Clinical oncology and cancer biology are challenged by the lack of assay platforms for measuring changes in cancer-cells'growth kinetics in response to therapeutic intervention. Cell growth kinetics can be measured in a number of ways, such as by DNA copy number, volume, mass, density, shape, or by expression of particular proteins. Here we define growth kinetics as changes in a cell's mass and density over time. We propose to develop an instrument for concurrently monitoring single cell growth kinetics and cell surface protein expression. We hypothesize that changes in growth kinetics and in cell surface protein expression can be used as a surrogate for response to pathway-directed therapeutic agents. As a validation of the instrument, we will monitor mass, density and cell-surface protein expression (determined by fluorescence) in single A431 cells in response to intervention with the pathway targeted therapy gefitinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), and appropriate controls. Single cell mass and density will be measured by a previously validated device known as the suspended microchannel resonator (SMR). The SMR can measure the mass of a mammalian cell with a resolution near 0.01% (1 Hz bandwidth). In order to achieve a continuous measurement of mass and density, silicon posts will be used for capturing a single cell within the sensitive region of the microchannel resonator. To enable simultaneous detection of protein expression, by fluorescence of coupled antibodies, the SMR will be modified to have optical transparency within the region where the cell is captured. The proposed instrument will leverage our prior proteomics work with the Center for Cancer Nanotechnology Excellence focused on Therapeutic Response (CCNE-TR) where we used proteomic methods to discover cell-surface protein biomarkers indicative of therapeutic response. Single-cell measurement of the abundance of these proteins, concurrently with cell mass and density kinetics will provide a new approach for characterizing and monitoring cell response to therapy on a physiological and molecular level.
The primary aim of this proposal is to develop an instrument for measuring the growth kinetics of single cells. We will accomplish this by developing a Cell Monitoring System (CMS) whereby single cells are trapped at the mass sensitive region of the SMR so the mass and density can be measured versus time and correlated with fluorescence readout from cell surface proteins. A key requirement for this instrument is the ability to control the fluidic environment of the cell so that growth can be perturbed by the delivery of various analytes. Once validated, we will determine if the CMS can be used to classify the response of cancer cells to pathway-directed therapeutic agents.
|Bryan, Andrea K; Engler, Alex; Gulati, Amneet et al. (2012) Continuous and long-term volume measurements with a commercial Coulter counter. PLoS One 7:e29866|
|Son, Sungmin; Tzur, Amit; Weng, Yaochung et al. (2012) Direct observation of mammalian cell growth and size regulation. Nat Methods 9:910-2|
|Weng, Yaochung; Delgado, Francisco Feijó; Son, Sungmin et al. (2011) Mass sensors with mechanical traps for weighing single cells in different fluids. Lab Chip 11:4174-80|