Radiotherapy (RT), which is involved in approximately 50% of all cancer treatments, is a pivotal therapeutic modality for killing cancer cells (Begg et al., 2011, Huang and Zhou, 2020). Owing to its precision and noninvasive nature, radiotherapy, which is mostly delivered in fractions of equal doses called fractionated radiotherapy (FRT), has been widely used in cancer treatment for more than a century (Schaue and McBride, 2015, Thariat et al., 2013). Whether FRT is employed independently or as part of sequential therapy, FRT is assumed to be a crucial component of treating oral cancer across different stages. With advances in radiotherapy techniques, the use of intensity-modulated radiation therapy (IMRT) and hyperfractionated radiotherapy has great potential to improve the survival of cancer patients while mitigating adverse reactions (De Felice et al., 2023, Lacas et al., 2017). Nonetheless, certain oral cancer patients still encounter local recurrence or metastasis. As reported, oral cancer patients following radiotherapy failure, indicated significantly deteriorated prognosis, exhibiting a shorter survival duration than other types of head and neck cancer patients (Leeman et al., 2017). This observation implies us if a heightened malignant tendency occurs within the residual tumors after radiotherapy failure, and how it leads to the dire prognosis.

The intrinsic radioresistance of cancer cells has been confirmed as one of the key factors leading to radiotherapy failure (Huang & Zhou, 2020). In addition, ionizing radiation (IR) can also induce some cancer cells, particularly radioresistant populations, to acquire stronger malignant characteristics, representing another core factor leading to tumor recurrence or metastasis (Lee et al., 2017). With respect to malignant tumors of epithelial origin (namely, carcinoma), epithelial–mesenchymal transition (EMT) allows cancer cells to acquire more malignant phenotypes; heightened EMT increases the ability of cancer cells to initiate tumors, increases invasive and metastatic potential, and increases drug resistance (Lamouille et al., 2014, Lu and Kang, 2019). In several carcinomas, scholars have provided evidence that the IR-induced EMT phenotype of cancer cells is linked to radioresistance (Kawamoto et al., 2012, Su et al., 2016, Wozny et al., 2019), characterized by the loss of epithelial morphology and markers and the acquisition of mesenchymal characteristics in cancer cells.

Cancer stem cells (CSCs), which act as cancer seeds, pose another formidable challenge for tumor eradication during conventional therapies. Due to their robust self-renewal capabilities and inherent resistance to therapy, these cells are responsible for tumor recurrence and metastasis (Chaudhary et al., 2023, Yang et al., 2020). We have previously proposed that radiotherapy can activate CSCs to initiate tumor relapse and metastasis in oral cancer (Liu et al., 2020). In other carcinoma types, CSCs can be enriched by radiotherapy because IR preferentially kills differentiated cancer cells and has the potential to dedifferentiate cancer cells to a stemness-like state (Ghisolfi et al., 2012, Lagadec et al., 2012, Salmina et al., 2010).

It is acknowledged that cancer cells undergoing EMT are endowed with aggressive properties, including invasion, migration and stemness (Shibue & Weinberg, 2017). Linking EMT and CSC phenotypes, specific metabolic reprogramming in cancer cells functions as an upstream regulator of cellular plasticity. Emerging research has confirmed that alterations in the patterns of multiple metabolic pathways are closely linked to EMT activation, highlighting the interdependence of EMT activation and metabolic reprogramming (Ji et al., 2023, Tian et al., 2020, Viswanathan et al., 2017). In contrast to differentiated progeny, CSCs exhibit distinctive and heterogeneous metabolic reprogramming patterns (Jagust et al., 2019, Jones et al., 2018, Pacifico et al., 2023). These findings suggest that whether IR can induce metabolic reprogramming in oral cancer cells is an important potential mechanism for enhancing malignant properties, including the EMT and CSC phenotypes, in oral radioresistant cancer cells.

Undoubtedly, comprehensive delineation of the map depicting the heightened malignant phenotypes of IR-induced oral cancer cells, such as cell invasion, EMT and CSC phenotype, and exploration of the underlying mechanism are highly important for improving the prognosis of oral cancer patients after radiotherapy. Therefore, in this study, we designed a fractionated dose of radiation strategy to simulate clinical radiotherapy, by subjecting two oral cancer cell lines, namely, Cal27 and Detroit 562, to a total of 60 Gy in 10 dose-escalating fractions to establish radioresistant oral cancer cells. Subsequently, we investigated their enhanced malignant behaviours involving the EMT process, cell invasion and migration, and CSC phenotype, as well as the specific metabolic reprogramming mechanism. Our findings provide experimental evidence that additional strategies targeting radioresistant cancer cells have the potential to increase the efficacy of radiotherapy for oral cancer treatment.