A controversial but well founded cancer cell model proposes that a small subset of tumor initiating cells (TIC) or cancer "stem-like" cells (CSC) are necessary to initiate and sustain cancer growth. They are resistant to traditional therapy and capable of division and differentiation to give rise to a heterogeneous population of tumor cells. Therapeutics which target TIC/CSC have the potential to drastically improve patient survival. However, there are several obstacles to the study of TIC/CSC, First, they are very rare, representing typically <5% of cells in cell lines and <1% of cells in tumors. Additionally, there i considerable evidence that several subpopulations of tumor initiating cells may exist within one tumor and their identification would require the use of many cell markers in combination with other identifying characteristics. Traditional screening methods typically focus on reduction of overall tumor cell number and will therefore miss these rare, transient cells. There is a clear need to provide tools to expedite the characterization of these rare but critical subtypes to aid i the development of more effective targeted therapies. This grant will focus on the development of high throughput single cell microfluidic platforms for the marker-free enrichment and study of cancer stem cells (CSC) or tumor initiating cells (TIC) using single-cell derived cancer spheroids. This platform will provide high efficiency single-cell capture (>90% capture) and long-term suspension and adherent culture from single cells. Suspended sphere culture of single cancer cells provides the ability to not only screen cancer heterogeneity at high throughput, but also provides the capability for label-free CSC/TIC drug screening. Preliminary Data: We have demonstrated a user-friendly microfluidic approach capable of automated capture (>80% rate) of single cells into high throughput arrays, using no external systems. With this platform we have successfully tracked captured single prostate cancer cells, grown clonal colonies, and analyzed their heterogeneous drug response. Additionally, we have investigated the capabilities of topographically patterned PDMS for non-adherent culture of cancer cells. These surfaces were integrated into our single cell microfluidic platform for the formation of single-cell derived spheres. Sphere forming efficiencies were measured for multiple breast cancer cell lines including SUM159, MCF7, and MDA-MB231.
Specific Aims :
In Specific Aim 1, we will characterize our novel patterned PDMS surface for non-adherent culture use, integrate them into an optimized single cell capture platform, and modify the device architecture to interface high throughput chemical screening libraries.
In Specific Aim 2, we will use our integrated system to characterize the sphere forming efficiency of multiple breast cancer lines, screen the CSC/TIC targeting efficiency of the NIH Clinical Collection chemical library, and perform a secondary dose response assay on those selected in the drug screen. Finally in Specific Aim 3, we will develop a method for harvesting spheres from our device and validate the top chemical agents from Aim 2 using a NOD/SCID mouse model.
This application will develop high throughput single cell microfluidic platforms for the marker-free enrichment and study of cancer stem cells (CSC) or tumor initiating cells (TIC) using single-cell derived cancer spheroids. Our approach can provide high efficiency single-cell capture (>90% capture) and long-term suspension and adherent culture from single cells. Suspended sphere culture of single cancer cells provides the ability to not only screen cancer heterogeneity in high throughput, but also provides the capability for label-free drug screening targeting cancer initiating cells.
|Chen, Yu-Chih; Cheng, Yu-Heng; Kim, Hong Sun et al. (2014) Paired single cell co-culture microenvironments isolated by two-phase flow with continuous nutrient renewal. Lab Chip 14:2941-7|