Developing an Immunofluorescence Assay for Detecting Rb and phospho-Rb on Circulating Tumor Cells in Breast Cancer

Endocrine therapies (ET) such as tamoxifen, fulvestrant, and aromatase inhibitors (AIs) are the standard-of-care first-line treatment in majority of estrogen receptor (ER)-positive breast cancers (BC). Recent clinical studies using cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) plus AIs or fulvestrant have shown significant improvement in survival outcomes in patients with ER+ metastatic BC compared to standalone ETs. CDK4/6i exert their action by inhibiting the phosphorylation of retinoblastoma (Rb) protein and consequently inducing cell cycle arrest. However, not all patients respond to this combination therapy and those, who initially respond, eventually develop resistance. Emerging studies suggest that the intrinsic resistance to CDK4/6i could be due to the loss of Rb or its mutations. Therefore, CDK4/6i resistance can be evaluated by measuring expression of total and phospho-Rb and Rb mutations. However, repeated biopsies to evaluate biomarkers in tumors is not feasible in patients. In this research, we aimed to develop an immunofluorescence assay to evaluate the expression of Rb and phospho-Rb using circulating tumor cells (CTCs) which can help predict response and resistance to CDK4/6i. CTCs serve as representative of the tumor bulk in patients and animal models and allow for less-invasive, frequent blood collection and real-time monitoring of treatment response. MCF7 cells treated with vehicle or abemaciclib (500 nM) for 48 hours were spiked into blood from non-tumor bearing mice. The MCF7 cells-spiked blood was processed using ScreenCell® device and the cells were transferred to slides and stained with DAPI, pan-cytokeratin, CD45, estrogen receptor, Rb and phospho-Rb. Tumor cells were defined as pan-cytokeratin-positive, CD45-negative, and nuclear stain-positive cells. Various antibody combinations were examined to increase the sensitivity of the IF assay for individual markers as well as the multiplexed assay. We also developed quantitative assessment approach to detect per-cell intensity of various markers. Treatment with abemaciclib reduced the intensity of phospho/Total Rb from 2.6 +/- 0.6 to 0.8 +/- 0.2 units (p < 0.05, t-test, n=8-9) in the vehicle-treated samples. There was no significant difference in the Rb intensity between the treatment groups. Ongoing studies focus on validation of the assay using preclinical models and clinical samples.