Multiplex molecular profiling of breast cancer cell lines with quantum dot-antibody conjugates

2173 Identification of biomarkers in breast tumors is of primary importance in predicting the likely benefit of systemic therapies. Conventional biomarkers, such as estrogen receptor (ER), progesterone receptor (PR) and HER2/neu (HER2) can be used to predict the outcome from hormonal therapies and trastuzumab. Techniques currently used to detect and quantify these conventional biomarkers such as immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are unable to perform multiplex antigen determination. Semiconductor nanoparticles such as quantum dots (QDs) have unique optical properties and can be conjugated with antibodies. QD-antibody conjugates allow the simultaneous detection of several proteins on a single specimen thereby avoiding problems with tumor heterogeneity. Methods: Antibodies conjugated to multicolor QDs were used to perform multiplex labeling of 5 breast cancer cell lines. Antibodies for HER2, ER and PR were conjugated to 565nm, 605nm and 655nm QDs (QDots Corp., CA) respectively. MCF-7, T47D, BT474, SKBR3 and MDA-MB-231 human breast cancer cell lines, and NH3T3 fibroblasts cells (used as controls) were plated in 8-wells chamber slides, fixed and stained simultaneously with anti-HER2, anti-ER and anti-PR antibody-QD conjugates. Quantitative analysis of the labeled antigens was then completed via single-cell spectroscopy using the SpectroPro 150 fluorescence microscope and a single-stage spectrograph. Results: We obtained a wavelength- resolved spectrum with three separated peaks representing expression levels of HER2, ER and PR. One hundred single cells from every cell line were analyzed and an average of intensity patterns was calculated. Estimated antigen amount is shown in the table (Fluorescent intensity -a.u.x10-2). We found an excellent correlation between biomarker expression levels using conjugated QDs and by Western blot, IHC and FISH. Conclusions: Using conjugated QDs, we have detected ER, PR and HER2 simultaneously on a single specimen. Conjugated QDs will ultimately allow the detection of up to 100 proteins simultaneously on a single specimen, thereby, opening exciting new possibilities in cancer molecular profiling using nanotechnology.