Hypofractionated Radiation Induced the Immunogenic Death of Bladder Cancer Cells Leading to the Immune Sensitization of Dendritic Cells

Purpose Radiotherapy is a commonly used method in the treatment of bladder cancer (BC). Radiation induced immunogenic death (ID) and antitumor immune response are related to the prognosis of radiotherapy. As the most powerful antigen-presenting cell in the body, the role of dendritic cells (DCs) is not very clear. Methods Apoptosis level, cell cycle analysis and expression levels of high mobility group protein 1 (HMGB1), calreticulin (CRT) and heat shock protein 70 (HSP70) were performed for bladder cancer cells after hypofractionated radiotherapy. The effects of the conditioned media on DCs for antitumor immune response activation were studied as well. The signicantly increased apoptosis level, G2/M phase cell cycle arrest and increased HMGB1, CRT and HSP70 expressions, and increased secretion of CCL5 and CCL21 in the supernatant of bladder cancer cells after hypofractionated radiotherapy. The expression of CD80, CD86, CCR5 and CCR7 on DCs was upregulated in the conditioned media of bladder cancer cells after hypofractionated radiotherapy.

Radiotherapy is one of the most important protocols in bladder preservation therapy. Radiotherapy not only directly kills tumor cells by damaging double-stranded DNA, blocking the cell cycle, and releasing reactive oxygen species, but also can induce the immune response against cancer through antigenpresenting cells (APCs), e.g. DCs, which capture cancer antigens in the local microenvironment and present them to naïve T cells in lymph node leading to cancer antigen-speci c T cell proliferation (Rompré-Brodeur et al. 2020, Siva et al. 2015).
ID is a regulated cell death induced by stressful pressure. It could enhance the antigen presentation capacity of DCs by releasing damage-associated molecular patterns (DAMPs), which activates the adaptive immune response of CD8 + T cells. DAMPs contained molecules including HMGB1, CRT, HSP70 and adenosine triphosphate (ATP) etc. HMGB1 can induce the maturation of DCs and promote the processing and presentation of tumor antigens by MHC-by binding to TLR9 and TLR4 (Yang et al. 2007).
CRT can act as a phagocytosis signal of DCs and send out a potential immunogenicity signal to recruit DCs and enhance their antigen presentation ability. HSP70 is rapidly recognized by APCs through CD91 and activates NF-κB, which promotes the release of proin ammatory cytokines by APCs and induces in ammatory responses (Zhou and Binder 2014).
Recently, several studies show that compared with conventional radiotherapy, hypofractionated radiation can induce cancer cell apoptosis and release more cancer antigens (Rodríguez-Barbeito et al. 2019) (Salimu et al. 2015). Moreover, it can also damage tumor blood vessels, making it easier for immune cells in the blood circulation to enter the tumor tissue and activate the immune response against cancer cells . Clinically, radiotherapy is the main treatment for BC patients with the goal of postoperative clearance of residual lesions and bladder preservation, but the roles of hypofractionated radiation-induced immune response and DCs in BC are still elusive. Therefore, in this study, human BC cells BT-B were radiated by different doses of X-rays, whose effect on their cell cycle and ID were investigated.
Preparation of DCs: Human peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors using the density gradient centrifugation method. After cells adhered to the wall of the culture ask, the non-adherent cells were removed. CD14 + monocytes were obtained from the collected adherent cells by immunomagnetic bead negative method. CD14 + monocytes were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco), 150 ng/ mL recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF, Peprotech) and 100 ng/ mL recombinant human interleukin-4 (rhIL-4, Peprotech) and maintained in a humi ed 5% CO 2 air incubator at 37 ℃ for 5 days to induce imDCs.

Radiation treatment
The BT-B cells in the logarithmic growth phase were irradiated with 160 kV,24 mA X-ray at the dose of 0 Gy,2 Gy, 4 Gy, 10 Gy, 18 Gy (RS-2000, Rad Source Technologies, Inc).

BT -B cells and dendritic cells co-culturing
Irradiated 5×10 6 BT -B cells were cultured for 48 h, and the supernatant of the culture media was collected. The conditioned media was prepared by removing the cell debris at 2000 rpm for 5 min and ltered by 0.22 um membrane (Millipore). Then, 2×10 6 imDCs were cultured in the conditioned media containing RPMI-1640 with 20% fetal bovine serum and 1 mL supernatants of irradiated BT -B cells. The control group was 2×10 6 imDCs suspension and 1 mL Phosphate Buffer solution (PBS). The cells were maintained in a humi ed 5% CO 2 air incubator at 37 ℃ for 48 h.

Cell cycle and apoptosis assay
After irradiation, BT-B cells were cultured routinely for 48 h. Cells were collected, xed overnight with 70% ethanol, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, washed with PBS three times, PI dye was added and incubated at room temperature for 15 min, and the cell cycle was detected by ow cytometry. The radiation treated cells were collected and xed with 70% ethanol for 2 h, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, washed with PBS three times, and Annexin V-FITC dye was added in the dark incubation for 30 min. PI dye was added before application, and the apoptosis of the cells was detected by ow cytometry.

Reverse transcription-polymerase chain reaction
After irradiation, BT -B cells were cultured routinely for 48 h to collect BT -B cells. The imDCs were cocultured with BT-B cells culture supernatant for 48 h, then the dendritic cells were collected. Total RNA of the cells was extracted by TRIzol Reagent kit (Invitrogen, USA), and the total RNA of three replicates was quanti ed and evaluated for quality control by ultramicro spectrophotometer. In the 20 uL PCR system, 1 µg total RNA was reversed to cDNA by Fastking gDNA Dispelling RT Supermix (Tiangen, Beijing). Realtime PCR was performed using SYBR® Premix Ex Taq TM (TaKaRa, Japan) kit. The mRNA expression level of the detected gene was determined by the ΔΔCT method. GAPDH was used as the internal reference gene, and the gene expression was calculated by the 2 −ΔΔCt method. The relative number of cells in the control group was set as 1, and the following speci c primers were used, including forward (F) and reverse (R) primers (5 'to 3'):

Western Blot
The BT-B cells were irradiated and cultured for 48 h. The cells were collected and the total protein of the cells was extracted with RIPA cell lysis solution (SolarBio, R0010) and PMSF (SolarBio, P0100). After the protein concentration was determined by the BCA method, 50 µg of total protein was extracted by 12% digestion SDS-PAGE separation and transferred to nitrocellulose membrane (Millipore, Boston, MA, USA). The nitrocellulose membrane was sealed with 5% skim milk powder for 1 h, washed three times with TBST, and speci c antibodies against GAPDH (Proteintech, 10494-1-AP), HMGB1 (BBI, D260488), CRT (Abcam, AB92516), HSP70 (Abcam, AB181606) were added, incubated overnight at 4 ℃, washed with TBST for three times, horseradish peroxidase-conjugated anti-rabbit IgG (ZSGB-BIO, ZB-2306) was added, incubated at room temperature for 1 h, washed with TBST for three times. Finally, the protein bands were detected with the ECL kit (Beyotime Biotechnology, P0018S). GAPDH was used as the internal reference protein.
Enzyme-linked immunosorbent assay (ELISA) Human CCL5 and CCL21(4A Biotech, CHE0092, CHE0140) were used for ELISA detection. We added 100 µL of standard or samples to the corresponding well of speci c antibody-coated ELISA plates and incubate for 90 min at 37 ℃. Each well was aspirated and washed with washing buffer (300 µL), and repeated the process three times. Next, 100 µL of biotin-antibody diluent was added to the corresponding well and incubated for 60 min at 37 ℃, followed by four washes with washing buffer. Enzyme binding diluent was added 100 µL to the corresponding well and incubated for 30 min at 37 ℃, followed by four washes with washing buffer. Added chromogenic agent 100 µL/well with protection from light and incubated for 20 min at 37 ℃. Finally, 100 µL of stop reagent was added to the corresponding well, the reagent was run on the microplate reader (Bio-Rad) and the measurement was immediately conducted at 450 nm.

Statistical analysis
Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software, Inc, USA) and image data were analyzed using Image J (NIH, USA). Statistic data were expressed as the mean ± standard deviation from at least three independent experiments, and comparisons between groups were made using Student's t-test. P values less than 0.05 were considered signi cant.

Result
Hypofractionated radiation promoted the apoptosis of bladder cancer cells After normal culture for 48 h, the apoptosis of BT-B cells was found closely correlated to the radiation dose (Fig. 1). The highest apoptosis level of BT-B cells was observed at the dose of 10 Gy. Meanwhile, we analyzed some apoptosis marker proteins expression for veri cation (Fig. 2). It was found that the expressions of apoptosis-related proteins in BT-B cells were up-regulated after irradiation.
Hypofractionated radiation altered the cell cycle of bladder cancer The cycle progression of BT-B cells showed a dose-dependent relationship with radiation (Fig. 3). With the rise of radiation dose, the ratio of BT-B cells in the G2/G1 phase increased, with signi cantly increased number of cells staying in the G2 phase.

Immunogenic death of bladder cancer cells induced by hypofractionated radiation
We veri ed the signature proteins of ID using by analyzing both mRNA levels and proteins levels. It was found that the expression of HMGB1 and CRT in BT-B cells rose to the highest expression after 4 Gy radiation (Fig. 4). The expression of HSP70 and HMGB1 in BT-B cells at the protein level were most expressed after 10 Gy radiation (Fig. 5). In general, radiation can induce the occurrence of the ID of BT-B cells.

Hypofractionated radiation increased the secretion of CCL5 and CCL21 in bladder cancer cells
After radiation, the concentration of CCL5 and CCL21 in the supernatant of BT-B cells increased gradually with hypofractionated radiation. In BT-B cells, the secretion of CCL5 and CCL21 both reached the highest level under 10 Gy radiation (Fig. 6).

Bladder cancer cells affected the function of immature dendritic cells after radiation
After co-cultured with BT-B cells supernatant after different radiation doses, the expression of CD80, CD86 and CCR7 on imDCs were upregulated and the expression of HLA-DR was down-regulated, the expression level of CCR5 peaked in 18 Gy group, while CD80 and CD86 peaked in 10 Gy group and CCR7 peaked in 4 Gy group, respectively (Fig. 7).

Discussion
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 ow cytometry (Fig. 1). Caspase The expression levels of Caspase-1, Caspase-3 and BAK1 increased after hypofractionated radiotherapy correlated well with the results from apoptosis assay detected by ow cytometry (Fig. 2), thereby con rmed 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 signi cantly, 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 ( . 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 signi cantly increased after co-culture with BT-B cells after hypofractionated radiation. This is . 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 ndings may contribute to the improvement of radiotherapy protocols for the most bene cial induction of anti-tumor immunity in bladder cancer treatments.