Stromal fibroblast senescence has been linked to ageing-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAFs) are frequently increased. Loss or ...downmodulation of the Notch effector CSL (also known as RBP-Jκ) in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumours. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as a direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is downmodulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas, whereas p53 expression and function are downmodulated only in the latter, with paracrine FGF signalling as the probable culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation-stromal co-evolution model under convergent CSL-p53 control.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
Heterogeneity of cancer-associated fibroblasts (CAFs) can result from activation of distinct signaling pathways. We show that in primary human dermal fibroblasts (HDFs), fibroblast growth factor ...(FGF) and transforming growth factor β (TGF-β) signaling oppositely modulate multiple CAF effector genes. Genetic abrogation or pharmacological inhibition of either pathway results in induction of genes responsive to the other, with the ETV1 transcription factor mediating the FGF effects. Duality of FGF/TGF-β signaling and differential ETV1 expression occur in multiple CAF strains and fibroblasts of desmoplastic versus non-desmoplastic skin squamous cell carcinomas (SCCs). Functionally, HDFs with opposite TGF-β versus FGF modulation converge on promoting cancer cell proliferation. However, HDFs with increased TGF-β signaling enhance invasive properties and epithelial-mesenchymal transition (EMT) of SCC cells, whereas HDFs with increased FGF signaling promote macrophage infiltration. The findings point to a duality of FGF versus TGF-β signaling in distinct CAF populations that promote cancer development through modulation of different processes.
Display omitted
•FGF and TGF-β signaling exert opposite control over multiple CAF effector genes•ETV1 transcription factor mediates FGF effects and suppresses those of TGF-β•Modulation of either pathway leads to different tumor-promoting CAF populations•TGF-β-activated CAFs promote EMT, but FGF-activated CAFs increase inflammation
Bordignon et al. show that activation of FGF and TGF-β control opposite key CAF effectors. Suppression of one pathway leads to activation of the other and results in tumor-promoting CAF populations that elicit EMT versus inflammation. FGF/TGF-β dualism applies to distinct CAF subsets in invading desmoplastic versus non-desmoplastic skin SCCs.
Cancer-associated fibroblasts (CAFs) are important for tumor initiation and promotion. CSL, a transcriptional repressor and Notch mediator, suppresses CAF activation. Like CSL, ATF3, a ...stress-responsive transcriptional repressor, is down-modulated in skin cancer stromal cells, and
knockout mice develop aggressive chemically induced skin tumors with enhanced CAF activation. Even at low basal levels, ATF3 converges with CSL in global chromatin control, binding to few genomic sites at a large distance from target genes. Consistent with this mode of regulation, deletion of one such site 2 Mb upstream of
induces expression of the gene. Observed changes are of translational significance, as bromodomain and extra-terminal (BET) inhibitors, unlinking activated chromatin from basic transcription, counteract the effects of ATF3 or CSL loss on global gene expression and suppress CAF tumor-promoting properties in an in vivo model of squamous cancer-stromal cell expansion. Thus, ATF3 converges with CSL in negative control of CAF activation with epigenetic changes amenable to cancer- and stroma-focused intervention.
Abstract
Cancer associated fibroblasts (CAFs) are a key component of the tumor microenvironment. Genomic alterations in these cells remain a point of contention. We report that CAFs from skin ...squamous cell carcinomas (SCCs) display chromosomal alterations, with heterogeneous
NOTCH1
gene amplification and overexpression that also occur, to a lesser extent, in dermal fibroblasts of apparently unaffected skin. The fraction of the latter cells harboring
NOTCH1
amplification is expanded by chronic UVA exposure, to which CAFs are resistant. The advantage conferred by
NOTCH1
amplification and overexpression can be explained by NOTCH1 ability to block the DNA damage response (DDR) and ensuing growth arrest through suppression of ATM-FOXO3a association and downstream signaling cascade. In an orthotopic model of skin SCC, genetic or pharmacological inhibition of
NOTCH1
activity suppresses cancer/stromal cells expansion. Here we show that
NOTCH1
gene amplification and increased expression in CAFs are an attractive target for stroma-focused anti-cancer intervention.
Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of ...tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.
Genomic instability is a hallmark of cancer. Whether it also occurs in Cancer Associated Fibroblasts (CAFs) remains to be carefully investigated. Loss of CSL/RBP-Jκ, the effector of canonical NOTCH ...signaling with intrinsic transcription repressive function, causes conversion of dermal fibroblasts into CAFs. Here, we find that CSL down-modulation triggers DNA damage, telomere loss and chromosome end fusions that also occur in skin Squamous Cell Carcinoma (SCC)-associated CAFs, in which CSL is decreased. Separately from its role in transcription, we show that CSL is part of a multiprotein telomere protective complex, binding directly and with high affinity to telomeric DNA as well as to UPF1 and Ku70/Ku80 proteins and being required for their telomere association. Taken together, the findings point to a central role of CSL in telomere homeostasis with important implications for genomic instability of cancer stromal cells and beyond.
Mitogen-Activated Protein Kinases (MAPKs) control a wide array of cellular functions by transducing extracellular information into defined biological responses. In order to understand how these ...pathways are regulated, dynamic single cell measurements are highly needed. Fluorescence microscopy is well suited to perform these measurements. However, more dynamic and sensitive biosensors that allow the quantification of signaling activity in living mammalian cells are required. We have engineered a synthetic fluorescent substrate for human MAPKs (ERK, JNK and p38) that relocates from the nucleus to the cytoplasm when phosphorylated by the kinases. We demonstrate that this reporter displays an improved response compared to other relocation biosensors. This assay allows to monitor the heterogeneity in the MAPK response in a population of isogenic cells, revealing pulses of ERK activity upon a physiological EGFR stimulation. We show applicability of this approach to the analysis of multiple cancer cell lines and primary cells as well as its application in vivo to developing tumors. Using this ERK biosensor, dynamic single cell measurements with high temporal resolution can be obtained. These MAPK reporters can be widely applied to the analysis of molecular mechanisms of MAPK signaling in healthy and diseased state, in cell culture assays or in vivo.
Cell biology; Systems biology; Biochemistry; Cancer research; MAPK signaling; Single cells; Fluorescent biosensor; Live-cell imaging
BackgroundSpatial biology has transformed our understanding of the tumor microenvironment (TME) by enabling the study of tissue composition and intercellular interactions at a single-cell level while ...preserving spatial context.1–3 Hyperplex immunofluorescence (IF) techniques allow the simultaneous detection of multiple protein biomarkers, enabling immune cell profiling in the TME.4 Similarly, RNA in situ hybridization (ISH) techniques have enabled the detection of RNA biomarkers, such as soluble factors with high sensitivity and specificity.5 Combining the detection of key RNA and protein targets can provide valuable insights into unique infiltrating immune cell populations and their activation states.In this study, we propose a novel approach that combines RNAscope™ and sequential immunofluorescence (seqIF™) protocols for the simultaneous detection of RNA and protein targets. The integrated same-slide multi-omics protocol is automated on the COMET™ platform, an advanced platform for tissue staining that uses precise temperature control and automation capabilities, ensuring reproducibility and efficiency in the workflow.MethodsThe RNAscope HiPlex assay was automated on COMET™ for RNA detection and combined with the seqIF™ protocol for the detection of protein biomarkers. By integrating the two, we could sequentially detect multiple RNAs and proteins in the same tissue sections, preserving the spatial relationship between different molecular species.ResultsWe developed an integrated protocol for RNA and protein detection with three cycles of RNA detection (four fluorescent channels per cycle), enabling a total of 12-plex RNA panel detection, followed by consecutive cycles of seqIF™, with two protein markers detected per cycle. We included antibodies to detect infiltration of T cells, B cells, macrophages, and other immune cells in combination with RNA probes for key biomarkers such as chemokines and cytokines. The automated process on COMET™ seamlessly synchronized all protocol steps, including imaging, and allowed multi-omics analysis without any user intervention. By combining RNA and protein codetection, we gained extensive insights into the TME molecular landscape, uncovering co-expression patterns and relationships between RNA and proteins within individual cells.ConclusionsOur results demonstrate the successful implementation of the combined RNAscope and seqIF™ protocols on COMET™. Preserving spatial context and intercellular relationships, this approach offers a more holistic understanding of the TME molecular landscape and the complex cellular interactions exhibited by different cell populations.Multi-omics analysis on the same slide will allow a better comprehension of the interplay between transcriptomics and proteomics information, opening new perspectives for personalized medicine and the discovery of novel therapeutic targets.ReferencesBosisio FM, Van Herck Y, Messiaen J, Bolognesi MM, Marcelis L, Van Haele M, Cattoretti G, Antoranz A, De Smet F. Next-Generation Pathology Using Multiplexed Immunohistochemistry: Mapping Tissue Architecture at Single-Cell Level. Front Oncol. 2022 Jul 29;12:918900.Binnewies M, Roberts EW, Kersten K, Chan V, Fearon DF, Merad M, Coussens LM, Gabrilovich DI, Ostrand-Rosenberg S, Hedrick CC, Vonderheide RH, Pittet MJ, Jain RK, Zou W, Howcroft TK, Woodhouse EC, Weinberg RA, Krummel MF. Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 2018 May;24(5):541–550.Vitale I, Shema E, Loi S, Galluzzi L. Intratumoral heterogeneity in cancer progression and response to immunotherapy. Nat Med. 2021 Feb;27(2):212–224.Hickey JW, Neumann EK, Radtke AJ, Camarillo JM, Beuschel RT, Albanese A, McDonough E, Hatler J, Wiblin AE, Fisher J, Croteau J, Small EC, Sood A, Caprioli RM, Angelo RM, Nolan GP, Chung K, Hewitt SM, Germain RN, Spraggins JM, Lundberg E, Snyder MP, Kelleher NL, Saka SK. Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging. Nat Methods. 2022 Mar;19(3):284–295.Palla G, Fischer DS, Regev A, Theis FJ. Spatial components of molecular tissue biology. Nat Biotechnol. 2022 Mar;40(3):308–318.
In this study, we extended co-indexing of transcriptomes and epitopes (CITE) to the spatial dimension and demonstrated high-plex protein and whole transcriptome co-mapping. We profiled 189 proteins ...and whole transcriptome in multiple mouse tissue types with spatial CITE sequencing and then further applied the method to measure 273 proteins and transcriptome in human tissues, revealing spatially distinct germinal center reactions in tonsil and early immune activation in skin at the Coronavirus Disease 2019 mRNA vaccine injection site.
BackgroundThe tumor and its microenvironment are distinguished by highly heterogeneous cell types in dynamic evolution1. In the past decades, the adoption of single-cell RNA sequencing technologies ...improved our understanding of intra- and inter-patient variations, refining cancer diagnosis and targeted therapy, while still lacking spatial context information2. The spatial analysis of single-cell gene expression provides previously missing information on cell interactions, crucial for innovative treatment opportunities. However, the large-scale implementation of these assays is hampered by the lack of automated and user-friendly workflow solutions3.Here, we aim at automating an in situ transcriptomic assay and integrating it with a proteomic workflow on the COMET™ platform for easy and comprehensive mapping of the tumor microenvironment.MethodsCOMET™ is a microfluidic-based instrument capable of automated sequential immunofluorescence (seqIF™) assays. The in situ RNA detection was based on DNA padlock probe hybridization and ligation, followed by rolling circle amplification and gene detection with fluorophore-tagged probes. A panel of probes targeting various immune-oncology genes was designed and the gene expression was evaluated in combination with a high-plex seqIF™ analysis.ResultsThe protocol for in situ RNA detection was automated and optimized on COMET™, resulting in specific detection of multiple transcripts (cell-type specific markers, transcription factors, secreted cytokines) on various tumor samples, with a signal quality that allows downstream computational analysis of gene expression signals. The transcriptomic assay was then integrated with a seqIF™ workflow for the co-detection of several target proteins for tumor microenvironment characterization on the same tissue section (figure 1). Double RNA and protein analysis allowed to examine the expression of biomarkers for which no good antibody clones are available, validation of new antibodies by detecting RNA and protein co-localization, in addition to the identification of the cellular source of secreted molecules. Compared to the manual protocol, the combined automated workflow resulted in a drastic time reduction being based on iterative fast cycles of detection and imaging of two transcripts or proteins each, lasting approximately 30 and 40 minutes, respectively.ConclusionsWe showed here that in situ spatial transcriptomics assays can be fully automated and combined with spatial proteomics on the COMET™ platform for a multi-omics approach with advantages in terms of time and complexity reduction with respect to manual protocols. This combinatorial automated detection of RNAs and proteins of pivotal biomarkers provides a powerful new tool for a simpler and better mapping of the tissue spatial context.ReferencesJaniszewska M. The microcosmos of intratumor heterogeneity: the space-time of cancer evolution. Oncogene. 2020 Mar;39(10):2031–2039.Rao A, Barkley D, França GS, Yanai I. Exploring tissue architecture using spatial transcriptomics. Nature. 2021 Aug;596(7871):211–220.Asp M, Bergenstråhle J, Lundeberg J. Spatially Resolved Transcriptomes-Next Generation Tools for Tissue Exploration. Bioessays. 2020 Oct;42(10):e1900221.Abstract 38 Figure 1Automated and simultaneous in situ detection of RNA and protRNA (ACTB, RPLP0, JUN, MYC) and protein (panCK and Vimentin) targets are co-detected on a human melanoma frozen section with a fully automated workflow on the COMET™ platform. Background autofluorescence was subtracted and brightness adjusted for visualization purposesFigure omitted. See PDF
PDF
