Subtype-specific differences in chromatin state markers and their morphometric predictors in breast cancer micronuclei

Abstract Micronuclei (MN) are indicators of chromosome instability and have been used for breast cancer (BC) screening, diagnosis, and prognosis. In this study, we explored the expression of transcription, replication, chromatin state, and DNA damage markers in BC MN using in vitro BC cell line (CL) models. Immunostaining using anti RNA Pol II, Lamin B1, emerin, H3K9ac, H3K27me3, cGAS, and γH2AX antibodies was performed, followed by confocal microscopy in MCF-7, T47D (hormone receptor+), SKBR3 (human epidermal growth factor receptor; HER2+), and MDA-MB-231 & 468 triple negative (TN) BC CLs. Quantitative analysis showed that 85–90% of MN were Lamin B1+ in all subtypes, with a predominance of disrupted staining (80–85%), indicating compromised micronuclear envelope integrity. Euchromatin (H3K9ac) and heterochromatin (H3K27me3) markers were expressed in 60–70% and 20–30%, MN respectively demonstrating enrichment of transcriptionally permissive chromatin. RNA Pol II positivity was observed in 75–85% of MN, with TNBC cells exhibiting a significantly higher frequency (~85%) compared to non-TNBC subtypes (p<0.001). Similarly, γ-H2AX and cGAS + MN were significantly more abundant in TNBC (55–60% and 40-45%) than in hormone receptor and HER2+ cells (45–50% and 32-35%) (γ-H2AX ; p<0.0001 and cGAS ; p<0.001). Logistic regression analysis revealed that MN morphometric and texture parameters, including area, fractal dimension, contrast, entropy, and angular second moment, significantly predicted RNA Pol II, Lamin B1, and γ-H2AX staining outcomes (p<0.01). Our findings indicate an active role of BC MN in chromatin remodeling which can be detected using a set of advanced image analysis parameters used in study. Differences in the frequency of MN expressing above markers in TNBC and Non-TNBC cells indicate their crucial role in etiopathogenesis, which needs to be studied further.