Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness‐induced CCN1 activates β‐catenin nuclear translocation and signaling and that this contributes to upregulate N‐cadherin levels on the surface of the endothelium, in vitro. This facilitates N‐cadherin‐dependent cancer cell–endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness‐induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis

Lung marginated and splenic murine resident neutrophils constitute pioneers in tissue-defense during systemic E. coli challenge

The rapid response of neutrophils throughout the body to a systemic challenge is a critical first step in resolution of bacterial infection such as Escherichia coli (E. coli). Here we delineated the dynamics of this response, revealing novel insights into the molecular mechanisms using lung and spleen intravital microscopy and 3D ex vivo culture of living precision cut splenic slices in combination with fluorescent labelling of endogenous leukocytes. Within seconds after challenge, intravascular marginated neutrophils and lung endothelial cells (ECs) work cooperatively to capture pathogens. Neutrophils retained on lung ECs slow their velocity and aggregate in clusters that enlarge as circulating neutrophils carrying E. coli stop within the microvasculature. The absolute number of splenic neutrophils does not change following challenge; however, neutrophils increase their velocity, migrate to the marginal zone (MZ) and form clusters. Irrespective of their location all neutrophils capturing heat-inactivated E. coli take on an activated phenotype showing increasing surface CD11b. At a molecular level we show that neutralization of ICAM-1 results in splenic neutrophil redistribution to the MZ under homeostasis. Following challenge, splenic levels of CXCL12 and ICAM-1 are reduced allowing neutrophils to migrate to the MZ in a CD29-integrin dependent manner, where the enlargement of splenic neutrophil clusters is CXCR2-CXCL2 dependent. We show directly molecular mechanisms that allow tissue resident neutrophils to provide the first lines of antimicrobial defense by capturing circulating E. coli and forming clusters both in the microvessels of the lung and in the parenchyma of the spleen

Multimodal decoding of human liver regeneration.

Acknowledgements: This work was supported by a Wellcome Trust Senior Research Fellowship in Clinical Science (ref. 219542/Z/19/Z) to N.C.H., a Chan Zuckerberg Initiative Seed Network Grant to N.C.H., and a Tenovus Scotland grant (E20-03) to K.P.M., S. J. Wallace and N.C.H. J.R.P. was supported by a Medical Research Council Precision Medicine PhD studentship. C.A.K. was a cross-disciplinary post-doctoral fellow (XDF) supported by funding from the University of Edinburgh and Medical Research Council (MC_UU_00009/2). T.G.B. was funded by the Wellcome Trust (ref. WT107492Z). S.M. was funded by Cancer Research UK core funding to the CRUK Beatson Institute (refs. A17196 and A31287). J.B.G.M. was supported by a CRUK programme grant (A23390). F.F., J.B.G.M. and L.M.C. were supported by CRUK core funding to the Beatson Institute (A31287) and CRUK core funding to L.M.C. (A23983; DRCRPG-Nov22/100007). D.J.M. was supported by a Medical Research Council Senior Clinical Fellowship (MR/P008887/1). P.R. was supported by a Medical Research Council Clinician Scientist Fellowship (MR/N008340/1) and Medical Research Council Senior Clinical Fellowship (MR/W015919/1). M.M.S. was supported by a Peter Samuel Fellowship. We thank the patients who donated liver tissue for this study; J. Davidson, J. Black, C. Ibbotson and A. Baird of the Scottish Liver Transplant Unit and the research nurses of the Wellcome Trust Clinical Research Facility for assistance with consenting patients for this study; the liver transplant coordinators and surgeons of the Scottish Liver Transplant Unit and the surgeons and staff of the Hepatobiliary Surgical Unit, Royal Infirmary of Edinburgh for assistance in procuring human liver samples; the US ALFSG network for assistance in procuring human liver samples; core facilities and services at the Beatson Institute, in particular the Biological Research Unit & the Beatson Advanced Imaging Resource (BAIR); G. Jacquemet for advice on Cellpose segmentation for IVM analysis; R. Insall and L. Machesky for helpful scientific discussions; W. Mungall for technical support; C. Nicol for help with manuscript illustrations; N. Pham for technical assistance with Incucyte; and C. Winchester and R. Li for critical reading of the manuscript. We acknowledge the contribution to this study made by the University of Birmingham’s Human Biomaterials Resource Centre, which has been supported through Birmingham Science City–Experimental Medicine Network of Excellence project. This research was funded in whole, or in part, by the Wellcome Trust (Wellcome Trust Senior Research Fellowship in Clinical Science to N.C.H.; ref. 219542/Z/19/Z). This publication is part of the Human Cell Atlas (www.humancellatlas.org/publications).The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine