Radiotherapy provides an alternative treatment for BC for patients who refuse or not allow to take RC (Storozynsky and Hitt 2020). In this study, we observed the highest BT-B cells apoptosis level in 10 Gy irradiation group using flow cytometry (Fig. 1). Caspase-1 promotes apoptosis by activating IL-1β and IL-18(Sun and Scott 2016, Miao, Rajan and Aderem 2011). BAK1 promotes apoptosis by leading to the reduction of membrane potential and the release of cytochrome C (Uren et al. 2017, Guttà et al. 2020). The protein encoded by Caspase-3 cleaves cellular proteins, such as cytoskeletal protein and proteins of repairing DNA, causing apoptosis (Zhou et al. 2018, Liu et al. 2017). The expression levels of Caspase-1, Caspase-3 and BAK1 increased after hypofractionated radiotherapy correlated well with the results from apoptosis assay detected by flow cytometry (Fig. 2), thereby confirmed the apoptosis of bladder cancer cells at the molecular and cellular levels. On the other hand, we found a dose-dependent relationship between BT-B cell cycle progression and radiotherapy dose (Fig. 3). After hypofractionated radiation, the number of BC cells remaining in the G2 phase increased significantly, indicating that the G2/M checkpoint blocked the cell cycle progression. Compared to G1 and S phases, cells in M and G2 phases have higher radiosensitivity (Kastan and Bartek 2004, Bhoora and Punchoo 2020, Williams and Stoeber 2012), indicating that hypofractionated radiation not only promoted the apoptosis of BC cells but also helped improve the sensitivity of BC cells to radiation.
Classic radiation oncology theory valued the radiation-induced DNA damage to malignant cells, while recent studies have shown that the immune response triggered in the irradiated tissue played the key roles in driving the efficacy of radiation therapy in vivo (Dar, Henson and Shiao 2018). Savage T et al. show that the expression of CRT and HMGB1 increase in breast cancer tumor tissues of mice after hypofractionated radiation, enhancing tumor immunogenicity (Savage, Pandey and Guha 2020, Golden et al. 2014). We found that the expressions of HSP70, HMGB1 and CRT in BT-B cells after hypofractionated radiation were all higher than those after conventional radiation (Fig. 4, Fig. 5). It showed that HSP70 can be rapidly recognized by APCs through cell surface receptors CD91 to promote the release of pro-inflammatory cytokines by APCs and trigger the Th17 response (Zhou and Binder 2014, Choudhury et al. 2021, Pawaria and Binder 2011). HMGB1 is a histone-chromatin binding protein released from radiation-damaged tumor cells, and its release amount is related to the degree of apoptosis (Venereau et al. 2012). Our results confirm the correctness of the previous research, that the HMGB1 expression and apoptosis of BC cells reached the highest level at 10 Gy irradiation dose. HMGB1 can also bind to TLR4 and TLR9 to up-regulate the expression of DCs surface molecules and promote the maturation of DCs (Kamo et al. 2013, Andersson and Tracey 2011, Yang et al. 2007). CRT exposure on the cell surface can act as DCs phagocytosis signal, promote DCs recruitment and enhance DCs antigen phagocytosis and presentation ability (Schcolnik-Cabrera et al. 2019, Venkateswaran et al. 2018). The increased expression of HSP70, HMGB1and CRT in BC cells after hypofractionated radiation indicated the occurrence of ID.
The occurrence of ID can induce DCs to mature and infiltrate into tumor tissue and activate the anti-tumor immune response (Zhou et al. 2019, Derer et al. 2015, Rapoport and Anderson 2019). Therefore, we analyzed the concentration of DCs chemokines CCL5 and CCL21 in the supernatant of BC cell culture after radiation, as well as the expression of DCs surface molecules after co-culture with the supernatant (Fig. 6, Fig. 7). Studies show that the concentration of CCL5 in the adult kidney is 97.51 pg/mL (Gawłowska-Marciniak and Niedzielski 2013), and that in the supernatant of human peripheral nerve cells is 359.2 pg/mL (Tianyi and Zhiyuan 2017). Our study found that hypofractionated radiation increased the secretion of CCL5. The highest concentration of CCL5 was secreted by BT-B cells at 10 Gy radiation, reaching 2430 pg/mL. When DCs receives antigenic stimulation, imDCs gradually transform into mDCs, which manifest as the upregulated expression of co-stimulatory molecule CD80, CD86 and chemokine receptor CCR7, and chemotactic migration to secondary lymphoid tissue along the concentration gradient of CCL19 and CCL21(Marsland et al. 2005). In our study, the expression of CD80, CD86 and CCR7 on imDCs was significantly increased after co-culture with BT-B cells after hypofractionated radiation. This is consistent with the findings of Kulzer L et al. in colorectal cancer after 5 Gy radiation (Kulzer et al. 2014, Rodríguez-Barbeito et al. 2019). At the same time, radiation increased the secretion of CCL21 in BC cells, which reached the highest at 10 Gy radiation. The above results suggested that the BC cells after hypofractionated radiation were more conducive to the maturation and of migration abilities DCs.
In conclusion, compared with conventional radiotherapy (2 Gy), hypofractionated radiation can induce stronger apoptosis, cell cycle arrest and immunogenic cell death of BT-B bladder cancer cells and promote maturation and migration abilities of DCs to serve better anti-tumor immunity effect. Such findings may contribute to the improvement of radiotherapy protocols for the most beneficial induction of anti-tumor immunity in bladder cancer treatments.