Ferroptosis is a type of regulated cell death driven by the iron-dependent accumulation of oxidized polyunsaturated fatty acid-containing phospholipids. There is no reliable way to selectively stain ...ferroptotic cells in tissue sections to characterize the extent of ferroptosis in animal models or patient samples. We address this gap by immunizing mice with membranes from lymphoma cells treated with the ferroptosis inducer piperazine erastin and screening ∼4,750 of the resulting monoclonal antibodies generated for their ability to selectively detect cells undergoing ferroptosis. We find that one antibody, 3F3 ferroptotic membrane antibody (3F3-FMA), is effective as a selective ferroptosis-staining reagent. The antigen of 3F3-FMA is identified as the human transferrin receptor 1 protein (TfR1). We validate this finding with several additional anti-TfR1 antibodies and compare them to other potential ferroptosis-detecting reagents. We find that anti-TfR1 and anti-malondialdehyde adduct antibodies are effective at staining ferroptotic tumor cells in multiple cell culture and tissue contexts.
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•3F3-FMA is identified in a screen as a selective ferroptosis-immunostaining reagent•The antigen of 3F3-FMA is identified as the transferrin receptor 1 protein (TfR1)•Anti-TfR1 antibodies can detect ferroptosis by immunofluorescence and flow cytometry•Anti-TfR1 and anti-MDA antibodies detect ferroptosis in xenograft cancer models
Feng et al. find that 3F3-FMA detects ferroptotic cells by screening ∼4,750 antibodies generated from mice immunized with membranes from DLBCL cells undergoing ferroptosis. The antigen of 3F3-FMA is the TfR1 protein. 3F3-FMA and other anti-TfR1 antibodies can be used to detect ferroptosis in cell culture and in cancer models.
Cancer-associated fibroblasts (CAF) are a poorly characterized cell population in the context of liver cancer. Our study investigates CAF functions in intrahepatic cholangiocarcinoma (ICC), a highly ...desmoplastic liver tumor. Genetic tracing, single-cell RNA sequencing, and ligand-receptor analyses uncovered hepatic stellate cells (HSC) as the main source of CAF and HSC-derived CAF as the dominant population interacting with tumor cells. In mice, CAF promotes ICC progression, as revealed by HSC-selective CAF depletion. In patients, a high panCAF signature is associated with decreased survival and increased recurrence. Single-cell RNA sequencing segregates CAF into inflammatory and growth factor-enriched (iCAF) and myofibroblastic (myCAF) subpopulations, displaying distinct ligand-receptor interactions. myCAF-expressed hyaluronan synthase 2, but not type I collagen, promotes ICC. iCAF-expressed hepatocyte growth factor enhances ICC growth via tumor-expressed MET, thus directly linking CAF to tumor cells. In summary, our data demonstrate promotion of desmoplastic ICC growth by therapeutically targetable CAF subtype-specific mediators, but not by type I collagen.
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•The majority of CAF in ICC are derived from hepatic stellate cells•Inflammatory CAF promote ICC through HGF and its receptor MET•myCAF promote ICC through Has2/hyaluronic acid•CAF-derived type I collagen contributes to stiffness but does not promote ICC growth
Intrahepatic cholangiocarcinoma (ICC) is an extraordinarily stiff liver tumor due to abundant scar-forming cancer-associated fibroblasts (CAF). Here, Affo et al. determine the origin and functions of CAF, and uncover distinct CAF subsets, promoting ICC growth via different therapeutically targetable mediators. Thus, CAF and their mediators may serve as therapeutic targets for ICC.
Spatial single-cell omics provides a readout of biochemical processes. It is challenging to capture the transient lipidome/metabolome from cells in a native tissue environment. We employed water gas ...cluster ion beam secondary ion mass spectrometry imaging (H2On>28K-GCIB-SIMS) at ≤3 μm resolution using a cryogenic imaging workflow. This allowed multiple biomolecular imaging modes on the near-native-state liver at single-cell resolution. Our workflow utilizes desorption electrospray ionization (DESI) to build a reference map of metabolic heterogeneity and zonation across liver functional units at tissue level. Cryogenic dual-SIMS integrated metabolomics, lipidomics, and proteomics in the same liver lobules at single-cell level, characterizing the cellular landscape and metabolic states in different cell types. Lipids and metabolites classified liver metabolic zones, cell types and subtypes, highlighting the power of spatial multi-omics at high spatial resolution for understanding celluar and biomolecular organizations in the mammalian liver.
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•Multimodal MSI integrates the spatial multi-omics at tissue and single-cell levels•Dual-SIMS images metabolic states of different cell types in the frozen liver•DESI reveals zonation across liver tissue functional units
Tian and Rajbhandari et al. integrated metabolites, lipids, and proteins profiling, using a multimodal MSI workflow, and identified the lipidomic/metabolic signatures in liver functional units and different cell types.
The transformation of molecular binding events into cellular decisions is the basis of most biological signal transduction. A fundamental challenge faced by these systems is that reliance on ...protein–ligand chemical affinities alone generally results in poor sensitivity to ligand concentration, endangering the system to error. Here, we examine the lipid-binding pleckstrin homology and Tec homology (PH-TH) module of Bruton’s tyrosine kinase (Btk). Using fluorescence correlation spectroscopy (FCS) and membranebinding kinetic measurements, we identify a phosphatidylinositol (3–5)-trisphosphate (PIP₃) sensing mechanism that achieves switchlike sensitivity to PIP₃ levels, surpassing the intrinsic affinity discrimination of PIP₃:PH binding. This mechanism employs multiple PIP3 binding as well as dimerization of Btk on the membrane surface. Studies in live cells confirm that mutations at the dimer interface and peripheral site produce effects comparable to that of the kinase-dead Btk in vivo. These results demonstrate how a single protein module can institute an allosteric counting mechanism to achieve high-precision discrimination of ligand concentration. Furthermore, this activation mechanism distinguishes Btk from other Tec family member kinases, Tec and Itk, which we show are not capable of dimerization through their PH-TH modules. This suggests that Btk plays a critical role in the stringency of the B cell response, whereas T cells rely on other mechanisms to achieve stringency.
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