Biology subjects

Huang, Y.

Publications and source records attributed to Huang, Y..

At least 685 records · Page 38Linked to original sources

bioRxiv2022

Osteocytes regulate organismal senescence of bone and bone marrow

The skeletal system contains a series of sophisticated cellular lineages arisen from the mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC), that determine the homeostasis of bone and bone marrow. Here we reasoned that osteocyte may exert a function in regulation of these lineage cell specifications and tissue homeostasis. Using a mouse model of conditional deletion of osteocytes by the expression of diphtheria toxin subunit (DTA) in dentin matrix protein 1 (DMP-1) positive osteocytes, we demonstrated that partial ablation of DMP-1positive osteocytes caused severe sarcopenia, osteoporosis and degenerative kyphosis, leading to shorter lifespan in these animals. Osteocyte reduction altered mesenchymal lineage commitment resulting in impairment of osteogenesis and induction of osteoclastogensis. Single cell RNA sequencing further revealed that hematopoietic lineage was mobilized towards myeloid lineage differentiation with expanded myeloid progenitors, neutrophils and monocytes, while the lymphopoiesis was impaired with reduced B cells in the osteocyte ablation mice. The acquisition of a senescence-associated secretory phenotype (SASP) in both osteoprogenic and myeloid lineage cells was the underlying cause. Together, we showed that osteocytes play critical roles in regulating of lineage cell specifications in bone and bone marrow through mediation of organismal senescence.

cell biology↗

bioRxiv2022

Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

ACE2 is a major receptor for cell entry of SARS-CoV-2. Despite advances in targeting ACE2 to inhibit SARS-CoV-2s binding, how to efficiently and flexibly control ACE2 levels for prevention of SARS-CoV-2 infection has not been explored. Here, we revealed Vitamin C (VitC) administration as an effective strategy to prevent SARS-CoV-2 infection. VitC reduced ACE2 protein levels in a dose-dependent manner, while partial reduction of ACE2 can greatly restrict SARS-CoV-2 infection. Further studies uncovered that USP50 is a crucial regulator of ACE2 protein levels, and VitC blocks the USP50-ACE2 interaction, thus promoting K48-linked polyubiquitination at Lys788 and degradation of ACE2, without disrupting ACE2 transcriptional expression. Importantly, VitC administration reduced host ACE2 and largely blocked SARS-CoV-2 infection in mice. This study identified an in vivo ACE2 balance controlled by both USP50 and an essential nutrient VitC, and revealed a critical role and application of VitC in daily protection from SARS-CoV-2 infection. HighlightsO_LIVitC reduces ACE2 protein levels in a dose-dependent manner C_LIO_LIVitC and USP50 regulate K48-linked ubiquitination at Lys788 of ACE2 C_LIO_LIVitC blocks the interaction between USP50 and ACE2 C_LIO_LIVitC administration lowers host ACE2 and prevents SARS-CoV-2 infection in vivo C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/499651v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@196682borg.highwire.dtl.DTLVardef@190f14dorg.highwire.dtl.DTLVardef@d22b59org.highwire.dtl.DTLVardef@1c0faa_HPS_FORMAT_FIGEXP M_FIG C_FIG The deubiquitinase USP50 controls ACE2 protein stability and levels, while Vitamin C blocks the USP50-ACE2 interaction and therefore results in ACE2 degradation, offering a flexible and efficient approach to protection of the host from SARS-CoV-2 infection.

microbiology↗

bioRxiv2022

Computational Enhanced Hi-C data reveals the function of structural geometry in genomic regulation

High-throughput chromosome conformation capture (Hi-C) technique profiles the genomic structure in a genome-wide fashion. The reproducibility and consistency of Hi-C data are essential in characterizing dynamics of genomic structures. We developed a diffusion-based method, CTG (Hi-C To Geometry), to deal with the technical bias induced by insufficient sampling in sequencing and obtain reliable gemeotric information of the chromatin. CTG properly quantifies dubiously weak or even undetected interactions and produces a consistent and reproducible framework for the 3D genomic structure. CTG allows for a reliable genome-wide insight on the alteration of genomic structures under different cellular conditions and reveals correlations between genomic-proximal genes at both transcriptional and translational levels. Cell-specific correspondence between gene-gene and corresponding protein-protein physical interactions, as well as that with the transcription correlation reveals the coordinated inter-molecular structural and regulatory information passage in the central dogma.

genomics↗

bioRxiv2022

Kynurenic acid promotes A1 to A2 astrocyte conversion against HIV-associated neurocognitive disorder

Although extensive astrocyte activation is present in the CNS of patients suffering from HIV-associated neurocognitive disorder (HAND), little is known about the contribution of reactive astrocytes to HAND neuropathology. Here, we report that the imbalance between neurotoxic (A1 astrocytes) and protective astrocytes (A2 astrocytes) occurring in HIV-1 gp120 transgenic mice impedes the resolution of inflammation in the CNS, perpetuating neuron damage and cognitive deficits. Kynurenic acid (KYNA), a tryptophan metabolite with 7 nicotinic acetylcholine receptors (7nAChR) inhibitory property, blunts gp120-induced A1 astrocyte activation while promoting A2 astrocyte formation through blockade of 7nAChR/JAK2/STAT3 signaling activation. Meanwhile, we provide evidence that 7nAChR deletion and tryptophan administration convert astrocyte phenotypes, ultimately facilitating neuronal and cognitive improvement in the gp120tg mice. These initial and determinant findings mark a turning point in our understanding of the role of 7nAChR in gp120-mediated astrocyte activation, which opens new opportunities to control reactive astrocyte generation through KYNA and tryptophan administration as a pharmacological approach for treating HAND.

microbiology↗

bioRxiv2022

Tetraspanin 4 mediates migrasome formation via a two-stage mechanism

Migrasomes are recently discovered signalling organelles, enriched with tetraspanin proteins (TSPAN)1. They form by local swelling of retraction fibers, the cylindrical protrusions of cell membranes that form as a result of cell migration along external substrates. Migrasomes can grow up to several micrometers in diameter2, and allow cells to release contents such as chemokines at specific locations, hence, transmitting signals to surrounding cells through the relevant chemokine receptors. Recently, evidence emerged showing that migrasomes play essential roles in fundamental cellular processes such transfer of mRNA and proteins3, organ morphogenesis4, and mitochondria quality control5. Thus, understanding the mechanism of migrasome biogenesis is of outstanding importance. Previously, it was established that the molecules crucial for migrasome formation are tetraspanin proteins and cholesterol forming macrodomains in the migrasome membrane, while the physical forces driving local swelling of the retraction fibers originate from membrane tension and bending rigidity1. Yet, it remained unknown how and in which time sequence these factors are involved in migrasome nucleation, growth, and stabilization, and what are the possible intermediate stages of migrasome biogenesis.

biophysics↗

bioRxiv2022

Making memories last: The peripheral effect of direct current stimulation on strengthening memories

Most memories that are formed are forgotten, while others are retained longer and are subject to memory stabilization. We show that non-invasive transcutaneous electrical stimulation of the greater occipital nerve (NITESGON) using direct current during learning elicited a long-term memory effect. However, it did not trigger an immediate effect on learning. A neurobiological model of long-term memory proposes a mechanism by which memories that are initially unstable can be strengthened through subsequent novel experiences. In a series of studies, we demonstrate NITESGONs capability to boost the retention of memories when applied shortly before, during or shortly after the time of learning by enhancing memory consolidation via activation and communication in and between the locus coeruleus pathway and hippocampus by modulating dopaminergic input. These findings may have a significant impact for neurocognitive disorders that inhibit memory consolidation such as Alzheimers disease.

neuroscience↗

bioRxiv2022

Noninvasive cancer detection by extracting and integrating multi-modal data from whole-methylome sequencing of plasma cell-free DNA

AbstractPlasma cell-free DNA (cfDNA) methylation and fragmentation signatures have been shown to be valid biomarkers for blood-based cancer detection. However, conventional methylation sequencing assays are inapplicable for fragmentomic profiling due to bisulfite-induced DNA damage. Here using enzymatic conversion-based low-pass whole-methylome sequencing (WMS), we developed a novel approach to comprehensively interrogate the genome-wide plasma methylation, fragmentation, and copy number profiles for sensitive and noninvasive multi-cancer detection. With plasma WMS data from a clinical cohort comprising 497 healthy controls and 780 patients with both early- and advanced-stage cancers of the breast, colorectum, esophagus, stomach, liver, lung, or pancreas, genomic features including methylation, fragmentation size, copy number alteration, and fragment end motif were extracted individually and subsequently integrated to develop an ensemble cancer classifier, called THEMIS, using machine learning algorithms. THEMIS outperformed individual biomarkers for differentiating cancer patients of all seven types from healthy individuals and achieved a combined area under the curve value of 0.971 in the independent test cohort, translating to a sensitivity of 86% and early-stage (I and II) sensitivity of 77% at 99% specificity. In addition, we built a cancer signal origin classifier with true-positive cancer samples at 100% specificity based on methylation and fragmentation profiling of tissue-specific accessible regulatory elements, which localized cancer-like signal to a limited number of clinically informative sites with 66% accuracy. Overall, this proof-of-concept work demonstrates the feasibility of extracting and integrating multi-modal biomarkers from a single WMS run for noninvasive detection and localization of common cancers across stages.

cancer biology↗

bioRxiv2022

CARD8 negatively regulates NLRP1 inflammasome activation level by interaction with NLRP1

NLRP1 inflammasome functions as canonical cytosolic sensor in response to intracellular infections and is implicated in auto-inflammatory diseases. But the regulation and signal transduction mechanisms of NLRP1 are incompletely understood. Here, we show that the T60 variant of CARD8, but not the canonical T48 isoform, negatively regulates NLRP1 inflammasome activation by directly interacting with the receptor molecule NLRP1 and inhibiting inflammasome assembly. Furthermore, our results suggest that the different ASC preference in three types of inflammasomes, namely ASC-indispensable NLRP1 inflammasome, ASC-dispensable mNLRP1b inflammasome and ASC-independent CARD8 inflammasome, is mainly caused by the CARD domain, not the UPA subdomain. Based on the systematic site-directed mutagenesis and structural analysis, we find that the signal transduction of NLRP1 inflammasome relies on multiple interaction surfaces on its death domain superfamily member CARD domain. Finally, our results partly explain the mechanism of the NLRP1 mutation-derived auto-inflammatory diseases caused by the overactivation of the NLRP1 inflammasome. In conclusion, our study not only reveals how CARD8 downregulates NLRP1 inflammasome activation, but also provides insights into the mechanisms of CARD-containing inflammasome assembly.

immunology↗

bioRxiv2022

Mechanoregulatory role of TRPV4 in prenatal skeletal development

Biophysical cues are essential for guiding skeletal development, but the mechanisms underlying the physical regulation of cartilage and bone formation are unknown. TRPV4 is a cell membrane ion channel responsible for transducing mechanical stimuli as a means of regulating skeletal cell homeostatic processes. Dysregulation of TRPV4 is associated with several skeletal developmental pathologies, indicating its involvement in cartilage and bone development, potentially in a mechanosensing capacity. In this study, we test the hypothesis that mechanically mediated prenatal skeletogenesis depends on TRPV4 activity. We first validate a novel model where we establish that dynamically loading embryonic mouse hindlimb explants cultured ex vivo promotes joint cartilage growth and morphogenesis, but not diaphyseal mineralization. We next reveal that TRPV4 protein expression is mechanically regulated and spatially localized to patterns of high biophysical stimuli in the femoral condyles of cultured limbs. Finally, we demonstrate that TRPV4 activity is crucial for the mechanical regulation of joint cartilage growth and shape, mediated via the control of cell proliferation and matrix biosynthesis, indicating a mechanism by which mechanical loading could direct morphogenesis during joint formation. We conclude that the regulatory pathways initiated by TRPV4 mechanotransduction are essential for the for the cartilage response to physical stimuli during skeletal development. Therefore, TRPV4 may be a valuable target for the development of therapeutic skeletal disease modifying drugs and developmentally-inspired tissue engineering strategies for skeletal repair.

developmental biology↗

bioRxiv2022

A knowledge integration strategy for the selection of a robust multi-stress biomarkers panel for Bacillus subtilis

Recent advances in high-throughput omics technologies have enhanced the identification of molecular biomarkers specific to phenotypes or states in bacteria. Using these biomarkers to monitor the state of bacteria used in biotechnological processes promises to increase process efficiency. However, live-cell monitoring systems applied to recognise bacterial cellular states in real time can only accommodate a small number of gene expression biomarkers. Computational methods are required to identify and prioritise robust biomarkers for experimental characterisation and verification. This study focused on designing a knowledge integration strategy for the selection of an optimal minimised gene expression biomarker panel to sense various stress states in Bacillus subtilis. We developed a computational method that ranks the candidate biomarker panels based on complementary information from machine learning model, gene regulatory network and co-expression network. We identified a recommended biomarker panel showing high stress sensing power for a variety of conditions included in both the dataset used for biomarker identification (mean f1-score achieved at 0.99) and the independent datasets from different sources (mean f1-score achieved at 0.98). We discovered a significant correlation between stress sensing power and evaluation metrics such as the number of associated regulators in a B. subtilis gene regulatory network (GRN) and the number of associated modules in a B. subtilis co-expression network (CEN). GRNs and CENs provide information relevant to the diversity of biological processes encoded by biomarker genes. We demonstrate that quantitatively relating meaningful evaluation metrics with stress sensing power has potential for recognising biomarkers that show better sensitivity and robustness to an extended set of stress conditions. We conclude that this approach is readily applicable to biomarker discovery model selection.

systems biology↗

bioRxiv2022

A beary good genome: Haplotype-resolved, chromosome-level assembly of the brown bear (Ursus arctos)

The brown bear (Ursus arctos) is the second largest and most widespread extant terrestrial carnivore on Earth and has recently emerged as a medical model for human metabolic diseases. Here, we report a fully-phased chromosome-level assembly of a male North American brown bear built by combining Pacific Biosciences (PacBio) HiFi data and publicly available Hi-C data. The final genome size is 2.47 Gigabases (Gb) with a scaffold and contig N50 length of 70.08 and 43.94 Mb, respectively. BUSCO analysis revealed that 94.5% of single-copy orthologs from mammalia were present in the genome (the highest of any ursid genome to date). Repetitive elements accounted for 44.48% of the genome and a total of 20,480 protein coding genes were identified. Based on whole genome alignment, the brown bear is highly syntenic with the polar bear, and our phylogenetic analysis of 7,246 single-copy BUSCOs supports the currently proposed species tree for Ursidae. This highly contiguous genome assembly will support future research on both the evolutionary history of the bear family and the physiological mechanisms behind hibernation, the latter of which has broad medical implications. SignificanceBrown bears (Ursus arctos) are the most widespread, large terrestrial carnivore on the planet and represent an interesting example of speciation through hybridization, as well as a medical model for sedentary lifestyle-related disease. Although a previous genome for a brown bear has been published, the reported contig N50 was low (only [~]530 kb), despite being scaffolded into putative chromosomes. Genomes of this quality limit the accuracy of analyses which rely on long contiguous stretches of the genome to be assembled (such as with many demographic analyses) as well as attempts at connecting genotype to phenotype (such as in association analyses). In order to support studies on both the complex hybridization history of the brown bear and investigations into medically-relevant phenotypes, we generated a fully-phased, chromosome-level assembly from a male grizzly bear. The genome has a total size of 2.47 Gb and 90% of the genome is contained in 36 scaffolds, roughly corresponding to one autosome per scaffold. This high-quality genome will enable studies across a variety of disciplines, including conservation, evolution, and medicine.

genomics↗

bioRxiv2022

Early and lifelong effects of APOE4 on neuronal gene expression networks relevant to Alzheimer's disease

Apolipoprotein E4 (APOE4) genotype and aging are critical risk factors for Alzheimers disease (AD). Aged APOE4 knock-in (APOE4-KI) mice have phenotypes reflecting features of AD. We conducted a large-scale single nucleus RNA-sequencing study to identify cell-type-specific effects of APOE4 on hippocampal gene expression during aging. APOE4-KI mice showed prominent alterations, relative to APOE3-KI mice, in neuronal transcriptome related to synaptic function, calcium signaling, and MAPK/Rap1/Pld signal transduction, starting by 5 months and persisting during aging. Mice with the APOE4 gene removed specifically from neurons failed to show most of these neuronal transcriptomic changes, suggesting a specific effect of neuron-derived APOE4 on the transcriptome. APOE4 affects similar cellular pathways in induced pluripotent stem cell-derived human neurons transplanted into APOE4-KI mouse hippocampus and in cortical neurons from aged human brains. Thus, neuronal APOE4 has early and persistent effects on neuronal transcriptomes, suggesting the requirement of early interventions for successfully treating APOE4-related AD.

neuroscience↗

bioRxiv2022

Increased α-ketoglutarate as a missing link from the C3-C4 intermediate state to C4 photosynthesis in the genus Flaveria

As a complex trait, C4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C3 towards C4 photosynthesis. However, the exact molecular mechanism underlying the transition between C2 photosynthesis towards C4 photosynthesis remains elusive. Here, we provide multiple evidence suggesting a role of higher -ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increases in the C3-C4 intermediate with a further increase in C4 species. Petiole feeding of AKG increased the concentrations of C4 related metabolites in C3-C4 and C4 species but not the activity of C4 related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C4 photosynthesis. Simulations with a constraint-based model for C3-C4 intermediate further show that decreasing the activity of Fd-GOGAT facilitates the transition from a C2-dominant to a C4-dominant CO2 concentrating mechanisms. All these provide an insight into the mechanistic switch from C3-C4 intermediate to C4 photosynthesis.

plant biology↗

bioRxiv2022

Orchestration of alternative splicing regulates bone marrow mesenchymal stem cells fate during aging

Senescence and change of differentiation direction in bone marrow stromal cells (BMSCs) are two of the most important causes of age-related bone loss. As an important post-transcriptional regulatory pathway, alternative splicing (AS) regulates diversity of gene expression. However, the role of AS in BMSCs during aging remains poorly defined. Here we identify AS in specific genes disrupt gene expression pattern and result in age-related debility of BMSCs. We demonstrate the deficiency of splicing factor Y-box protein 1 (YBX1) result in mis-splicing in genes such as Fn1, Taz, Sirt2 and Sp7, further contributing to senescence and shift in differentiation direction of BMSCs during aging. Deletion or over-expression of YBX1 in BMSCs accelerate bone loss or stimulate bone formation in mice. Notably, we identify a small compound sciadopitysin which attenuate the degradation of YBX1 and attenuate bone loss in old mice. Our study demonstrates elaborately controlled RNA splicing governs cell fate of BMSCs and provides a potential therapeutic target for age-related osteoporosis. SummaryThis study demonstrates that YBX1 deficiency induces pre-mRNA mis-splicing and causes senescence and shift in differentiation direction of BMSCs and further accelerates aging-related bone loss. This study identifies Sciadopitysin could reverse this process by targeting YBX1.

cell biology↗

bioRxiv2022

Stable High-Concentration Monoclonal Antibody Formulations Enabled by an Amphiphilic Copolymer Excipient

Monoclonal antibodies are a staple in modern pharmacotherapy. Unfortunately, these biopharmaceuticals are limited by their tendency to aggregate in formulation, resulting in poor stability and often requiring low concentration drug formulations. Moreover, existing excipients designed to stabilize these formulations are often limited by their toxicity and tendency to form particles such as micelles. Here, we demonstrate the ability of a simple "drop-in", amphiphilic copolymer excipient to enhance the stability of high concentration formulations of clinically-relevant monoclonal antibodies without altering their pharmacokinetics or injectability. Through interfacial rheology and surface tension measurements, we demonstrate that the copolymer excipient competitively adsorbs to formulation interfaces. Further, through determination of monomeric composition and retained bioactivity through stressed aging, we show that this excipient confers a significant stability benefit to high concentration antibody formulations. Finally, we demonstrate that the excipient behaves as an inactive ingredient, having no significant impact on the pharmacokinetic profile of a clinically relevant antibody in mice. This amphiphilic copolymer excipient demonstrates promise as a simple formulation additive to create stable, high concentration antibody formulations, thereby enabling improved treatment options such as a route-of-administration switch from low concentration intravenous (IV) to high concentration subcutaneous (SC) delivery while reducing dependence on the cold chain.

bioengineering↗

bioRxiv2022

SARS-CoV-2 Omicron BA.2.12.1, BA.4, and BA.5 subvariants evolved to extend antibody evasion

SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 have surged dramatically to become dominant in the United States and South Africa, respectively1,2. These novel subvariants carrying additional mutations in their spike proteins raise concerns that they may further evade neutralizing antibodies, thereby further compromising the efficacy of COVID-19 vaccines and therapeutic monoclonals. We now report findings from a systematic antigenic analysis of these surging Omicron subvariants. BA.2.12.1 is only modestly (1.8-fold) more resistant to sera from vaccinated and boosted individuals than BA.2. However, BA.4/5 is substantially (4.2-fold) more resistant and thus more likely to lead to vaccine breakthrough infections. Mutation at spike residue L452 found in both BA.2.12.1 and BA.4/5 facilitates escape from some antibodies directed to the so-called class 2 and 3 regions of the receptor-binding domain3. The F486V mutation found in BA.4/5 facilitates escape from certain class 1 and 2 antibodies but compromises the spike affinity for the viral receptor. The R493Q reversion mutation, however, restores receptor affinity and consequently the fitness of BA.4/5. Among therapeutic antibodies authorized for clinical use, only bebtelovimab retains full potency against both BA.2.12.1 and BA.4/5. The Omicron lineage of SARS-CoV-2 continues to evolve, successively yielding subvariants that are not only more transmissible but also more evasive to antibodies.

microbiology↗

bioRxiv2022

Striatal Mu-Opioid Receptor Activation Triggers Direct-Pathway GABAergic Plasticity to Induce Negative Affect

Withdrawal from chronic opioid use often causes hypodopaminergic states and negative affect, which drives relapse. Direct-pathway medium spiny neurons (dMSNs) in the striatal patch compartment contain high levels of {micro}-opioid receptors (MORs). It remains unclear how chronic opioid exposure affects these MOR-expressing dMSNs and their striatopallidal and striatonigral outputs to induce negative emotions and relapse. Here, we report that MOR activation acutely suppressed GABAergic striatopallidal transmission in habenula-projecting globus pallidus neurons. Notably, repeated administrations of a MOR agonist (morphine or fentanyl) potentiated this GABAergic transmission. We also discovered that intravenous self-administration of fentanyl enhanced GABAergic striatonigral transmission and reduced the firing activity of midbrain dopaminergic neurons. Importantly, fentanyl withdrawal caused depression-like behaviors and promoted the reinstatement of fentanyl-seeking behaviors. These data suggest that chronic opioid use triggers GABAergic striatopallidal and striatonigral plasticity to induce a hypodopaminergic state, promoting negative emotions and leading to relapse. HighlightsO_LIRepeated administration of morphine potentiates IPSCdMSN{lozenge}GPh neurotransmission. C_LIO_LIRepeated administration of fentanyl potentiates IPSCdMSN{lozenge}SNc neurotransmission. C_LIO_LIFentanyl withdrawal induces negative emotional states, which drive relapse. C_LI

neuroscience↗

bioRxiv2022

Characterizing metabolism from bulk and single-cell RNA-seq data using METAFlux

Cells often alter metabolic strategies under nutrient-deprived conditions to support their survival and growth. Characterizing metabolic reprogramming in the TME (Tumor Microenvironment) is of emerging importance in ongoing cancer research and therapy development. Recent developments in mass spectrometry (MS)-based technologies allow simultaneous characterization of metabolic features of tumor, stroma, and immune cells in the TME. However, they only measure a subset of metabolites and cannot provide in situ measurements. Computational methods such as flux balance analysis (FBA) have been developed to estimate metabolic flux from bulk RNA-seq data and have recently been extended to single-cell RNA-seq (scRNA-seq) data. However, it is unclear how reliable the results are, particularly in the context of tissue TME characterization. To investigate this question and fill the analytical gaps, we developed a computational program METAFlux (METAbolic Flux balance analysis), which extends the FBA framework to infer metabolic fluxes from either bulk or single-cell transcriptomic TME data. We benchmarked the prediction accuracy of METAFlux using the exometabolomics data generated on the NCI-60 cell lines and observed significant improvement over existing approaches. We tested METAFlux in bulk RNA-seq data obtained from various tumor types including those in the TCGA. We validated previous knowledge, e.g., lung squamous cell carcinoma (LUSC) has higher glucose uptake than lung adenocarcinoma (LUAD). We also found a novel subset of LUAD samples with unique metabolic profiles and distinct survival outcome. We further examined METAFlux on scRNA-seq data obtained from coculturing tumor cells with CAR-NK cells and observed high consistency between the predicted and the experimental (i.e., Seahorse extracellular) flux measurements. Throughout our investigation, we discovered various modes of metabolic cooperation and competition between various cell-types in TMEs, which could lead to further target discovery and development.

bioinformatics↗