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Separated Pancreatic stellate cells (PSCs) by functional separation using modified Matrigel invasion assay

ABSTRACT: We separated PSCs by functional separation using moidfied Matrigel invasion assay our previously reported (Fujiwara, PLoS One, 2012). In this culture system, we evaluated two primary cultures of human PSCs. PSCs in the upper chambers were incubated for 72 hours in culture with SUIT-2 pancreatic cancer cells in the lower chambers, or as controls without pancreatic cancer cells in the lower chambers, and were collected from the top and bottom sides of the upper chambers separately. The top side-derived PSCs were cells that did not migrate toward the cancer cells, and the bottom side-derived PSCs were cells that migrated toward the pancreatic cancer cells. We then performed a DNA microarray analysis using these cells. The quality of the RNA samples was evaluated using Experion™ Automated Electrophoresis Station (Bio-Rad Laboratories, Hercules, CA). We used a HumanHT-12v4 Expression BeadChip (Illumina, San Diego, CA) for these analyses. The data were analyzed using Bead Studio software version 3.2.3 (Illumina).

ORGANISM(S): Homo sapiens

SUBMITTER: Fujiwara Kenji

PROVIDER: S-ECPF-GEOD-55300 | biostudies-other |

REPOSITORIES: biostudies-other

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Separated Pancreatic stellate cells (PSCs) by functional separation using modified Matrigel invasion assay

Project description:We separated PSCs by functional separation using moidfied Matrigel invasion assay our previously reported (Fujiwara, PLoS One, 2012). In this culture system, we evaluated two primary cultures of human PSCs. PSCs in the upper chambers were incubated for 72 hours in culture with SUIT-2 pancreatic cancer cells in the lower chambers, or as controls without pancreatic cancer cells in the lower chambers, and were collected from the top and bottom sides of the upper chambers separately. The top side-derived PSCs were cells that did not migrate toward the cancer cells, and the bottom side-derived PSCs were cells that migrated toward the pancreatic cancer cells. We then performed a DNA microarray analysis using these cells. The quality of the RNA samples was evaluated using Experion™ Automated Electrophoresis Station (Bio-Rad Laboratories, Hercules, CA). We used a HumanHT-12v4 Expression BeadChip (Illumina, San Diego, CA) for these analyses. The data were analyzed using Bead Studio software version 3.2.3 (Illumina).

2014-02-25 | E-GEOD-55300 | biostudies-arrayexpress

Separated Pancreatic stellate cells (PSCs) by functional separation using modified Matrigel invasion assay

2014-02-25 | GSE55300 | GEO

Pancreatic Stellate Cells Facilitate Perineural Invasion of Pancreatic Cancer via HGF/c-Met Pathway.

Project description:Pancreatic cancer (PC) is a highly lethal cancer that has a strong ability for invasion and metastasis, poor prognosis, and a stubbornly high death rate due to late diagnosis and early metastasis. Therefore, a better understanding of the mechanisms of metastasis should provide novel opportunities for therapeutic purposes. As a route of metastasis in PC, perineural invasion (PNI) occurs frequently; however, the molecular mechanism of PNI is still poorly understood. In this study, we show that the hepatocyte growth factor (HGF)/c-Met pathway plays a vital role in the PNI of PC. We found that HGF promotes PC cell migration and invasion by activating the HGF/c-Met pathway, and enhances the expression of nerve growth factor (NGF) and matrix metalloproteinase-9 (MMP9) in vitro. Furthermore, HGF significantly increased PC cell invasion of the dorsal root ganglia (DRG) and promoted the outgrowth of DRG in cocultured models of PC cells and DRG. In contrast, the capacity for invasion and the phenomenon of PNI in PC cells were reduced when the HGF/c-Met pathway was blocked by siRNA. In conclusion, PSCs facilitate PC cell PNI via the HGF/c-Met pathway. Targeting the HGF/c-Met signaling pathway could be a promising therapeutic strategy for PC.

| S-EPMC6767883 | biostudies-literature

Whole genome functional characterization of RE1 silencers using a modified massively parallel reporter assay

Project description:Both upregulation and downregulation by cis-regulatory elements help establish precise gene expression. Our understanding of how elements repress transcriptional activity is far more limited than activating elements. To address this gap, we characterized RE1, a group of transcriptional silencers bound by REST, on a genome-wide scale using an optimized massively parallel reporter assay (MPRAduo). MPRAduo empirically defined a minimal binding strength of REST required by silencer (REST m-value), above which multiple cofactors colocalize and act to directly silence transcription. We identified 1,500 human variants that alter RE1 silencing and found their effect sizes are predictable when they overlap with REST binding sites above the m-value. In addition, we demonstrate that non-canonical REST binding motifs exhibit silencer function only if they precisely align two half sites with specific spacer length. Our results show mechanistic insights into RE1 silencer which allows us to predict its activity and effect of variants on RE1, providing a paradigm for performing genome-wide functional characterization transcription factors binding sites.

2022-02-17 | GSE196171 | GEO

Nuclear translocation of FGFR1 and FGF2 in pancreatic stellate cells facilitates pancreatic cancer cell invasion.

Project description:Pancreatic cancer is characterised by desmoplasia, driven by activated pancreatic stellate cells (PSCs). Over-expression of FGFs and their receptors is a feature of pancreatic cancer and correlates with poor prognosis, but whether their expression impacts on PSCs is unclear. At the invasive front of human pancreatic cancer, FGF2 and FGFR1 localise to the nucleus in activated PSCs but not cancer cells. In vitro, inhibiting FGFR1 and FGF2 in PSCs, using RNAi or chemical inhibition, resulted in significantly reduced cell proliferation, which was not seen in cancer cells. In physiomimetic organotypic co-cultures, FGFR inhibition prevented PSC as well as cancer cell invasion. FGFR inhibition resulted in cytoplasmic localisation of FGFR1 and FGF2, in contrast to vehicle-treated conditions where PSCs with nuclear FGFR1 and FGF2 led cancer cells to invade the underlying extra-cellular matrix. Strikingly, abrogation of nuclear FGFR1 and FGF2 in PSCs abolished cancer cell invasion. These findings suggest a novel therapeutic approach, where preventing nuclear FGF/FGFR mediated proliferation and invasion in PSCs leads to disruption of the tumour microenvironment, preventing pancreatic cancer cell invasion.

| S-EPMC3992074 | biostudies-literature

Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumour invasion.

Project description:In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) promotes fibrotic, therapy-resistant tumours. Conversely, suppression of CAFs can result in aggressive metastatic tumours. Here we show that the Rho-effector kinase protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 was associated with reduced PSC proliferation and contractility, retention of lipid droplets and decreased a-SMA stress fibres. PKN2 loss was also associated with a myofibroblast CAF to -like inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. In spheroid co-cultures with PDAC cells, loss of PKN2 prevented PSC invasion but, counter-intuitively, promoted invasive cancer cell outgrowth. Further, deletion of PKN2 in the pancreatic stroma induced more locally invasive, orthotopic pancreatic tumours. Finally, we demonstrated that a PKN2KO PKN2 KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast differentiation function can limit important stromal tumour suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.

2021-11-19 | GSE189027 | GEO

Whole genome functional characterization of RE1 silencers using a modified massively parallel reporter assay BRB-seq

2022-09-30 | GSE212253 | GEO

Cells assemble invadopodia-like structures and invade into matrigel in a matrix metalloprotease dependent manner in the circular invasion assay.

Project description:The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the process of cancer cell invasion through basement membrane or in the stroma. We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion. Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process. We find that cells invade in a protease dependent manner in this assay and that they assemble focal adhesions and invadopodia that resemble structures visualized in 3D embedded cells. We propose that this is a useful assay for routine and medium throughput analysis of invasion of cancer cells in vitro and the study of cells migrating in a 3D environment.

| S-EPMC3275555 | biostudies-literature

Nuclear FGFR1 promotes pancreatic stellate cell-driven invasion through up-regulation of Neuregulin 1.

Project description:Pancreatic stellate cells (PSCs) are key to the treatment-refractory desmoplastic phenotype of pancreatic ductal adenocarcinoma (PDAC) and have received considerable attention as a stromal target for cancer therapy. This approach demands detailed understanding of their pro- and anti-tumourigenic effects. Interrogating PSC-cancer cell interactions in 3D models, we identified nuclear FGFR1 as critical for PSC-led invasion of cancer cells. ChIP-seq analysis of FGFR1 in PSCs revealed a number of FGFR1 interaction sites within the genome, notably NRG1, which encodes the ERBB ligand Neuregulin. We show that nuclear FGFR1 regulates transcription of NRG1, which in turn acts in autocrine fashion through an ERBB2/4 heterodimer to promote invasion. In support of this, recombinant NRG1 in 3D model systems rescued the loss of invasion incurred by FGFR inhibition. In vivo we demonstrate that, while FGFR inhibition does not affect the growth of pancreatic tumours in mice, local invasion into the pancreas is reduced. Thus, FGFR and NRG1 may present new stromal targets for PDAC therapy.

| S-EPMC9918430 | biostudies-literature

Hypoxic pancreatic stellate cell-derived exosomal mirnas promote proliferation and invasion of pancreatic cancer through the PTEN/AKT pathway.

Project description:Pancreatic stellate cells (PSCs) are important components of the tumor microenvironment in pancreatic cancer (PC) and contribute to its development and metastasis through mechanisms that remain incompletely characterized. Tumor hypoxia affects the function and behavior of PC and stromal cells, and can alter exosomal content to modify cell-cell communication. The present study explored the effects of exosomal miRNAs produced by hypoxia-preconditioned PSCs on the growth and metastatic potential of PC cells. Subcutaneous xenografts and liver metastasis mouse models revealed increased tumorigenic potential upon co-implantation of PC cells and PSCs as compared to PC cells alone. Screening miRNA profiles of mouse plasma exosomes and cultured PSCs, followed by miRNA overexpression and inhibition assays, enabled us to identify miR-4465 and miR-616-3p as prominent hypoxia-induced, PSC-derived, exosomal miRNAs promoting PC cell proliferation, migration, and invasion. Proteomics analysis of PC cells incubated with exosomes derived from hypoxic PSCs showed significant downregulation of PTEN. Dual-luciferase reporter assays and western blotting showed that both miR-4465 and miR-616-3p target PTEN and activate AKT signaling in PC cells. We conclude that hypoxia upregulates miR-4465 and miR-616-3p expression in PSC-derived exosomes. Following exosome uptake, these miRNAs promote PC progression and metastasis by suppressing the PTEN/AKT pathway.

| S-EPMC7993707 | biostudies-literature

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