RESUMO
Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.
Assuntos
Heterogeneidade Genética , Genômica , Imageamento Tridimensional , Neoplasias Pancreáticas , Lesões Pré-Cancerosas , Análise de Célula Única , Adulto , Feminino , Humanos , Masculino , Células Clonais/metabolismo , Células Clonais/patologia , Sequenciamento do Exoma , Aprendizado de Máquina , Mutação , Pâncreas/anatomia & histologia , Pâncreas/citologia , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Fluxo de Trabalho , Progressão da Doença , Detecção Precoce de Câncer , Oncogenes/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer for which few effective therapies exist. Immunotherapies specifically are ineffective in pancreatic cancer, in part due to its unique stromal and immune microenvironment. Pancreatic intraepithelial neoplasia, or PanIN, is the main precursor lesion to PDAC. Recently it was discovered that PanINs are remarkably abundant in the grossly normal pancreas, suggesting that the vast majority will never progress to cancer. Here, through construction of 48 samples of cm 3 -sized human pancreas tissue, we profiled the immune microenvironment of 1,476 PanINs in 3D and at single-cell resolution to better understand the early evolution of the pancreatic tumor microenvironment and to determine how inflammation may play a role in cancer progression. We found that bulk pancreatic inflammation strongly correlates to PanIN cell fraction. We found that the immune response around PanINs is highly heterogeneous, with distinct immune hotspots and cold spots that appear and disappear in a span of tens of microns. Immune hotspots generally mark locations of higher grade of dysplasia or locations near acinar atrophy. The immune composition at these hotspots is dominated by naïve, cytotoxic, and regulatory T cells, cancer associated fibroblasts, and tumor associated macrophages, with little similarity to the immune composition around less-inflamed PanINs. By mapping FOXP3+ cells in 3D, we found that regulatory T cells are present at higher density in larger PanIN lesions compared to smaller PanINs, suggesting that the early initiation of PanINs may not exhibit an immunosuppressive response. This analysis demonstrates that while PanINs are common in the pancreases of most individuals, inflammation may play a pivotal role, both at the bulk and the microscopic scale, in demarcating regions of significance in cancer progression.
RESUMO
The ability of stem cells to build and replenish tissues depends on support from their niche. Although niche architecture varies across organs, its functional importance is unclear. During hair follicle growth, multipotent epithelial progenitors build hair via crosstalk with their remodeling fibroblast niche, the dermal papilla, providing a powerful model to functionally interrogate niche architecture. Through mouse intravital imaging, we show that dermal papilla fibroblasts remodel individually and collectively to form a morphologically polarized, structurally robust niche. Asymmetric TGF-ß signaling precedes morphological niche polarity, and loss of TGF-ß signaling in dermal papilla fibroblasts leads them to progressively lose their stereotypic architecture, instead surrounding the epithelium. The reorganized niche induces the redistribution of multipotent progenitors but nevertheless supports their proliferation and differentiation. However, the differentiated lineages and hairs produced by progenitors are shorter. Overall, our results reveal that niche architecture optimizes organ efficiency but is not absolutely essential for organ function.
Assuntos
Folículo Piloso , Cabelo , Camundongos , Animais , Diferenciação Celular , Epitélio , Fator de Crescimento Transformador betaRESUMO
The classic network of mitogen-activated protein kinases (MAPKs) is highly interconnected and controls a diverse array of biological processes. In multicellular eukaryotes, the MAPKs ERK, JNK, and p38 control opposing cell behaviors but are often activated simultaneously, raising questions about how input-output specificity is achieved. Here, we use multiplexed MAPK activity biosensors to investigate how cell fate control emerges from the connectivity and dynamics of the MAPK network. Through chemical and genetic perturbation, we systematically explore the outputs and functions of all the MAP3 kinases encoded in the human genome and show that MAP3Ks control cell fate by triggering unique combinations of MAPK activity. We show that these MAPK activity combinations explain the paradoxical dual role of JNK signaling as pro-apoptotic or pro-proliferative kinase. Overall, our integrative analysis indicates that the MAPK network operates as a unit to control cell fate and shifts the focus from MAPKs to MAP3Ks to better understand signaling-mediated control of cell fate.