ABSTRACT
The wide possibilities opened by the developments of multi-parametric cytometry are limited by the inadequacy of the classical methods of analysis to the multi-dimensional characteristics of the data. While new computational tools seemed ideally adapted and were applied successfully, their adoption is still low among the flow cytometrists. In the purpose to integrate unsupervised computational tools for the management of multi-stained samples, we investigated their advantages and limits by comparison to manual gating on a typical sample analyzed in immunomonitoring routine. A single tube of PBMC, containing 11 populations characterized by different sizes and stained with 9 fluorescent markers, was used. We investigated the impact of the strategy choice on manual gating variability, an undocumented pitfall of the analysis process, and we identified rules to optimize it. While assessing automatic gating as an alternate, we introduced the Multi-Experiment Viewer software (MeV) and validated it for merging clusters and annotating interactively populations. This procedure allowed the finding of both targeted and unexpected populations. However, the careful examination of computed clusters in standard dot plots revealed some heterogeneity, often below 10%, that was overcome by increasing the number of clusters to be computed. MeV facilitated the identification of populations by displaying both the MFI and the marker signature of the dataset simultaneously. The procedure described here appears fully adapted to manage homogeneously high number of multi-stained samples and allows improving multi-parametric analyses in a way close to the classic approach. © 2016 International Society for Advancement of Cytometry.
Subject(s)
Algorithms , Data Interpretation, Statistical , Flow Cytometry/methods , Immunophenotyping/statistics & numerical data , Leukocytes, Mononuclear/cytology , Cell Lineage/immunology , Fluorescent Dyes/chemistry , Humans , Immunophenotyping/methods , Leukocytes, Mononuclear/classification , Leukocytes, Mononuclear/immunology , Software , Staining and Labeling/methodsABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is a critical health issue in the field of cancer, with few therapeutic options. Evidence supports an implication of the intratumoral microenvironment (stroma) on PDA progression. However, its contribution to the role of neuroplastic changes within the pathophysiology and clinical course of PDA, through tumor recurrence and neuropathic pain, remains unknown, neglecting a putative, therapeutic window. Here, we report that the intratumoral microenvironment is a mediator of PDA-associated neural remodeling (PANR), and we highlight factors such as 'SLIT2' (an axon guidance molecule), which is expressed by cancer-associated fibroblasts (CAFs), that impact on neuroplastic changes in human PDA. We showed that 'CAF-secreted SLIT2' increases neurite outgrowth from dorsal root ganglia neurons as well as from Schwann cell migration/proliferation by modulating N-cadherin/ß-catenin signaling. Importantly, SLIT2/ROBO signaling inhibition disrupts this stromal/neural connection. Finally, we revealed that SLIT2 expression and CAFs are correlated with neural remodeling within human and mouse PDA. All together, our data demonstrate the implication of CAFs, through the secretion of axon guidance molecule, in PANR. Furthermore, it provides rationale to investigate the disruption of the stromal/neural compartment connection with SLIT2/ROBO inhibitors for the treatment of pancreatic cancer recurrence and pain.
Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Animals , Axons/drug effects , Axons/metabolism , Cadherins/metabolism , Cell Communication/drug effects , Cell Compartmentation/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Culture Media/pharmacology , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mice, Nude , Models, Biological , Neurons/drug effects , Neurons/metabolism , Pancreatic Neoplasms/genetics , Schwann Cells/drug effects , Schwann Cells/metabolism , Schwann Cells/pathology , Signal Transduction/drug effects , Stromal Cells/drug effects , Stromal Cells/metabolism , Stromal Cells/pathology , Transcriptome/genetics , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , beta Catenin/metabolism , Pancreatic NeoplasmsSubject(s)
DNA Methylation , Epigenesis, Genetic , Histones/genetics , Leukemia, Myeloid, Acute/genetics , Multigene Family , Aged , Biomarkers/metabolism , Chromatin/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation , Gene Expression Regulation, Leukemic , Humans , Male , Middle Aged , Mutation , Nuclear Proteins/genetics , NucleophosminABSTRACT
Stem cells represent not only a potential source of treatment for degenerative diseases but can also shed light on developmental biology and cancer. It is believed that stem cells differentiation and fate is triggered by a common genetic program that endows those cells with the ability to differentiate into specialized progenitors and fully differentiated cells. To extract the stemness signature of several cells types at the transcription level, we integrated heterogeneous datasets (microarray experiments) performed in different adult and embryonic tissues (liver, blood, bone, prostate and stomach in Homo sapiens and Mus musculus). Data were integrated by generalization of the hematopoietic stem cell hierarchy and by homology between mouse and human. The variation-filtered and integrated gene expression dataset was fed to a single-layered neural network to create a classifier to (i) extract the stemness signature and (ii) characterize unknown stem cell tissue samples by attribution of a stem cell differentiation stage. We were able to characterize mouse stomach progenitor and human prostate progenitor samples and isolate gene signatures playing a fundamental role for every level of the generalized stem cell hierarchy.
