In Situ Molecular Profiling of Breast Cancer Biomarkers with Multicolor Quantum Dots

Identification of potential diagnostic markers and target molecules among the plethora of tumor oncoproteins requires enabling technology that is capable of analyzing multiple biomarkers in tumor cells and tissues quantitatively. Diagnostic and prognostic classifications of human tumors are currently based on immunohistochemistry (IHC), a technique that has been used in clinical medicine for over 80 years. However, the immunoenzyme (HRP-based) IHC method has a singlecolor nature and is unable to perform multiplexed molecular profiling. Moreover, IHC remains semi-quantitative and subjective, resulting in considerable inter-observer variation of the results. With new molecular profiling technologies, it is possible to read the molecular signatures of an individual patient’s tumor, and to correlate a panel of cancer biomarkers with clinical outcome for personalized therapy. A major difficulty in molecular profiling is that most cancer tumors (especially breast and prostate cancers) are highly heterogeneous, containing a mixture of benign, cancerous, and stroma cells. Current technologies such as RT-PCR, gene chips, protein chips, two-dimensional gel electrophoresis, biomolecular mass spectrometry (e.g., MALDI-MS, ES-MS, and SELDI-MS) are not designed to handle this type of heterogeneous samples. Furthermore, a limitation shared by all these technologies is that they require destructive preparation of cells or tissue specimens into a homogeneous solution, leading to a loss of valuable 3D cellular and tissue morphological information associated with the original tumor. In comparison, the development of nanotechnology, especially bioconjugated nanoparticles, can provide an essential link by which biomarkers could be functionally correlated with cancer behavior. Indeed, several groups recently reported the use of quantum dot (QD) probes for immunostaining of fixed cells and tissue specimens enabled by their unique optical properties such as improved brightness, simultaneous excitation of multiple colors, stability against photobleaching, and extremely large Stokes shift. However, translational research of the QD-based immunostaining has not received widespread adaptation by clinical studies. A major problem is the lack of technology validation using conventional methods and the validation in large-scale clinical studies. In this Communication, we report the use of multicolor QDs for quantitative and simultaneous profiling of multiple biomarkers using intact breast cancer cells and clinical tissue specimens. We also compare and validate the new QD-based molecular profiling technology with standard western blotting (WB) and fluorescence in situ hybridization (FISH). This new technology could become the first clinical applications of QDs and open a new avenue in molecular pathology. Multicolor QDs are directly conjugated with antibodies through covalent bonds. Compared with our previously reported carbodiimide-mediated carboxylate and amine condensation, the QDs and antibodies are linked to each other via active ester maleimide-mediated amine and sulfhydryl coupling. Because free sulfhydryl groups are rare in native antibodies and are often unstable in the presence of oxygen, antibodies were first treated with reducing reagents such as dithiothreitol (DTT) to generate free thio groups in the ‘hinge’ region of antibodies as shown in Figure 1. This procedure results in less nanoparticle aggregation in comparison with carbodiimide-mediated COOH-NH2 condensation. On the other hand, chemical treatment of antibodies affects their stability and, as a consequence, could impede the antigen-recognition activity. To demonstrate the feasibility of multiplexed labeling, QDs emitting at 525 nm, 565 nm, 605 nm, 655 nm and 705 nm were directly conjugated to primary Abs against HER2 (QD-HER2), ER (QD-ER), PR (QD-PR), EGFR (QD-EGFR) and mTOR (QD-mTOR). The multicolor bioconjugates were used for simultaneous detection of the five clinically significant tumor markers in breast cancer cells, MCF-7 and BT-474. These two cell lines were selected because they have different expression levels of the five protein C O M M U N IC A TI O N