Simultaneous, quantitative detection of multiple biomarkers in human breast cancers using multicolor nanoparticles

619 Background: Conventional methods of detecting breast cancer biomarkers are hampered by a lack of adequate quantification and/or an inability to detect multiple targets on small quantities of tissue. We have previously demonstrated that estrogen receptor (ER), progesterone receptor (PR) and HER2/neu (HER2) can be detected and quantified simultaneously using antibodies (Abs) directly conjugated to nanoparticles, called quantum dots (QDs), on single breast cancer sections (ASCO 2005). We have expanded our assay to use multicolored QDs conjugated directly to Abs (QD-Abs) to detect and quantify simultaneously ER, PR, and HER2, along with 3 putative biomarkers, epidermal growth factor receptor (EGFR), mammalian target of Rapamycin (mTOR), and insulin-like growth factor receptor (IGFR), in breast cancer cell lines and human breast cancers. Methods: We used multicolored QDs directly conjugated to primary Abs to detect the 6 proteins in breast cancer cell lines (MCF-7, BT474, MDA-231) and single sections of human breast cancers. The 6 proteins were quantified using spectral separation microscopy, and compared to Western blotting. Results: We detected all 6 proteins simultaneously using QD-Abs in breast cancer cell lines and breast tumors. Using hyper-spectral imaging and wavelength-resolved spectroscopy, we separated all 6 fluorescent signals, and quantified the expression of each protein detected using QD-Abs. Quantification of the biomarkers showed good correlation with Western blotting. Conclusions: These results are proof of principle that 6 proteins can be simultaneously quantified using QD-Abs in single breast cancer sections. The use of multiplex QDs offers a novel method of determining the proteome of an individual breast cancer on single breast cancer sections. With the expanding use of targeted therapies in breast cancer, the ability to detect multiple proteins on small breast cancer specimens using QD-Abs, could allow not only the accurate selection of therapy, but a unique method of determining the activity of specific targeted agents. No significant financial relationships to disclose.