Yi Feng

Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation.

Live imaging and genetic studies of the initial interactions between leukocytes and transformed cells in zebrafish larvae indicate an attractant role for H2O2 and suggest that blocking these early interactions reduces expansion of transformed cell clones.

Multicomponent Reactions for de Novo Synthesis of BODIPY Probes: In Vivo Imaging of Phagocytic Macrophages

Multicomponent reactions are excellent tools to generate complex structures with broad chemical diversity and fluorescent properties. Herein we describe the adaptation of the fluorescent BODIPY scaffold to multicomponent reaction chemistry with the synthesis of BODIPY adducts with high fluorescence quantum yields and good cell permeability. From this library we identified one BODIPY derivative (PhagoGreen) as a low-pH sensing fluorescent probe that enabled imaging of phagosomal acidification in activated macrophages. The fluorescence emission of PhagoGreen was proportional to the degree of activation of macrophages and could be specifically blocked by bafilomycin A, an inhibitor of phagosomal acidification. PhagoGreen does not impair the normal functions of macrophages and can be used to image phagocytic macrophages in vivo.

Live Imaging of Tumor Initiation in Zebrafish Larvae Reveals a Trophic Role for Leukocyte-Derived PGE2

Summary Epidemiology studies and clinical trials have suggested that the use of non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, can significantly reduce the incidence of and mortality associated with many cancers [1–3], and upregulation of the COX2-PGE2 pathway in tumor microenvironments might drive several aspects of cancer progression [4–6]. For these reasons, the mechanisms linking COX blockade and cancer prevention have long been an area of active investigation [7]. During carcinogenesis, COX-2 is expressed both by malignant epithelial cells [8, 9] and by tumor-associated stromal cells, including macrophages [10–12], but the observation that NSAIDs are most effective in cancer prevention in APCmin/+ mice if the mice are treated from conception [13] suggests that the COX-2/PGE2 pathway might also be critical at the earliest stages of tumor development. In this study we take advantage of the translucency and genetic tractability of zebrafish larvae to investigate the involvement of inflammatory cells at cancer initiation, when transformed cells first arise in tissues. We previously showed that innate immune cells supply early transformed cells with proliferative cues [14] and, by using complementary pharmacological and genetic experiments, we now show that prostaglandin E2 (PGE2) is the trophic signal required for this expansion of transformed cells. Our in vivo observations at these early stages of cancer initiation provide a potential mechanistic explanation for why long-term use of low doses of NSAIDs, including aspirin, might reduce cancer onset.

The wound inflammatory response exacerbates growth of pre-neoplastic cells and progression to cancer

There is a long‐standing association between wound healing and cancer, with cancer often described as a “wound that does not heal”. However, little is known about how wounding, such as following surgery, biopsy collection or ulceration, might impact on cancer progression. Here, we use a translucent zebrafish larval model of RasG12V‐driven neoplasia to image the interactions between inflammatory cells drawn to a wound, and to adjacent pre‐neoplastic cells. We show that neutrophils are rapidly diverted from a wound to pre‐neoplastic cells and these interactions lead to increased proliferation of the pre‐neoplastic cells. One of the wound‐inflammation‐induced trophic signals is prostaglandin E2 (PGE2). In an adult model of chronic wounding in zebrafish, we show that repeated wounding with subsequent inflammation leads to a greater incidence of local melanoma formation. Our zebrafish studies led us to investigate the innate immune cell associations in ulcerated melanomas in human patients. We find a strong correlation between neutrophil presence at sites of melanoma ulceration and cell proliferation at these sites, which is associated with poor prognostic outcome.

Inflammation drives wound hyperpigmentation in zebrafish by recruiting pigment cells to sites of tissue damage

SUMMARY In humans, skin is the largest organ and serves as a barrier between our body and the outside world. Skin protects our internal organs from external pathogens and other contaminants, and melanocytes within the skin protect the body from damage by ultraviolet light. These same pigment cells also determine our skin colour and complexion. Skin wounding triggers a repair response that includes a robust recruitment of inflammatory cells, which function to kill invading microbes and clear away cell and matrix debris. Once at the wound site, these innate immune cells release a barrage of cytokines that direct the activities of other cells during the repair process. Tissue damage and repair also frequently lead to alterations in skin pigmentation, in particular to wound hyperpigmentation. In this study, we describe a model of wound hyperpigmentation in the translucent zebrafish larva, where we can live-image the recruitment of melanocytes and their precursors, melanoblasts, to the wound site. We show that these pigment cells are drawn in after the initial recruitment of innate immune cells and that the inflammatory response is essential for wound hyperpigmentation. This new model will allow us to uncover the molecular link between immune and pigment cells during tissue repair and to screen for potential therapeutics to dampen wound hyperpigmentation.

Imaging innate immune responses at tumour initiation: new insights from fish and flies

Recent imaging studies in genetically tractable and translucent zebrafish and Drosophila melanogaster models have opened a window on the earliest stages of tumorigenesis, when pre-neoplastic cells first arise in tissues before they progress into full-blown cancers. Innate immune cells often find these cells soon after they develop, but this efficient surveillance is not always good for the host because although immune cells have phagocytic capacity, they can also nurture the growing clones of pre-neoplastic cells. We describe these newly observed early interactions between immune cells and cancer cells and speculate on their potential clinical implications.

Pivotal role of hmx2 and hmx3 in zebrafish inner ear and lateral line development.

hmx2 (nkx5.2) and hmx3 (nkx5.1) are highly conserved homeobox transcription factors required for mouse inner ear development. We have identified four hmx genes that are expressed in developing mechanosensory organs in zebrafish. Knockdown of both hmx2 and hmx3 disrupts formation of the mechanosensory neuromasts and also leads to impaired vestibular function in which utricular maculae fail to develop and the utricular otolith gradually fuses with the saccular otolith. We demonstrate that pax5, known to be required for development of the utricular maculae, is expressed downstream of hmx2 and hmx3. In addition, we show that FGF signaling regulates expression of hmx2 and hmx3 in the otic vesicle, and conversely, hmx2 and hmx3 maintain the expression of fgf ligands, thus revealing a novel tissue-specific feedback mechanism. Our data suggest that hmx2 and hmx3 act as cell autonomous factors required redundantly for cell fate specification and differentiation during inner ear and lateral line development.

Stimulation of hepatocarcinogenesis by neutrophils upon induction of oncogenic kras expression in transgenic zebrafish

Background & Aims Chronic inflammation is a major etiological factor for hepatocellular carcinoma (HCC), but how immune cells respond in the initiation of hepatocarcinogenesis remains uncharacterized. This study aims to investigate the response and roles of neutrophils in early hepatocarcinogenesis. Methods By inducible expression of oncogenic krasV12 in hepatocytes in transgenic zebrafish combined with live imaging of neutrophils in transparent larvae, the response of neutrophils to oncogenic liver was characterized and their roles investigated by pharmaceutical and genetic manipulations. Results We found a rapid recruitment of neutrophils to the liver upon induction of krasV12 expression. Pharmaceutical stimulation of neutrophils resulted in further increases of neutrophils in oncogenic livers, liver size and tumor severity, while inhibition of neutrophils caused decreases of liver-associated neutrophils and liver size. Time-lapse video indicated that neutrophils had a stagnant migratory pattern meandering along the tumor edge but became relatively stationary upon entering the krasV12-expressing liver. Both oncogenic hepatocytes and tumor-associated neutrophils (TANs) were isolated via fluorescence-activated cell sorting. Molecular analyses indicated a pro-inflammatory microenvironment, as marked by increased tgfβ1a expression in krasV12-expressing hepatocytes and a loss of anti-tumor activities in TANs. Depletion of Tgf-β significantly reduced the number of TANs and the size of oncogenic liver. Conclusions An inflammatory cue from oncogenic hepatocytes upon induction of krasV12 expression causes a rapid recruitment of neutrophils to oncogenic liver and the neutrophils play a promoting role in early hepatocarcinogenesis.

Endostatin promotes the anabolic program of rabbit chondrocyte

ABSTRACTEndostatin is a natural occurred angiogenesis inhibitor derived from collagenXVIII. So far its function during the angiogenesis process of bone formation and arthropathy has not been well studied yet. The present study addresses the function of endostatin in rabbit articular chondrocytes (RAC). We found that endostatin can promote RAC adhesion and spreading as well as its proliferation. In monolayer cultured RAC, CollagenII, TIMP1 and collagenXVIII transcription were up regulated by endostatin while collagenI and MMP9 were down regulated. Moreover collagenXVIII and endostatin antigens are present at synovial fluid. These findings indicate new function of endostatin as a homeostatic factor in cartilage metabolism.

Inflammation: Wound healing in zebrafish.

What is the first signal that directs the rapid influx of immune cells to a wound to stave off potential infection? A study in the zebrafish reveals an unusual but well-qualified candidate. A study of the role of hydrogen peroxide (H2O2) during the early events of wound responses in zebrafish larvae — a popular vertebrate model for inflammatory and regenerative responses to wounds — reveals a that tissue gradient of H2O2 forms in response to injury of the zebrafish tail fin. The gradient, made visible by the use of a genetically encoded fluorescent sensor protein, is created by the activity of dual oxidase (DUOX) and acts to attract leukocytes to the wound margin during the initial phase of inflammation. This is the first report of a role for H2O2 in signalling to leukocytes, in addition to its established role as an antiseptic.