Abstract B67: Establishment of a high-content assay to assess the affects of gene silencing on inflammatory breast cancer cell motility

Inflammatory breast cancer (IBC) is an aggressive, highly invasive tumor with one of the worst clinical outcome among breast cancers with rapid clinical progression and resistance to chemotherapy and radiotherapy. Epidemiologic data show that IBC affects women at a younger age (58.8 years vs. 66.2 years for locally advanced breast cancer (LABC)) and minorities (African Americans, Hispanics, and Native Americans) are disproportionately affected. Taken together, the clinical and epidemiological data demonstrate that IBC is in fact a distinct entity from LABC, and therefore more work is required to elucidate the unique molecular characteristics of IBC that will allow for targeted treatment regimens. We have previously shown that elevated expression of Gli1 mRNA, typically a marker for Hh pathway activation, was observed in the SUM149 IBC cell line, compared to a panel of other IBC and non-IBC cell lines, and that Gli1 silencing in SUM149 using siRNA resulted in decreased proliferation and increased apoptosis. SUM149 cells were generated from an aggressive inflammatory breast cancer and have been characterized as a (ErbB2-, ER- and PR-) basal B subtype of breast cancer cell, which is frequently more invasive than other breast cancer cell types. Hence, we also wanted to investigate whether the high levels of Gli1 in SUM149 cells play a role in its unique migratory phenotype. In our study, we first used a conventional in vitro wound healing assay as a measure of migration and found that silencing of Gli1 in SUM149 cells dramatically reduces SUM149 cell migration over 24 hours. To glean more quantitative information from the motility wound healing assay, we were interested in developing high content imaging assays that would also extract other critical parameters defining migration behavior. The advantages of the wound healing assay are that it can be miniaturized and is compatible with microscopy making it amenable to high throughput imaging. Such a high content automated microscopy system allows the analysis of both population and single-cell data on a heterogeneous population of cells and potentially link phenotypic variability to genetic differences. To obtain a more quantitative assessment and further assess the role of Gli1 in the migratory phenotype of SUM149, we tagged SUM149 cells with green fluorescent protein-histone2B (GFP-H2B) to fluorescently label nuclei of living cells and using a high content live cell fluorescence-based imaging system demonstrated that we can track not only cell migration but additional parameters of motility such as linearity of movement and, integrated distance traveled. Many features of each cellular trajectory were tabulated and compared for the experimental conditions and both average linearity and distance traveled were reduced in SUM149 cells with Gli1 silenced. We expect this type of multi-parameter analysis of cell migration after GFP-H2B tagging to be generally applicable to many cell types to assess affects on motility. Grant Support: NIH grant CA137844 (K. P. Williams) and American Cancer Society grant RSG-08-290-01 -CCE (G. R. Devi). K. P. W. and G. R. D. co-senior authors. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B67.