Hepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8(+) T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8(+) T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8(+) and NKT cells cooperatively induce liver damage. Hepatocellular LTβR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development.

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

SummaryMelanoma cells can switch between an elongated mesenchymal-type and a rounded amoeboid-type migration mode. The rounded 'amoeboid' form of cell movement is driven by actomyosin contractility resulting in membrane blebbing. Unlike elongated A375 melanoma cells, rounded A375 cells do not display any obvious morphological front-back polarisation, although polarisation is thought to be a prerequisite for cell movement. We show that blebbing A375 cells are polarised, with ezrin (a linker between the plasma membrane and actin cytoskeleton), F-actin, myosin light chain, plasma membrane, phosphatidylinositol (4,5)-bisphosphate and b1-integrin accumulating at the cell rear in a uropod-like structure. This structure does not have the typical protruding shape of classical leukocyte uropods, but, as for those structures, it is regulated by protein kinase C. We show that the ezrin-rich uropod-like structure (ERULS) is an inherent feature of polarised A375 cells and not a consequence of cell migration, and is necessary for cell invasion. Furthermore, we demonstrate that membrane blebbing is reduced at this site, leading to a model in which the rigid ezrin-containing structure determines the direction of a moving cell through localised inhibition of membrane blebbing. Journal of Cell ScienceIn confined spaces, blebbing cells can move forward without adhesion by a process termed 'chimneying' (Hawkins et al., 2009;Malawista and de Boisfleury Chevance, 1997). In fibrillar threedimensional matrices, such as collagen gels, blebs can expand into pores in the matrix, causing the squeezing phenotype observed in amoeboid lymphocytes and cancer cells in collagen gels (Haston and Shields, 1984;Wolf et al., 2003a). Cells that form small blebs can use them to 'elbow' their way through the meshwork of the ECM (Friedl and Wolf, 2009). The site of bleb formation determines the direction of cell movement (Keller and Bebie, 1996). However, some cells, such as A375 melanoma cells, form multiple blebs over the membrane and would therefore not be able to move directionally. However, A375 melanoma cells are able to invade into a collagen matrix and move in vitro and in vivo (Sahai and Marshall, 2003;Sanz-Moreno et al., 2008). As asymmetry is a prerequisite of cell movement (Petrie et al., 2009), this suggests some form of cellular polarisation. To understand cell movement driven by multiple blebs, we investigated polarisation in blebbing 'amoeboid' A375 melanoma cells. We show that A375 cells form an ezrin-rich uropod-like structure (which we call the ERULS) at their cell rear, and that this is required for invasion into a three-dimensional matrix. We present a model whereby accumulation of ezrin, membrane and actin in this structure reflects strong connections between the plasma membrane and the submembrane cortex, leading to the formation of a rigid structure that inhibits blebbing at the back of the cell. This leads to net movement of the cell in the direction opposite to the uropod-like structure. Our model explains how cells can move ...