Abstract 7023: Application of exclusive liquid repellency for low input CUT&Tag histone analyses in prostate cancer spheroids
Authors: Kauffman Zachary J. , Koesser Kevin , Helzer Kyle T. , Sperger Jamie M. , Sharifi Marina N. , Li Chao , Heninger Erika , Hazelberg Xavier , Gilsdorf Cole , Zhao Shuang G. , Juang Duane S. , Beebe David J. , Lang Joshua M. 
Published: March 2024

Abstract
Introduction/Background: Epigenetic alterations, including histone modifications and DNA methylation, drive treatment resistance and lethal prostate cancer. However, epigenetic assays typically require thousands of cells and are difficult to scale for patient tumor biopsies. While identifying these alterations is possible in cell lines, these analytes are more difficult to follow in clinical settings. Patient-derived organoids have emerged as viable intermediaries to investigate the changing chromatin landscape during the progression toward treatment-emergent prostate cancer. 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 prostate cancer spheroids.
Methods: The Oil Immersed Lossless Total Analysis System (OIL-TAS) is an exclusive liquid repellency (ELR) platform that passes 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 LNCaP prostate cancer cell lines harvested from either 2D cell culture or 3D spheroids was performed to evaluate the sensitivity and specificity of this integrated assay.
Results: The macroscale CUT&Tag assay has been performed with inputs of 25,000 nuclei from LNCaP spheroids with greater yields than similar inputs from 2D LNCaP cell culture. Yield increases have also been seen when performing the assay with inputs &lt;10,000 nuclei in microscale on the OIL-TAS relative to the macroscale. Utilizing the OIL-TAS platform with spheroid inputs will allow for more sensitive investigation of histone modifications.
Conclusions: Integration of 3D, humanized models of tumor growth into the CUT&Tag assay will enable discovery of differential epigenetic regulation within patient-derived organoids or complex co-culture studies. Establishing baseline epigenetic landscapes among these intermediary models will enable a new platform for drug testing. Utilization of the OIL-TAS platform will allow for miniaturization of the assay down to 1,000 cell inputs and enable multiparametric RNA-seq, EM-seq, and CUT&Tag-seq of the same patient samples for longitudinal study.
Citation Format: Zachary J. Kauffman, Kevin Koesser, Kyle T. Helzer, Jamie M. Sperger, Marina N. Sharifi, Chao Li, Erika Heninger, Xavier Hazelberg, Cole Gilsdorf, Shuang G. Zhao, Duane S. Juang, David J. Beebe, Joshua M. Lang. Application of exclusive liquid repellency for low input CUT&Tag histone analyses in prostate cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7023.