Genetic alterations in signaling pathways and processes, including mutations, deletions, and amplifications, have been found in many pancreatic cancers. Prominent among these pathways is the phosphoinositide 3-kinase (PI3-K) pathway, which regulates multiple functions, including: proliferation; stress response; and apoptosis. Akt (also known as protein kinase B or PKB) is a serine/threonine kinase in the PI3-K pathway whose activity has been linked to providing cancer cells with anti-apoptotic properties. In pancreatic cancer, constitutive PI3-K/Akt activity appears to be an indicator of tumor aggressiveness, with high levels of active Akt associated with decreased patient survival. Currently, the gold standard for assessment of Akt activity is Western blot, where cellular homogenates are probed with anti-Akt antibodies to measure active Akt. However, this technology lacks single-cell resolution and yields an average result for a population of cells. Heterogeneity of Akt activity in single cells is thought to be an important mechanism in chemotherapy resistance, and analysis of multiple cell types suggests that Akt activity may be bimodal. A population average-based measurement would fail to capture the existence of high and low states of activity, whereas single-cell interrogation should reveal this behavior.

We describe the single-cell measurement of Akt activity in single human pancreatic cancer cells. An optimized peptide substrate incorporating non-native residues for peptidase resistance was used to measure Akt activity within cells. Briefly, the peptide was microinjected into a single cell, where it was available for phosphorylation by Akt. Single cells were lysed and their contents loaded into an overlying fused-silica capillary for electrophoretic separation of the non-phosphorylated and phosphorylated peptide. We first validated the system in 3 model pancreatic cancer cell lines before applying it to primary cells from human pancreatic cancer xenografts. Some tumor cell lines exhibited statistically significant bimodal behavior in the level of Akt activity. In the primary cells, Akt exhibited highly variable activity at the single-cell level, with some cells showing little to no activity while others demonstrated very high levels of activity as indicated by nearly complete phosphorylation of the substrate peptide. Single-cell Akt activity was blocked in the presence of wortmannin, a PI3-K inhibitor. This system also enabled characterization of peptidase action on the substrate with primary cells demonstrating a 10-fold lower level of peptidase activity. Future work focuses on utilizing this technology for higher-throughput analysis of Akt activity in primary patient tissue from multiple cancer types.

Citation Format: Angela Proctor, S. Gabriela Herrera-Loeza, Jen Jen Yeh, Nancy L. Allbritton. Measurement of Akt activity in single primary human pancreatic cancer cells using chemical cytometry. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3345. doi:10.1158/1538-7445.AM2014-3345