Platelets favor the outgrowth of established metastases.

Despite abundant evidence demonstrating that platelets foster metastasis, anti-platelet agents have low therapeutic potential due to the risk of hemorrhages. In addition, whether platelets can regulate metastasis at the late stages of the disease remains unknown. In this study, we subject syngeneic models of metastasis to various thrombocytopenic regimes to show that platelets provide a biphasic contribution to metastasis. While potent intravascular binding of platelets to tumor cells efficiently promotes metastasis, platelets further support the outgrowth of established metastases via immune suppression. Genetic depletion and pharmacological targeting of the glycoprotein VI (GPVI) platelet-specific receptor in humanized mouse models efficiently reduce the growth of established metastases, independently of active platelet binding to tumor cells in the bloodstream. Our study demonstrates therapeutic efficacy when targeting animals bearing growing metastases. It further identifies GPVI as a molecular target whose inhibition can impair metastasis without inducing collateral hemostatic perturbations.

Evidence and therapeutic implications of biomechanically regulated immunosurveillance in cancer and other diseases.

Disease progression is usually accompanied by changes in the biochemical composition of cells and tissues and their biophysical properties. For instance, hallmarks of cancer include the stiffening of tissues caused by extracellular matrix remodelling and the softening of individual cancer cells. In this context, accumulating evidence has shown that immune cells sense and respond to mechanical signals from the environment. However, the mechanisms regulating these mechanical aspects of immune surveillance remain partially understood. The growing appreciation for the 'mechano-immunology' field has urged researchers to investigate how immune cells sense and respond to mechanical cues in various disease settings, paving the way for the development of novel engineering strategies that aim at mechanically modulating and potentiating immune cells for enhanced immunotherapies. Recent pioneer developments in this direction have laid the foundations for leveraging 'mechanical immunoengineering' strategies to treat various diseases. This Review first outlines the mechanical changes occurring during pathological progression in several diseases, including cancer, fibrosis and infection. We next highlight the mechanosensitive nature of immune cells and how mechanical forces govern the immune responses in different diseases. Finally, we discuss how targeting the biomechanical features of the disease milieu and immune cells is a promising strategy for manipulating therapeutic outcomes.

Organelle morphology and positioning orchestrate physiological and disease-associated processes.

In cells, organelles are distributed nonrandomly to regulate cells' physiological and disease-associated processes. Based on their morphology, position within the cell, and contacts with other organelles, they exert different biological functions. Endo-lysosomes are critical cell metabolism and nutrient-sensing regulators modulating cell growth and cellular adaptation in response to nutrient availability. Their spatial distribution is intimately linked to their function. In this review, we will discuss the role of endolysosomes under physiological conditions and in the context of cancer progression, with a special focus on their morphology, the molecular mechanisms determining their subcellular position, and the contacts they form with other organelles. We aim to highlight the relationship between cell architecture and cell function and its impact on maintaining organismal homeostasis.

Blood flow diverts extracellular vesicles from endothelial degradative compartments to promote angiogenesis.

Extracellular vesicles released by tumors (tEVs) disseminate via circulatory networks and promote microenvironmental changes in distant organs favoring metastatic seeding. Despite their abundance in the bloodstream, how hemodynamics affect the function of circulating tEVs remains unsolved. We demonstrated that efficient uptake of tEVs occurs in venous endothelial cells that are subjected to hemodynamics. Low flow regimes observed in veins partially reroute internalized tEVs toward non-acidic and non-degradative Rab14-positive endosomes, at the expense of lysosomes, suggesting that endothelial mechanosensing diverts tEVs from degradation. Subsequently, tEVs promote the expression of pro-angiogenic transcription factors in low flow-stimulated endothelial cells and favor vessel sprouting in zebrafish. Altogether, we demonstrate that low flow regimes potentiate the pro-tumoral function of circulating tEVs by promoting their uptake and rerouting their trafficking. We propose that tEVs contribute to pre-metastatic niche formation by exploiting endothelial mechanosensing in specific vascular regions with permissive hemodynamics.

Active Remodeling of Capillary Endothelium via Cancer Cell-Derived MMP9 Promotes Metastatic Brain Colonization.

Crossing the blood-brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it. SIGNIFICANCE: Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell-derived MMP9, which might enable the development of approaches to prevent brain metastasis. See related commentary by McCarty, p. 1167.

Inhibition of the pseudokinase MLKL alters extracellular vesicle release and reduces tumor growth in glioblastoma.

Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells.

Circulating extracellular vesicles and tumor cells: sticky partners in metastasis.

The intravascular behavior of tumor-derived extracellular vesicles (EVs) and circulating tumor cells (CTCs) lies at the heart of the metastatic cascade. Their capacity to disseminate and stop at specific vascular regions precedes and determines the formation of metastatic foci. We discuss in detail the central role of cellular adhesion molecules (CAMs) that are present on EV/CTC surface, as well as their endothelial ligands, in dictating their arrest site and their capacity to exit the vasculature. We focus on the differences and similarities between CAMs on CTCs and EVs, and speculate about their role in the organotropism of different cancer types. Better understanding of the binding mechanisms might pinpoint potential targets for novel therapies.

Liquid Biopsies: Flowing Biomarkers.

Metastatic dissemination accounts for most of the death in patients during cancer progression. There is thus an urge to identify specific biomarkers as proxies for cancer progression and assessment of treatment efficiency. Cancer is a systemic disease involving the shuttling of tumor cells and tumor secreted factors to distant organs, mostly via biofluids. During this transfer, these factors are accessible for easy sampling and therefore constitute a unique source of information witnessing the presence and the evolution of the disease. Hence, liquid biopsies offer multiple advantages, including simple and low-invasive sampling procedures, low cost, and higher compliance. Importantly, liquid biopsies are adapted to personalized medicine allowing a longitudinal follow-up to monitor treatment efficiency or resistance, and risk of relapse.The evolution of methodologies to isolate circulating tumor cells (CTCs) and extracellular vesicles (EVs) from blood samples associated with the characterization of their membrane surface repertoire and content have been instrumental in the emergence of liquid biopsies as an easy and non-invasive alternative as opposed to classical surgery-mediated tumor biopsies.In this chapter, we comment on CTCs and EVs carrying features with great potential as cancer biomarkers. More specifically, we focus on the adhesive and mechanical properties of CTCs as metastatic markers. We also consider the recent development of EVs isolation methods and the identification of new biomarkers. Finally, we discuss their relevance as cancer prognosis tools.

Circulating tumor cells: Towards mechanical phenotyping of metastasis.

During cancer progression, metastatic dissemination accounts for ∼90% of death in patients. Metastasis occurs upon dissemination of circulating tumor cells (CTC) through body fluids, in particular the bloodstream, and several key steps remain elusive. Although the majority of CTCs travel as single cells, they can form clusters either with themselves (homoclusters) or with other circulating cells (heteroclusters) and thereby increase their metastatic potential. In addition, cancer cell mechanics and mechanical cues from the microenvironment are important factors during metastatic progression. Recent progress in intravital imaging technologies, biophysical methods, and microfluidic-based isolation of CTCs allow now to probe mechanics at single cell resolution while shedding light on key steps of the hematogenous metastatic cascade. In this review, we discuss the importance of CTC mechanics and their correlation with metastatic success and how such development could lead to the identification of therapeutically relevant targets.

Optimal Physicochemical Properties of Antibody-Nanoparticle Conjugates for Improved Tumor Targeting.

Tumor-targeted antibody (mAb)/fragment-conjugated nanoparticles (NPs) represent an innovative strategy for improving the local delivery of small molecules. However, the physicochemical properties of full mAb-NPs and fragment-NPs-that is, NP material, size, charge, as well as the targeting antibody moiety, and the linker conjugation strategies-remain to be optimized to achieve an efficient tumor targeting. A meta-analysis of 161 peer-reviewed studies is presented, which describes the use of tumor-targeted mAb-NPs and fragment-NPs from 2009 to 2021. The use of these targeted NPs is confirmed to result in significantly greater tumor uptake of NPs than that of naked NPs (7.9 ± 1.9% ID g-1 versus 3.2 ± 0.6% ID g-1 , respectively). The study further demonstrates that for lipidic NPs, fragment-NPs provide a significantly higher tumor uptake than full mAb-NPs. In parallel, for both polymeric and organic/inorganic NPs, full mAb-NPs yield a significant higher tumor uptake than fragment-NPs. In addition, for both lipidic and polymeric NPs, the tumor uptake is improved with the smallest sizes of the conjugates. Finally, the pharmacokinetics of the conjugates are demonstrated to be driven by the NPs and not by the antibody moieties, independently of using full mAb-NPs or fragment-NPs, confirming the importance of optimizing the NP design to improve the tumor uptake.

Tumor extracellular vesicles drive metastasis (it's a long way from home).

Among a plethora of functions, extracellular vesicles released by primary tumors spread in the organism and reach distant organs where they can induce the formation of a premetastatic niche. This constitutes a favorable microenvironment for circulating tumor cells which facilitates their seeding and colonization. In this review, we describe the journey of extracellular vesicles (EVs) from the primary tumor to the future metastatic organ, with a focus on the mechanisms used by EVs to target organs with a specific tropism (i.e., organotropism). We then highlight important tumor EV cargos in the context of premetastatic niche formation and summarize their known effects on extracellular matrix remodeling, angiogenesis, vessel permeabilization, resident cell activation, recruitment of foreign cells, and ultimately the formation of a pro-inflammatory and immuno-tolerant microenvironment. Finally, we discuss current experimental limitations and remaining opened questions in light of metastatic diagnosis and potential therapies targeting PMN formation.

Intravital imaging technology guides FAK-mediated priming in pancreatic cancer precision medicine according to Merlin status.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow­induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.

The power of imaging to understand extracellular vesicle biology in vivo.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

Impairing flow-mediated endothelial remodeling reduces extravasation of tumor cells.

Tumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.

Probing Intravascular Adhesion and Extravasation of Tumor Cells with Microfluidics.

Cancer metastasis is a multistep process during which tumor cells leave the primary tumor mass and form distant secondary colonies that are lethal. Circulating tumor cells (CTCs) are transported by body fluids to reach distant organs, where they will extravasate and either remain dormant or form new tumor foci. Development of methods to study the behavior of CTCs at the late stages of the intravascular journey is thus required to dissect the molecular mechanisms at play. Using recently developed microfluidics approaches, we have demonstrated that CTCs arrest intravascularly, through a two-step process: (a) CTCs stop using low energy and rapidly activated adhesion receptors to form transient metastable adhesions and (b) CTCs stabilize their adhesions to the endothelial layer with high energy and slowly activated adhesion receptors. In this methods chapter, we describe these easy-to-implement quantitative methods using commercially available microfluidic channels. We detail the use of fast live imaging combined to fine-tuned perfusion to measure the adhesion potential of CTC depending on flow velocities. We document how rapidly engaged early metastable adhesion can be discriminated from slower activated stable adhesion using microfluidics. Finally, CTC extravasation potential can be assessed within this setup using long-term cell culture under flow. Altogether, this experimental pipeline can be adapted to probe the adhesion (to the endothelial layer) and extravasation potential of any circulating cell.

Fluorescent nanocarriers targeting VCAM-1 for early detection of senescent endothelial cells.

Endothelial senescence has been identified as an early event in the development of endothelial dysfunction, a hallmark of cardiovascular disease. This study developed theranostic nanocarriers (NC) decorated with VCAM-1 antibodies (NC-VCAM-1) in order to target cell surface VCAM-1, which is overexpressed in senescent endothelial cells (ECs) for diagnostic and therapeutic purposes. Incubation of Ang II-induced premature senescent ECs or replicative senescent ECs with NC-VCAM-1 loaded with lipophilic fluorescent dyes showed higher fluorescence signals than healthy EC, which was dependent on the NC size and VCAM-1 antibodies concentration, and not observed following masking of VCAM-1. NC loaded with omega 3 polyunsaturated fatty acid (NC-EPA:DHA6:1) were more effective than native EPA:DHA 6:1 to prevent Ang II-induced VCAM-1 and p53 upregulation, and SA-ß-galactosidase activity in coronary artery segments. These theranostic NC might be of interest to evaluate the extent and localization of endothelial senescence and to prevent pro-senescent endothelial responses.

The NANOTUMOR consortium - Towards the Tumor Cell Atlas.

Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.

Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.