Irradiation induces an epigenetically plastic CD44low persister cell population associated with tumor regrowth in colorectal cancer

Radiotherapy is clinically established in rectal cancer and is also used in selected colon cancer settings. However, treatment resistance and post-irradiation tumor regrowth remain major clinical challenges, and the residual cellular states that survive irradiation and later drive tumor regrowth remain poorly defined. We investigated irradiation-surviving persister populations in colorectal cancer (CRC), with a particular focus on cancer stem cell-associated changes, using CRC cell lines and patient-derived CRC organoids, including radiation-resistant models. Cells were analyzed before and after irradiation by flow cytometry, cell-tracing experiments, proliferation and drug sensitivity assays, xenograft studies, RNA sequencing, and DNA methylation profiling. In conventional CRC cell lines, CD44 and CD133 distributions remained largely unchanged after irradiation, even in radiation-resistant models. In contrast, patient-derived CRC organoids revealed the emergence of a CD44low persister population after irradiation. Cell-tracing experiments demonstrated that this population arose predominantly from parental CD44high cells rather than from expansion of pre-existing CD44low cells. Surviving CD44low cells regenerated CD44high cells, reconstituted parental-like heterogeneity, and exhibited enhanced proliferative capacity, chemoresistance, and tumor-forming ability with more prominent mucinous features compared with the surviving CD44high fraction. In radiation-resistant organoids, the CD44low/SSEA-4high persister population was associated with global hypomethylation and activation of pluripotency-related programs, including upregulation of NANOG, POU5F1, and SOX2 with reduced promoter-region methylation. These findings support the presence of an irradiation-associated, reversible CD44low/SSEA-4high persister state with epigenetic alterations linked to tumor regrowth and therapeutic resistance. Treatment-induced plasticity and associated epigenetic alterations may represent potential avenues for therapeutic intervention to achieve more durable radiotherapy responses in CRC.