Discipline: Biological Sciences
Subcategory: Cancer Research
Alexis Ruiz - University of Houston
Co-Author(s): Noor Abdulkareem, University of Houston College of Pharmacy, Houston, TX; Meghna Trivedi, University of Houston College of Pharmacy, Houston, TX
Current statistics show that 1 out of 8 women will develop breast cancer (BC) in her lifetime. The most aggressive form of breast cancer is triple-negative breast cancer (TNBC), which lacks expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 and constitutes 10-15% of all BC cases. TNBC subtype has the lowest survival rate and worst prognosis. Higher mortality rate in TNBC is mainly due to higher risk of metastasis. Metastasis occurs when the cancer spreads to other parts of the body and forms secondary tumors. Circulating tumor cells (CTCs) from the primary breast tumor break off and travel to other parts of the body through the bloodstream. Therefore, CTCs are the building blocks of metastasis. CTCs exist either as clusters or single cells. CTC clusters have a higher likelihood of forming metastasis than single CTCs. Previous studies from our laboratory have found high expression of B-cell lymphoma 2 (Bcl2) in tumors associated with CTC clusters. Bcl2 is an oncogene that promotes survival and regulates pro- and anti-apoptotic pathways in cancer. The expression of Bcl2 is a poor prognostic marker in TNBC patients. However, whether Bcl2 expression is higher in CTC clusters compared to single CTCs is not known. Therefore, we aim to develop an Immunofluorescence (IF) assay to detect Bcl2 expression and determine Bcl2 expression in single CTCs vs CTC clusters in Patient Derived Xenograft (PDX) models and patients. To develop the assay, we will test various antibodies and assay conditions and determine the fluorescence intensity of the staining using a TNBC cell line and a control cell line. We will also use Bcl2 siRNAs to knockdown the gene to ensure the specificity of the assay for Bcl2 expression. Using the optimized assay, we will test the expression of Bcl2 on CTC clusters versus single CTCs from PDX models and patients. For this, CTCs will be detected as cells that are positive for DAPI (nuclear stain) and pan-cytokeratin (epithelial cell marker) and negative for CD45 (leukocyte marker). If we see a higher Bcl2 expression on CTC clusters versus single CTCs, we will test if an FDA-approved Bcl2 inhibitor, venetoclax, can break down CTC clusters. This research will allow us to target CTC clusters and hence metastasis in TNBC, which is an unmet clinical need.References: Thangavel H., et al., Journal of clinical medicine, 2019
Funder Acknowledgement(s): This study was supported by the University of Houston GEAR Grant.
Faculty Advisor: Meghna Trivedi,
Role: As part of this project, I perform cell culture and plate cells on slides. After, I perform a 2 day immunofluorescence (IF) protocol. The IF protocol requires blocking for an hour to remove nonspecific targets. A primary antibody is added overnight, then a secondary antibody is added for an hour to stain the targets. The Leica STED 8 confocal is used to image each slide and perform analysis of the expression of each target using ImageJ. I also perform the Bcl2 knockdown using small interfering RNAs (siRNA) to reduce the Bcl2 expression.