Abstract 5383: Design, optimization, and validation of multiplex immunofluorescence assay for detecting biomarker expression on circulating tumor cells in breast cancer
Authors: Kumar Tanya , Thangavel Hariprasad , Bhat Raksha , Abdulkareem Noor M. , Dobrolecki Lacey E. , Lewis Michael T. , Trivedi Meghana V. 
Published: September 2020

Majority of breast cancer-related deaths occur due to metastasis. In the metastatic process, tumor cells travel from the primary site to distant organs. Tumor cells traveling in the blood stream are called circulating tumor cells (CTCs), which can serve as liquid biopsies as representative of the tumor bulk in patients. A major advantage of liquid biopsies for the analysis of tumor cells is its less-invasive collection, which allows more frequent and real-time monitoring of treatment response over conventional tissue biopsies. CTCs are being evaluated for early diagnosis and screening, treatment selection, and monitoring of treatment response. In our research, we are investigating CTCs as a tool to evaluate presence of prognostic and predictive markers in breast cancer. The purpose of this study was to design, optimize, and validate a multiplex immunofluorescence (IF) assay to identify a panel of unique biomarkers for CTCs. The panel of biomarkers to be tested on CTCs included estrogen receptor (ER), B-cell lymphoma (Bcl2), retinoblastoma protein (RB), and phosphorylated-RB (pRB). CTCs were defined as pan-cytokeratin-positive, CD45-negative, and nuclear stain-positive cells. First, antibodies for individual protein markers were tested on breast cancer cells spiked into control mouse blood. Positive and negative controls were used to test the selectivity and specificity of the antibodies against the key biomarkers. Multiple parameters including different fixative reagents, direct and indirect antibody conjugation, and varying antibody incubation time were examined to increase the sensitivity of the IF assay for individual markers as well as multiplexed assay. Our results indicated that various antigen staining was similar with both fixation reagents, 4% paraformaldehyde and 2% formaldehyde. In comparing the direct (single step) and indirect (two-step) antibody additions, a more intense signal for the CTCs and leukocytes were observed for the latter. Additionally, overnight incubation with primary antibodies allowed for better binding between the antibody and the antigen on cells as indicated by strong fluorescence signal with less exposure time. On the other hand, incubation for only one hour with primary antibodies had a weaker signal with high non-specific background from other blood components. The validated assay has been successfully applied to our ongoing CTC investigation using the blood from breast cancer patient-derived xenograft models.

Citation Format: Tanya Kumar, Hariprasad Thangavel, Raksha Bhat, Noor M. Abdulkareem, Lacey E. Dobrolecki, Michael T. Lewis, Meghana V. Trivedi. Design, optimization, and validation of multiplex immunofluorescence assay for detecting biomarker expression on circulating tumor cells in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5383.