RESUMO
Transmigration of circulating monocytes from the bloodstream to tissues represents an early hallmark of inflammation. This process plays a pivotal role during viral neuroinvasion, encephalitis, and HIV-associated neurocognitive disorders. How monocytes locally unzip endothelial tight junction-associated proteins (TJAPs), without perturbing impermeability, to reach the central nervous system remains poorly understood. Here, we show that human circulating monocytes express the TJAP Occludin (OCLN) to promote transmigration through endothelial cells. We found that human monocytic OCLN (hmOCLN) clusters at monocyte-endothelium interface, while modulation of hmOCLN expression significantly impacts monocyte transmigration. Furthermore, we designed OCLN-derived peptides targeting its extracellular loops (EL) and show that transmigration of treated monocytes is inhibited in vitro and in zebrafish embryos, while preserving vascular integrity. Monocyte transmigration toward the brain is an important process for HIV neuroinvasion and we found that the OCLN-derived peptides significantly inhibit HIV dissemination to cerebral organoids. In conclusion, our study identifies an important role for monocytic OCLN during transmigration and provides a proof-of-concept for the development of mitigation strategies to prevent monocyte infiltration and viral neuroinvasion.
Assuntos
Células Endoteliais , Monócitos , Ocludina , Migração Transendotelial e Transepitelial , Peixe-Zebra , Ocludina/metabolismo , Ocludina/genética , Humanos , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/virologia , Animais , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Células Endoteliais/efeitos dos fármacos , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , HIV-1/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/metabolismo , Encéfalo/metabolismo , Encéfalo/virologiaRESUMO
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.
Assuntos
Plaquetas , Metástase Neoplásica , Glicoproteínas da Membrana de Plaquetas , Animais , Plaquetas/metabolismo , Plaquetas/efeitos dos fármacos , Humanos , Camundongos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/genética , Linhagem Celular Tumoral , Feminino , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Vesículas Extracelulares , Células Neoplásicas Circulantes , Moléculas de Adesão Celular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Ligantes , Células Neoplásicas Circulantes/patologiaRESUMO
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.
Assuntos
Vesículas Extracelulares , Células Neoplásicas Circulantes , Biomarcadores Tumorais , Vesículas Extracelulares/química , Humanos , Biópsia Líquida , Recidiva Local de Neoplasia , Células Neoplásicas Circulantes/patologiaRESUMO
During cancer progression, metastatic dissemination accounts for â¼90% 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.
RESUMO
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.
Assuntos
Endotélio Vascular/fisiologia , Hemorreologia , Migração Transendotelial e Transepitelial , Animais , Animais Geneticamente Modificados , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/fisiologia , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Microscopia Intravital , Microfluídica , Microscopia Confocal , Células Neoplásicas Circulantes , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Transdução de Sinais/fisiologia , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra/embriologiaRESUMO
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.
Assuntos
Adesão Celular , Ensaios de Migração Celular/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/metabolismo , Migração Transendotelial e Transepitelial , Animais , Linhagem Celular Tumoral , Ensaios de Migração Celular/instrumentação , Humanos , Microfluídica/instrumentaçãoRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Exossomos/patologia , GTP Fosfo-Hidrolases/metabolismo , Metástase Neoplásica/genética , Animais , Neoplasias da Mama/secundário , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Corpos Multivesiculares/fisiologia , Peixe-ZebraRESUMO
Clustering of tumor cells is known to grant superior metastatic efficiency compared with single cells. However, the mechanisms involved remain elusive. Reporting in Cell, Wrenn et al. describe how sealed intercellular compartments, nanolumina, are used as growth factor reservoirs within tumor cell clusters to regulate tumor cell proliferation.
Assuntos
Neoplasias , Proliferação de Células , Humanos , Transdução de SinaisRESUMO
Metastasis is a dynamic succession of events involving the dissemination of tumour cells to distant sites within the body, ultimately reducing the survival of patients with cancer. To colonize distant organs and, therefore, systemically disseminate within the organism, cancer cells and associated factors exploit several bodily fluid systems, which provide a natural transportation route. Indeed, the flow mechanics of the blood and lymphatic circulatory systems can be co-opted to improve the efficiency of cancer cell transit from the primary tumour, extravasation and metastatic seeding. Flow rates, vessel size and shear stress can all influence the survival of cancer cells in the circulation and control organotropic seeding patterns. Thus, in addition to using these fluids as a means to travel throughout the body, cancer cells exploit the underlying physical forces within these fluids to successfully seed distant metastases. In this Review, we describe how circulating tumour cells and tumour-associated factors leverage bodily fluids, their underlying forces and imposed stresses during metastasis. As the contribution of bodily fluids and their mechanics raises interesting questions about the biology of the metastatic cascade, an improved understanding of this process might provide a new avenue for targeting cancer cells in transit.
Assuntos
Líquidos Corporais/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patologia , Microambiente Tumoral , Animais , Biomarcadores , Líquidos Corporais/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Terapia de Alvo Molecular , Metástase Neoplásica , Neoplasias/etiologia , Neoplasias/terapia , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Cancer progression to metastatic dissemination is responsible for â¼90% of deaths in patients. Tremendous efforts have been made to understand primary tumor growth, cancer genetics, and clonal evolution, but also secondary sites of colonization. Intravital imaging technologies are instrumental in understanding key steps of the metastasis cascade, which are believed to be therapeutically relevant targets. However, these remain cumbersome in mouse models. Recent work has demonstrated the zebrafish's unique ability as an experimental metastasis model for the dynamic study of cancer progression at the single-cell level. Its compatibility with state-of-the art imaging techniques and biophysical approaches allows probing of the interaction of tumor cells with their microenvironment and monitoring of fast and rare cellular events at high spatiotemporal resolution. In this review, we highlight the multiple benefits of the zebrafish as an alternative metastasis preclinical model from an imaging standpoint.
Assuntos
Metástase Neoplásica/diagnóstico por imagem , Microambiente Tumoral/genética , Animais , Modelos Animais de Doenças , Progressão da Doença , Peixe-ZebraRESUMO
Cancer metastasis is a process whereby a primary tumor spreads to distant organs. We have demonstrated previously that blood flow controls the intravascular arrest of circulating tumor cells (CTCs) through stable adhesion to endothelial cells. We now aim to define the contribution of cell adhesion potential and identify adhesion receptors at play. Early arrest is mediated by the formation of weak adhesion, depending on CD44 and integrin αvß3. Stabilization of this arrest uses integrin α5ß1-dependent adhesions with higher adhesion strength, which allows CTCs to stop in vascular regions with lower shear forces. Moreover, blood flow favors luminal deposition of fibronectin on endothelial cells, an integrin α5ß1 ligand. Finally, we show that only receptors involved in stable adhesion are required for subsequent extravasation and metastasis. In conclusion, we identified the molecular partners that are sequentially exploited by CTCs to arrest and extravasate in vascular regions with permissive flow regimes.
Assuntos
Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Estresse Mecânico , Animais , Adesão Celular , Linhagem Celular Tumoral , Embrião não Mamífero/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrinas/metabolismo , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Peixe-Zebra/embriologiaRESUMO
Metastatic seeding is driven by cell-intrinsic and environmental cues, yet the contribution of biomechanics is poorly known. We aim to elucidate the impact of blood flow on the arrest and the extravasation of circulating tumor cells (CTCs) in vivo. Using the zebrafish embryo, we show that arrest of CTCs occurs in vessels with favorable flow profiles where flow forces control the adhesion efficacy of CTCs to the endothelium. We biophysically identified the threshold values of flow and adhesion forces allowing successful arrest of CTCs. In addition, flow forces fine-tune tumor cell extravasation by impairing the remodeling properties of the endothelium. Importantly, we also observe endothelial remodeling at arrest sites of CTCs in mouse brain capillaries. Finally, we observed that human supratentorial brain metastases preferably develop in areas with low perfusion. These results demonstrate that hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth.
Assuntos
Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Adesão Celular , Hemodinâmica , Neoplasias Pulmonares/patologia , Melanoma/patologia , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/metabolismo , Ciclo Celular , Circulação Cerebrovascular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Neoplásicas Circulantes/metabolismo , Estudos Retrospectivos , Células Tumorais Cultivadas , Peixe-ZebraRESUMO
Most cancers end up with the death of patients caused by the formation of secondary tumors, called metastases. However, how these secondary tumors appear and develop is only poorly understood. A fine understanding of the multiple steps of the metastasis cascade requires in vivo models allowing high spatiotemporal analysis of the behavior of metastatic cells. Zebrafish embryos combine several advantages such as transparency, small size, stereotyped anatomy, and easy handling, making it a very powerful model for cell and cancer biology, and in vivo imaging analysis. In the following chapter, we describe a complete procedure allowing in vivo imaging methods, at high throughput and spatiotemporal resolution, to assess the behavior of circulating tumor cells (CTCs) in an experimental metastasis assay. This protocol provides access, for the first time, to the earliest steps of tumor cell seeding during metastasis formation.
Assuntos
Embrião não Mamífero/patologia , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes/patologia , Peixe-Zebra/embriologia , Animais , Análise Espaço-TemporalRESUMO
Hemidesmosomes (HDs) are epithelial-specific cell-matrix adhesions that stably anchor the intracellular keratin network to the extracellular matrix. Although their main role is to protect the epithelial sheet from external mechanical strain, how HDs respond to mechanical stress remains poorly understood. Here we identify a pathway essential for HD remodeling and outline its role with respect to α6ß4 integrin recycling. We find that α6ß4 integrin chains localize to the plasma membrane, caveolae, and ADP-ribosylation factor-6+ (Arf6+) endocytic compartments. Based on fluorescence recovery after photobleaching and endocytosis assays, integrin recycling between both sites requires the small GTPase Arf6 but neither caveolin1 (Cav1) nor Cavin1. Strikingly, when keratinocytes are stretched or hypo-osmotically shocked, α6ß4 integrin accumulates at cell edges, whereas Cav1 disappears from it. This process, which is isotropic relative to the orientation of stretch, depends on Arf6, Cav1, and Cavin1. We propose that mechanically induced HD growth involves the isotropic flattening of caveolae (known for their mechanical buffering role) associated with integrin diffusion and turnover.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Caveolina 1/metabolismo , Hemidesmossomos/metabolismo , Integrina beta4/metabolismo , Queratinócitos/metabolismo , Fator 6 de Ribosilação do ADP , Linhagem Celular , Membrana Celular/metabolismo , Hemidesmossomos/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Microscopia ImunoeletrônicaRESUMO
Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond-like phenotype.
Assuntos
Doenças da Medula Óssea/genética , Insuficiência Pancreática Exócrina/genética , Lipomatose/genética , Neutropenia/congênito , Partícula de Reconhecimento de Sinal/genética , Animais , Criança , Síndrome Congênita de Insuficiência da Medula Óssea , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Modelos Moleculares , Neutropenia/genética , Pâncreas Exócrino/metabolismo , Fenótipo , Domínios Proteicos , Síndrome de Shwachman-Diamond , Partícula de Reconhecimento de Sinal/química , Peixe-ZebraRESUMO
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.
Assuntos
Biologia Celular , Imageamento Tridimensional , Análise Espaço-Temporal , Animais , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos BiológicosRESUMO
Epithelial cells constitute the main barrier between the inside and outside of organs, acting as gatekeepers of their structure and integrity. Hemidesmosomes and desmosomes are respectively cell-matrix and cell-cell adhesions coupled to the intermediate filament cytoskeleton. These adhesions ensure mechanical integrity of the epithelial barrier. Although desmosomes and hemidesmosomes are essential in maintaining strong cell-cell and cell-matrix adhesions, there is an emerging view that they should be remodeled in order to maintain epithelial homeostasis. Here we review the adhesion properties of desmosomes and hemidesmosomes, as well as the mechanisms driving their remodeling. We also discuss recent data suggesting that keratin-coupled adhesion complexes can sense the biomechanical cellular environment and participate in the cellular response to such external cues.
Assuntos
Adesão Celular , Desmossomos/fisiologia , Queratinas/metabolismo , Animais , Fenômenos Biomecânicos , Junções Célula-Matriz/metabolismo , Citoesqueleto/metabolismo , Desmossomos/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , HumanosRESUMO
Adhesion between cells and the extracellular matrix is mediated by different types of transmembraneous proteins. Their associations to specific partners lead to the assembly of contacts such as focal adhesions and hemidesmosomes. The spatial overlap between both contacts within cells has however limited the study of each type of contact. Here we show that with "stampcils" focal contacts and hemidesmosomes can be spatially separated: cells are plated within the cavities of a stencil and the grids of the stencil serve as stamps for grafting an extracellular matrix protein-fibronectin. Cells engage new contacts on stamped zones leading to the segregation of adhesions and their associated cytoskeletons, i.e., actin and intermediate filaments of keratins. This new method should provide new insights into cell contacts compositions and dynamics.