Biology subjects

Cheng, E.

Publications and source records attributed to Cheng, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Natural compounds magnolol and dicoumarol enhance adipogenesis for future food applications

Edible adipose tissue can enhance the sensory and nutritional qualities of cultivated and plant-based meats, yet efficient adipogenic differentiation remains a major bottleneck and synthetic PPAR{gamma} agonists are not approved for use in food production. Here, we report a natural compound screen in 3T3-L1 adipocytes that identifies magnolol and dicoumarol as enhancers of adipogenesis; this combination also robustly promotes lipid accumulation in primary porcine dedifferentiated fat cells and ovine preadipocytes. Transcriptomic analyses show that magnolol and dicoumarol induce adipogenesis in murine and porcine cell systems through canonical adipogenic pathways with a narrower transcriptional footprint than the potent PPAR{gamma} agonist rosiglitazone. These findings support the potential of naturally occurring compounds magnolol and dicoumarol as enhancers of adipogenesis for both mechanistic studies and food-relevant applications. More broadly, our findings establish a generalizable screening framework and identify small-molecule combinations that accelerate adipose tissue engineering across murine, porcine, and ovine culture systems.

bioengineering↗

bioRxiv2026

PRMT1 arginine methylation of MCM4 restricts ssDNA gap formation during DNA replication

Replication-associated single-stranded DNA (ssDNA) gaps are increasingly recognized as major sources of genome instability, but how replisome-associated mechanisms suppress their formation remains unclear. Here we identify protein arginine methyltransferase 1 (PRMT1) as a replisome-associated methyltransferase that promotes DNA synthesis and fork integrity. PRMT1 localizes to active forks and its depletion or inhibition slows fork progression in multiple human cell types. We identify MCM4 as a direct PRMT1 substrate and show that methylation-deficient MCM4 causes spontaneous replication stress, elevated RPA, and nuclease-sensitive nascent DNA tracts, indicating persistent daughter-strand gaps. Despite these defects, methylation-deficient MCM4 cells exhibit apparent fork acceleration, consistent with discontinuous DNA synthesis. By contrast, PRMT1 depletion slows forks and selectively reduces fork association of SMC5/6 without broadly disrupting core replisome components. Together, these data support a model in which PRMT1-dependent methylation of MCM4 suppresses gap-prone DNA synthesis, while fork-associated SMC5/6 stabilizes vulnerable gap-containing intermediates.

molecular biology↗

bioRxiv2026

Expanding the C. elegans toolkit with gonad explants

Animal development is a complex process that requires the coordination of a plethora of pathways in space and time. In several species, the availability of tissue explants has provided a simplified context that facilitates mechanistic investigations, particularly into dynamic events. Here, we demonstrate that extruded C. elegans gonads are a viable tissue explant system for this model organism. Using live-cell imaging, we show that C. elegans gonad explants retain many tissue properties that have been documented in vivo, including mitosis, meiosis, apoptosis and gametogenesis. We further show that C. elegans explants are acutely responsive to treatment by the microtubule depolymerizing drug nocodazole. Our work thus reveals C. elegans gonad explants as a new system in which live-cell imaging and acute drug treatment can be combined to decipher the mechanisms governing germline development.

developmental biology↗

bioRxiv2026

Apical Localization of RNA Polymerases Modulate Transcription Dynamics and Supercoiling Domains Revealed by Cryo-ET

Protein interactions with canonical B-form DNA are well-characterized, yet the effect on these interactions of torsionally constrained DNA--ubiquitous in cells--remains underexplored. Using cryo-electron tomography (cryo-ET), we 3D-reconstructed entire negatively supercoiled DNA substrates bound to active RNA polymerase (RNAP), revealing diverse DNA supercoiling conformations and their interplay with transcription. RNAP preferentially localizes at plectoneme apices in a swiveled, pause-prone state. RNAP, along with other DNA-melting proteins like dCas9, can act as torsional roadblocks that segregate "twin-supercoiling domains" during active transcription, independent of external DNA/RNAP tethering. Co-transcribing RNAPs further intensify this domain separation: tandem oriented RNAPs relieve negative supercoiling more effectively than opposing ones, promote greater RNAP accumulation and enhanced elongation, both in vitro and in vivo. Topoisomerase I relieves torsional stress and facilitates RNAP escape from apical stalls, thereby supporting apical transcription regulation. Together, these findings support a load-and-release mechanism at plectoneme apices that may underlie supercoiling-dependent transcriptional bursting.

biophysics↗

bioRxiv2026

TEsingle enables locus-specific transposable element expression analysis at single-cell resolution

Transposable elements (TEs) are mobile genetic sequences that can generate new copies of themselves via insertional mutations. These viral-like sequences comprise nearly half the human genome and are present in most genome wide sequencing assays. While only a small fraction of genomic TEs have retained their ability to transpose, TE sequences are often transcribed from their own promoters or as part of larger gene transcripts. Accurately assessing TE expression from each individual genomic TE locus remains an open problem in the field, due to the highly repetitive nature of these multi-copy sequences. These issues are compounded in single-cell and single-nucleus transcriptome experiments, where additional complications arise due to sparse read coverage and unprocessed mRNA introns. Here we present our tool for single-cell TE and gene expression analysis, TEsingle. Using synthetic datasets, we show the problems that arise when not properly accounting for intron retention events, failing to address uncertainty in alignment scoring, and failing to make use of unique molecular identifiers for transcript resolution. Addressing these challenges has enabled an accurate TE analysis suite that simultaneously tracks gene expression as well as locus-specific resolution of expressed TEs. We showcase the performance of TEsingle using single-nucleus profiles from substantia nigra (SN) tissues of Parkinsons Disease (PD) patients. We find examples of young and intact TEs that mark dopaminergic neurons (DA) as well as many young TEs from the LINE and ERV families that are elevated in PD neurons and glia. These results demonstrate that TE expression is highly cell-type and cellular-state specific and elevated in particular subsets of neurons, astrocytes, and microglia from PD patients.

genomics↗

bioRxiv2026

Divergent phenotypic and functional roles of human T follicular helper cells from infancy to adulthood

Antibody responses to T-dependent antigens are suboptimal in young children, yet the evolution of T follicular helper cell (Tfh) function across the human lifespan remains poorly defined. Using human tonsils, a physiologically relevant and abundant source of Tfh, we investigated age-associated differences in their repertoire and functional programs. Pediatric tonsils were enriched for cytokine-expressing Tfh subsets with increased clonal diversity and phenotypic plasticity. However, in response to influenza antigens, they exhibited reduced Th1 polarization, diminished IL-21 production, and limited B cell help. Across ages, high neutralizing flu antibody responses were associated with robust Tfh1 activation, which was ICOS dependent in adults but not in children. Interestingly, Tfh depletion strategies revealed enhanced Tfh differentiation from distinct precursors in pediatric donors, yet antibody responses during early life were less reliant on Tfh help. Together, these findings define developmentally programmed differences in Tfh differentiation and function with implications for pediatric vaccine design.

immunology↗

bioRxiv2025

Microglia coordinate activity-dependent protein synthesis in neurons through metabolic coupling

De novo protein synthesis is required for long-lasting synaptic plasticity and memory, but it comes with a great metabolic cost. In the mammalian brain, it remains unclear which cell types and biological mechanisms are critical for sensing and responding to increased metabolic demand. Here we demonstrate that microglia, resident macrophages of the brain, coordinate metabolic coupling between endothelial cells, astrocytes, and neurons to fuel protein synthesis in active neurons. Increasing metabolic demand via a motor task stimulates microglia to secrete the hypoxia-responsive protein CYR61, increasing glucose transporter expression in brain vasculature. Depleting microglia reduces training-induced metabolic fluxes and neuronal protein synthesis, which can be reproduced by blocking CYR61 signaling. Thus, we define a neuroimmune metabolic circuit required for on-demand protein synthesis in mouse motor cortex.

neuroscience↗

bioRxiv2025

SPIN: Inkjet-Driven Nanowell Workflow for Scalable and Sensitive Single-Cell Proteomics

Single-cell proteomics is emerging as a powerful approach to resolve cellular heterogeneity, yet sample processing remains challenging due to limited input material and the absence of protein amplification. We present a protocol centered on an image-guided, machine-learning-driven inkjet single-cell printer integrated with a dew-point-controlled nanowell chip to reduce loss, increase throughput, and improve reproducibility. The system dispenses single cells at >1 Hz into sealed nanoliter wells with minimal surface contact, virtually eliminating evaporation; a high-thermal-conductivity aluminum substrate and precise environmental control further ensure exceptional reproducibility. Relative to a commercial dispenser, the workflow yields significantly higher protein and peptide recovery without bias toward high-abundance species, delivering uniformly deep coverage. Biological pathway analysis emphasizes the robustness of this workflow, as there is a near 100% protein completeness detected among the enzymes in the Kreb cycle in both A549 and astrocytes, suggesting the consistency across all samples evaluated. This platform addresses core processing bottlenecks and enables reliable, scalable single-cell proteomics.

systems biology↗

bioRxiv2024

A Reproducibility Focused Meta-Analysis Method for Single-Cell Transcriptomic Case-Control Studies Uncovers Robust Differentially Expressed Genes

We assessed the reproducibility of differentially expressed genes (DEGs) in previously published Alzheimers (AD), Parkinsons (PD), Huntingtons (HD), Schizophrenia (SCZ), and COVID-19 scRNA-seq studies. While transcriptional scores from DEGs of individual PD, HD, and COVID-19 datasets had moderate predictive power for case-control status of other datasets, genes from individual AD and SCZ datasets had poor predictive power. We developed a non-parametric meta-analysis method, SumRank, based on reproducibility of relative differential expression ranks across datasets, and found DEGs with improved predictive power. By multiple metrics, specificity and sensitivity of these genes were substantially higher than those discovered by dataset merging and inverse variance weighted p-value aggregation methods and had significant enrichment in snATAC-seq peaks and human disease gene associations. The DEGs revealed known and novel biological pathways, such as up-regulation of chaperone-mediated protein processing in PD glia and lipid transport in AD and PD microglia, and down-regulation of glutamatergic processes in AD astrocytes and glutamatergic neurons and synaptic processing and neuron projection genes in HD FOXP2 neurons. We find 56 DEGs shared amongst AD, PD, and HD, and validate BCAT1 as down-regulated in AD mouse oligodendrocytes. Lastly, we evaluate factors influencing reproducibility of individual studies as a prospective guide for experimental design.

genomics↗

bioRxiv2024

Non-autonomous insulin signaling regulates the duration of mitosis in C. elegans germline stem and progenitor cells

Stem and progenitor cell mitosis is essential for tissue development and homeostasis. How these cells ensure proper chromosome segregation, and thereby maintain mitotic fidelity, in the complex physiological environment of a living animal is poorly understood. Here we use in situ live-cell imaging of C. elegans germline stem and progenitor cells (GSPCs) to ask how the signaling environment influences stem and progenitor cell mitosis in vivo. Through a candidate screen we identify a new role for the insulin/IGF receptor (IGFR), daf-2, during GSPC mitosis. Mitosis is delayed in daf-2/IGFR mutants, and these delays require canonical, DAF-2/IGFR to DAF-16/FoxO insulin signaling, here acting cell non-autonomously from the soma. Interestingly, mitotic delays in daf-2/IGFR mutants depend on the spindle assembly checkpoint but are not accompanied by a loss of mitotic fidelity. Correspondingly, we show that caloric restriction, which delays GSPC mitosis and compromises mitotic fidelity, does not act via the canonical insulin signaling pathway, and instead requires AMP-activated kinase (AMPK). Together this work demonstrates that GSPC mitosis is influenced by at least two genetically separable signaling pathways and highlights the importance of signaling networks for proper stem and progenitor cell mitosis in vivo. Author SummaryStem and progenitor cells drive tissue development and sustain adult tissue turnover by producing new daughter cells via cell division, the success of which depends on proper chromosome segregation during mitosis. Stem and progenitor cells perform mitosis in the complex environment of a living animal, yet relatively little is known about how events during mitosis are influenced by this in vivo context. In particular, whether signaling pathways that coordinate other aspects of stem and progenitor cell behavior with animal physiology also play a role during mitosis is poorly understood. Here we took advantage of the germline stem and progenitor cells of the model nematode C. elegans to address this question. Through live-cell imaging of germline stem and progenitor cell mitosis, we uncover a new role for the insulin signaling pathway. We find that reducing insulin signaling delays germline stem and progenitor cell mitosis, but, surprisingly, these delays are not accompanied by a loss of mitotic fidelity. In addition, we find that reducing insulin signaling in somatic tissues is sufficient to delay germline stem and progenitor mitosis, indicating that the pathway acts non-autonomously. Finally, while insulin signaling is known to link cell division with nutritional status in many species, we found that it did not mediate the effects of caloric restriction on germline stem and progenitor cell mitosis. Instead, caloric restriction acts via the conserved energy-sensing regulator AMPK. These results uncover new regulators of germline stem and progenitor cell mitosis and emphasize the importance of signaling pathways for proper stem and progenitor cell mitosis in vivo.

developmental biology↗

bioRxiv2024

The BLM-TOP3A-RMI1-RMI2 proximity map reveals that RAD54L2 suppresses sister chromatid exchanges

Homologous recombination is a largely error-free DNA repair mechanism conserved across all domains of life and is essential for the maintenance of genome integrity. Not only are the mutations in homologous recombination repair genes probable cancer drivers, some also cause genetic disorders. In particular, mutations in the Bloom (BLM) helicase cause Bloom Syndrome, a rare autosomal recessive disorder characterized by increased sister chromatid exchanges and predisposition to a variety of cancers. The pathology of Bloom Syndrome stems from the impaired activity of the BLM-TOP3A-RMI1-RMI2 (BTRR) complex which suppresses crossover recombination to prevent potentially deleterious genome rearrangements. We provide a comprehensive BTRR proximity interactome, revealing proteins that suppress crossover recombination. We find that RAD54L2, a SNF2-family protein, physically interacts with BLM and suppresses sister chromatid exchanges. RAD54L2 is important for recruitment of BLM to chromatin and requires an intact ATPase domain to promote non-crossover recombination. Thus, the BTRR proximity map identifies a regulator of recombination.

molecular biology↗

bioRxiv2023

Kinome focused CRISPR-Cas9 screens in African ancestry patient-derived breast cancer organoids identifies essential kinases and synergy of EGFR and FGFR1 inhibition.

Precision medicine approaches to cancer treatment aim to exploit genomic alterations that are specific to individual patients to tailor therapy strategies. These alterations are usually revealed via next generation sequencing of the tumor tissue. Yet, it is clear that some targetable genes and pathways are essential for tumor cell viability even in the absence of direct genomic alterations. This is especially important in under-represented populations, whose mutational landscape and determinants of response to existing therapies are poorly characterized due to limited inclusion in clinical trials and studies. One way to reveal tumor essential genes is with genetic screens. Most screens are conducted on cell lines that bear little resemblance to patient tumors, after years of culture in non-physiological conditions. To address this problem, we aimed to develop a CRISPR screening pipeline in 3D-grown patient-derived tumor organoid (PDTO) models. We focused on identifying essential kinases that may translate to options for targeted therapies, including combination therapies. We first established a breast cancer PDTO biobank focused on underrepresented populations, including West African patients. We then performed a negative selection kinome-focused CRISPR screen to identify kinases essential for organoid growth and potential targets for combination therapy with EGFR or MEK inhibitors. We identified several previously unidentified kinase targets and showed that combination of FGFR1 and EGFR inhibitors synergizes to block organoids proliferation. Together these data demonstrate feasibility of CRISPR-based genetic screens in patient-derived tumor models, including PDTOs from under-represented cancer patients, and identify new targets for cancer therapy.

cancer biology↗

bioRxiv2023

Patient-derived tumor organoids with p53 mutations, and not wild-type p53, are sensitive to synergistic combination PARP inhibitor treatment

Poly (ADP-ribose) polymerase inhibitors (PARPi) are used for patients with BRCA1/2 mutations, but patients with other mutations may benefit from PARPi treatment. Another mutation that is present in more cancers than BRCA1/2 is mutation to the TP53 gene. In 2D breast cancer cell lines, mutant p53 (mtp53) proteins tightly associate with replicating DNA and Poly (ADP-ribose) polymerase (PARP) protein. Combination drug treatment with the alkylating agent temozolomide and the PARPi talazoparib kills mtp53 expressing 2D grown breast cancer cell lines. We evaluated the sensitivity to the combination of temozolomide plus PARPi talazoparib treatment to breast and lung cancer patient-derived tumor organoids (PDTOs). The combination of the two drugs was synergistic for a cytotoxic response in PDTOs with mtp53 but not for PDTOs with wtp53. The combination of talazoparib and temozolomide induced more DNA double-strand breaks in mtp53 expressing organoids than in wild-type p53 expressing organoids as shown by increased {psi}-H2AX protein expression. Moreover, breast cancer tissue microarrays (TMAs) showed a positive correlation between stable p53 and high PARP1 expression in sub-groups of breast cancers, which may indicate sub-classes of breast cancers sensitive to PARPi therapy. These results suggest that mtp53 could be a biomarker to predict response to the combination of PARPi talazoparib-temozolomide treatment.

cancer biology↗

bioRxiv2022

Rad51-mediated interhomolog recombination during budding yeast meiosis is promoted by the meiotic recombination checkpoint and the conserved Pif1 helicase

During meiosis, recombination between homologous chromosomes (homologs) generates crossovers that promote proper segregation at the first meiotic division. Recombination is initiated by Spo11-catalyzed double strand breaks (DSBs). 5 end resection of the DSBs creates 3 single strand tails that two recombinases, Rad51 and Dmc1, bind to form presynaptic filaments that search for homology, mediate strand invasion and generate displacement loops (D-loops). D-loop processing then forms crossover and non-crossover recombinants. Meiotic recombination occurs in two temporally distinct phases. During Phase 1, Rad51 is inhibited and Dmc1 mediates the interhomolog recombination that promotes homolog synapsis. In Phase 2, Rad51 becomes active and functions with Rad54 to repair residual DSBs, making increasing use of sister chromatids. The transition from Phase 1 to Phase 2 is controlled by the meiotic recombination checkpoint through the meiosis-specific effector kinase Mek1. This work shows that constitutive activation of Rad51 in Phase 1 results in a subset of DSBs being repaired by a Rad51-mediated interhomolog recombination pathway that is distinct from that of Dmc1. Strand invasion intermediates generated by Rad51 require more time to be processed into recombinants, resulting in a meiotic recombination checkpoint delay in prophase I. Without the checkpoint, Rad51-generated intermediates are more likely to be repaired using a sister chromatid, thereby increasing Meiosis I chromosome nondisjunction. This Rad51 interhomolog recombination pathway is specifically promoted by the conserved 5-3 helicase PIF1 and its paralog, RRM3 and requires Pif1 helicase activity and its interaction with PCNA. This work demonstrates that (1) inhibition of Rad51 during Phase 1 is important to prevent competition with Dmc1 for DSB repair, (2) Rad51-mediated meiotic recombination intermediates are initially processed differently than those made by Dmc1, (3) the meiotic recombination checkpoint provides time during prophase 1 for processing of Rad51-generated recombination intermediates. AUTHOR SUMMARYTo sexually reproduce, cells containing two copies of each chromosome must undergo the specialized cell division of meiosis to sort the chromosomes into gametes containing a single copy of each chromosome. But how do homologous chromosomes know who is who? The answer is by recombination, a process in which double strand breaks on one chromosome are converted to single stranded ends that can search for the complementary sequence on the homolog. In yeast and mammals, this homology search involves binding of single strand ends by two highly conserved recombinases, Rad51 and the meiosis specific Dmc1. Rad51 is used in mitotic cells to repair breaks, primarily using sister chromatids as templates, while Dmc1 functions in meiosis to generate interhomolog crossovers. In budding yeast, Rad51 strand exchange activity is normally inhibited while Dmc1 is active. We show here that when Rad51 and Dmc1 are active at the same time, Rad51 competes with Dmc1 to mediate interhomolog recombination of a subset of double strand breaks. However, because Rad51- generated recombination intermediates take longer to process, there is a need to keep Rad51 inactive while interhomolog recombination is occurring.

genetics↗

bioRxiv2022

Assignment of structural transitions during mechanical unwrapping of nucleosomes and their disassembly products

Nucleosome DNA unwrapping and its disassembly into hexasomes and tetrasomes is necessary for genomic access and plays an important role in transcription regulation. Previous single-molecule mechanical nucleosome unwrapping revealed a low- and a high-force transitions, and force-FRET pulling experiments showed that DNA unwrapping is asymmetrical occurring always first from one side before the other. However, the assignment of DNA segments involved in these transitions remains controversial. Here, using high-resolution optical tweezers with simultaneous single-molecule FRET detection we show that the low-force transition corresponds to the undoing of the outer-wrap of one side of the nucleosome (~27 bp), a process that can occur either cooperatively or non-cooperatively, whereas the high-force transition corresponds to the simultaneous unwrapping of ~76 bp from both sides. This process may give rise stochastically to the disassembly of nucleosomes into hexasomes and tetrasomes whose unwrapping/rewrapping trajectories we establish. In contrast, nucleosome rewrapping does not exhibit asymmetry. To rationalize all previous nucleosome unwrapping experiments, it is necessary to invoke that mechanical unwrapping involves two nucleosome reorientations: one that contributes to the change in extension at the low-force transition, and another that coincides but does not contribute to the high-force transition. Significance statementNucleosomes, the fundamental structural unit of chromatin, consists of ~147 DNA base pairs wrapped around a histone protein octamer. Determining the forces required to unwrap the DNA from the core particle and the stepwise transitions involved in the process are essential to characterize the strength of the nucleosomal barrier and its contribution as a mechanism of control of gene expression. Here, we performed combined optical tweezers and single-molecule fluorescence measurements to annotate the specific DNA segments unwrapping during the force transitions observed in mechanical unwrapping of nucleosomes. Furthermore, we characterize the mechanical signatures of subnucleosomal particles: hexasomes and tetrasomes. The characterization performed in this work is essential for the interpretation of ongoing studies of chromatin remodelers, polymerases, and histone chaperones.

biophysics↗

bioRxiv2022

A comprehensive phenotypic screening strategy to identify modulators of cargo translocation by the bacterial Type IVB secretion system

Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.7 mouse macrophages. Using a fluorescence resonance energy transfer (FRET)-based detection assay in a bacteria/macrophage co-culture format, we screened a library of over 18,000 compounds and, upon vetting compound candidates in a variety of in vitro and cell-based secondary screens, isolated several hits that efficiently interfered with biological processes that depend on a functional T4SS, such as intracellular bacterial proliferation or lysosomal avoidance, but had no detectable effect on L. pneumophila growth in culture medium, conditions under which the T4SS is dispensable. Notably, the same hit compounds also attenuated, to varying degrees, effector delivery by the closely related T4SS from Coxiella burnetii, notably without impacting growth of this organism within synthetic media. Together, these results support the idea that interference with T4SS function is a possible therapeutic intervention strategy, and the emerging compounds provide tools to interrogate at a molecular level the regulation and dynamics of these virulence-critical translocation machines. ImportanceMulti-drug-resistant pathogens are an emerging threat to human health. Since conventional antibiotics target not only the pathogen but also eradicate the beneficial microbiota, they often cause additional clinical complications. Thus, there is an urgent need for the development of "smarter" therapeutics that selectively target pathogens without affecting beneficial commensals. The bacterial type IV secretion system (T4SS) is essential for the virulence of a variety of pathogens but dispensable for bacterial viability in general and can, thus, be considered a pathogens Achilles heel. By identifying small molecules that interfere with cargo delivery by the T4SS from two important human pathogens, Legionella pneumophila and Coxiella burnetii, our study represents the first step in our pursuit towards precision medicine by developing pathogen-selective therapeutics capable of treating the infections without causing harm to commensal bacteria.

microbiology↗

bioRxiv2022

Spatial positioning and matrix programs of cancer-associated fibroblasts promote T cell exclusion in human lung tumors

It is currently accepted that activated cancer-associated fibroblasts (CAF) participate in T cell exclusion from tumor nests, but it remains unclear how they promote barrier phenotypes, and whether specific subsets are involved. Here, using single-cell RNA sequencing coupled with multiplex imaging on a large cohort of lung tumors, we identify four main CAF populations, of which only two are associated with T cell exclusion: (i) MYH11+SMA+ CAF, which are present in early-stage tumors and form a single-cell layer lining cancer aggregates, and (ii) FAP+SMA+ CAF, which appear in more advanced tumors and organize in patches within the stroma or in multiple layers around tumor nests. Both CAF populations show a contractility phenotype together with dense and aligned matrix fiber deposition compared to the T cell-permissive CAF. Yet they express distinct matrix genes, including COL4A1/COL9A1 (MYH11+SMA+ CAF) and COL11A1/COL12A1 (FAP+SMA+ CAF). Hereby, we uncovered unique molecular programs of CAF driving T cell marginalization, whose targeting should increase immunotherapy efficacy in patients bearing T cell-excluded tumors. SIGNIFICANCEThe cellular and molecular programs driving T cell marginalization in solid tumors remain unclear. Here, we describe two CAF populations associated with T cell exclusion in human lung tumors. We demonstrate the importance of pairing molecular and spatial analysis of the tumor microenvironment, a prerequisite to develop new strategies targeting T cell-excluding CAF.

cancer biology↗

bioRxiv2020

Cell-type specific gene expression profiling in heterogeneous in vitro cultures using epitope-tagged RPL22

Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease, but also challenge our ability to resolve cell-type specific perturbations. Here we report an extension of the RiboTag system, first developed to achieve cell-type restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables efficient depletion of off-target RNA in mixed species primary co-cultures and in hiPSC-derived neural progenitor cells, motor neurons, and GABAergic neurons. Nonetheless, depletion efficiency varies across independent experimental replicates. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

molecular biology↗