Abstract 3303: Identifying a functional correlation between cancer stem cells and 3D clonogenic growth: testing rational combinations of PI3K pathway inhibitors in subtype-specific BC

Abstract

Abstract Introduction: Studying clonogenic behavior of tumor cells in the context of physiologically relevant ECM offers the potential to explore signaling processes which more closely approximate the in vivo microenvironment. This approach is valuable for identification and dissection of the key molecular signaling of cancer cells to test targeted drugs. 3D cell cultures have been recognized as the method of choice for the physiologically relevant modeling of many aspects of malignant cell behavior ex vivo. Kenny et al., 2007 identified that morphologies of breast cancer cell lines in 3D assays correlate with their gene expression profiles and protein expression patterns reflecting the underlying distinct morphologies which were associated with tumor cell invasiveness and metastases-associated phenotypes. Study of the molecular characteristics and clinical significance of CSC in the context of prognostic relevance as well as the understanding the scope of therapeutically targeting CSC within distinct tumor cell populations have revealed that CD44+ cell-specific genes represents SC markers and correlated with decreased patient survival (Fillmore and Kuperwasser, 2008). Hypothesis: Since 3D clonogenic growth of epithelial tumor cells represents the most appropriate model in vitro, we hypothesized that there is a functional association between phenotypic 3D clonogenic growth and cancer stem cells (CSC) proportion. Methods: To study the role of CSCs in the 3D clonogenic growth of tumor cells we have used BC and ovarian cell lines.The cells were challenged to form 3D colonies in matrigel and cells were recovered from the 3D colonies after de-gelling using PBS-EDTA buffer. Relative distribution of CD24+, CD44+, CD24- and CD44- expression in cells were determined by flow cytometry (Accuri C6) following recovery of the cells from the colony and compared to cells grown in 2D. We have tested the effect of a rational combination of pan-PI3K pathway inhibitor, GDC-0941 or isoform-specific inhibitors along with AI, trastuzumab, or HRD inhibitors (PARP) in ER+/PIK3CA mutated, HER2+/PIK3CA mutated or PTEN-null TNBC cells. Results: CD44/CD24 expression level changes between 2D and 3D growth was characterized in individual cell lines. Overall 3D growth led to a reduction in CD44 levels. Rational drug combinations blocked proliferative signals and enhanced apoptosis (cleaved caspase3) as demonstrated by WB, AnnexinV staining, proliferation (Incucyte). The mechanistic details of the effect of this combination on the CD44H/CD24L stem cell populations are being worked out, the results of which will be presented in the meeting. Significance: Our study reports a methodological breakthrough to determine CD24L/CD44H CSC fraction from live cells following 3D clonogenic transformation. Citation Format: Jennifer H. Carlson, Nandini Dey, Pradip De, Lori Friedman, Casey Williams, Brian Leyland-Jones. Identifying a functional correlation between cancer stem cells and 3D clonogenic growth: testing rational combinations of PI3K pathway inhibitors in subtype-specific BC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3303.