Stem cells face various challenges in their differentiation process including external perturbations, such as cell-to-cell signaling and internal perturbations, such as genetic mutations. To robustly differentiate, stem cells must integrate and respond to cues, yet be tolerant of destructive ones. Previous work shows that chromatin landscape is determinant of cellular activity, hence, it may infer the cellular sensitivity to perturbations as well. However, how the dynamics of the chromatin landscape reflects the cell’s sensitivity, is still missing. My hypothesis is that sensitivity to perturbations during differentiation is facilitated by a more accessible chromatin landscape. Here, I set to (a) define the temporal sensitivity of differentiating cells to various perturbations, and (b) define how the chromatin landscape reflects the sensitivity and robustness of the cells. First, we will grow and differentiate stem cells along several trajectories while characterizing their differentiation in-depth, utilizing sequence-based technologies such as chromatin accessibility and conformation, methylation and gene expression. Second, we will perturb the several differentiation trajectories, imposing external and internal perturbations, defining the perturbations’ effect on differentiation. Finally, we will perturb the cells at dense time points in the differentiation process, generating sequence-based time-specific profiles of perturbed vs normal differentiation. This work will characterize several differentiation trajectories in great details and provide the necessary high-throughput data to investigate the causality between chromatin landscape and sensitivity to perturbations.

Liquid-liquid phase separation is key for organising cellular content dynamically without the need for membrane confinement. Phase separation is an intriguing concept to explain as yet puzzling observations in chromatin organisation. It is known to be involved in the condensation of nucleoli, heterochromatin and long-range chromatin-chromatin contacts such as super-enhancers. The cellular mechanisms that control these phenomena are still poorly understood. Studies so far were focussing mainly on biophysical characterisation rather than function or regulation. Preliminary data in the host lab points to the dual-specificity kinase DYRK3, a known regulator of condensation, to be a regulator of novel nuclear condensates containing the chromatin remodeler Set1/COMPASS complex and other factors involved in gene expression control. On this basis, I will combine proteomics, chromatin and transcriptome profiling, cell biology and high-throughput multiscale imaging to determine the composition of transcriptional condensates and how they are controlled by DYRK3. Targeted perturbations will reveal the impact of these condensates on gene expression. The mechanisms discovered for DYRK3 will form a basis for understanding kinase-controlled transcription regulation more generally. Understanding the regulation of these condensates will not only shed light on the basic biology of cellular organisation but may enable therapeutic intervention in protein-aggregation diseases and cancer.

Isopeptide bonds - covalent amide bonds between amino acid side chains - stabilize and crosslink protein folds, notably in gram-positive bacterial pili, or fibrin clots. In contrast to disulfide bonds, far less is known about the generation of isopeptides. Here, I propose to de novo design and characterize proteins that can autocatalytically form both intra- and intermolecular isopeptides. To study a family of pili subunits in gram-positive bacteria that forms intramolecular isopeptides without enzymes, corresponding autocatalytic conformations will be constructed de novo. By controlling the orientation and hydrogen bonding of catalytic amino acids in the protein, I will dissect and optimize the mechanism of isopeptide formation. Resulting protein designs will serve as minimal models to investigate the stabilizing biological functions of isopeptides. Pathogenic bacteria use proteins containing thioester bonds to attach to their host via intermolecular isopeptides - a poorly understood process that I propose to elucidate with de novo design. A reactive Cysteine-Glutamine thioester will be confined onto a designed simplified protein scaffold. Docking de novo binders into proximity of the thioester will enable me to isolate key variables that govern autocatalytic intermolecular isopeptide formation in a controlled environment. Computational design of autocatalytic reactions in proteins enables radical new ways to covalently stabilize and crosslink them, currently lacking in the Rosetta software suite. Fundamentally, I aim to reveal what drives autocatalytic isopeptide formation and what constitutes their biological function by deconstructing their de novo design principles.

Although growing evidence suggesting that typical immune molecules contribute to shaping the synaptic plasticity and regulating the neuronal activity in the brain, the cellular and molecular mechanisms of these molecules, remains unclear. Moreover, the spatial and temporal expression of these molecules in the brain during development as well as in adults remains unresolved. Here I aim to use the most advanced approaches and techniques to investigate the role of such immune molecules for regulating the synaptic plasticity, neuronal circuits, and brain function. For that, I am interested in tackling fundamental and unsolved questions in the field: Does neuron in the brain express typical immune cytokine receptors or another immune molecule? If it so, does such receptor express within the synaptic cleft? Does it express in pre or post-synaptic or both? What is the molecular function of such receptor? Does it bind their classical cytokine and mediate intracellular signaling? What is the molecular and cellular consequence of such receptor activation? Does it affect synaptic transmission, plasticity, and strength? Does it have any impacts on the neuronal function or circuits? Could the activation of such receptors influence the animal behavior? What are the molecular mechanisms regulating such receptor and cytokine expression? Tackling these fundamental questions could help us better understand the regulation of neuronal function by the immune system in the brain which could be critical for processes such as learning, memory, and behavior during development as well as in pathophysiological conditions.

The intestinal microbiome is a species-rich, spatially heterogeneous ecosystem and core determinant of overall host health. An explosion of metagenomic studies interrogating the functions of microbiome composition in health and disease has revolutionized our view of intestinal microbial communities, but many important gaps remain in our understanding of these ecosystems. Chief among these are the combined contributions of viruses - particularly bacteriophages - to microbiome homeostasis, and of the spatial and temporal structure of the microbiome. Spatially structured communities, or biofilms, are the norm rather than the exception among microbes in the wild, including the intestinal tract, qualitatively altering their ecological and evolutionary dynamics across species but also with their host or predators. Here, we propose to fill these gaps by performing a mechanistic characterization of the physical and ecological forces driving host-microbiome-phageome ecosystem stability and dynamics. To achieve this, we will use an interdisciplinary approach combining tools and concepts form ecology, physics and engineering. First, we will develop a novel experimental framework built around the miniGut, a tube-shaped organoid that recapitulates critical biological and physical features of the intestine, including mucus secretion. We will use miniGuts to identify and quantify the critical factors governing the steady states and stability of a model microbiome, including the presence of a lysogenic phage engineered to allow us to track bacteria-phage-intestinal spatial dynamics in live samples at single-cell resolution. Underlying all aims will be the development of a dynamical systems perspective on the two- and three-way interactions where we will record ecosystem equilibrium points and trajectories in phase diagrams. Finally, using this knowledge, we will engineer lambda phage to manipulate ecosystem steady-states within miniGuts. To do so, we will transmit new functions to host microbial species via lysogeny, for example altering the bacterium’s ability to adhere to mucus, to tolerate antibiotics, and to either cooperate or antagonize other members of the model microbiome. Altogether, our interdisciplinary study will set the stage for mechanistic explorations of the role of phage and microbiome spatial structure in host physiology.

In host-microbe interaction studies, genomic technologies have significantly advanced our understanding of host responses and structure of host-associated microbial communities (microbiota). However, spatial and physiological heterogeneity in host-microbe interactions has been more challenging to study. Plants are comprised of various cell types, each of which may interact with a distinct set of microbes and, at the same time, accomplish other tasks such as growth and nutrient acquisition/utilization. To date, interactions between individual plant cell types and the microbiota are poorly understood due to our lack of knowledge about cell type-specific responses of plants and the composition of associated microbes at the cellular level. Here, I employ high-throughput single cell trasncriptomics to analyze plant cell type-specific responses to the microbiota under nutrient (phosphate) replete and starvation conditions. I will mainly investigate immune response and phosphate starvation response and crosstalks between them in individual cell types. I will also develop a method for in situ microbiota community profiling by imaging-based sequencing of bacterial 16S rRNA, which provides a spatial map of the plant microbiota at the strain-level of resolution. By linking plant gene expression and microbiota distribution at the single cell resolution, I will investigate the roles of plant genes and microbiota members that show plant cell-type specificity. This project attempts to reveal plant cell type-specific responses to biotic and abiotic environmental signals and microbiota assembly that are significant and biologically meaningful.

The proposed project aims to implement a new technology based on functional ultrasound (fUS) imaging and Ca2+ imaging using acoustic sensors of calcium (ASC). Functional ultrasound imaging is a new neuroimaging modality providing highly sensitive functional imaging of the brain by measuring cerebral blood flow. Ultrasound has excellent penetration depth, providing whole-brain coverage. However, the reliance of existing fUS methods on imaging blood flow results in an indirect readout of neuronal activity. Recently, it has become possible to connect ultrasound more directly to cellular function using genetically encoded acoustic reporters based on air-filled proteins derived from buoyant bacteria. In this project, I will use new versions of these proteins engineered to serve as dynamic sensors of Ca2+, which I will express in neurons and perform functional calcium imaging in the mouse brain using ultrasound. In addition to the ultrasonic detection of ASCs during task-evoked and resting state acquisition, I will complement the technology with simultaneous hemodynamic measurements to understand how the two signals relate to each other. This Whole-brain Acoustic Tracking of Calcium and Hemodynamics (WATCH) will provide valuable insights on neurovascular coupling and allow the interpretation of hemodynamics signals when models cannot be modified genetically with ASCs.

Because of its ancient origins, little is known about the first steps in the transition to multicellularity. Perhaps the largest gap in our knowledge concerns the origin of cellular differentiation. Our proposed research will examine how aging (a nearly-universal feature of cellular life) can be co-opted by simple multicellular organisms to drive the emergence of novel multicellular development. The proposal combines the PIs’ complementary expertise in aging and protein homeostasis (Saarikangas) and evolutionary biology (Ratcliff). Our research uses a unique approach: we evolve multicellularity from a single-celled yeast in the lab, examining how, over thousands of generations of evolution, they become more complex. Following our preliminary results, we focus on the role of aging and the chaperone protein Hsp90, mechanistically examining their role in the origin of simple cellular differentiation in our model system. We contextualize these results with mathematical modeling, examining the potential for age-dependent development to arise in different lineages of multicellular organisms. By showing how multicellular development can arise de novo, our research illuminates a problem of profound biological importance, and removes one of the last conceptual hurdles remining in our understanding of the origin of multicellular life.

Biomolecules in cells can self-organize into immiscible fluid droplets via liquid-liquid phase separation (LLPS). These liquid-like domains act as organizational hubs and play important physiological functions, including facilitation of biochemical reactions, transport, and signaling. Alteration of their size, internal structure, and coalescence dynamics have been linked to neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and ALS. Despite the continuous discoveries of new liquid-like compartments in cells, a biophysical understanding of their formation and resulting properties is still missing. Here, I will describe experiments designed to study: i) how intermolecular interactions control the assembly, material properties such as viscosity and surface tension, and dynamical arrest of liquid-like droplets; and ii) how non-equilibrium processes in cells, such as mechanical stresses exerted by the cytoskeleton, affect LLPS and regulate the size distribution and fluidity of the liquid domains. I will develop an in vitro system based on phase separating mixtures of DNA oligomers suspended in an active cytoskeletal network composed of purified filamentous proteins that will model the liquid condensates and cell interior. DNA oligomers are an ideal system to relate molecular structure to phase behavior because of the specificity and determinism of the base pairing interactions. Active cytoskeletal networks will mimic the non-equilibrium features of the cell interior. By exploiting the unique features of these systems, I will build a biophysical framework to decipher the roles of non-equilibrium pathways to LLPS and irreversible protein aggregation in living cells.

The ribosome which functionally establishes all proteomes across all life kingdoms, has historically been considered as a homogenous molecular machine that only passively contributes to gene expression. In contrast, there is growing evidence that ribosomes are heterogeneous and actively regulate gene expression through selective mRNA translation. In humans, there are hundreds of ribosomal DNA (rDNA) copies spread across multiple chromosomal loci, with recent studies showing copy number and sequence variations with unknown effects. In this project, I will characterize for the first time how variant rDNA alleles give rise to heterogeneous ribosomes that contribute to human physiology and disease. Using full genome sequencing data for 120,000 healthy and diseased individuals, I will (1) map population-wide rDNA variability in sequence and copy number variation and (2) identify rDNA variation that predicts human disorders. To get to a mechanistic understanding of rDNA variability I will (3) employ reporter assays to characterize the impact of ribosomal RNA (rRNA) variation on ribosome functionality and (4) use super resolution microscopy to test if rRNAs display expression specificity within a tissue and between different tissues including cancer samples. rDNA sequence variants reflect a hidden facet to our genomes and human health with the potential to change our understanding of cellular physiology at a global populations level. By studying the genetic variability of human rDNAs and its role in health and disease we aim to find how this diversity controls gene expression at the translation level and detect disease associated variants for early disease prognostics.

The regeneration of the optic nerve in the adult mammalian brain is limited [1,2]. During embryonic development different guidance molecules direct retinal ganglion cell (RGC) axons from the retina to the dorsal lateral geniculate nucleus (dLGN) [3]. Within the dLGN certain receptor/ligand gradients [4,5] and spontaneous patterned neural activity [6] assure correct retinotopic mapping. However, little is known on how these developmental mechanisms could be exploited to functionally reconnect RGC axons to the adult brain. Thus, we will ask: 1) How could axons of in vitro grown RGCs reinnervate and functionally reconnect to the dLGN in the adult rodent? 2) How does the axonal innervation pattern in the adult dLGN adapt in response to stimulation or genetic modification of the RGCs? To tackle these challenging questions, we will build a novel biohybrid platform in which RGCs are cultured on an implantable stretchable multi-chamber multielectrode array (MEA) [7] that is connected to a bioabsorbable axon-guiding nanofiber tube. In contrast to traditional organoid or explant based axonal rewiring approaches the new biohybrid system offers three unique advantages: First, we can modulate gene expression in a spatially defined subpopulation of RGCs. Second, we can spatiotemporally control the activity patterns of the RGCs. Third, we can target axons to deep brain regions and stimulate at an almost single cell resolution. This is significantly better than the mm3 size stimulation of any conventional deep brain stimulation electrode and has the potential to restore functional vision in blind patients without eyes.

A number of transformative molecular tools for biomedical research and therapeutics have been developed. However, a lack of safe, efficient delivery methods has precluded the widespread therapeutic application of these tools. One promising route to overcoming this limitation is to investigate the diverse biomolecule exchange systems found in nature and develop delivery tools based on these systems. Recently, a novel membrane vesicle (MV)-mediated horizontal gene transfer system was found in archaea. In this system, a relatively large 50 kbp plasmid is enclosed in a MV and delivered to other archaea. These plasmid vesicles (PVs) use a viral-like plasmid, pR1SE, that encodes the proteins required to assemble PVs and transfer the plasmid to other cells. As intercellular communication by MV is a universal process among the three domains of life, these systems may be exploited to deliver tools in human cells or the human microbiome. However, the mechanisms of archaeal MV and PV formation are largely unknown. I propose to identify the key proteins in the PV system. I will narrow down the candidates by remote homology detection among pR1SE and related viruses, and validate them by biochemical experiments. Furthermore, I will use genome-wide CRISPR screening to identify key proteins of host archaeal cells. Ultimately, I aim to reconstruct the PV system towards programmable synthetic vesicles production. This proposal will elucidate basic archaeal biology and provide a hint to the origin of viruses with a long-term objective of harnessing archaeal vesicle systems for delivery tools. Finally, this approach will serve as a paradigm for engineering archaea for molecular technologies.

Some animals can regenerate lost organs and tissues with high efficiency. In many such species, cellular dedifferentiation plays a major role in this process. How dedifferentiation is induced in vivo in the correct context is unknown and represents a major question in regenerative biology. The cnidarian Hydractinia can regenerate any lost body part in a tissue-specific manner. Regeneration of some body parts is mediated by adult, migratory stem cells called i-cells that are absent from the intact head. Surprisingly, host lab found that isolated heads can, nevertheless, regenerate a fully functional animal including i-cells that arise by dedifferentiation. Following amputation, isolated heads transiently display features consistent with cellular senescence. Here, I propose to study the mechanisms accompanying injury-induced senescence in Hydractinia dedifferentiation-dependent whole-body regeneration. My approach includes the mechanistic characterization of the molecular/cellular events that occur between decapitation and the appearance of new i-cells through dedifferentiation. For this, I will perform transcriptomic analyses, gene function experiments, and in vivo imaging. The strength of my proposal rests upon the usage of a unique animal model that is highly regenerative, displays natural dedifferentiation, and allows performing in vivo experiments that are impossible to conduct in other animals.

Many bacteria have evolved protein machineries to inject proteins into eukaryotic or prokaryotic cells. The type III secretion system (T3SS), widely distributed among gram-negative bacteria, is made up of a needle complex, an export apparatus and a sorting platform (SP). Although, the whole injectosome was characterized in a recent study, the assembly process of the SP is poorly understood. It has been shown that SpaO (a core component of the SP) localizes in two spatial populations, one at the bacterial membrane and another one located within the bacterial cytosol. This observation of the presence of SpaO-associated clusters away from the bacterial membrane suggests the presence of fully or partially assembled SPs in the bacterial cytoplasm. Furthermore, there are two existing versions of SpaO, a short (SpaOS) and a long (SpaOL) version. It has been postulated that SpaOS plays a role in chaperoning SpaOL during the assembly process of the SP but is not a structural part of the SP itself. If this holds true, SpaOS should only be localized in the (partly assembled) SPs located in the bacterial cytosol. In order to understand the assembly of the SP and the role of SpaOS in this process, I will image all proteins of the SP using recently developed 4Pi microscopy together with DNA-PAINT super-resolution microscopy. This novel combination, will enable me to image all proteins of the SP with molecular resolution (5 nm isotropic resolution). This will be an ambitious step towards understanding the sequence of events leading to the assembly of the SP in the T3SS. I anticipate that the tools developed for this work will bring new technologies to the field of bacterial physiology.

Identical genomes in social insects generate castes with distinct morphologies and behaviors. The Harpegnathos ants have a remarkable lifestyle, whereby the queen’s death triggers transition of a subset of workers to become her substitutes, called gamergates. This entails minor changes in morphology, yet a pronounced shift in behavior, and remarkably, a fivefold extension of lifespan. This process is reversible if the gamergate is placed in a colony with a real queen. The capability of such a dramatic transition in adult life provides a unique context to study neuronal plasticity and aging. Moreover, social insects challenge major aging theories as their calorie intake and fecundity positively correlate with lifespan, in contrast to most animals. I aim to provide an extensive mechanistic description of the worker-to-gamergate transition in H. saltator, and to characterize the mechanisms of the resultant aging deceleration. I will study the brain as it regulates caste transition and produces hormones implicated in aging, while also being affected by both processes. First, I will perform single-cell RNA-sequencing of the worker and nascent gamergate brains to describe changes in genes controlling synaptic wiring and cellular signaling. Next, I will test whether deceleration of aging in gamergates is accompanied by decreased rates of molecular damage, protein synthesis, transposon activity, and telomere shortening. Finally, I will combine both datasets to link transcriptional changes during the caste transition with aging-related molecular processes. Deciphering the paradoxical aging mechanism in social insects may uncover previously unknown strategies to prolong lifespan.

Glycerophospholipids are one of the major lipid species in cellular membranes of eukaryotic cells. Among glycerophospholipids, phosphatidylethanolamine (PE) is a highly conserved lipid, found in all organelles but mainly synthesized in the endoplasmic reticulum (ER) and in mitochondria. If it has been shown to play essential roles in membrane protein structure and function, its distribution and metabolism have not been fully elucidated. Indeed, little is known about lipid transport in terms of quantitative and qualitative contribution of each pathway, because methods to measure lipid transport between organelles in cells are lacking. Unlike proteins which are relatively easily manipulated by genetic techniques, individual lipid species cannot be readily modified in vivo. Here, I propose to develop and use chemical tools to study PE transport and metabolism in living cells with high spatiotemporal resolution. I will synthesize photocaged PE which can be localized with precision to mitochondria (or ER) within cells and then released with irradiation of light. Using isotope-labelling, I will trace their transport and quantify the metabolites inside living cells by lipidomics and mass spectrometry approaches. Moreover, mass spectrometry imaging could allow visualizing the de novo labeled phospholipids. This interdisciplinary approach will give us the spatiotemporal resolution that is needed to create for the first time a 4D map of PE metabolism in cells. It will allow us to elucidate its metabolism related to its dependence on intracellular localization and prove that PE can or cannot be transported, for instance, bidirectionally between ER and mitochondria.

Touch the sensitive plant Mimosa pudica and it rapidly changes shape, folding its leaves to deter herbivory and dislodge pests. This and other feats of rapid plant motion have fascinated scientists for centuries, but the physiological mechanisms underlying them remain poorly understood. I propose a collaborative, interdisciplinary project to elucidate two key aspects of rapid plant motion: sensation of mechanical stimuli and rapid generation of force. I hypothesize that knowledge of the sensory and motor strategies of motile animals can inform our approach to the study of rapid plant motion, and outline a novel experimental approach that draws tools and theory from the fields of biomechanics, neurophysiology, and materials science to explore rapid motion in Mimosa pudica, a model system for the study of rapid plant movement. I have identified an ideal host laboratory in which to carry out this work situated at a nexus of expertise in mechanotransduction, plant physiology, and mechanical modeling. To this environment I will bring expertise in skeletal muscle physiology and the biomechanics of force production. The proposed work forges a connection between seemingly disparate areas of physiological research, will provide unique training that increases my technical and theoretical breadth, and has great potential to elucidate basic principles underlying sensory and motor strategies in biological systems.

Protein degradation systems maintain protein homeostasis. A failure of these systems causes various diseases, such as neurodegenerative diseases and cancers. In eukaryotic cells, ubiquitin is a general marker for selective degradation and determines protein lifetime in vivo. In selective degradation, E3 ubiquitin ligases determine target proteins. Although there are ~800 E3 ligases in human, only a handful of them have already shown to recognize specific short peptide motifs called “degrons”. Moreover, the feature(s) for unstable or misfolded structures recognized by E3 ligases remain unclear. In part, this is because we lack a comprehensive approach to investigate the global relationship between protein structural stability, ubiquitination status, and lifetime of the protein. To reveal the effect of protein folding stability (and other features) on ubiquitination and biological lifetime in vivo, I propose to measure these parameters for thousands of designed mini-proteins, whose folding stability has been previously characterized in detail. First, I will measure biological lifetime for these mini-proteins by flow cytometry, and monitor their ubiquitination status by using top-down proteomics approach. Then, I will analyze these data by using in silico analysis and decipher what factor(s) determine ubiquitination states and biological lifetime. This highly innovative and comprehensive approach using thousands of designed proteins will allow me to uncover the fundamental principle for protein lifetime in vivo and provide a mechanistic basis for designing better tools to manipulate protein lifetime.

During gastrulation, embryonic stem cells are primed towards three germ layers: the ectoderm, the mesoderm and the endoderm. While acquiring their fate, the cells re-arrange massively and the embryo polarizes with the appearance of an antero-posterior axis. The signals that are necessary for this patterning and the role of the mechanical forces remain poorly understood, in particular for the human embryo. Synthetic biology has thus developed new models of embryos using human stem cell lines (hESCs). We propose to study the effect of differential mechanical tension on the appearance of a primitive streak in a synthetic human embryo. We will investigate the relationship between the WNT pathway activation and mechanical instability, following recent results on a biomechanical regulation of this pathway. We will first work on the correlation between the substrate induced curvature and WNT expression in 2D cultures of human stem cells. Secondly, we will map the resulting stress field of the cells to this expression pattern. Thirdly, we will study cellular self-organization under mechanical load in a 3D model synthetic embryo. We will eventually focus on the inducer role of a second cell population in the polarization of this synthetic embryo. In the four cases we will correlate the resulting dynamic features with the expression of markers relative to the three germ layers. We hope to provide a first quantitative understanding of the relationship between the force field and the differentiation of stem cells in a model synthetic embryo. This project will be co-supervised by Dr. Eric Siggia and Dr. Ali Brivanlou. The experiments will be performed in the Brivanlou lab.

Metabolic transformation is a key characteristic of cancer cells and considered a promising avenue for therapeutic intervention. Nevertheless, metabolic lethalities identified in laboratories often fail to translate to the clinic. This may be because the complexities of tumor metabolism cannot be recapitulated by studying cancer cell autonomous metabolism alone: tumors are not solid masses of cancer cells, but include host tissue, stroma, and immune cells that together shape the tumor metabolic environment. This insight highlights the need to observe and interrogate tumor metabolism in vivo. Metabolic studies rely heavily on mass spectrometry-based analysis of metabolites extracted from cells or tissues, but this approach lacks spatial resolution to resolve metabolic heterogeneity between cells. To address this unmet need, I will develop a transformative platform for single-cell tissue metabolomics using mass spectrometry imaging and use it to study redox metabolism in liver metastatic cancer to investigate a major unanswered question in cancer research: how does the host tissue support metastatic tumor growth? This work contributes a new strategy to incorporate spatial resolution into metabolomics and provides the first comprehensive analysis of the metabolic symbiosis between host and cancer cells in vivo. It aims to redefine the traditional approach to metabolic studies that can progress our understanding of metabolic heterogeneity. Focusing on liver metastatic cancer, I aim to provide novel mechanistic understanding of redox-metabolic crosstalk between cells in the tumor-host niche and explore strategies to interfere with redox metabolism to combat metastasis.