Abstract A030: Lossless altered histone modification analysis system to investigate patient derived cancer organoids and circulating tumor cells from patients with prostate cancer

Abstract Introduction Aberrant histone modifications and chromatin structure alter gene expression that drive phenotypic and functional behavior of aggressive prostate cancer. Technological limitations have constrained comprehensive analyses of epigenomic alterations to less relevant immortalized cell lines and pre-clinical models, as many current epigenomic assay techniques require hundreds of thousands of cells, thus limiting interrogation of clinical samples. To facilitate rare cell epigenetic analysis in patient derived cancer organoids (PDCOs) and circulating tumor cells (CTCs) from patients with prostate cancer, we miniaturized the Cleavage Under Targets and Tagmentation (CUT&Tag) assay with an Exclusive Liquid Repellency (ELR)-enabled “Lossless Altered Histone Modification Analysis System” (LAHMAS) device. CUT&Tag was developed to investigate protein-DNA interactions with high resolution, lower background and decreased cellular input requirements relative to ChIP but performance is less reliable for low inputs, making analysis of rare cell samples difficult for cell number <5000. Methods: Organoids were grown from primary tumor samples from 3 patient undergoing prostatectomy. There were a total of 5 loci collected. These samples were digested to single cells prior to assessment in the Cut&Tag assay with an antibody to H3K27Me3 or control IgG. Each sample was paired with RNAseq to assess gene expression. Results: Performing CUT&Tag on the LAHMAS device increases the signal to for very low input samples, enabling the investigation of chromatin dynamics in rare cell sample types, including PDCOs and CTCs. We observed significantly higher FRiP scores using the microscale method compared to macroscale method (average 30.3% vs 18.2%, p<0.0001) facilitating accurate chromatin profiling of low input and rare cell samples. H3K27Me3 peaks are conserved from 3000 down to 50 nuclei in cell line models on the LAHMAS device. We are evaluated H3K27Me3 histone modifications from PDCOs grown from prostatectomy samples from 3 patiens using the LAHMAS device. We obtained high quality results from these samples, obtaining yields (LNCaP: 0.147-5.15 [median: 1.13] vs. PDCO: 0.144-5.36 [0.445]), and MACS2 FRiP scores (LNCaP: 1.79-29.91 [median: 4.91] vs. PDCO: 0.73-10.21 [5.76]) similar to cell line sample. We have also leveraged the LAHMAS device to evaluate circulating tumor cells from a patient with prostate adenocarcinoma progressing on chemotherapy. This patient demonstrated high expression of ASCL1 (0 tpm) and low expression KLK3 (16237.6 tpm) by RNAseq. Concordantly, we detected H3K27Me3 peaks at the promoter of ASCL1, consistent with the repression of transcription. Conclusions: The pairing of this LAHMAS microfluidic device with a highly sensitive CUT&Tag assay has enabled the investigation of chromatin dynamics in rare cells such as PDCOs and CTCs to evaluate the contribution of these molecular alterations in tumorigenesis and treatment resistance. Citation Format: Zachary J. Kauffman, Kevin Koesser, Kyle T. Helzer, Marina N. Sharifi, Erika Heninger, Emma E. Recchia, David Kosoff, David F. Jarrard, Shuang G. Zhao, Jamie M. Sperger, David J. Beebe, Joshua M. Lang. Lossless altered histone modification analysis system to investigate patient derived cancer organoids and circulating tumor cells from patients with prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr A030.