Macrophages abundantly found in the tumor microenvironment enhance malignancy1. At metastatic sites a distinct population of metastasis associated macrophages (MAMs) promote tumor cell extravasation, seeding and persistent growth2. Our study has defined the origin of these macrophages by showing Gr1+ inflammatory monocytes (IMs) are preferentially recruited to pulmonary metastases but not primary mammary tumors, a process also found for human IMs in pulmonary metastases of human breast cancer cells. The recruitment of these CCR2 (receptor for chemokine CCL2) expressing IMs and subsequently MAMs and their interaction with metastasizing tumor cells is dependent on tumor and stromal synthesized CCL2 (FigS1). Inhibition of CCL2/CCR2 signaling using anti-CCL2 antibodies blocks IM recruitment and inhibits metastasis in vivo and prolongs the survival of tumor-bearing mice. Depletion of tumor cell-derived CCL2 also inhibits metastatic seeding. IMs promote tumor cell extravasation in a process that requires monocyte-derived VEGF. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer (Fig S2)3-6. Our data provides the mechanistic link between these two clinical associations and indicates new therapeutic targets for treating metastatic breast disease.

The ability of non-invasive monitoring of deep-tissue developmental, metabolic, and pathogenic processes will advance modern biotechnology. Imaging of live mammals using fluorescent probes is more feasible within a “near-infrared optical window” (NIRW)1. Here we report a phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm. Bright fluorescence in a living mouse proved iRFP to be a superior probe for non-invasive imaging of internal mammalian tissues. Its high intracellular stability, low cytotoxicity, and lack of the requirement to add external biliverdin-chromophore makes iRFP as easy to use as conventional GFP-like proteins. Compared to earlier phytochrome-derived fluorescent probes, the iRFP protein has better in vitro characteristics and performs well in cells and in vivo, having greater effective brightness and photostability. Compared to the far-red GFP-like proteins, iRFP has substantially higher signal to background ratio in a mouse model owing to its infra-red shifted spectra.

SUMMARY We utilized a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a novel rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic background such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which were converted into blue probes. Further improvement of a blue variant of TagRFP using random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescence proteins with a histidine in the chromophore. Detailed biochemical and photochemical analysis indicates mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging as well as the outstanding donor for green fluorescent proteins in FRET applications.

WNT/β-catenin signalling is crucial for intestinal homoeostasis. The intestinal epithelium and stroma are the major source of WNT ligands but their origin and role in intestinal stem cell (ISC) and epithelial repair remains unknown. Macrophages are a major constituent of the intestinal stroma. Here, we analyse the role of macrophage-derived WNT in intestinal repair in mice by inhibiting their release using a macrophage-restricted ablation of Porcupine, a gene essential for WNT synthesis. Such Porcn-depleted mice have normal intestinal morphology but are hypersensitive to radiation injury in the intestine compared with wild-type (WT) littermates. Porcn-null mice are rescued from radiation lethality by treatment with WT but not Porcn-null bone marrow macrophage-conditioned medium (CM). Depletion of extracellular vesicles (EV) from the macrophage CM removes WNT function and its ability to rescue ISCs from radiation lethality. Therefore macrophage-derived EV-packaged WNTs are essential for regenerative response of intestine against radiation.

To produce blood platelets, megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P 2 , a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P 2 in the DMS occurs in proximity to PI-5-P-4-kinase ␣ (PIP4K␣), and short hairpin (sh) RNAmediated loss of PIP4K␣ impairs both DMS development and expansion of megakaryocyte size. Thus, PI-4,5-P 2 is a marker and possibly essential component of internal membranes. PI-4,5-P 2 is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P 2 in the megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P 2 might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis. IntroductionMammals synthesize blood platelets as functional cell particles within a precursor cell, the megakaryocyte (MK). Terminally differentiated MKs acquire a significantly polyploid DNA content and enlarge to 50 to 100 m in diameter before releasing their platelet load. In the release phase, the MK cytoplasm converts into long branched protrusions, and disc-shaped platelets are assembled de novo within these proplatelet extensions. 1 Microtubule bundles form the core of each proplatelet, and their distal tips organize into the repetitively coiled structure of the platelet marginal band. [1][2][3] The actin cytoskeleton also participates actively in thrombopoiesis, judged mainly by the effects of inhibitors of actin polymerization on proplatelet morphology. 1,[4][5][6] Although these studies reveal cytoskeletal aspects of thrombopoiesis, little is known about the corresponding regulation of nascent platelet membranes or how membrane and cytoskeletal morphogenesis are coordinated.The conversion of a single MK into thousands of platelets is accompanied by a large increase in total surface area. MK ultrastructure reveals an abundant pool of cytoplasmic membranes that constitute the demarcation membrane system (DMS). By virtue of its origin in tubular invaginations of the plasma membrane, the DMS maintains continuity with the extracellular space, 7,8 and whole-cell patch-clamp recordings reveal the DMS to be a single electrophysiologic entity. 9 DMS functions, however, remain controversial, and its name recalls early theories on platelet biogenesis. Prior to proplatelet-based models of thrombopoiesis, nascent platele...