RESUMEN
We have recently shown that cancer cells of various origins take up extracellular citrate through the plasma membrane citrate carrier (pmCiC), a specific plasma membrane citrate transporter. Extracellular citrate is required to support cancer cell metabolism, in particular fatty acid synthesis, mitochondrial activity, protein synthesis and histone acetylation. In addition, cancer cells tend to acquire a metastatic phenotype in the presence of extracellular citrate. Our recent study also showed that cancer-associated stromal cells synthesise and release citrate and that this process is controlled by cancer cells. In the present study, we evaluated the expression of pmCiC, fibroblast activation protein-α (FAP) and the angiogenesis marker cluster of differentiation 31 (CD31) in human cancer tissues of different origins. In the cohort studied, we found no correlation between disease stage and the expression of FAP or CD31. However, we have identified a clear correlation between pmCiC expression in cancer cells and cancer-associated stroma with tumour stage. It can be concluded that pmCiC is increased in cancer cells and in cancer-supporting cells in the tumour microenvironment at the later stages of cancer development, particularly at the metastatic sites. Therefore, pmCiC expression has the potential to serve as a prognostic marker, although further studies are needed.
RESUMEN
Citrate is a key metabolite of the Krebs cycle that can also be exported in the cytosol, where it performs several functions. In normal cells, citrate sustains protein acetylation, lipid synthesis, gluconeogenesis, insulin secretion, bone tissues formation, spermatozoid mobility, and immune response. Dysregulation of citrate metabolism is implicated in several pathologies, including cancer. Here we discuss how cancer cells use citrate to sustain their proliferation, survival, and metastatic progression. Also, we propose two paradoxically opposite strategies to reduce tumour growth by targeting citrate metabolism in preclinical models. In the first strategy, we propose to administer in the tumor microenvironment a high amount of citrate, which can then act as a glycolysis inhibitor and apoptosis inducer, whereas the other strategy targets citrate transporters to starve cancer cells from citrate. These strategies, effective in several preclinical in vitro and in vivo cancer models, could be exploited in clinics, particularly to increase sensibility to current anti-cancer agents.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Ácido Cítrico/metabolismo , Neoplasias/patología , Glucólisis/fisiología , Ciclo del Ácido Cítrico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Microambiente TumoralRESUMEN
Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin with a poor prognosis. The factors driving this cancer must be better understood in order to discover novel targets for more effective therapies. In the search for targets, we followed our interest in citrate as a central and critical metabolite linked to fatty acid synthesis in cancer development. A key to citrate uptake in cancer cells is the high expression of the plasma membrane citrate transporter (pmCiC), which is upregulated in the different adenocarcinoma types tested so far. In this study, we show that the pmCiC is also highly expressed in Merkel cell carcinoma cell lines by western blot and human tissues by immunohistochemistry staining. In the presence of extracellular citrate, MCC cells show an increased proliferation rate in vitro; a specific pmCiC inhibitor (Na+-gluconate) blocks this citrate-induced proliferation. Furthermore, the 3D in vivo Chick Chorioallantoic Membrane (CAM) model showed that the application of Na+-gluconate also decreases Merkel cell carcinoma growth. Based on our results, we conclude that pmCiC and extracellular citrate uptake should be considered further as a potential novel target for the treatment of Merkel cell carcinoma.
RESUMEN
Cancer metabolism has been of interest for decades; however, the recent development of sophisticated techniques such as metabolomics or lipidomics have significantly increased our understanding of processes taking place in tumour cells [...].
Asunto(s)
Metabolismo de los Lípidos , Neoplasias , Humanos , Lipidómica , Metabolómica/métodosRESUMEN
Brain tissue is known to have elevated citrate levels, necessary to regulate ion chelation, neuron excitability, and are also necessary for the supply of necessary energy substrates to neurons. Importantly, citrate also acts as a central substrate in cancer metabolism. Recent studies have shown that extracellular citrate levels in the brain undergo significant changes during tumor development and may play a dual role in tumor progression, as well as cancer cell aggressiveness. In the present article, we review available literature describing changes of citrate levels in brain tissue, blood, and cerebrospinal fluid, as well as intracellular alterations during tumor development before and after metastatic progression. Based on the available literature and our recent findings, we hypothesize that changes in extracellular citrate levels may be related to the increased consumption of this metabolite by cancer cells. Interestingly, cancerassociated cells, including reactive astrocytes, might be a source of citrate. Extracellular citrate uptake mechanisms, as well as potential citrate synthesis and release by surrounding stroma, could provide novel targets for anti-cancer treatments of primary brain tumors and brain metastases.
Asunto(s)
Neoplasias Encefálicas , Ácido Cítrico , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Citratos , Ácido Cítrico/metabolismo , Humanos , Neuronas/metabolismoRESUMEN
It is well established that cancer cells acquire energy via the Warburg effect and oxidative phosphorylation. Citrate is considered to play a crucial role in cancer metabolism by virtue of its production in the reverse Krebs cycle from glutamine. Here, we review the evidence that extracellular citrate is one of the key metabolites of the metabolic pathways present in cancer cells. We review the different mechanisms by which pathways involved in keeping redox balance respond to the need of intracellular citrate synthesis under different extracellular metabolic conditions. In this context, we further discuss the hypothesis that extracellular citrate plays a role in switching between oxidative phosphorylation and the Warburg effect while citrate uptake enhances metastatic activities and therapy resistance. We also present the possibility that organs rich in citrate such as the liver, brain and bones might form a perfect niche for the secondary tumour growth and improve survival of colonising cancer cells. Consistently, metabolic support provided by cancer-associated and senescent cells is also discussed. Finally, we highlight evidence on the role of citrate on immune cells and its potential to modulate the biological functions of pro- and anti-tumour immune cells in the tumour microenvironment. Collectively, we review intriguing evidence supporting the potential role of extracellular citrate in the regulation of the overall cancer metabolism and metastatic activity.
Asunto(s)
Ácido Cítrico , Neoplasias , Citratos , Ácido Cítrico/metabolismo , Ciclo del Ácido Cítrico , Humanos , Neoplasias/metabolismo , Fosforilación Oxidativa , Microambiente Tumoral/fisiologíaRESUMEN
Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.
Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Ácido Cítrico/metabolismo , Neoplasias Pancreáticas/metabolismo , Fibroblastos Asociados al Cáncer/fisiología , Línea Celular Tumoral , Epigénesis Genética , Humanos , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Neoplasias Pancreáticas/patología , Células del Estroma/metabolismo , Microambiente Tumoral/fisiología , Neoplasias PancreáticasRESUMEN
Cancer cells need excess energy and essential nutrients/metabolites not only to divide and proliferate but also to migrate and invade distant organs for metastasis. Fatty acid and cholesterol synthesis, considered a hallmark of cancer for anabolism and membrane biogenesis, requires citrate. We review here potential pathways in which citrate is synthesized and/or supplied to cancer cells and the impact of extracellular citrate on cancer cell metabolism and growth. Cancer cells employ different mechanisms to support mitochondrial activity and citrate synthesis when some of the necessary substrates are missing in the extracellular space. We also discuss the different transport mechanisms available for the entry of extracellular citrate into cancer cells and how citrate as a master metabolite enhances ATP production and fuels anabolic pathways. The available literature suggests that cancer cells show an increased metabolic flexibility with which they tackle changing environmental conditions, a phenomenon crucial for cancer cell proliferation and metastasis.
RESUMEN
We have recently discovered that cancer cells take up extracellular citrate through plasma membrane citrate transporter (pmCiC) and advantageously use citrate for their metabolism. Citrate uptake can be blocked with gluconate and this results in decreased tumor growth and altered metabolic characteristics of tumor tissue. Interestingly, gluconate, considered to be physiologically neutral, is incidentally used in medicine as a cation carrier, but not as a therapeutically active substance. In this review we discuss the results of our recent research with available literature and suggest that gluconate may be useful in the treatment of cancer.
RESUMEN
Glycolysis and fatty acid synthesis are highly active in cancer cells through cytosolic citrate metabolism, with intracellular citrate primarily derived from either glucose or glutamine via the tricarboxylic acid cycle. We show here that extracellular citrate is supplied to cancer cells through a plasma membrane-specific variant of the mitochondrial citrate transporter (pmCiC). Metabolomic analysis revealed that citrate uptake broadly affected cancer cell metabolism through citrate-dependent metabolic pathways. Treatment with gluconate specifically blocked pmCiC and decreased tumor growth in murine xenografts of human pancreatic cancer. This treatment altered metabolism within tumors, including fatty acid metabolism. High expression of pmCiC was associated with invasion and advanced tumor stage across many human cancers. These findings support the exploration of extracellular citrate transport as a novel potential target for cancer therapy.Significance: Uptake of extracellular citrate through pmCiC can be blocked with gluconate to reduce tumor growth and to alter metabolic characteristics of tumor tissue. Cancer Res; 78(10); 2513-23. ©2018 AACR.
Asunto(s)
Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/metabolismo , Proliferación Celular/efectos de los fármacos , Ácido Cítrico/metabolismo , Gluconatos/farmacología , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias de la Próstata/patología , Animales , Línea Celular Tumoral , Células Epiteliales/metabolismo , Ácidos Grasos/biosíntesis , Glucólisis/fisiología , Humanos , Masculino , Ratones , Transportadores de Anión Orgánico , Próstata/citología , Próstata/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
The effects of continuous water infusion on efficiency and repeatability of atmospheric pressure chemical ionization of both methyl chloroformate (MCF) and methoxime-trimethylsilyl (MO-TMS) derivatives of metabolites were evaluated using gas chromatography-time-of-flight mass spectrometry. Water infusion at a flow-rate of 0.4 mL/h yielded not only an average 16.6-fold increase in intensity of the quasimolecular ion for 20 MCF-derivatized metabolite standards through suppression of in-source fragmentation but also the most repeatable peak area integrals. The impact of water infusion was the greatest for dicarboxylic acids and the least for (hetero-) aromatic compounds. Water infusion also improved the ability to detect reliably fold changes as small as 1.33-fold for the same 20 MCF-derivatized metabolite standards spiked into a human serum extract. On the other hand, MO-TMS derivatives were not significantly affected by water infusion, neither in their fragmentation patterns nor with regard to the detection of differentially regulated compounds. As a proof of principle, we applied MCF derivatization and GC-APCI-TOFMS to the detection of changes in abundance of metabolites in pancreatic cancer cells upon treatment with 17-DMAG. Water infusion increased not only the number of metabolites identified via their quasimolecular ion but also the reproducibility of peak areas, thereby almost doubling the number of significantly regulated metabolites (false discovery rate < 0.05) to a total of 23.
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Formiatos/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Metabolómica , Agua/química , Área Bajo la Curva , Presión Atmosférica , Benzoquinonas/química , Línea Celular Tumoral , Formiatos/análisis , Humanos , Lactamas Macrocíclicas/química , Oximas/análisis , Oximas/metabolismo , Análisis de Componente Principal , Curva ROCRESUMEN
The prognosis of patients suffering from pancreatic cancer is still poor and novel therapeutic options are urgently needed. Recently, the transcription factor signal transducer and activator of transcription 5b (STAT5b) was associated with tumor progression in human solid cancer. Hence, we assessed whether STAT5b might serve as an anticancer target in ductal pancreatic adenocarcinoma (DPAC). We found that nuclear expression of STAT5b can be detected in approximately 50% of DPAC. Blockade of STAT5b by stable shRNA-mediated knockdown showed no effects on tumor cell growth in vitro. However, inhibition of tumor cell motility was found even in response to stimulation with epidermal growth factor or interleukin-6. These findings were paralleled by a reduction of prometastatic and proangiogenic factors in vitro. Subsequent in vivo experiments revealed a strong growth inhibition on STAT5b blockade in subcutaneous and orthotopic models. These findings were paralleled by impaired tumor angiogenesis in vivo. In contrast to the subcutaneous model, the orthotopic model revealed a strong reduction of tumor cell proliferation that emphasizes the meaning of assessing targets in an appropriate microenvironment. Taken together, our results suggest that STAT5b might be a potential novel target for human DPAC.
Asunto(s)
Adenocarcinoma Mucinoso/secundario , Carcinoma Ductal Pancreático/secundario , Carcinoma Papilar/secundario , Neovascularización Patológica , Neoplasias Pancreáticas/patología , Factor de Transcripción STAT5/metabolismo , Adenocarcinoma Mucinoso/irrigación sanguínea , Adenocarcinoma Mucinoso/metabolismo , Animales , Western Blotting , Carcinoma Ductal Pancreático/irrigación sanguínea , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Papilar/irrigación sanguínea , Carcinoma Papilar/metabolismo , Movimiento Celular , Proliferación Celular , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Clasificación del Tumor , Invasividad Neoplásica , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/metabolismo , Fosforilación , ARN Interferente Pequeño/genética , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/genética , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Activation of receptor tyrosine kinases, such as fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), and VEGF receptor (VEGFR), has been implicated in tumor progression and metastasis in human pancreatic cancer. In this study, we investigated the effects of TKI258, a tyrosine kinase inhibitor to FGFR, PDGFR, and VEGFR on pancreatic cancer cell lines (HPAF-II, BxPC-3, MiaPaCa2, and L3.6pl), endothelial cells, and vascular smooth muscle cells (VSMC). Results showed that treatment with TKI258 impaired activation of signaling intermediates in pancreatic cancer cells, endothelial cells, and VSMCs, even upon stimulation with FGF-1, FGF-2, VEGF-A, and PDGF-B. Furthermore, blockade of FGFR/PDGFR/VEGFR reduced survivin expression and improved activity of gemcitabine in MiaPaCa2 pancreatic cancer cells. In addition, motility of cancer cells, endothelial cells, and VSMCs was reduced upon treatment with TKI258. In vivo, therapy with TKI258 led to dose-dependent inhibition of subcutaneous (HPAF-II) and orthotopic (L3.6pl) tumor growth. Immunohistochemical analysis revealed effects on tumor cell proliferation [bromodeoxyuridine (BrdUrd)] and tumor vascularization (CD31). Moreover, lymph node metastases were significantly reduced in the orthotopic tumor model when treatment was initiated early with TKI258 (30 mg/kg/d). In established tumors, TKI258 (30 mg/kg/d) led to significant growth delay and improved survival in subcutaneous and orthotopic models, respectively. These data provide evidence that targeting FGFR/PDFGR/VEGFR with TKI258 may be effective in human pancreatic cancer and warrants further clinical evaluation.
Asunto(s)
Antineoplásicos/farmacología , Neovascularización Patológica/metabolismo , Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The prostate is a highly specialized mammalian organ that produces and releases large amounts of citrate. However, the citrate release mechanism is not known. Here, we present the results of molecular cloning of a citrate transporter from human normal prostate epithelial PNT2-C2 cells shown previously to express such a mechanism. By using rapid amplification of cDNA ends PCR, we determined that the prostatic carrier is an isoform of the mitochondrial transporter SLC25A1 with a different first exon. We confirmed the functionality of the clone by expressing it in human embryonic kidney cells and performing radiotracer experiments and whole-cell patch-clamp recordings. By using short interfering RNAs targeting different parts of the sequence, we confirmed that the cloned protein is the main prostatic transporter responsible for citrate release. We also produced a specific antibody and localized the cloned transporter protein to the plasma membrane of the cells. By using the same antibody, we have shown that the cloned transporter is expressed in non-malignant human tissues.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Evolución Molecular , Próstata/citología , Secuencia de Aminoácidos , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/metabolismo , Transporte Biológico , Proteínas Portadoras/química , Línea Celular , Ácido Cítrico/metabolismo , Células Epiteliales/citología , Silenciador del Gen , Humanos , Inmunohistoquímica , Iones/metabolismo , Masculino , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Transportadores de Anión Orgánico , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
The endocytic membrane activities of two human breast cancer cell lines (MDA-MB-231 and MCF-7) of strong and weak metastatic potential, respectively, were studied in a comparative approach. Uptake of horseradish peroxidase was used to follow endocytosis. Dependence on ionic conditions and voltage-gated sodium channel (VGSC) activity were characterized. Fractal methods were used to analyze quantitative differences in vesicular patterning. Digital quantification showed that MDA-MB-231 cells took up more tracer (i.e., were more endocytic) than MCF-7 cells. For the former, uptake was totally dependent on extracellular Na(+) and partially dependent on extracellular and intracellular Ca(2+) and protein kinase activity. Analyzing the generalized fractal dimension (D(q )) and its Legendre transform f(alpha) revealed that under control conditions, all multifractal parameters determined had values greater for MDA-MB-231 compared with MCF-7 cells, consistent with endocytic/vesicular activity being more developed in the strongly metastatic cells. All fractal parameters studied were sensitive to the VGSC blocker tetrodotoxin (TTX). Some of the parameters had a "simple" dependence on VGSC activity, if present, whereby pretreatment with TTX reduced the values for the MDA-MB-231 cells and eliminated the differences between the two cell lines. For other parameters, however, there was a "complex" dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.
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Neoplasias de la Mama/fisiopatología , Membrana Celular , Endocitosis , Activación del Canal Iónico , Canales de Sodio/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Fractales , HumanosRESUMEN
Citrate, an organic trivalent anion, is a major substrate for generation of energy in most cells. It is produced in mitochondria and used either in the Krebs' cycle or released into cytoplasm through a specific mitochondrial carriers. Citrate can also be taken up from blood through different plasma membrane transporters. In the cytoplasm, citrate can be used ultimately for fatty acid synthesis, which is increased in cancer cells. Here, we review the ways in which citrate can be transported and discuss the changes in transport and metabolism that occur in cancer cells. The primary focus is on the prostate gland, which is known to produce and release large amounts of citrate during its normal secretory function. The significant changes that occur in citrate-related metabolism and transport in prostate cancer are the second focus. This review strives to relate these mechanisms to molecular biology on the one hand and to clinical applications on the other.
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Ácido Cítrico/metabolismo , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Animales , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Ácidos Grasos/metabolismo , Humanos , Hidrógeno/química , Litio/química , Masculino , Modelos BiológicosRESUMEN
An important goal in many branches of science, especially in molecular biology and medicine is the quantitative analysis of the structures and their morphology. The morphology can be analyzed in many ways, in particular by the fractal analysis. Apart from the fractal dimension, an important part of the fractal analysis is the lacunarity measurement which, roughly speaking, characterizes the distribution of gaps in the fractal: a fractal with high lacunarity has large gaps. In this paper, we present an extension of the lacunarity measure to objects with nonregular shapes that enables us to provide a successful discrimination of cancer cell lines. The cell lines differ in the shape of vacuole (the gaps in their body) which is perfectly suited for the lacunarity analysis.
Asunto(s)
Línea Celular Tumoral/citología , Fractales , Vacuolas/ultraestructura , Línea Celular Tumoral/clasificación , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The "single cell adhesion measuring apparatus" (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as "detachment negative pressure" (DNP) or "DNP relative to cell size" (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na(+) channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 microM), the adhesion values for the Mat-LyLu cells increased significantly 4.3-fold; there was no effect on the AT-2 cells. For the strongly metastatic PC-3M cells, TTX treatment caused a significant (approximately 30%) increase in adhesion. The adhesion of PNT2-C2 ("normal") cells was not affected by the TTX pretreatment. The TTX-induced increase in the adhesiveness of the strongly metastatic cells was consistent with the functional VGSC expression in these cells and the proposed role of VGSC activity in metastatic cell behaviour. In conclusion, the SCAMA, which can be constructed easily and cheaply, offers a simple and effective method to characterise single-cell adhesion and its modulation.