Fundamental characteristics of cells important to human health and development, such as the metastatic potential of cancer stem cells and the differentiation of normal embryonic cells, are controlled by precisely regulated activity of specific sets of genes. The exquisite control of gene activity in normal cells is regulated through epigenetic factors that determine which genes are expressed and which are repressed. Dysregulation of epigenetic factors can lead to activation of inappropriate signaling pathays, uncontrolled growth, and faulty cellular differentiation. Discovering the epigenetic characteristics of specific cell types may allow us to develop strategies to control their fates.
Epigenetic mechanisms that regulate gene activity include DNA methylation, modification of DNA-binding proteins, and factors that control the translation of protein-coding genes such as microRNAs.
We have previously developed microarray-based tools that enable the high-throughput analysis of two epigenetic mechanisms, microRNAs expression and DNA methylation, in a variety of cell types. Although these are powerful analytic tools, the current methods require samples containing at least 10,000-20,000 cells.
While these platforms are adequate for certain cell types, they are not sufficiently sensitive for analysis of rare stem cell populations, such as cancer stem cells, biopsies of engrafted cells from transplantation trials, small subpopulations of human embryonic stem cell-derived differentiated cells, and cells from early embryos.
We propose to develop assays that will enable quick, cost-effective epigenetic analysis of samples as small as 100-1,000 cells on two of our existing high-performance analytical systems, the [REDACTED] DNA Methylation Assay and the [REDACTED] microRNA Assay. To develop the assays, we will use well-studied human embryonic stem cell populations, using the existing larger-scale assays as gold standards.
Finally, we will apply the assays to a real-world clinical situation, by characterizing rare populations of circulating tumor cells found in the blood of cancer patients.
Since the human embryonic stem cell line we will use is not NIH-approved, this study is not eligible for NIH funding.
Cancer stem cells, the cells that seed and fuel metastatic tumors, are likely to be responsible for recurrence in many different forms of cancer. These cells are just beginning to be identified in some forms of leukemia, but it has been extremely challenging to devise tools to study them because they are present in such low numbers. We know that fundamental characteristics of cells important to human health are controlled by precisely regulated activity of specific sets of genes. The exquisite control of gene activity in normal cells is regulated through regulatory molecules collectively known as “epigenetic” factors because they determine what genes are active in the DNA sequence without altering the sequence itself. Uncontrolled growth of cells, including cancer cells, is believed to be influenced by loss of epigenetic controls.
Since current technology for analyzing the epigenetic state of cells requires thousands of cells, it has been impossible to study this important characteristic in rare populations, such as the few hundred cancer stem cells that are believed to be circulating in a patient’s bloodstream. We have developed sensitive tools for analysis of DNA methylation, which is a major form of epigenetic control, and of microRNAs, which are powerful regulators of gene activity. While these assayswork well if samples contain thousands of cells, they have not been optimized for very small cell populations.
We propose to systematically modify and tune our existing technology so that it becomes sensitive enough to analyze rare cells that number in the hundreds. We will test the assays on cancer cells isolated from the blood of patients. Understanding the epigenetic state of cancer stem cells will help in development of strategies to inactivate them to prevent metastasis. The tool we will develop may also have application in cancer diagnosis, which would greatly benefit Californians, both by improving healthcare and encouraging development of the biotechnology industry.