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

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.

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.

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.

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.