Abstract 2477: Evaluating the effects of Bcl2 inhibitor, venetoclax, on cellular growth and clustering in triple-negative breast cancer cells
Authors: Ruiz Alexis Jordan , Park Nicolas , Abdulkareem Noor , Trivedi Meghna 
Published: April 2023

Abstract
Most mortality associated with breast cancer (BC) is caused by metastasis. The metastatic process involves dissemination of tumor cells from the primary site to distant organs through the bloodstream. The tumor cells found in the bloodstream are called circulating tumor cells (CTCs). The number of CTCs is an independent predictor of poor survival in BC patients. We and others have found that CTCs exist as single cells or in clusters. CTC clusters have up to 100 times more metastatic potential, higher cell survival, and more resistance to chemotherapy than single CTCs in BC, regardless of the subtype or prior treatment. Our published transcriptomic and proteomic studies have recently identified high B-cell lymphoma 2 (Bcl2) expression and downregulation of apoptosis pathways in triple-negative BC tumors associated with CTC clusters using patient-derived xenograft (PDX) models. Bcl2 is an oncogene that promotes survival and regulates pro- and anti-apoptotic pathways in cancer. Approximately 75% of breast tumors are positive for Bcl2. Also, the expression of Bcl2 is a poor prognostic marker in TNBC patients, mainly in the absence of adjuvant therapy. However, the exact role of Bcl2 in clustering of cells needs further investigation. In the current study, we aimed to evaluate concentration-dependent effects of the Bcl2 inhibitor, venetoclax, on cellular growth in TNBC cells. We first evaluated the cell growth patterns over time of two different cell numbers plated per well. We also generated 8-point concentration-response curves (10nM - 30μM) of venetoclax in MDA-MB-231 cell line to assess cell growth. The cells were plated in 96-well plates overnight followed by the addition of venetoclax and daily image analysis to obtain cell count using EnSight multimode plate reader. We found that 500 cells per well allowed us to obtain cell growth data for up to 6 days and 2,000 cells per well for up to 3 days. Therefore, 500 cells per well were selected to determine the effects of venetoclax over a 6-day period. On day 6, venetoclax treatment inhibited cell growth in a concentration-dependent manner. The half-maximal inhibitory concentration (IC50) of venetoclax was 40 μM. Next, we evaluated the effects of venetoclax on disruption of cellular clustering in MDA-MB-231 cells. We plated 10,000 cells per well in 6-well plates and incubated for overnight attachment. The cells were treated with venetoclax (10μM and 30μM) and trypsinized for a short-time (one minute) after 72 hours of treatment. The number of clusters with 3 or more cells clumped together were counted in 5 independent images using Image J. We found that there was a significant decrease in the number of clusters with venetoclax at 30μM. Our ongoing studies focus on exploring the effects of Bcl2 knockdown by siRNAs on cell growth and on disruption of cellular clustering in TNBC cells.
Citation Format: Alexis Jordan Ruiz, Nicolas Park, Noor Abdulkareem, Meghna Trivedi. Evaluating the effects of Bcl2 inhibitor, venetoclax, on cellular growth and clustering in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2477.