Background: SCNP is a multiparametric flow cytometry-based assay that simultaneously provides measurements, at the single cell level, of extracellular surface markers and quantitative changes in the activation levels of intracellular signaling proteins in response to extracellular modulators (Kornblau et al. Clin Cancer Res 2010). Studies in hematologic malignancies have shown the value of quantitatively measuring single cell signaling networks under modulated conditions as a basis for the development of prognostic and predictive tests. The aim of this study was to assess the feasibility of applying SCNP to the examination of signaling networks in an epithelial cancer, using bladder washings as a BC sample source and EGF +/− the PI3K inhibitor GDC-0941 as modulators. Method: Bladder washes from non-cancer (NC) patients (pts) (n=8) and confirmed/suspected BC pts (n=20) were collected using standard practice and shipped overnight on ice for processing within 24 hours. Antibodies against CD45, CytoKeratin (CK), EpCam, and cleaved-PARP (cPARP) were used to differentiate non-apoptotic epithelial cells from leukocytes, while measurements of DNA aneuploidy (DAPI stain) allowed for distinction between tumor and normal epithelial cells. Signaling activity in the PI3K and MAPK pathways was assessed by measuring intracellular levels of p-AKT and p-ERK both at baseline and in response to pathway modulation. Upon delivery cells were pelleted, counted and incubated for 1 hr at 37°C. GDC-0941 (200nM) or vehicle control was added for 1 hr prior to stimulation with EGF (5 min) or vehicle control. After cell fixation and permeabilization, samples were stained with fluorophore-conjugated antibodies/DAPI cocktail, and data acquired using multi-parametric flow cytometry. The epithelial bladder carcinoma cell line HT-1376 was used as a control for the epithelial phenotype and EGF signaling. DNA index was determined using the lymphoid population in the pt samples as a diploid G0/G1 reference.

Results: 50% (N=10) of BC samples and 25% (N=2) of NC samples met the “evaluable” criteria i.e., at least 400,000 total cells upon sample receipt and >2% of cells acquired containing an epithelial phenotype (DAPI+, CD45Low, CK+, and EpCam+). The majority of epithelial cells detected in BC samples were non-apoptotic (i.e. >77% cPARPneg) and therefore suitable for functional pathway analysis. In 3/10 BC samples a quantifiable increase in EGF-induced p-AKT and p-ERK signaling was identified (2–3 fold over baseline) and was preventable by GDC-0941 pre-incubation, whereas no EGF-induced signaling was observed in the NC specimens.

Conclusion: This study demonstrates the feasibility of applying SCNP, using multi-parametric flow cytometry, to the functional characterization of BC. Ongoing studies are currently focused on correlation of the individual patient in vitro network signaling profile with clinical outcome to develop prognostic and predictive tools for improved BC management to inform treatment options. The feasibility of extending such SCNP analyses to other solid tumor indications, using alternative tissue sources is also under investigation, e.g., using circulating tumor cells or cells obtained from body fluids such as pleural effusions, ascites.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C92.