RESUMEN
Bacillus subtilis can form various types of spatially organised communities on surfaces, such as colonies, pellicles and submerged biofilms. These communities share similarities and differences, and phenotypic heterogeneity has been reported for each type of community. Here, we studied spatial transcriptional heterogeneity across the three types of surface-associated communities. Using RNA-seq analysis of different regions or populations for each community type, we identified genes that are specifically expressed within each selected population. We constructed fluorescent transcriptional fusions for 17 of these genes, and observed their expression in submerged biofilms using time-lapse confocal laser scanning microscopy (CLSM). We found mosaic expression patterns for some genes; in particular, we observed spatially segregated cells displaying opposite regulation of carbon metabolism genes (gapA and gapB), indicative of distinct glycolytic or gluconeogenic regimes coexisting in the same biofilm region. Overall, our study provides a direct comparison of spatial transcriptional heterogeneity, at different scales, for the three main models of B. subtilis surface-associated communities.
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Bacillus subtilis , Biopelículas , Bacillus subtilis/metabolismo , Microscopía Confocal , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
The formation of biofilm at the air-liquid interface of a still flask is related to the emergence of exopolysaccharides (EPS) overproducers. These mutants have the ability to remain near the surface, where oxygen is abundant. Yet, it is still unclear what role oxygen plays in cellular metabolism under this condition. A. Besse, M.-C. Groleau and E. Déziel (mSphere e00057-23, 2023; https://doi.org/10.1128/msphere.00057-23) explains that the redox state of cells is key in understanding the emergence of EPS overproducers. They found that the spatial distribution of oxidizing agent (not oxygen specifically) controls the advantage of remaining near the air-liquid interface, and hence the advantage that EPS-overproduction confers. All together this research paves the way for a deeper comprehension of the relationship between the environment's spatial structure and population dynamics.
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Biopelículas , Oxígeno , Oxidación-ReducciónRESUMEN
Pseudomonas aeruginosa makes and secretes massive amounts of rhamnolipid surfactants that enable swarming motility over biogel surfaces. But how these rhamnolipids interact with biogels to assist swarming remains unclear. Here, I use a combination of optical techniques across scales and genetically engineered strains to demonstrate that rhamnolipids can induce agar gel swelling over distances >10,000× the body size of an individual cell. The swelling front is on the micrometric scale and is easily visible using shadowgraphy. Rhamnolipid transport is not restricted to the surface of the gel but occurs through the whole thickness of the plate and, consequently, the spreading dynamics depend on the local thickness. Surprisingly, rhamnolipids can cross the whole gel and induce swelling on the opposite side of a two-face Petri dish. The swelling front delimits an area where the mechanical properties of the surface properties are modified: water wets the surface more easily, which increases the motility of individual bacteria and enables collective motility. A genetically engineered mutant unable to secrete rhamnolipids (ΔrhlA), and therefore unable to swarm, is rescued from afar with rhamnolipids produced by a remote colony. These results exemplify the remarkable capacity of bacteria to change the physical environment around them and its ecological consequences.
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Glucolípidos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Mutación , Glucolípidos/metabolismo , Tensoactivos/metabolismoRESUMEN
In nature, bacteria form biofilms in very diverse environments, involving a range of specific properties and exhibiting competitive advantages for surface colonization. However, the underlying mechanisms are difficult to decipher. In particular, the contribution of cell flagellar motility to biofilm formation remains unclear. Here, we examined the ability of motile and nonmotile E. coli cells to form a biofilm in a well-controlled geometry, both in a simple situation involving a single-species biofilm and in the presence of co-colonizers. Using a millifluidic channel, we determined that motile cells have a clear disadvantage in forming a biofilm, exhibiting a long delay as compared to nonmotile cells. By monitoring biofilm development in real time, we observed that the decisive impact of flagellar motility on biofilm formation consists in the alteration of surface access time potentially highly dependent on the geometry of the environment to be colonized. We also report that the difference between motile and nonmotile cells in the ability to form a biofilm diminishes in the presence of co-colonizers, which could be due to motility inhibition through the consumption of key resources by the co-colonizers. We conclude that the impact of flagellar motility on surface colonization closely depends on the environment properties and the population features, suggesting a unifying vision of the role of cell motility in surface colonization and biofilm formation.
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Biopelículas , Escherichia coli , Escherichia coli/metabolismo , FlagelosRESUMEN
Much of our understanding of bacterial behavior stems from studies in liquid culture. In nature, however, bacteria frequently live in densely packed spatially-structured communities. How does spatial structure affect bacterial cooperative behaviors? In this work, we examine rhamnolipid production-a cooperative and virulent behavior of Pseudomonas aeruginosa. Here we show that, in striking contrast to well-mixed liquid culture, rhamnolipid gene expression in spatially-structured colonies is strongly associated with colony specific growth rate, and is impacted by perturbation with diffusible quorum signals. To interpret these findings, we construct a data-driven statistical inference model which captures a length-scale of bacterial interaction that develops over time. Finally, we find that perturbation of P. aeruginosa swarms with quorum signals preserves the cooperating genotype in competition, rather than creating opportunities for cheaters. Overall, our data demonstrate that the complex response to spatial localization is key to preserving bacterial cooperative behaviors.
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Interacciones Microbianas/fisiología , Modelos Biológicos , Proteínas Bacterianas/genética , Biomasa , Recuento de Colonia Microbiana , Regulación Bacteriana de la Expresión Génica , Glucolípidos/genética , Glucolípidos/metabolismo , Locomoción , Interacciones Microbianas/genética , Mutación , Imagen Óptica , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiología , Percepción de Quorum , Análisis Espacio-TemporalRESUMEN
Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.
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Análisis por Conglomerados , Melanoma/metabolismo , Metástasis de la Neoplasia/patología , Células Neoplásicas Circulantes/patología , Animales , Regulación Neoplásica de la Expresión Génica/fisiología , Melanoma/patología , Cresta Neural/patología , Pez CebraRESUMEN
In the last decade Open Science principles have been successfully advocated for and are being slowly adopted in different research communities. In response to the COVID-19 pandemic many publishers and researchers have sped up their adoption of Open Science practices, sometimes embracing them fully and sometimes partially or in a sub-optimal manner. In this article, we express concerns about the violation of some of the Open Science principles and its potential impact on the quality of research output. We provide evidence of the misuses of these principles at different stages of the scientific process. We call for a wider adoption of Open Science practices in the hope that this work will encourage a broader endorsement of Open Science principles and serve as a reminder that science should always be a rigorous process, reliable and transparent, especially in the context of a pandemic where research findings are being translated into practice even more rapidly. We provide all data and scripts at https://osf.io/renxy/ .
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COVID-19 , Pandemias , Humanos , Pandemias/prevención & control , Publicaciones , Investigadores , SARS-CoV-2RESUMEN
Predicting the evolution of expanding populations is critical to controlling biological threats such as invasive species and cancer metastasis. Expansion is primarily driven by reproduction and dispersal, but nature abounds with examples of evolution where organisms pay a reproductive cost to disperse faster. When does selection favor this "survival of the fastest"? We searched for a simple rule, motivated by evolution experiments where swarming bacteria evolved into a hyperswarmer mutant that disperses â¼100% faster but pays a growth cost of â¼10% to make many copies of its flagellum. We analyzed a two-species model based on the Fisher equation to explain this observation: the population expansion rate (v) results from an interplay of growth (r) and dispersal (D) and is independent of the carrying capacity: v=2(rD)1/2 . A mutant can take over the edge only if its expansion rate (v2) exceeds the expansion rate of the established species (v1); this simple condition ( v2>v1 ) determines the maximum cost in slower growth that a faster mutant can pay and still be able to take over. Numerical simulations and time-course experiments where we tracked evolution by imaging bacteria suggest that our findings are general: less favorable conditions delay but do not entirely prevent the success of the fastest. Thus, the expansion rate defines a traveling wave fitness, which could be combined with trade-offs to predict evolution of expanding populations.
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Evolución Biológica , Modelos Teóricos , Pseudomonas aeruginosa/crecimiento & desarrollo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutación , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Selección GenéticaRESUMEN
By consuming and producing environmental resources, organisms inevitably change their habitats. The consequences of such environmental modifications can be detrimental or beneficial not only to the focal organism but also to other organisms sharing the same environment. Social evolution theory has been very influential in studying how social interactions mediated by public 'goods' or 'bads' evolve by emphasizing the role of spatial structure. The environmental dimensions driving these interactions, however, are typically abstracted away. We propose here a new, environment-mediated taxonomy of social behaviors where organisms are categorized by their production or consumption of environmental factors that can help or harm others in the environment. We discuss microbial examples of our classification and highlight the importance of environmental intermediates more generally.
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Ambiente , Invertebrados/fisiología , Conducta Social , Vertebrados/fisiología , Animales , Evolución Biológica , EcosistemaRESUMEN
Confinement and substrate topology strongly affect the behavior of cell populations and, in particular, their collective migration. In vitro experiments dealing with these aspects require strategies of surface patterning that remain effective over long times (typically several days) and ways to control the surface topology in three dimensions. Here, we describe protocols addressing these two aspects. High-resolution patterning of a robust cell-repellent coating is achieved by etching the coating through a photoresist mask patterned directly on the coated surface. Out-of-plane curvature can be controlled using glass wires or corrugated "wavy" surfaces.
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Movimiento Celular/fisiología , Animales , Línea Celular , Humanos , Polietilenglicoles/químicaRESUMEN
Bacteria of many species rely on a simple molecule, the intracellular secondary messenger c-di-GMP (Bis-(3'-5')-cyclic dimeric guanosine monophosphate), to make a vital choice: whether to stay in one place and form a biofilm, or to leave it in search of better conditions. The c-di-GMP network has a bow-tie shaped architecture that integrates many signals from the outside world-the input stimuli-into intracellular c-di-GMP levels that then regulate genes for biofilm formation or for swarming motility-the output phenotypes. How does the 'uninformed' process of evolution produce a network with the right input/output association and enable bacteria to make the right choice? Inspired by new data from 28 clinical isolates of Pseudomonas aeruginosa and strains evolved in laboratory experiments we propose a mathematical model where the c-di-GMP network is analogous to a machine learning classifier. The analogy immediately suggests a mechanism for learning through evolution: adaptation though incremental changes in c-di-GMP network proteins acquires knowledge from past experiences and enables bacteria to use it to direct future behaviors. Our model clarifies the elusive function of the ubiquitous c-di-GMP network, a key regulator of bacterial social traits associated with virulence. More broadly, the link between evolution and machine learning can help explain how natural selection across fluctuating environments produces networks that enable living organisms to make sophisticated decisions.
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GMP Cíclico/análogos & derivados , Aprendizaje Automático , Modelos Biológicos , Transducción de Señal/fisiología , Biopelículas , Movimiento Celular , Biología Computacional , GMP Cíclico/metabolismo , Fenotipo , Pseudomonas aeruginosa/fisiologíaRESUMEN
How does metabolism influence social behavior? This fundamental question at the interface of molecular biology and social evolution is hard to address with experiments in animals, and therefore, we turned to a simple microbial system: swarming in the bacterium Pseudomonas aeruginosa. Using genetic engineering, we excised a locus encoding a key metabolic regulator and disrupted P. aeruginosa's metabolic prudence, the regulatory mechanism that controls expression of swarming public goods and protects this social behavior from exploitation by cheaters. Then, using experimental evolution, we followed the joint evolution of the genome, the metabolome and the social behavior as swarming re-evolved. New variants emerged spontaneously with mutations that reorganized the metabolome and compensated in distinct ways for the disrupted metabolic prudence. These experiments with a unicellular organism provide a detailed view of how metabolism-currency of all physiological processes-can determine the costs and benefits of a social behavior and ultimately influence how an organism behaves towards other organisms of the same species.
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Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Evolución Molecular Dirigida/métodos , Metabolómica/métodos , Mutación , Pseudomonas aeruginosa/genética , Conducta Social , Factores de Transcripción/genéticaRESUMEN
The genetic and phenotypic diversity of cells within tumors is a major obstacle for cancer treatment. Because of the stochastic nature of genetic alterations, this intratumoral heterogeneity is often viewed as chaotic. Here we show that the altered metabolism of cancer cells creates predictable gradients of extracellular metabolites that orchestrate the phenotypic diversity of cells in the tumor microenvironment. Combining experiments and mathematical modeling, we show that metabolites consumed and secreted within the tumor microenvironment induce tumor-associated macrophages (TAMs) to differentiate into distinct subpopulations according to local levels of ischemia and their position relative to the vasculature. TAMs integrate levels of hypoxia and lactate into progressive activation of MAPK signaling that induce predictable spatial patterns of gene expression, such as stripes of macrophages expressing arginase 1 (ARG1) and mannose receptor, C type 1 (MRC1). These phenotypic changes are functionally relevant as ischemic macrophages triggered tube-like morphogenesis in neighboring endothelial cells that could restore blood perfusion in nutrient-deprived regions where angiogenic resources are most needed. We propose that gradients of extracellular metabolites act as tumor morphogens that impose order within the microenvironment, much like signaling molecules convey positional information to organize embryonic tissues. Unearthing embryology-like processes in tumors may allow us to control organ-like tumor features such as tissue repair and revascularization and treat intratumoral heterogeneity.
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Neoplasias/metabolismo , Neoplasias/patología , Microambiente Tumoral , Línea Celular Tumoral , Análisis por Conglomerados , Metabolismo Energético , Espacio Extracelular/metabolismo , Perfilación de la Expresión Génica , Humanos , Hipoxia/metabolismo , Ácido Láctico/metabolismo , Sistema de Señalización de MAP Quinasas , Macrófagos/metabolismo , Macrófagos/patología , Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Oxígeno/metabolismo , Fenotipo , Transcriptoma , Microambiente Tumoral/genéticaRESUMEN
Aquaporin 0 (AQP0) is a transmembrane protein specific to the eye lens, involved as a water carrier across the lipid membranes. During eye lens maturation, AQP0s are truncated by proteolytic cleavage. We investigate in this work the capability of truncated AQP0 to conduct water across membranes. We developed a method to accurately determine water permeability across lipid membranes and across proteins from the deflation under osmotic pressure of giant unilamellar vesicles (GUVs) deposited on an adhesive substrate. Using reflection interference contrast microscopy (RICM), we measure the spreading area of GUVs during deswelling. We interpret these results using a model based on hydrodynamic, binder diffusion towards the contact zone, and Helfrich's law for the membrane tension, which allows us to relate the spread area to the vesicle internal volume. We first study the specific adhesion of vesicles coated with biotin spreading on a streptavidin substrate. We then determine the permeability of a single functional AQP0 and demonstrate that truncated AQP0 is no more a water channel.
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Acuaporinas/metabolismo , Proteínas del Ojo/metabolismo , Animales , Acuaporinas/química , Acuaporinas/aislamiento & purificación , Proteínas del Ojo/química , Proteínas del Ojo/aislamiento & purificación , Cinética , Cristalino/metabolismo , Microscopía de Interferencia , Presión Osmótica , Permeabilidad , Porosidad , Ovinos , Succinimidas/química , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo , Agua/químicaRESUMEN
How populations of growing cells achieve cell-size homeostasis remains a major question in cell biology. Recent studies in rod-shaped bacteria support the "incremental rule" where each cell adds a constant length before dividing. Although this rule explains narrow cell-size distributions, its mechanism is still unknown. We show that the opportunistic pathogen Pseudomonas aeruginosa obeys the incremental rule to achieve cell-length homeostasis during exponential growth but shortens its cells when entering the stationary phase. We identify a mutant, called frik, which has increased antibiotic sensitivity, cells that are on average longer, and a fraction of filamentous cells longer than 10 µm. When growth slows due to entry in stationary phase, the distribution of frik cell sizes decreases and approaches wild-type length distribution. The rare filamentous cells have abnormally large nucleoids, suggesting that a deficiency in DNA segregation prevents cell division without slowing the exponential elongation rate.
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Ciclo Celular , Homeostasis , Pseudomonas aeruginosa/citología , Antibacterianos/farmacología , Mutación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologíaRESUMEN
Tissue fusion eliminates physical voids in a tissue to form a continuous structure and is central to many processes in development and repair. Fusion events in vivo, particularly in embryonic development, often involve the purse-string contraction of a pluricellular actomyosin cable at the free edge. However, in vitro, adhesion of the cells to their substrate favors a closure mechanism mediated by lamellipodial protrusions, which has prevented a systematic study of the purse-string mechanism. Here, we show that monolayers can cover well-controlled mesoscopic nonadherent areas much larger than a cell size by purse-string closure and that active epithelial fluctuations are required for this process. We have formulated a simple stochastic model that includes purse-string contractility, tissue fluctuations, and effective friction to qualitatively and quantitatively account for the dynamics of closure. Our data suggest that, in vivo, tissue fusion adapts to the local environment by coordinating lamellipodial protrusions and purse-string contractions.
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Organogénesis , Actomiosina/metabolismo , Animales , Adhesión Celular , Perros , Células Epiteliales/citología , Epitelio/fisiología , Terapia por Láser , Células de Riñón Canino Madin Darby , Modelos Biológicos , Procesos Estocásticos , Propiedades de Superficie , Cicatrización de HeridasRESUMEN
Many unicellular organisms live in multicellular communities that rely on cooperation between cells. However, cooperative traits are vulnerable to exploitation by non-cooperators (cheaters). We expand our understanding of the molecular mechanisms that allow multicellular systems to remain robust in the face of cheating by dissecting the dynamic regulation of cooperative rhamnolipids required for swarming in Pseudomonas aeruginosa. We combine mathematical modeling and experiments to quantitatively characterize the integration of metabolic and population density signals (quorum sensing) governing expression of the rhamnolipid synthesis operon rhlAB. The combined computational/experimental analysis reveals that when nutrients are abundant, rhlAB promoter activity increases gradually in a density dependent way. When growth slows down due to nutrient limitation, rhlAB promoter activity can stop abruptly, decrease gradually or even increase depending on whether the growth-limiting nutrient is the carbon source, nitrogen source or iron. Starvation by specific nutrients drives growth on intracellular nutrient pools as well as the qualitative rhlAB promoter response, which itself is modulated by quorum sensing. Our quantitative analysis suggests a supply-driven activation that integrates metabolic prudence with quorum sensing in a non-digital manner and allows P. aeruginosa cells to invest in cooperation only when the population size is large enough (quorum sensing) and individual cells have enough metabolic resources to do so (metabolic prudence). Thus, the quantitative description of rhlAB regulatory dynamics brings a greater understating to the regulation required to make swarming cooperation stable.
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Regulación Bacteriana de la Expresión Génica , Lípidos/química , Pseudomonas aeruginosa/fisiología , Percepción de Quorum , Algoritmos , Proteínas Bacterianas/metabolismo , Biomasa , Alimentos , Genes Bacterianos , Proteínas Fluorescentes Verdes/química , Hierro/química , Cinética , Redes y Vías Metabólicas , Microscopía Fluorescente , Modelos Teóricos , Nitrógeno/química , Operón , Regiones Promotoras Genéticas , Programas InformáticosRESUMEN
Pseudomonas aeruginosa is a monoflagellated bacterium that can use its single polar flagellum to swim through liquids and move collectively over semisolid surfaces, a behavior called swarming. Previous studies have shown that experimental evolution in swarming colonies leads to the selection of hyperswarming bacteria with multiple flagella. Here we show that the advantage of such hyperswarmer mutants cannot be explained simply by an increase in the raw swimming speed of individual bacteria in liquids. Cell tracking of time-lapse microscopy to quantify single-cell swimming patterns reveals that both wild-type and hyperswarmers alternate between forward and backward runs, rather than doing the run-and-tumble characteristic of enteric bacteria such as E. coli. High-throughput measurement of swimming speeds reveals that hyperswarmers do not swim faster than wild-type in liquid. Wild-type reverses swimming direction in sharp turns without a significant impact on its speed, whereas multiflagellated hyperswarmers tend to alternate fast and slow runs and have wider turning angles. Nonetheless, macroscopic measurement of swimming and swarming speed in colonies shows that hyperswarmers expand faster than wild-type on surfaces and through soft agar matrices. A mathematical model explains how wider turning angles lead to faster spreading when swimming through agar. Our study describes for the first time the swimming patterns in multiflagellated P. aeruginosa mutants and reveals that collective and individual motility in bacteria are not necessarily correlated. Understanding bacterial adaptations to surface motility, such as hyperswarming, requires a collective behavior approach.
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Adaptación Fisiológica , Adhesión Bacteriana , Pseudomonas aeruginosa/fisiología , Modelos Biológicos , Mutación , Pseudomonas aeruginosa/genética , Percepción de QuorumRESUMEN
Drastic metabolic alterations, such as the Warburg effect, are found in most if not all types of malignant tumors. Emerging evidence shows that cancer cells benefit from these alterations, but little is known about how they affect noncancerous stromal cells within the tumor microenvironment. Here we show that cancer cells are better adapted to metabolic changes in the microenvironment, leading to the emergence of spatial structure. A clear example of tumor spatial structure is the localization of tumor-associated macrophages (TAMs), one of the most common stromal cell types found in tumors. TAMs are enriched in well-perfused areas, such as perivascular and cortical regions, where they are known to potentiate tumor growth and invasion. However, the mechanisms of TAM localization are not completely understood. Computational modeling predicts that gradients--of nutrients, gases, and metabolic by-products such as lactate--emerge due to altered cell metabolism within poorly perfused tumors, creating ischemic regions of the tumor microenvironment where TAMs struggle to survive. We tested our modeling prediction in a coculture system that mimics the tumor microenvironment. Using this experimental approach, we showed that a combination of metabolite gradients and differential sensitivity to lactic acid is sufficient for the emergence of macrophage localization patterns in vitro. This suggests that cancer metabolic changes create a microenvironment where tumor cells thrive over other cells. Understanding differences in tumor-stroma sensitivity to these alterations may open therapeutic avenues against cancer.