Conditioned medium from brachytherapy-irradiated hepatocellular carcinoma cells drives SASP-mediated senescence in naïve cellular counterparts.

Background and purpose: Local ablation, including high-dose radiation brachytherapy (HDR-BT), provides a minimally invasive treatment for cancers such as hepatocellular carcinoma (HCC), achieving effective tumor targeting with reduced peri-interventional risk and morbidity. Despite benefits, these treatments face limitations due to tumor recurrence. Cellular senescence might play a key role in therapy resistance by way of tumor cell evasion. This study investigates whether HDR-BT induces cellular senescence in vitro, potentially linking these processes to tumor recurrence in HCC.

Material and methods: HCC cell lines (HepG2, Huh7, and Hep3B) were irradiated with 7.5 Gy using an in vitro irradiation device. Culture supernatant was collected and transferred to non-irradiated naïve cells. Cell proliferation and senescence were assessed kinetically using BrdU incorporation, Ki-67 immunostaining, and clonogenic assay. Senescence was confirmed by beta-galactosidase staining. Secretome analysis was conducted using a high-throughput proteomic assay.

Results: After irradiation, HCC cells show a transient increase in DNA synthesis, peaking before 72 h without leading to cell division. Exposure of naïve cells to supernatant from irradiated cells replicates these effects, suggesting that the conditioned medium alone can mimic radiation-induced responses. Molecular analysis reveals reduced Ki-67 expression and increased senescence in naïve, incubated cells. Proteomic profiling shows an enrichment of senescence-associated secretory phenotype (SASP) proteins in conditioned medium with exposed naïve cells producing a similar SASP-enriched secretome.

Conclusion: In vitro brachytherapy triggers a bystander effect in HCC cells via SASP-associated proteins inducing senescence in neighboring cells. Modulating senescence or its associated secretory phenotype may offer a novel target for therapy in future trials.