Abstract
Abstract Pre-clinical models have identified diverse epigenetic alterations in prostate cancer that associate with treatment resistance and may also be therapeutic targets. However, these assays typically require thousands of cells and are difficult to scale for patient tumor biopsies. This report seeks to adapt the Cleavage Under Targets and Tagmentation (CUT&Tag) assay for use on a microfluidic technology to eliminate cell loss associated with complex, multi-step epigenetic assays to facilitate analysis of low input primary tumor samples. Exclusive Liquid Repellant (ELR) platforms pass magnetic bead bound analytes through immiscible phases to separate and extract analytes of interest while eliminating traditional centrifugation and wash steps that lead to sample loss. This platform enables adaptation of complex molecular assays such as CUT&Tag for lossless sample manipulation. We focus on the analysis of histone modifications in rare cells utilizing antibodies for H3K27 acetylation and methylation. Comparison of prostate cancer cell lines with LNCaP, V16D, 49F, 42D, and H660s with serial dilutions was performed to evaluate the sensitivity and specificity of this integrated assay. The CUT&Tag assay was performed in standard fashion on a serial dilution of 50-100,000 cells from LNCaP and V16D calls and showed dropout of recoverable DNA at 50 cells. Sequencing of DNA at these dilutions confirmed identification of differential histone modifications consistent with previously published data. CUT&Tag was adapted to ELR through the serial patterning of each buffer in distinct wells connected by an oil interface. In order to optimize the assay for reproducibility and increased sensitivity, we optimized antibody binding/washing to minimize the protein A-Transposase inhibition. This optimization showed increased antibody binding by 36% compared to protocol defined concentrations. We have now successfully performed this assay with a sensitivity of ~100 cells with recoverable DNA for sequencing to identify heterogenous histone patterns in each cell line. CUT&Tag has improved sensitivity to detect epigenetic alterations that associate with aggressive prostate cancer. The integration of CUT&Tag with ELR improves both sensitivity and reproducibility for epigenetic analysis at low input cell numbers. This assay is now being utilized to analyze patient tumor samples and prostate cancer heterogeneity. Citation Format: Zachary J. Kauffman, Kevin Koesser, Kyle T. Helzer, Jamie M. Sperger, Marina N. Sharifi, Chao Li, Duane S. Juang, Cole S. Gilsdorf, Shuang G. Zhao, David J. Beebe, Joshua M. Lang. Microscale analysis of histone modifications in rare cell populations in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A057.
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