Single-cell RNA sequencing combined with multiplex immunofluorescence probes the role of MFAP5+ fibroblasts in the microenvironment of pancreatic ductal adenocarcinoma
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ABSTRACT:
PROVIDER: PRJNA1202135 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31814477_1.fastq.gz | Fastqsanger.gz | |||
| SRR31814477_2.fastq.gz | Fastqsanger.gz | |||
| SRR31814478_1.fastq.gz | Fastqsanger.gz | |||
| SRR31814478_2.fastq.gz | Fastqsanger.gz | |||
| SRR31814479_1.fastq.gz | Fastqsanger.gz |
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Project description:we employed single-cell RNA sequencing to examine the biological characteristics of MFAP5+ fibroblasts in PDAC and their interaction with vascular endothelial cells within tumors. We then utilized a proposed temporal sequencing analysis technique to infer the evolution of cellular subtypes of cancer-associated fibroblasts. To verify our hypothesis, we employed a multiplex immunofluorescence technique to observe the spatial distribution of MFAP5+ fibroblasts and endothelial cells. This study investigated the tumor heterogeneity of PDAC, elucidating intricate cellular interactions within the tumor microenvironment through a comprehensive analysis of 23,905 cells. The biology of MFAP5+ fibroblasts in pancreatic ductal adenocarcinoma was investigated, and it was found that MFAP5+ fibroblasts engage in active biosignaling exchanges with FABP4+ endothelial cells and other endothelial cells through the activation of key tumor-promoting signaling pathways, such as TGF-β, VEGF, and FGF. These pathways provide support for the tumor's invasive and malignant behavior. Our hypothesis was validated by multiplex immunofluorescence and subsequent semi-quantitative analysis, which demonstrated a notable increase in the prevalence of FABP4+ and VWF+ endothelial cells in the area with elevated MFAP5+ fibroblast expression. Furthermore, our study revealed that vascular endothelial growth factor and FGF2 signaling were highly expressed in the region with high MFAP5+ fibroblast expression.
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Project description:we employed single-cell RNA sequencing to examine the biological characteristics of MFAP5+ fibroblasts in PDAC and their interaction with vascular endothelial cells within tumors. We then utilized a proposed temporal sequencing analysis technique to infer the evolution of cellular subtypes of cancer-associated fibroblasts. To verify our hypothesis, we employed a multiplex immunofluorescence technique to observe the spatial distribution of MFAP5+ fibroblasts and endothelial cells.
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Project description:Single-cell RNA sequencing combined with multiplex immunofluorescence probes the role of MFAP5+ fibroblasts in the microenvironment of pancreatic ductal adenocarcinoma.
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Project description:MFAP5, also known as microfibrillar-associated glycoprotein-2 (MAGP2), may influence parameters of skin wound healing related to scar formation. To further elucidate its role in skin wound healing, we assessed skin wound repair in Mfap5-/- mice. Loss of MFAP5 significantly reduced wound closure rates and angiogenesis while enhancing neutrophil and macrophage influx into wounds. Loss of MFAP5 also reduced the deposition of total and mature collagen in uninjured normal skin (NS), but not in wounds. Furthermore, NS dermis of Mfap5-/- mice was thinner without any reduction in tensile strength. Single-cell RNA-sequencing of NS and wounds from Mfap5+/+ and Mfap5-/- mice revealed two fibroblast subclusters that express MFAP5 more highly than other subclusters. Enrichment analysis of the differentially expressed genes (DEGs) in these two subclusters suggests these fibroblasts engage in extracellular matrix (ECM) deposition and angiogenesis. Mfap5+/+ and Mfap5-/- fibroblasts also exhibit transcriptomic differences throughout in vivo wound healing, though as healing progressed, fewer differences were evident. To examine the direct effect of MFAP5 on fibroblasts outside of the wound space, fibroblasts were isolated from Mfap5+/+ and Mfap5-/- mice for in vitro analysis. MRNA-sequencing of Mfap5+/+ and Mfap5-/- fibroblasts found genes involved in cellular migration and proliferation, ECM synthesis, and angiogenesis to be downregulated in Mfap5-/- fibroblasts vs Mfap5+/+ fibroblasts. Functionally, Mfap5-/- fibroblasts exhibited reduced migration, contractility, proliferation, and ECM deposition. Our findings indicate that MFAP5 is a multifunctional glycoprotein in skin wound healing as it promotes angiogenesis and collagen deposition, inhibits inflammatory cell influx, and promotes pro-scarring fibroblast behavior.
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Project description:MFAP5, also known as microfibrillar-associated glycoprotein-2 (MAGP2), may influence parameters of skin wound healing related to scar formation. To further elucidate its role in skin wound healing, we assessed skin wound repair in Mfap5-/- mice. Loss of MFAP5 significantly reduced wound closure rates and angiogenesis while enhancing neutrophil and macrophage influx into wounds. Loss of MFAP5 also reduced the deposition of total and mature collagen in uninjured normal skin (NS), but not in wounds. Furthermore, NS dermis of Mfap5-/- mice was thinner without any reduction in tensile strength. Single-cell RNA-sequencing of NS and wounds from Mfap5+/+ and Mfap5-/- mice revealed two fibroblast subclusters that express MFAP5 more highly than other subclusters. Enrichment analysis of the differentially expressed genes (DEGs) in these two subclusters suggests these fibroblasts engage in extracellular matrix (ECM) deposition and angiogenesis. Mfap5+/+ and Mfap5-/- fibroblasts also exhibit transcriptomic differences throughout in vivo wound healing, though as healing progressed, fewer differences were evident. To examine the direct effect of MFAP5 on fibroblasts outside of the wound space, fibroblasts were isolated from Mfap5+/+ and Mfap5-/- mice for in vitro analysis. MRNA-sequencing of Mfap5+/+ and Mfap5-/- fibroblasts found genes involved in cellular migration and proliferation, ECM synthesis, and angiogenesis to be downregulated in Mfap5-/- fibroblasts vs Mfap5+/+ fibroblasts. Functionally, Mfap5-/- fibroblasts exhibited reduced migration, contractility, proliferation, and ECM deposition. Our findings indicate that MFAP5 is a multifunctional glycoprotein in skin wound healing as it promotes angiogenesis and collagen deposition, inhibits inflammatory cell influx, and promotes pro-scarring fibroblast behavior.
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Project description:Ovarian cancer is the fifth most common form of cancer in women in the United States. Among different types of ovarian cancer, epithelial ovarian cancer is the most common and is highly lethal, however, prognostic and predictive markers, which can be used to predict chemoresponse and patient survival, have not been thoroughly explored. One critically important yet often overlooked component to the tumor progression process is the tumor microenvironment. Primarily composed of fibroblasts and extracellular matrix proteins (ECM) as well as endothelial cells and lymphocytic infiltrate, the tumor microenvironment has been shown to directly affect cell growth, migration, and differentiation through secreted proteins, cell-cell interactions and matrix remodeling (Tlsty and Coussens, 2006). The tumor microenvironment has the potential to promote tumor initiation of normal epithelial cells and facilitate progression of malignant cells, thereby, presenting a unique approach to diagnosing, understanding and treating cancer. Using a whole-genome oligonucleotide array platform to perform transcriptome profiling on the fibroblastic stromal component microdissected from a series of advanced stage high-grade serous ovarian adenocarcinomas, we identified a transcriptome signature for the ovarian cancer associated fibroblast (CAF). We further functionally characterized one of the identified genes, MFAP5, and we showed that stromal MFAP5 is a prognostic marker associated with poor patient survival. In addition to that, to investigate the signaling machanism and the effect of MFAP5 treatment on ovarian cancer cells, transcriptome profiling of MFAP5 treated OVCA432 high-grade serous ovarian cancer cells was performed. Further functional studies showed that stromal MFAP5 modulated ovarian cancer cell motility and invasion potential. High grade serous ovarian cancer cell line OVCA432 was used. Total RNA was isolated from control samples and MFAP5 treated cancer cell samples at 48 hours post-treatment. Followed by cDNA synthesis, IVT and biotin labeling, samples were then hybridized onto Affymetrix Human genome U133 plus 2.0 microarrays. For each treatment group, three independent samples were prepared for the microarray experiment.
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Project description:Ovarian cancer is the fifth most common form of cancer in women in the United States. Among different types of ovarian cancer, epithelial ovarian cancer is the most common and is highly lethal, however, prognostic and predictive markers, which can be used to predict chemoresponse and patient survival, have not been thoroughly explored. One critically important yet often overlooked component to the tumor progression process is the tumor microenvironment. Primarily composed of fibroblasts and extracellular matrix proteins (ECM) as well as endothelial cells and lymphocytic infiltrate, the tumor microenvironment has been shown to directly affect cell growth, migration, and differentiation through secreted proteins, cell-cell interactions and matrix remodeling (Tlsty and Coussens, 2006). The tumor microenvironment has the potential to promote tumor initiation of normal epithelial cells and facilitate progression of malignant cells, thereby, presenting a unique approach to diagnosing, understanding and treating cancer. Using a whole-genome oligonucleotide array platform to perform transcriptome profiling on the fibroblastic stromal component microdissected from a series of advanced stage high-grade serous ovarian adenocarcinomas, we identified a transcriptome signature for the ovarian cancer associated fibroblast (CAF). We further functionally characterized one of the identified genes, MFAP5, and we showed that stromal MFAP5 is a prognostic marker associated with poor patient survival. In addition to that, to investigate the signaling machanism and the effect of MFAP5 treatment on ovarian cancer cells, transcriptome profiling of MFAP5 treated OVCA432 high-grade serous ovarian cancer cells was performed. Further functional studies showed that stromal MFAP5 modulated ovarian cancer cell motility and invasion potential.
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