Abstract 5080: Investigation of exome-wide tumor heterogeneity on colorectal tissue-based single cells

Abstract The progression of colorectal cancer is highly influenced by various environmental and genetic conditions, however, we can highlight tumor heterogeneity presenting serious resistance to targeted therapies and having crucial effects on disease outcomes. Motivated by this issue, we aimed to develop an end-to-end single-cell sequencing workflow starting from morphology-derived sample isolation from different regions of a tissue slide continue with exome sequencing to investigate the patterns of tumor heterogeneity. Colonic biopsy specimens were collected during the endoscopic procedure, then cryosections (15 μm) were prepared and mounted on MMI MembraneSlides. Hematoxylin-eosin staining was applied to visualize the tissue morphology. Single-cell samples were obtained from 12 individual regions of negative control (NEG), tumor-associated normal (NAT), and colorectal cancer (CRC) tissues. Each sample had a diameter of 30-40 μm and a height of approximately 10-20 μm and was cut from a membrane frame slide by laser microdissection method. Following the single-cell DNA extraction and library preparation (Qiagen GmbH, CA, US), we performed paired-end next generation sequencing using NextSeq High Output kit on a NextSeq 500/550 instrument (IIllumina Inc., San Diego, CA, USA). Finally, we implemented bioinformatics analysis focusing on the patterns of tissue heterogeneity in the samples illustrated by CRC-specific mutated genes. We obtained sequencing data with an average of 30X coverage. We investigated the mutually exclusive and co-occurring gene pairs and found that the co-occurrence of genes corresponding to near-tumor tissues was more significantly detected (such as TTN-ADGRE1 in CRC with p<0.05). Our observations on single-cell tumor heterogeneity revealed that the different regions associated with the tumor - including negative control tissues - displayed altered mutation and heterogeneity profiles. Eventually, we presented the onco-mutation motifs of tissue regions which showed that the occurrence of mutations in genes BRAF (4/12), FBXW7 (2/12), and PCDH17 (1/12) in CRC samples are linked to resistance to EGFR inhibitor therapy. Additionally, the non-zero mutation rate of COL5A1 (1/12) gene in NAT indicates poorer survival outcome of patients. Supplementary, we aimed to present additional therapy resistant mutations characteristic to antiangiogenic, 5-fluorouracil (5-FU), chemo- and cetuximab therapies. In our study, we identified region-specific heterogeneity motifs and demonstrated the individual and cumulative heterogeneity patterns of CRC and NAT single-cell-like samples. Additionally, we successfully detected high impact actionable mutations that influence therapy response and survival chances. Citation Format: Nikolett Szakállas, Alexandra Kalmár, Barbara Kinga Barták, Béla Molnár, István Takács. Investigation of exome-wide tumor heterogeneity on colorectal tissue-based single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5080.