With the genetic portraits of all major human malignancies now available, we next face the challenge of characterizing the function of mutated genes, their downstream targets, interactions and ...molecular networks. Moreover, poorly understood at the functional level are also non-mutated but dysregulated genomes, epigenomes or transcriptomes. Breakthroughs in manipulative mouse genetics offer new opportunities to probe the interplay of molecules, cells and systemic signals underlying disease pathogenesis in higher organisms. Herein, we review functional screening strategies in mice using genetic perturbation and chemical mutagenesis. We outline the spectrum of genetic tools that exist, such as transposons, CRISPR and RNAi and describe discoveries emerging from their use. Genome-wide or targeted screens are being used to uncover genomic and regulatory landscapes in oncogenesis, metastasis or drug resistance. Versatile screening systems support experimentation in diverse genetic and spatio-temporal settings to integrate molecular, cellular or environmental context-dependencies. We also review the combination of in vivo screening and barcoding strategies to study genetic interactions and quantitative cancer dynamics during tumour evolution. These scalable functional genomics approaches are transforming our ability to interrogate complex biological systems.
Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM ...pathology are poorly understood. Here we show that the somatic PIK3CA
mutation, resulting in constitutive activation of the p110α PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CA
-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110α activation determining the LM subtype. In the postnatal vasculature, PIK3CA
promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways.
Two studies by Lodestijn et al. in Cell Stem Cell and Cell Reports reveal a lack of stem cell hierarchies in acinar cell-derived tissue renewal and host instructed clonogenic growth of pancreatic ...cancer, thereby elucidating determinants of pancreas regeneration and cancer.
Two studies by Lodestijn et al. in Cell Stem Cell and Cell Reports reveal a lack of stem cell hierarchies in acinar cell-derived tissue renewal and host instructed clonogenic growth of pancreatic cancer, thereby elucidating determinants of pancreas regeneration and cancer.
Adaptive fear responses to external threats rely upon efficient relay of computations underlying contextual encoding to subcortical circuits. Brain-wide analysis of highly coactivated ensembles ...following contextual fear discrimination identified the dorsolateral septum (DLS) as a relay of the dentate gyrus-CA3 circuit. Retrograde monosynaptic tracing and electrophysiological whole-cell recordings demonstrated that DLS somatostatin-expressing interneurons (SST-INs) receive direct CA3 inputs. Longitudinal in vivo calcium imaging of DLS SST-INs in awake, behaving mice identified a stable population of footshock-responsive SST-INs during contextual conditioning whose activity tracked and predicted non-freezing epochs during subsequent recall in the training context but not in a similar, neutral context or open field. Optogenetic attenuation or stimulation of DLS SST-INs bidirectionally modulated conditioned fear responses and recruited proximal and distal subcortical targets. Together, these observations suggest a role for a potentially hard-wired DLS SST-IN subpopulation as arbiters of mobility that calibrate context-appropriate behavioral fear responses.
Memories about sensory experiences are tightly linked to the context in which they were formed. Memory contextualization is fundamental for the selection of appropriate behavioral reactions needed ...for survival, yet the underlying neuronal circuits are poorly understood. By combining trans-synaptic viral tracing and optogenetic manipulation, we found that the ventral hippocampus (vHC) and the amygdala, two key brain structures encoding context and emotional experiences, interact via multiple parallel pathways. A projection from the vHC to the basal amygdala mediates fear behavior elicited by a conditioned context, whereas a parallel projection from a distinct subset of vHC neurons onto midbrain-projecting neurons in the central amygdala is necessary for context-dependent retrieval of cued fear memories. Our findings demonstrate that two fundamentally distinct roles of context in fear memory retrieval are processed by distinct vHC output pathways, thereby allowing for the formation of robust contextual fear memories while preserving context-dependent behavioral flexibility.
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•vHC projects to the BA and the CEA via separate pathways with distinct behavioral roles•vHC inputs to the CEA contact output cells that target PAG and NST•vHC projection to the BA is required for contextual fear memory retrieval•vHC projection to the CEA is necessary for context-dependent cue fear memory retrieval
Two parallel hippocampal circuits link sensory memories with the context in which they are formed, helping the selection of appropriate behavioral responses to fear.
Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer ...metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage.
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•DeepMACT is a deep learning-based pipeline for comprehensive analysis of metastases•DeepMACT identifies micrometastases and single cancer cells in full-body 3D scans•DeepMACT reveals the efficacy of antibody-drug targeting in the entire body•DeepMACT indicates that the tumor microenvironment affects drug targeting efficacy
Deep learning-based automated detection and quantification of micrometastases and therapeutic antibody targeting down to the level of single disseminated cancer cells provides unbiased analysis of multiple metastatic cancer models at the full-body scale.
The incidence and lethality of pancreatic ductal adenocarcinoma (PDAC) will continue to increase in the next decade. For most patients, chemotherapeutic combination therapies remain the standard of ...care. The development and successful implementation of precision oncology in other gastrointestinal tumor entities point to opportunities also for PDAC. Therefore, markers linked to specific therapeutic responses and important subgroups of the disease are needed. The MYC oncogene is a relevant driver in PDAC and is linked to drug resistance and sensitivity. Here, we update recent insights into MYC biology in PDAC, summarize the connections between MYC and drug responses, and point to an opportunity to image MYC non-invasively. In sum, we propose MYC-associated biology as a basis for the development of concepts for precision oncology in PDAC.
Employing inducible genetically engineered and orthotopic mouse models, we demonstrate a key role for transcriptional regulator Yap in maintenance of Kras-mutant pancreatic tumors. Integrated ...transcriptional and metabolomics analysis reveals that Yap transcribes Myc and cooperates with Myc to maintain global transcription of metabolic genes. Yap loss triggers acute metabolic stress, which causes tumor regression while inducing epigenetic reprogramming and Sox2 upregulation in a subset of pancreatic neoplastic cells. Sox2 restores Myc expression and metabolic homeostasis in Yap-deficient neoplastic ductal cells, which gradually re-differentiate into acinar-like cells, partially restoring pancreatic parenchyma in vivo. Both the short-term and long-term effects of Yap loss in inducing cell death and re-differentiation, respectively, are blunted in advanced, poorly differentiated p53-mutant pancreatic tumors. Collectively, these findings reveal a highly dynamic and interdependent metabolic, transcriptional, and epigenetic regulatory network governed by Yap, Myc, Sox2, and p53 that dictates pancreatic tumor metabolism, growth, survival, and differentiation.
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•Yap maintains the global metabolic transcriptional program in conjunction with Myc•Yap ablation triggers metabolic crisis and regresses early-stage pancreatic tumors•Yap ablation de-represses Sox2 and acinar lineage genes via DNA demethylation•Sox2 restores Myc and metabolic homeostasis in Yap-null pancreatic tumor cells
Murakami et al. demonstrate the stage-dependent requirement for Yap in PDAC maintenance. Yap knockout causes tumor regression and regeneration of pancreatic parenchyma in early-stage PDAC but only temporarily halts tumor growth in advanced p53 mutant PDAC tumors due to de-repression of Sox2, which substitutes for Yap in maintaining metabolic homeostasis.
Type 2 diabetes mellitus (T2DM) is associated with hyperglycemia and a risk to develop pancreatic ductal adenocarcinoma (PDAC), one of the most fatal malignancies. Cancer stem cells (CSC) are ...essential for initiation and maintenance of tumors, and acquisition of CSC-features is linked to epithelial-mesenchymal-transition (EMT). The present study investigated whether hyperglycemia promotes EMT and CSC-features in premalignant and malignant pancreatic ductal epithelial cells (PDEC). Under normoglycemia (5 mM d-glucose), Panc1 PDAC cells but not premalignant H6c7-kras cells exhibited a mesenchymal phenotype along with pronounced colony formation. While hyperglycemia (25 mM d-glucose) did not impact the mesenchymal phenotype of Panc1 cells, CSC-properties were aggravated exemplified by increased Nanog expression and Nanog-dependent formation of holo- and meroclones. In H6c7-kras cells, high glucose increased secretion of Transforming-Growth-Factor-beta1 (TGF-β1) as well as TGF-β1 signaling, and in a TGF-β1-dependent manner reduced E-cadherin expression, increased Nestin expression and number of meroclones. Finally, reduced E-cadherin expression was detected in pancreatic ducts of hyperglycemic but not normoglycemic mice. These data suggest that hyperglycemia promotes the acquisition of mesenchymal and CSC-properties in PDEC by activating TGF-β signaling and might explain how T2DM facilitates pancreatic tumorigenesis.
•Elevated glucose levels aggravate cancer stemness in pancreatic cancer cells.•Hyperglycemia essentially impacts already on pancreatic ductal epithelial cells.•Hyperglycemia-induced mesenchymal and CSC-properties are TGF-β1 dependent.•This study gives insight how hyperglycemia contributes to pancreatic tumorigenesis.
Pathological lymphatic diseases mostly affect vessels in specific tissues, yet little is known about organ-specific regulation of the lymphatic vasculature. Here, we show that the vascular ...endothelial growth factor receptor 3 (VEGFR-3)/p110α PI3-kinase signaling pathway is selectively required for the formation of mesenteric lymphatic vasculature. Using genetic lineage tracing, we demonstrate that part of the mesenteric lymphatic vasculature develops from cKit lineage cells of hemogenic endothelial origin through a process we define as lymphvasculogenesis. This is contrary to the current dogma that all mammalian lymphatic vessels form by sprouting from veins. Our results reveal vascular-bed-specific differences in the origin and mechanisms of vessel formation, which may critically underlie organ-specific manifestation of lymphatic dysfunction in disease. The progenitor cells identified in this study may be exploited to restore lymphatic function following cancer surgery, lymphedema, or tissue trauma.
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•VEGFR-3/PI3K signaling regulates organ-specific mesenteric lymphatic development•Different lymphatic vascular beds have different origins•Hemogenic endothelium-derived cells contribute to mesenteric lymphatic vessels•Mesenteric lymphatic vessels develop by a process defined as lymphvasculogenesis
Stanczuk et al. show that lymphatic vessels in different organs are of different origins. Lymphatic vessels in the mesentery develop from hemogenic endothelium-derived cells, unlike lymphatic vessels in other organs that form via sprouting from veins.
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