Nano-Fluidic Detection of Oncoprotein Signaling in Preclinical and Patient Lymphoma Samples.

Inactivation of oncogenes can be an effective cancer therapy. Determining precise levels of oncogene expression is important in the development of drugs to target oncogenes. We have developed a novel automated nano-fluidic Western-blot-like technology to detect and quantify oncogene expression in small numbers of mouse and human hematopoietic tumor cells. To detect different levels of oncogene expression, we generated transgenic mice in which the MYC or BCL2 oncogenes are regulated conditionally via the Tetracycline Regulatory System (Tet-system). Using lymphoma- derived cell lines from these mice, we titrated the level of oncogene expression by adding different concentrations of doxycycline in vitro. We were able to distinguish among different levels of oncogene expression in cell lysates, with high sensitivity in as few as 400 cells by nano-fluidic detection. Next, lymphoma derived cell lines were injected subcutaneously into syngeneic mice. Upon tumor development, MYC or BCL2 oncogenes were inactivated in vivo. Both MYC and BCL2 levels decreased in serial fine needle aspirations (FNAs) of tumor nodules upon parallel analysis with nano-fluidic detection and Western blot. Finally, we used nano-fluidic detection to determine levels of MYC, BCL2, AKT and ERK in lymph node samples from patients with follicular, transformed DLBC, Burkitt’s, and mantle cell lymphomas. BCL2 was overexpressed in mantle cell and follicular lymphoma patients, confirmed by Western analysis, whereas MYC was found to be overexpressed in Burkitt’s lymphoma. These results demonstrate that nano-fluidic detection technology may be used both as a preclinical tool for the assessment of changes in oncoprotein signaling and as a clinical diagnostic modality on microscopic clinical specimens.