PROFILING SIGNALING ACTIVITY AND GENE EXPRESSION IN SINGLE, PANCREATIC ADENOCARCINOMA CELLS USING CE-RNA-SEQ

Abstract
Pancreatic ductal adenocarcinoma is a devastating disease in dire need of improved therapies
targeted at specific signaling pathways. Strategies to molecularly profile aberrant pancreatic
tissue and inform targeted therapeutic decisions would be of immense value in patient
treatment. However, molecular profiling is extremely challenging since biopsied patient tissue
is a complex mixture of normal and malignant pancreatic cells. Furthermore, there is a growing
understanding that mutations and gene expression alone do not tightly correlate with clinical
response. In the current application, a multidisciplinary research team proposes to develop a
state-of-the-art, single-cell, platform technology to measure the catalytic activity of sentinel
kinases within the KRAS pathway and gene expression through RNA sequencing. The
investigators will optimize and validate microsampling and microelectrophoresis methods to
assay single cells, simultaneously overcoming the challenges of cellular heterogeneity and
sample-size limits. Novel reporters of kinase activity within KRAS-outflow signaling pathways
will be designed and new methods and instrumentation combining single-cell capillary
electrophoresis with efficient RNA capture will be pioneered. Human tumor samples
maintained in murine xenografts will be assayed to gain unique insights into tumor properties
not currently addressable. The work will directly link mRNA production with the catalytic
activity of kinases in individual tumor cells derived from patients. The technology will enable
questions such as which kinase signaling patterns drive the classical vs the basal phenotypes of
pancreatic adenocarcinoma and whether a single tumor possesses a mixture of classical and
basal-type cells. The data and the insights gained from implementation of this technology will
provide a new approach for clinical assays with the potential for a profound impact on
therapeutic strategies in the emerging field of precision medicine.