Detection Of Multiple Biomarkers Simultaneously Using Multi-Colored Nanoparticles In Breast Cancer Tissues.

9525 Background: Available methods of molecular profiling of tumors are unable to detect biomarkers simultaneously on single specimens, and do not take into account tumor heterogeneity. Additionally many of these methods require tissue destruction restricting investigation of tumor microenvironment and single cell profiling. We investigated a nanotechnology-based approach to profile cancer cell biomarkers simultaneously utilizing multicolor nanoparticles, called quantum dots (QDs). QDs are photon-emitting nanoparticles, which can be engineered to emit across a wide spectrum, by altering the size of the particle. We have developed a novel method of multiplex QD-based labeling (MQDL) for in-situ analysis of biomarkers in breast cancer cell lines and xenograft tumors. Methods: Antibodies against human ER, PR, HER2 and Ki-67 were conjugated with multicolor QDs (605nm, 655nm, 565nm and 705nm respectively) and incubated with MCF-7, BT474 and MDA-MB-231 human breast cancer cell lines. Labeled antigens were visualized by laser-scanning microscopy and level of their expression was established by spectral microscopy. For in-situ tumor diagnostics frozen tumor tissues from xenografts of breast cancer origin were stained with QDs-antibody conjugates and analyzed by spectroscopy. Results: We were able to identify and quantify ER, PR, HER2 and Ki67 using conjugated QDs simultaneously on single slides of breast cancer cell lines and breast xenograft tumors. Spectroscopy revealed a curve with 4 separate peaks representing the level of expression of the analyzed antigens with each peak corresponding to the coloured identity specifically assigned to each antigen of interest in the breast cancer cells. Quantifying analysis of ER, PR, HER2 and Ki-67 expression by MQDL showed excellent correlation with routine methods. Conclusion: Simultaneous multiplex QD labeling is a new method of molecular profiling that allows determination and measurement of expression of analyzed biomarkers in single specimens of cell lines and tumor samples. MQDL can facilitate in identifying individual biomolecular protein signatures of tumors, a step necessary to design selective targeting therapy. No significant financial relationships to disclose.