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression. Therefore, we investigated the relevance of HPSE duality in malignant melanoma in vitro as well as in mouse melanoma models basing on the intradermal injection of transfected melanoma cells. In line with its extracellular action, HPSE-deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of VEGF affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-and most prominently MMP-9 upon HPSE knockdown. In vivo, HPSE-deficiency was related to increased lymph node metastasis. While inhibition of its extracellular function heparin was unable to block the gene regulatory impact of HPSE we proposed an intracellular mechanism. Immunostainings revealed a counter-staining of HPSE and NF-B in the nucleus suggesting a close relationship between both proteins. This finding was further supported by the discovery of a direct charge-driven molecular interaction between HPSE and DNA by using atomic force microscopy and a co-precipitation approach. Our findings are novel and point towards a dual identity of HPSE in malignant melanoma with a protumorigenic extracellular activity and a tumor suppressive nuclear action. Identification of molecular strategies to shuttle extracellular HPSE into the nuclei of cancer cells envisions new therapeutic options. AbstractHeparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and glycocalyx thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression.Therefore, we investigated the relevance of HPSE duality in malignant melanoma in vitro as well as in mouse melanoma models basing on the intradermal injection of transfected melanoma cells. In line with its extracellular action, HPSE-deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of VEGF affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-α and most prominently MMP-9 upon HPSE knockdown. In vivo, HPSE-deficiency was related to increased lymph node metastasis.While inhibition of its extracellular function heparin was unable to block the gene regulatory impact of HPSE we proposed an intracellular mechanism. Immunostainings revealed a counter-staining of HPSE and NF-κB in the nucleus suggesting a close relationship between both proteins. This finding was further supported by the discovery of a dir...

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva’s vertebrate ortholog, MFSD1, rescues the minerva mutant’s migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis.

Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation; but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2ecKO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2ecKO and Ccr2fl/fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2ecKO and Ccr2fl/fl littermates the absence of vascular permeability induction was observed only in Ccr2ecKO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction and vascular leakiness that was blocked by an addition of CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. Implications Findings provide mechanistic insight into how CCL2-CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation resulting in endothelial retraction and enhanced tumor cell migration and metastasis.

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