ABSTRACT
UNLABELLED: An integrated ß-camera and microfluidic chip was developed that is capable of quantitative imaging of glycolysis radioassays using (18)F-FDG in small cell populations down to a single cell. This paper demonstrates that the integrated system enables digital control and quantitative measurements of glycolysis in B-Raf(V600E)-mutated melanoma cell lines in response to specific B-Raf inhibition. METHODS: The ß-camera uses a position-sensitive avalanche photodiode to detect charged particle-emitting probes within a microfluidic chip. The integrated ß-camera and microfluidic chip system was calibrated, and the linearity was measured using 4 different melanoma cell lines (M257, M202, M233, and M229). Microfluidic radioassays were performed with cell populations ranging from hundreds of cells down to a single cell. The M229 cell line has a homozygous B-Raf(V600E) mutation and is highly sensitive to a B-Raf inhibitor, PLX4032. A microfluidic radioassay was performed over the course of 3 days to assess the cytotoxicity of PLX4032 on cellular (18)F-FDG uptake. RESULTS: The ß-camera is capable of imaging radioactive uptake of (18)F-FDG in microfluidic chips. (18)F-FDG uptake for a single cell was measured using a radioactivity concentration of 37 MBq/mL during the radiotracer incubation period. For in vitro cytotoxicity monitoring, the ß-camera showed that exposure to 1 µM PLX4032 for 3 days decreased the (18)F-FDG uptake per cell in highly sensitive M229 cells, compared with vehicle controls. CONCLUSION: The integrated ß-camera and microfluidic chip can provide digital control of live cell cultures and allow in vitro quantitative radioassays for multiple samples simultaneously.
Subject(s)
Beta Particles , Glycolysis , Microfluidic Analytical Techniques , Molecular Imaging/methods , Systems Integration , Biological Transport/drug effects , Calibration , Cell Line, Tumor , Fluorodeoxyglucose F18/metabolism , Glucose/metabolism , Humans , Indoles/pharmacology , Molecular Imaging/instrumentation , Radiometry , Single-Cell Analysis , Sulfonamides/pharmacology , Time Factors , VemurafenibSubject(s)
Breast Neoplasms/chemistry , Complex Mixtures/chemistry , Microfluidics/instrumentation , Nanostructures/chemistry , Neoplastic Cells, Circulating/chemistry , Prostatic Neoplasms/chemistry , Silicon/chemistry , Breast Neoplasms/blood , Breast Neoplasms/pathology , Equipment Design , Female , Humans , Male , Microfluidics/methods , Molecular Imaging , Neoplastic Cells, Circulating/pathology , Prostatic Neoplasms/blood , Prostatic Neoplasms/pathology , Surface PropertiesABSTRACT
The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform-microfluidic image cytometry (MIC)-capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine.
