Endothelium Barrier Function Helps Mitigate Metastasis

Cancer Biology: Learnings from Transcriptomics Analysis

Human Umbilical Vein Endothelial Cells, or HUVECs as they are more commonly known, are widely utilized as an in vitro model system for studying physiological and pathological processes of the vascular system. Endothelial cells line the inside of blood vessels and have a central role in angiogenesis, the formation of new blood vessels. Endothelial cells, connected by tight junctions also form a defensive barrier between blood vessels and tissue, regulating the movement of nutrients and cells.

In fact, endothelial cells are of great interest in cancer biology because they are a prime target for cancer cells. During pathogenesis, cancer cells bind to endothelial cells and release soluble factors into the microenvironment that activate endothelial cells to form new blood vessels to the tumor, increasing its access to nutrient and oxygen-rich blood to support its growth. Additionally, the breakdown of tight junctions between the endothelial cells is implicated in cancer cell extravasation and tumor metastasis to distal parts of the body through the blood vessels. Utilizing sophisticated “-omics” approaches, scientists are studying changes in HUVECs by co-culturing them with cancer cells in vitro to gain essential insights into the complex interplay between the cell types in hopes of developing more effective cancer therapeutics.

The Lifeline® catalog has a variety of high-quality endothelial and cardiac cells from a variety of tissue sources, which include the following:

• Cardiac microvascular endothelial cells
• Cardiac fibroblasts
• Pulmonary artery endothelial cells
• Aortic endothelial cells
• Coronary artery endothelial cells
• Lung microvascular endothelial cells
• Dermal microvascular endothelial cells (adult and neonatal)
• Iliac artery endothelial cells
• Umbilical cord endothelial cells (HUVECs; primary and 10-donor pool)

Tumor Cells Modulate Gene Expression in Endothelial Cells

Recently, Pan and Colleagues published a paper investigating the endothelium and the importance of its barrier function to mitigate tumor invasion and metastasis. When tumor cells adhere to the endothelial cells, changes are induced in both cell types where endothelial barrier integrity is impacted and angiogenesis and metastasis of tumor cells is activated. However, the changes that happen in endothelial cells when tumor–endothelium interactions occur are still unclear. To look specifically at changes within the endothelial cells, the authors utilized an in vitro coculture model system with Lifeline’s Human Umbilical Vein Endothelial Cells (HUVEC’s) and the prostate tumor cell line PC-3M to study the underlying molecular mechanisms and biological processes triggered by tumor cell adhesion to the endothelium.

In recent years, genomics, transcriptomics, and proteomics analyses have been widely used in cancer-related research. Among them, transcriptomics, which analyzes RNA expression showed that long non-coding RNAs (lncRNAs) play key roles in the initiation and progression of cancer. lncRNAs are > 200 nucleotides long and interact with other cellular components, such as protein, RNA, and DNA to modulate their function. Here, the authors used the Affymetrix Gene Chip Human Transcriptome Array 2.0. and quantitative real-time PCR (qPCR) to map the genetic changes in endothelial cells induced by prostate tumor cell attachment compared to baseline endothelial cells. A total of 504 differentially expressed mRNAs and 444 long non-coding RNAs (lncRNAs) were obtained through chip data analysis demonstrating that the adhesion of tumor cells significantly dysregulated the mRNAs and lncRNAs in endothelial cells.

From the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the differentially expressed genes in HUVECs resulting from tumor cell (PC-3M) adhesion were found to be involved in a wide range of biological functions like DNA replication and repair, energy metabolism (especially purine and pyrimidine), and growth-related signaling pathways. In looking at the interaction networks between the RNA populations (mRNA, miRNA and lncRNA), PRKAA2 was identified as one of the integral genes connecting the functions identified by bioinformatics analysis. The authors found that PRKAA2 mRNA expression was significantly higher in cocultured HUVECs/PC-3M than HUVECs alone. PRKAA2 is a catalytic subunit of the AMP-activated protein kinase (AMPK), which is important in maintaining the cells’ energy homeostasis by regulating catabolic pathways. In the context of endothelial cells, upregulation of PRKAA2 and AMPK caused by PC-3M adhesion results in increased catabolic pathways, such as glycolysis, fatty acid oxidation, and glutamine metabolism, which have essential roles during angiogenesis.

Further examination of the interaction network modeled by the authors identified the miRNA miR-124-3p and several lncRNAs (n339695, n378130, and n410438) that are involved in regulating PRKAA2 expression. miR-124-3p specifically regulates the expression of PRKAA2 where its downregulation in HUVECs cocultured with PC-3M cells resulting in increased PRKAA2 expression. Research suggests that lncRNAs act as an “miRNA sponge” to modulate levels of miRNA within the cells, thus leading the authors to speculate that the lncRNAs identified in this network could regulate the “miR-124-3p/PRKAA2” axis in endothelial cells when activated by tumor cell attachment and that modulation of this pathway plays a pivotal role in tumor–endothelium interactions.

Together, this transcriptomics approach was able to better define the underlying molecular mechanisms and biological processes tumor–endothelium interaction is important to better our understanding of, and perhaps help identify therapeutic strategies to control angiogenesis and tumor metastasis.

Are you using Lifeline cells or media in your research? Let us know and your study could be featured in a future blog post!