Abstract 3890: Novel miFISH single-cell approach gives new insights into chromosomal instability and evolution of colorectal cancers

Abstract Introduction: Chromosomal instability (CIN) is the predominant form of genomic instability in cancers, characterized by both numerical and structural alterations of chromosomes. CIN is crucial for the most threatening aspect of cancers: their ability to evolve, allowing them to circumvent immune detection, metastasize, develop resistance to therapies, and lead to treatment failure. At the same time, the CIN phenotype could provide valuable targets for cancer therapy. We developed a multiplex FISH (miFISH) method to study heterogeneity and ongoing CIN in cancer cells. The objective of our research was to determine whether high-grade aneuploidy develops through sequential or abrupt changes in chromosomal content, a question previously unexplored due to the absence of comprehensive methods for simultaneously detecting different forms of ongoing CIN on a single-cell level. Experimental Procedure: Sequential hybridizations of miFISH panels allow the assessment of up to 35 probes per single cell. Each panel was designed to include the centromeric probe and gene-specific probes on the p- and q-arm for a given chromosome. This approach was applied to colorectal cancer cell lines (with and without microsatellite instability) and colorectal cancer samples. A total of 10000-25000 targets were collected from each slide. Analysis was done by counting of signals in mononucleated cells, binucleated/dividing cells and metaphase spreads. Results: The simultaneous evaluation of centromeric and gene-specific sets of miFISH probes led to unprecedented insights into the patterns of distribution of chromosomal content among individual cells, and, especially, in dividing cells. We compared different forms of ongoing CIN: structural rearrangements, formation of micronuclei with whole chromosomes or chromosomal fragments, non-disjunctions of chromosomes, grossly asymmetrical cell divisions, including divisions with ploidy changes and multipolar mitoses. We observed that gradual changes in chromosomal/genomic content occur primarily due to frequent structural rearrangements, less frequent chromosome or fragment losses in micronuclei, and very rare single chromosome non-disjunctions. Of note, grossly abnormal cell divisions, characterized by massive non-disjunctions and structural rearrangements, occur with relatively high frequency. The same patterns of chromosomal heterogeneity and instability have been observed in cancer cell lines and in tumor samples. Conclusions: The novel miFISH approach applied to studies of chromosomal instability in colorectal cancers and cell lines revealed multiple paths leading to chromosomal diversification of cancer cell populations. Grossly abnormal cell divisions are likely to contribute to high-level aneuploidies and ploidy changes, and could underlie the rapid evolution of cancer under selective pressure. Citation Format: Anna Roschke, Darawalee Wangsa, Nina Gutzeit, Richard Korshkov, Tim Thoerner, Kerstin Heselmeyer-Haddad, Paul Meltzer. Novel miFISH single-cell approach gives new insights into chromosomal instability and evolution of colorectal cancers [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 3890.