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We present a protocol to generate highly multiplexed spatial data at cellular and subcellular resolutions using iterative indirect immunofluorescence imaging (4i). We describe streamlined steps for using 4i across fixed cultured cells, formalin-fixed paraffin-embedded (FFPE) tissue sections, and metaphase chromosome spreads. We detail procedures for sample preparation, antibody and DNA staining, immunofluorescence imaging, antibody elution, and image processing. This protocol is adapted for high-throughput analysis of fixed cultured cells and addresses sample-specific challenges such as intrinsic tissue autofluorescence and chromosome fragility. For complete details on the use and execution of this protocol for fixed cultured cells, please refer to Comandante-Lou et al.1.
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Viruses elicit long-term adaptive responses in the tissues they infect. Understanding viral adaptions in humans is difficult in organs such as the heart, where primary infected material is not routinely collected. In search of asymptomatic infections with accompanying host adaptions, we mined for cardio-pathogenic viruses in the unaligned reads of nearly one thousand human hearts profiled by RNA sequencing. Among virus-positive cases (~20%), we identified three robust adaptions in the host transcriptome related to inflammatory NFκB signaling and post-transcriptional regulation by the p38-MK2 pathway. The adaptions are not determined by the infecting virus, and they recur in infections of human or animal hearts and cultured cardiomyocytes. Adaptions switch states when NFκB or p38-MK2 are perturbed in cells engineered for chronic infection by the cardio-pathogenic virus, coxsackievirus B3. Stratifying viral responses into reversible adaptions adds a targetable systems-level simplification for infections of the heart and perhaps other organs.
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Background: Primary luminal breast cancer cells lose their identity rapidly in standard tissue culture, which is problematic for testing hormone interventions and molecular pathways specific to the luminal subtype. Breast cancer organoids are thought to retain tumor characteristics better, but long-term viability of luminal-subtype cases is a persistent challenge. Our goal was to adapt short-term organoids of luminal breast cancer for parallel testing of genetic and pharmacologic perturbations. Methods: We freshly isolated patient-derived cells from luminal tumor scrapes, miniaturized the organoid format into 5 µl replicates for increased throughput, and set an endpoint of 14 days to minimize drift. Therapeutic hormone targeting was mimicked in these "zero-passage" organoids by withdrawing ß-estradiol and adding 4-hydroxytamoxifen. We also examined sulforaphane as an electrophilic stress and commercial neutraceutical with reported anti-cancer properties. Downstream mechanisms were tested genetically by lentiviral transduction of two complementary sgRNAs and Cas9 stabilization for the first week of organoid culture. Transcriptional changes were measured by RT-qPCR or RNA sequencing, and organoid phenotypes were quantified by serial brightfield imaging, digital image segmentation, and regression modeling of cellular doubling times. Results: We achieved >50% success in initiating luminal breast cancer organoids from tumor scrapes and maintaining them to the 14-day zero-passage endpoint. Success was mostly independent of clinical parameters, supporting general applicability of the approach. Abundance of ESR1 and PGR in zero-passage organoids consistently remained within the range of patient variability at the endpoint. However, responsiveness to hormone withdrawal and blockade was highly variable among luminal breast cancer cases tested. Combining sulforaphane with knockout of NQO1 (a phase II antioxidant response gene and downstream effector of sulforaphane) also yielded a breadth of organoid growth phenotypes, including growth inhibition with sulforaphane, growth promotion with NQO1 knockout, and growth antagonism when combined. Conclusions: Zero-passage organoids are a rapid and scalable way to interrogate properties of luminal breast cancer cells from patient-derived material. This includes testing drug mechanisms of action in different clinical cohorts. A future goal is to relate inter-patient variability of zero-passage organoids to long-term outcomes.
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Basal-like breast cancer (BLBC) is highly aggressive, and often characterized by BRCA1 and p53 deficiency. Although conventional mouse models enabled the investigation of BLBC at malignant stages, its initiation and pre-malignant progression remain understudied. Here, we leveraged a mouse genetic system known as mosaic analysis with double markers (MADM) to study BLBC initiation by generating rare GFP+Brca1, p53-deficient mammary cells alongside RFP+ wild-type sibling cells. After confirming the close resemblance of mammary tumors arising in this model to human BLBC at both transcriptomic and genomic levels, we focused our studies on the pre-malignant progression of BLBC. Initiated GFP+ mutant cells showed a stepwise pre-malignant progression trajectory from focal expansion to hyper-alveolarization and then to micro-invasion. Furthermore, despite morphological similarities to alveoli, hyper-alveolarized structures actually originate from ductal cells based on twin-spot analysis of GFP-RFP sibling cells. Finally, luminal-to-basal transition occurred exclusively in cells that have progressed to micro-invasive lesions. Our MADM model provides excellent spatiotemporal resolution to illuminate the pre-malignant progression of BLBC, and should enable future studies on early detection and prevention for this cancer.
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Neoplasias da Mama , Neoplasias Mamárias Animais , Camundongos , Animais , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteína Supressora de Tumor p53/genética , Neoplasias Mamárias Animais/genética , Mama/patologiaRESUMO
Genotoxic stress in mammalian cells, including those caused by anti-cancer chemotherapy, can induce temporary cell-cycle arrest, DNA damage-induced senescence (DDIS), or apoptotic cell death. Despite obvious clinical importance, it is unclear how the signals emerging from DNA damage are integrated together with other cellular signaling pathways monitoring the cell's environment and/or internal state to control different cell fates. Using single-cell-based signaling measurements combined with tensor partial least square regression (t-PLSR)/principal component analysis (PCA) analysis, we show that JNK and Erk MAPK signaling regulates the initiation of cell senescence through the transcription factor AP-1 at early times after doxorubicin-induced DNA damage and the senescence-associated secretory phenotype (SASP) at late times after damage. These results identify temporally distinct roles for signaling pathways beyond the classic DNA damage response (DDR) that control the cell senescence decision and modulate the tumor microenvironment and reveal fundamental similarities between signaling pathways responsible for oncogene-induced senescence (OIS) and senescence caused by topoisomerase II inhibition. A record of this paper's transparent peer review process is included in the supplemental information.
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Senescência Celular , DNA Topoisomerases Tipo II , Animais , DNA Topoisomerases Tipo II/genética , Senescência Celular/genética , Transdução de Sinais , Sistema de Sinalização das MAP Quinases , Dano ao DNA , MamíferosRESUMO
Protein copy numbers constrain systems-level properties of regulatory networks, but absolute proteomic data remain scarce compared to transcriptomics obtained by RNA sequencing. We addressed this persistent gap by relating mRNA to protein statistically using best-available data from quantitative proteomics-transcriptomics for 4366 genes in 369 cell lines. The approach starts with a central estimate of protein copy number and hierarchically appends mRNA-protein and mRNA-mRNA dependencies to define an optimal gene-specific model that links mRNAs to protein. For dozens of independent cell lines and primary prostate samples, these protein inferences from mRNA outmatch stringent null models, a count-based protein-abundance repository, and empirical protein-to-mRNA ratios. The optimal mRNA-to-protein relationships capture biological processes along with hundreds of known protein-protein interaction complexes, suggesting mechanistic relationships are embedded. We use the method to estimate viral-receptor abundances of CD55-CXADR from human heart transcriptomes and build 1489 systems-biology models of coxsackievirus B3 infection susceptibility. When applied to 796 RNA sequencing profiles of breast cancer from The Cancer Genome Atlas, inferred copy-number estimates collectively reclassify 26% of Luminal A and 29% of Luminal B tumors. Protein-based reassignments strongly involve a pharmacologic target for luminal breast cancer (CDK4) and an α-catenin that is often undetectable at the mRNA level (CTTNA2). Thus, by adopting a gene-centered perspective of mRNA-protein covariation across different biological contexts, we achieve accuracies comparable to the technical reproducibility limits of contemporary proteomics. The collection of gene-specific models is assembled as a web tool for users seeking mRNA-guided predictions of absolute protein abundance (http://janeslab.shinyapps.io/Pinferna).
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Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.
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Different cellular compartments within a tissue present distinct cancer-initiating capacities. Current approaches to dissect such heterogeneity require cell-type-specific genetic tools based on a well-understood lineage hierarchy, which are lacking for many tissues. Here, we circumvented this hurdle and revealed the dichotomous capacity of fallopian tube Pax8+ cells in initiating ovarian cancer, utilizing a mouse genetic system that stochastically generates rare GFP-labeled mutant cells. Through clonal analysis and spatial profiling, we determined that only clones founded by rare, stem/progenitor-like Pax8+ cells can expand on acquiring oncogenic mutations whereas vast majority of clones stall immediately. Furthermore, expanded mutant clones undergo further attrition: many turn quiescent shortly after the initial expansion, whereas others sustain proliferation and manifest a bias toward Pax8+ fate, underlying early pathogenesis. Our study showcases the power of genetic mosaic system-based clonal analysis for revealing cellular heterogeneity of cancer-initiating capacity in tissues with limited prior knowledge of lineage hierarchy.
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Basal-like breast cancer is an aggressive breast cancer subtype, often characterized by a deficiency in BRCA1 function and concomitant loss of p53 . While conventional mouse models enable the investigation of its malignant stages, one that reveals its initiation and pre-malignant progression is lacking. Here, we leveraged a mouse genetic system known as M osaic A nalysis with D ouble M arkers (MADM) to generate rare GFP-labeled Brca1 , p53 -deficient cells alongside RFP+ wildtype sibling cells in the mammary gland. The mosaicism resembles the sporadic initiation of human cancer and enables spatially resolved analysis of mutant cells in comparison to paired wildtype sibling cells. Mammary tumors arising in the model show transcriptomic and genomic characteristics similar to human basal-like breast cancer. Analysis of GFP+ mutant cells at interval time points before malignancy revealed a stepwise progression of lesions from focal expansion to hyper-alveolarization and then to micro-invasion. These stereotyped morphologies indicate the pre-malignant stage irrespective of the time point at which it is observed. Paired analysis of GFP-RFP siblings during focal expansion suggested that hyper-alveolarized structures originate from ductal rather than alveolar cells, despite their morphological similarities to alveoli. Evidence for luminal-to-basal transition at the pre-malignant stages was restricted to cells that had escaped hyper-alveoli and progressed to micro-invasive lesions. Our MADM-based mouse model presents a useful tool for studying the pre-malignancy of basal-like breast cancer. Summary statement: A mouse model recapitulates the process of human basal-like breast tumorigenesis initiated from sporadic Brca1, p53 -deficient cells, empowering spatially-resolved analysis of mutant cells during pre-malignant progression.
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Activation of HER2/ErbB2 coincides with escape from ductal carcinoma in situ (DCIS) premalignancy and disrupts 3D organization of cultured breast-epithelial spheroids. The 3D phenotype is infrequent, however, and mechanisms for its incomplete penetrance have been elusive. Using inducible HER2/ErbB2-EGFR/ErbB1 heterodimers, we match phenotype penetrance to the frequency of co-occurring transcriptomic changes and uncover a reconfiguration in the karyopherin network regulating ErbB nucleocytoplasmic transport. Induction of the exportin CSE1L inhibits nuclear accumulation of ErbBs, whereas nuclear ErbBs silence the importin KPNA1 by inducing miR-205. When these negative feedbacks are incorporated into a validated systems model of nucleocytoplasmic transport, steady-state localization of ErbB cargo becomes ultrasensitive to initial CSE1L abundance. Erbb2-driven carcinomas with Cse1l deficiency outgrow less irregularly from mammary ducts, and NLS-attenuating mutants or variants of HER2 favor escape in 3D culture. We conclude here that adaptive nucleocytoplasmic relocalization of HER2 creates a systems-level molecular switch at the premalignant-to-malignant transition.
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Carcinoma Intraductal não Infiltrante , Humanos , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/patologia , Transporte Ativo do Núcleo Celular , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Perfilação da Expressão Gênica , TranscriptomaRESUMO
Leading researchers at the intersection of infectious disease and systems biology speak about how systems approaches have influenced modern infectious disease research and what these tools can offer for the future of the field.
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Doenças Transmissíveis , Humanos , Doenças Transmissíveis/terapia , Biologia de SistemasRESUMO
Many lncRNAs have been discovered using transcriptomic data; however, it is unclear what fraction of lncRNAs is functional and what structural properties affect their phenotype. MUNC lncRNA (also known as DRReRNA) acts as an enhancer RNA for the Myod1 gene in cis and stimulates the expression of other promyogenic genes in trans by recruiting the cohesin complex. Here, experimental probing of the RNA structure revealed that MUNC contains multiple structural domains not detected by prediction algorithms in the absence of experimental information. We show that these specific and structurally distinct domains are required for induction of promyogenic genes, for binding genomic sites and gene expression regulation, and for binding the cohesin complex. Myod1 induction and cohesin interaction comprise only a subset of MUNC phenotype. Our study reveals unexpectedly complex, structure-driven functions for the MUNC lncRNA and emphasizes the importance of experimentally determined structures for understanding structure-function relationships in lncRNAs.
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Desenvolvimento Muscular/genética , RNA Longo não Codificante/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Diferenciação Celular/genética , Linhagem Celular , Feminino , Genoma , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Conformação de Ácido Nucleico , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Longo não Codificante/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de SequênciaRESUMO
We describe how to use a publicly available computational model for coxsackievirus B3 (CVB3) infection that we recast as a graphical user interface (GUI). The GUI-based implementation enables non-computationalists to incorporate systems-biology modeling into their research and teaching. The model simulates the full life cycle of CVB3, including the host antiviral response, and includes 44 alterable parameters. The model simplifies some viral life cycle processes to improve interpretability and utility when performing in silico experiments. For complete details on the use and execution of this protocol, please refer to Lopacinski et al. (2021).
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Simulação por Computador , Infecções por Coxsackievirus/virologia , Enterovirus Humano B , Biologia de Sistemas/métodos , Interface Usuário-Computador , Enterovirus Humano B/patogenicidade , Enterovirus Humano B/fisiologia , Humanos , Cinética , Software , Vírion/patogenicidade , Vírion/fisiologiaRESUMO
Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.
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Enterovirus Humano B/genética , Interações Hospedeiro-Patógeno/genética , Humanos , CinéticaRESUMO
The heterogeneous composition of solid tumors is known to impact disease progression and response to therapy. Malignant cells coexist in different regulatory states that can be accessed transcriptomically by single-cell RNA sequencing, but these methods have many caveats related to sensitivity, noise, and sample handling. We revised a statistical fluctuation analysis called stochastic profiling to combine with 10-cell RNA sequencing, which was designed for laser-capture microdissection (LCM) and extended here for immuno-LCM. When applied to a cohort of late-onset, early-stage luminal breast cancers, the integrated approach identified thousands of candidate regulatory heterogeneities. Intersecting the candidates from different tumors yielded a relatively stable set of 710 recurrent heterogeneously expressed genes (RHEG), which were significantly variable in >50% of patients. RHEGs were not strongly confounded by dissociation artifacts, cell-cycle oscillations, or driving mutations for breast cancer. Rather, RHEGs were enriched for epithelial-to-mesenchymal transition genes and, unexpectedly, the latest pan-cancer assembly of driver genes across cancer types other than breast. These findings indicate that heterogeneous transcriptional regulation conceivably provides a faster, reversible mechanism for malignant cells to evaluate the effects of potential oncogenes or tumor suppressors on cancer hallmarks. SIGNIFICANCE: Profiling intratumor heterogeneity of luminal breast carcinoma cells identifies a recurrent set of genes, suggesting sporadic activation of pathways known to drive other types of cancer.See related articles by Schaff and colleagues, p. 1853 and Sutcliffe and colleagues, p. 1868.
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Neoplasias da Mama , Neoplasias Pulmonares , Mama , Neoplasias da Mama/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Oncogenes , Microambiente TumoralRESUMO
Cancer evolves from premalignant clones that adopt unusual cell states to achieve transformation. We previously pinpointed the oligodendrocyte precursor cell (OPC) as a cell of origin for glioma, but the early changes of mutant OPCs during premalignancy remained unknown. Using mice engineered for inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection, determined global gene-expression changes by bulk RNA sequencing, and compared with cell-state fluctuations at the single-cell level by stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. At 12 days after Nf1-Trp53 deletion, bulk differences were mostly limited to mitotic hallmarks and genes for ribosome biosynthesis, and stochastic profiling revealed a spectrum of stem-progenitor (Axl, Aldh1a1), proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days, bulk sequencing detected few differentially expressed transcripts, whereas stochastic profiling revealed cell states for neurons and mural cells that do not give rise to glial tumors, suggesting cellular dead-ends for gliomagenesis. Importantly, mutant OPCs that strongly expressed key effectors of nonsense-mediated decay (Upf3b) and homology-dependent DNA repair (Rad51c, Slx1b, Ercc4) were identified along with DNA-damage markers, suggesting transcription-associated replication stress. Analysis of 10-cell transcriptomes at 90 days identified a locus of elevated gene expression containing an additional repair endonuclease (Mus81) and Rin1, a Ras-Raf antagonist and possible counterbalance to Nf1 loss, which was microdeleted or downregulated in gliomas at 150 days. These hidden cell-state variations uncover replication stress as a potential bottleneck that must be resolved for glioma initiation. SIGNIFICANCE: Profiling premalignant cell states in a mouse model of glioma uncovers regulatory heterogeneity in glioma cells-of-origin and defines a state of replication stress that precedes tumor initiation.See related articles by Singh and colleagues, p. 1840 and Schaff and colleagues, p. 1853.
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Neoplasias da Mama , Glioma , Células Precursoras de Oligodendrócitos , Animais , Transformação Celular Neoplásica , Proteínas de Ligação a DNA , Endonucleases , Feminino , Glioma/genética , Humanos , Camundongos , Oligodendroglia , Proteínas de Ligação a RNARESUMO
Small-cell lung cancers derive from pulmonary neuroendocrine cells, which have stem-like properties to reprogram into other cell types upon lung injury. It is difficult to uncouple transcriptional plasticity of these transformed cells from genetic changes that evolve in primary tumors or secondary metastases. Profiling of single cells is also problematic if the required sample dissociation activates injury-like signaling and reprogramming. Here we defined cell-state heterogeneities in situ through laser capture microdissection-based 10-cell transcriptomics coupled with stochastic-profiling fluctuation analysis. In labeled cells from a small-cell lung cancer mouse model initiated by neuroendocrine deletion of Rb1-Trp53, variations in transcript abundance revealed cell-to-cell differences in regulatory state in vitro and in vivo. Fluctuating transcripts in spheroid culture were partly shared among Rb1-Trp53-null models, and heterogeneities increased considerably when cells were delivered intravenously to colonize the liver. Colonization of immunocompromised animals drove a fractional appearance of alveolar type II-like markers and poised cells for paracrine stimulation from immune cells and hepatocytes. Immunocompetency further exaggerated the fragmentation of tumor states in the liver, yielding mixed stromal signatures evident in bulk sequencing from autochthonous tumors and metastases. Dozens of transcript heterogeneities recurred irrespective of biological context; their mapped orthologs brought together observations of murine and human small-cell lung cancer. Candidate heterogeneities recurrent in the liver also stratified primary human tumors into discrete groups not readily explained by molecular subtype but with prognostic relevance. These data suggest that heterotypic interactions in the liver and lung are an accelerant for intratumor heterogeneity in small-cell lung cancer. SIGNIFICANCE: These findings demonstrate that the single-cell regulatory heterogeneity of small-cell lung cancer becomes increasingly elaborate in the liver, a common metastatic site for the disease.See related articles by Singh and colleagues, p. 1840 and Sutcliffe and colleagues, p. 1868.
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Neoplasias da Mama , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Animais , Feminino , Humanos , Pulmão , Neoplasias Pulmonares/genética , Camundongos , Recidiva Local de Neoplasia , Carcinoma de Pequenas Células do Pulmão/genética , Microambiente TumoralRESUMO
Caloric restriction (CR) improves health span and life span of organisms ranging from yeast to mammals. Understanding the mechanisms involved will uncover future interventions for aging-associated diseases. In budding yeast, Saccharomyces cerevisiae, CR is commonly defined by reduced glucose in the growth medium, which extends both replicative and chronological life span (CLS). We found that conditioned media collected from stationary-phase CR cultures extended CLS when supplemented into nonrestricted (NR) cultures, suggesting a potential cell-nonautonomous mechanism of CR-induced life span regulation. Chromatography and untargeted metabolomics of the conditioned media, as well as transcriptional responses associated with the longevity effect, pointed to specific amino acids enriched in the CR conditioned media (CRCM) as functional molecules, with L-serine being a particularly strong candidate. Indeed, supplementing L-serine into NR cultures extended CLS through a mechanism dependent on the one-carbon metabolism pathway, thus implicating this conserved and central metabolic hub in life span regulation.