Jacky G. Goetz

Endothelial Cilia Mediate Low Flow Sensing during Zebrafish Vascular Development

VIDEO ABSTRACT The pattern of blood flow has long been thought to play a significant role in vascular morphogenesis, yet the flow-sensing mechanism that is involved at early embryonic stages, when flow forces are low, remains unclear. It has been proposed that endothelial cells use primary cilia to sense flow, but this has never been tested in vivo. Here we show, by noninvasive, high-resolution imaging of live zebrafish embryos, that endothelial cilia progressively deflect at the onset of blood flow and that the deflection angle correlates with calcium levels in endothelial cells. We demonstrate that alterations in shear stress, ciliogenesis, or expression of the calcium channel PKD2 impair the endothelial calcium level and both increase and perturb vascular morphogenesis. Altogether, these results demonstrate that endothelial cilia constitute a highly sensitive structure that permits the detection of low shear forces during vascular morphogenesis.

Fluid flows and forces in development: functions, features and biophysical principles

Throughout morphogenesis, cells experience intracellular tensile and contractile forces on microscopic scales. Cells also experience extracellular forces, such as static forces mediated by the extracellular matrix and forces resulting from microscopic fluid flow. Although the biological ramifications of static forces have received much attention, little is known about the roles of fluid flows and forces during embryogenesis. Here, we focus on the microfluidic forces generated by cilia-driven fluid flow and heart-driven hemodynamics, as well as on the signaling pathways involved in flow sensing. We discuss recent studies that describe the functions and the biomechanical features of these fluid flows. These insights suggest that biological flow determines many aspects of cell behavior and identity through a specific set of physical stimuli and signaling pathways.

Fast and precise targeting of single tumor cells in vivo by multimodal correlative microscopy

ABSTRACT Intravital microscopy provides dynamic understanding of multiple cell biological processes, but its limited resolution has so far precluded structural analysis. Because it is difficult to capture rare and transient events, only a few attempts have been made to observe specific developmental and pathological processes in animal models using electron microscopy. The multimodal correlative approach that we propose here combines intravital microscopy, microscopic X-ray computed tomography and three-dimensional electron microscopy. It enables a rapid (c.a. 2 weeks) and accurate (<5 µm) correlation of functional imaging to ultrastructural analysis of single cells in a relevant context. We demonstrate the power of our approach by capturing single tumor cells in the vasculature of the cerebral cortex and in subcutaneous tumors, providing unique insights into metastatic events. Providing a significantly improved throughput, our workflow enables multiple sampling, a prerequisite for making correlative imaging a relevant tool to study cell biology in vivo. Owing to the versatility of this workflow, we envision broad applications in various fields of biological research, such as cancer or developmental biology. Highlighted Article: We provide here a novel correlative workflow combining intravital microscopy, microCT and 3D electron microscopy to reveal metastatic events in mouse brain and skin tissue at high resolution.

Intravital Correlative Microscopy: Imaging Life at the Nanoscale.

Studying key biological events within complex model systems relies on dynamic and functional imaging at optimum spatial and temporal resolutions. Intravital correlative light and electron microscopy (intravital CLEM) combines imaging living multicellular model systems with electron microscopy, and offers full ultrastructural details of dynamic or transient events in vivo. However, routine use of intravital CLEM is hindered by multiple technological challenges faced when targeting a micron-size object (e.g., single cells or organelles) in a complex living organism. Recently, various approaches have been developed to overcome these limitations. In this review we outline the current methods and present the power of intravital CLEM in different fields of research. Finally, we describe approaches that will make intravital CLEM a routine, quantitative method for high-resolution cell biology in vivo.

Correlating intravital multi-photon microscopy to 3D electron microscopy of invading tumor cells using anatomical reference points.

Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.

Metastasis of circulating tumor cells: Favorable soil or suitable biomechanics, or both?

Metastasis is the end product of a multistep process where cancer cells disseminate and home themselves in distant organs. Tumor cell extravasation is a rare, inefficient and transient event in nature and makes its studies very difficult. Noteworthy, little is known about how cancer cells arrest, adhere and pass through the endothelium of capillaries. Moreover, the key events driving metastatic growth in specific organs are not well understood. Thus, although metastasis is the leading cause of cancer-related death, how cancer cells acquire their abilities to colonize distant organs and why they do so in specific locations remain central questions in the understanding of this deadly disease. In this review, we would like to confront 2 concepts explaining the efficiency and location of metastatic secondary tumors. While the “seed and soil” hypothesis states that metastasis occurs at sites where the local microenvironment is favorable, the “mechanical” concept argues that metastatic seeding occurs at sites of optimal flow patterns. In addition, recent evidence suggests that the primary event driving tumor cell arrest before extravasation is mostly controlled by blood circulation patterns as well as mechanical cues during the process of extravasation. In conclusion, the organ tropism displayed by cancer cells during metastatic colonization is a multi-step process, which is regulated by the delivery and survival of circulating tumor cells (CTCs) through blood circulation, the ability of these CTCs to adhere and cross the physical barrier imposed by the endothelium and finally by the suitability of the soil to favor growth of secondary tumors.

Seeing is believing: multi-scale spatio-temporal imaging towards in vivo cell biology.

ABSTRACT Life is driven by a set of biological events that are naturally dynamic and tightly orchestrated from the single molecule to entire organisms. Although biochemistry and molecular biology have been essential in deciphering signaling at a cellular and organismal level, biological imaging has been instrumental for unraveling life processes across multiple scales. Imaging methods have considerably improved over the past decades and now allow to grasp the inner workings of proteins, organelles, cells, organs and whole organisms. Not only do they allow us to visualize these events in their most-relevant context but also to accurately quantify underlying biomechanical features and, so, provide essential information for their understanding. In this Commentary, we review a palette of imaging (and biophysical) methods that are available to the scientific community for elucidating a wide array of biological events. We cover the most-recent developments in intravital imaging, light-sheet microscopy, super-resolution imaging, and correlative light and electron microscopy. In addition, we illustrate how these technologies have led to important insights in cell biology, from the molecular to the whole-organism resolution. Altogether, this review offers a snapshot of the current and state-of-the-art imaging methods that will contribute to the understanding of life and disease. Summary: This Commentary provides an overview of the imaging technologies currently available for performing cell biology in vivo.

The microenvironment controls invadosome plasticity

ABSTRACT Invadosomes are actin-based structures involved in extracellular matrix degradation. Invadosomes is a term that includes podosomes and invadopodia, which decorate normal and tumour cells, respectively. They are mainly organised into dots or rosettes, and podosomes and invadopodia are often compared and contrasted. Various internal or external stimuli have been shown to induce their formation and/or activity. In this Commentary, we address the impact of the microenvironment and the role of matrix receptors on the formation, and dynamic and degradative activities of invadosomes. In particular, we highlight recent findings regarding the role of type I collagen fibrils in inducing the formation of a new linear organisation of invadosomes. We will also discuss invadosome plasticity more generally and emphasise its physio-pathological relevance. Summary: This Commentary defines invadosomes and illustrates their plasticity, which depends on various factors, including the matrix microenvironment.

Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity.

The ability of cellular fibronectin, found in the vessel wall in a fibrillar conformation, to regulate platelet functions and trigger thrombus formation remains largely unknown. In this study, we evaluated how parietal cellular fibronectin can modulate platelet responses under flow conditions. A fibrillar network was formed by mechanically stretching immobilised dimeric cellular fibronectin. Perfusion of anticoagulated whole blood over this surface resulted in efficient platelet adhesion and thrombus growth. The initial steps of platelet adhesion and activation, as evidenced by filopodia extension and an increase in intracellular calcium levels (419 ± 29 nmol/l), were dependent on integrins α5β1 and αIIbβ3. Subsequent thrombus growth was mediated by these integrins together with the GPIb-V-IX complex, GPVI and Toll-like receptor 4. The involvement of Toll-like receptor 4 could be conveyed via its binding to the EDA region of cellular fibronectin. Upon thrombus formation, the platelets became procoagulant and generated fibrin as revealed by video-microscopy. This work provides evidence that fibrillar cellular fibronectin is a strong thrombogenic surface which supports efficient platelet adhesion, activation, aggregation and procoagulant activity through the interplay of a series of receptors including integrins α5β1 and αIIbβ3, the GPIb-V-IX complex, GPVI and Toll-like receptor 4.

Integrity of lipid nanocarriers in bloodstream and tumor quantified by near-infrared ratiometric FRET imaging in living mice

Lipid nanocarriers are considered as promising candidates for drug delivery and cancer targeting because of their low toxicity, biodegradability and capacity to encapsulate drugs and/or contrasting agents. However, their biomedical applications are currently limited because of a poor understanding of their integrity in vivo. To address this problem, we report on fluorescent nano-emulsion droplets of 100 nm size encapsulating lipophilic near-infrared cyanine 5.5 and 7.5 dyes with a help of bulky hydrophobic counterion tetraphenylborate. Excellent brightness and efficient Förster Resonance Energy Transfer (FRET) inside lipid NCs enabled for the first time quantitative fluorescence ratiometric imaging of NCs integrity directly in the blood circulation, liver and tumor xenografts of living mice using a whole-animal imaging set-up. This unique methodology revealed that the integrity of our FRET NCs in the blood circulation of healthy mice is preserved at 93% at 6 h of post-administration, while it drops to 66% in the liver (half-life is 8.2 h). Moreover, these NCs show fast and efficient accumulation in tumors, where they enter in nearly intact form (77% integrity at 2 h) before losing their integrity to 40% at 6 h (half-life is 4.4 h). Thus, we propose a simple and robust methodology based on ratiometric FRET imaging in vivo to evaluate quantitatively nanocarrier integrity in small animals. We also demonstrate that nano-emulsion droplets are remarkably stable nano-objects that remain nearly intact in the blood circulation and release their content mainly after entering tumors.