Use of copper-cysteamine nanoparticles to simultaneously enable radiotherapy, oxidative therapy and immunotherapy for melanoma treatment.

Dear Editor, Melanoma, squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) are three major types of skin cancer. Among them, melanoma is the most severe form and accounts for ~4% of all newly diagnosed cancers annually in the United States. It is estimated that approximately 9500 people are diagnosed with skin cancer every day, and more than 1 million Americans are living with melanoma. Melanoma treatment is still a major challenge in the clinic. Photodynamic therapy (PDT) is composed of targeted ablation and immune activation, is less invasive than other therapies and has been widely used in the treatment of various cancers. However, the limitation of light penetration is an issue in PDT for deep cancer treatment. To overcome this limitation and enable PDT for deep cancer treatment, researchers have proposed X-ray-induced PDT and nanoparticle self-lighting PDT, and these techniques have become intensively studied topics. Recently, Chen et al. invented a new sensitizer called copper-cysteamine (Cu–Cy) that can be activated by UV, X-rays, microwave, and ultrasound to generate reactive oxygen species (ROS) to destroy cancer cells as well as bacteria. As ROS generation by Cu–Cy nanoparticles (NPs) is not solely activated by regular light, it is more appropriate to call it oxidative therapy (OT) rather than PDT. Cu–Cy NPs of an average size of 96 nm have been tested for skin cancer treatment. It was found that these Cu–Cy NP-based XPDT exhibited a strong antitumor effect towards SCC. However, B16F10 melanoma was resistant to these Cu–Cy NP-based X-PDT, both in vitro and in vivo. Size is known to be a sensitive factor influencing nanomaterial properties and performance. To further evaluate the effect of Cu–Cy NP-based X-PDT on melanoma, we applied particles with an average size of ~40 nm for the treatment of melanoma, as the 40 nm Cu–Cy NPs have a larger surface area than other NPs, thereby producing more ROS. In addition, the cell uptake is higher for the 40 nm NPs. As expected, the 40 nm Cu–Cy NPs were very effective in inhibiting melanoma under X-ray stimulation. These observations confirmed that the combination of Cu–Cy and X-rays facilitated cell apoptosis and/or necrosis of B16 cells. More interestingly, this combination promoted the formation of the antitumor immune response. These results suggest that Cu–Cy NPs can simultaneously facilitate radiotherapy, oxidative therapy, and immunotherapy for melanoma treatment, as illustrated in Fig. 1a. The distribution of Cu–Cy was assessed by confocal fluorescence microscopy. As shown in Supplementary Fig. S1, the uptake of Cu–Cy NPs in the nucleus after 6 h was substantially increased compared to that after 2 and 4 h of incubation. Next, the cytotoxicity was measured to assess the efficacy of Cu–Cy on B16 cells by the CCK8 viability assay. After incubation with various amounts of Cu–Cy for 24 h, the cells were irradiated with X-rays at 0 or 2.5 Gy. The results showed that the viability of the cells in the control group (Cu–Cy only) had no obvious reduction. In contrast, a dramatic reduction in cell viability was observed in a dosedependent manner in the 2.5 Gy group (Fig. 1b, c), suggesting that the Cu–Cy NPs had low toxicity towards cells but could easily be activated by X-rays to induce substantial cytotoxicity. As shown in Fig. 1d and Supplementary Fig. S2a, the Cu–Cy+X-ray group exhibited substantially higher green fluorescence of DCF than the PBS, Cu–Cy, and PBS+X-ray groups, indicating the generation of significant levels of ROS in the Cu–Cy+X-ray group. To further investigate the effect of Cu–Cy-based PDT on B16 cells, we performed a cell apoptosis assay. The apoptosis rate was only 24.1% in the Cu–Cy group (100 μg/mL). In contrast, a significant killing effect was observed in the B16 cells after the combined treatment of Cu–Cy (100 μg/mL) and X-rays with a cell apoptosis rate of ~84.7% (Fig. 1e, f). To assess the PDT therapeutic efficacy of Cu–Cy in vivo, we subcutaneously injected mice with B16 cells and randomly divided them into four groups. When the tumor volumes reached approximately 300 mm, all mice were intratumorally injected with PBS or Cu–Cy. At 6 h post-injection, the tumor-bearing mice were irradiated by X-rays (5 Gy) in the tumor location. The results demonstrated that the PBS and Cu–Cy groups showed a negligible effect on tumor growth, while the PBS+X-ray and Cu–Cy+X-ray groups showed significant inhibitory efficiency from day 12. Notably, the most significant inhibitory efficiency was found in the Cu–Cy+X-ray group (Fig. 1g, h, and Supplementary Fig. S3c). However, the body weights of the mice did not show obvious changes, and pathological injury to the spleen was not observed in any group (Supplementary Fig. S3b and d). Thus, these results showed that the antitumor activity of the Cu–Cy NPs could be significantly magnified after irradiation with X-rays. The generation of ROS was the major process in PDT, which could lead to the destruction of tumor cells and further induce the immune response. Considering the obvious antitumor effect of Cu–Cy-based PDT in vivo, we then evaluated whether Cu–Cy-based PDT could trigger an immune response and influence the proportion of immunocytes in the tumor and spleen. Our results revealed that only treatment with Cu–Cy+X-rays triggered the enhancement of CD4T and CD8T cells in the spleen (Fig. 1i and Supplementary Fig. S4a), while no noticeable change was observed in DCs, macrophages, neutrophils, NK cells, and γδT cells in the spleen or MDSCs with slight changes (Supplementary Fig. S6). The recruitment of immune cells into the tumor microenvironment (TME) is an important event associated with antitumor immune responses. Thus, we investigated whether the antitumor effect of Cu–Cy-based PDT was facilitated by an increase in the infiltration of immune cells, including DCs, M1 macrophages, CD4T cells, and NK cells, in tumors. Flow cytometric analysis showed that DCs,


Cu-Cy nanoparticles and cell culture
Cu-Cy nanoparticles were synthesized in Wei Chen's lab at The University of Texas at Arlington, USA. 1 B16 mouse melanoma cell line was obtained from the Cell Resource Center, Peking Union Medical College. All cells were cultured in Dulbecco modified Eagle medium (DMEM) (Hyclone, USA) supplemented with 10 % FBS (HyClone, GE) penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 ℃ with 5 % CO2.

X-ray irradiation
The X-ray system was obtained from Faxitron X-Ray Corp (IL, USA). X-ray irradiation (160 kV and 25 mA) was performed at a dose rate of 2.5 Gy in vitro or 5 Gy in vivo.

Cellular uptake of Cu-Cy
B16 cells (2×10 5 cells/well) were seeded into 12-well plates and incubated at 37 ℃ for 12 h. Cells were incubated with Cu-Cy at the concentration of 100 µg/mL for the indicated times (2, 4, and 6 h).
Subsequently, the medium was removed and the cells were washed with PBS three times. Cells were then fixed with 4 % paraformaldehyde for 10 min at 4 ℃ and stained with DAPI for 1 h in dark condition. The images were taken on an Olympus FluoView FV1000 microscope.

X-ray treatment
When the density of cells reached about 70 %, they were pretreated using various amounts of Cu-Cy (0, 6.25, 12.5, 25, 50, and 100 µg/mL) in the complete culture medium. After 6 h of incubation, the Supporting Information 3 medium was replaced with fresh culture medium and cells were irradiated with 0 or 2.5 Gy X-ray and incubated for another 24 h.
B16 cells were plated in 24-well plates at 2×10 4 cells/well for 12 h at 37 °C. The old medium was removed and fresh media containing different concentrations of Cu-Cy (0, 6.25, 12.5, 25, 50, and 100 µg/mL) were added into the wells and incubated for 24 h. Afterward, the cells were washed with PBS three times to remove the free nanoparticles. Then, the cells were treated with X-ray (0 or 2.5 Gy) and incubated for another 24 h. Finally, the medium was replaced with 100 µL mixed medium (DMEM: CCK8 = 9:1) and incubated for 4 h. The absorbance was measured at 490 nm via scanning multiwell spectrophotometer.

ROS detection in vitro
The intracellular ROS was detected by DCFH-DA using an Olympus FluoView FV1000 microscope and flow cytometry following the manufacturer's protocol. For flow cytometry analysis of ROS, B16 cells were seeded into 12-well plates at 2×10 4 cells/well for 12 h and then incubated with PBS and Cu-Cy (100 µg/mL) for 6 h. Then, cells were irradiated with X-ray (2.5 Gy) and co-cultured with DCFH-DA (10 µM) diluted with serum-free medium (1:1000) for 30 min. The culture medium was then removed and washed for three times with serum-free medium. Afterward, cells were collected and the fluorescence signals of ROS intensity were analyzed by flow cytometry or Olympus FluoView FV1000 microscope.

Apoptosis assay
The cellular apoptosis was assessed by the Annexin V-FITC/PI Apoptosis Detection Kit (BD Pharmingen, San Jose, CA, USA). 2×10 5 cells/well were cultured in 12-well plates for 12 h. After the Cu-Cy (0 and 100 μg/mL ) and X-ray treatments, the cells were harvested and washed with PBS for two times, then resuspended with 1 mL of 1x binding buffer, stained with 5 μL Annexin V-FITC for 10 min at room temperature in the dark, and then stained with 5 μL of PI for 5 min in the dark. Finally, Supporting Information 4 these cells were analyzed by a FACSCalibur instrument (BD Biosciences). The results were analyzed with FlowJo software (Tree Star, Ashland, OR).

Western blot analysis
After PDT, B16 cells were lysed in RIPA buffer containing 1 % phosphatase inhibitor cocktail and 1 mM PMSF for 30 min. 20 μg protein was subjected to SDS-PAGE and transferred onto PVDF membranes for western blot analysis using rabbit p-STAT3(705), STAT3, Bcl2, Bax, Cytochrome C, mouse, anti-GAPDH antibodies (Cell Signaling Technology). All of the antibodies were diluted with 1:1000. The ECL Western Blotting Detection System (Millipore Corporation, Billerica, MA, USA) was used to detect immunoreactive bands.

Therapeutic efficacy analysis
B16 cells (2×10 5 ) were injected subcutaneously into the mice (6-8 weeks old, n = 6). The growth of the tumor was measured every 2 days. When tumor volume reached 300 mm 3 , the mice were randomly divided into four groups: PBS, Cu-Cy, PBS+X-ray, and Cu-Cy+X-ray groups. Afterward, mice were anesthetized and injected 50 μL PBS or 50 μL Cu-Cy (50 μg/mL) intratumorally. In X-ray groups, at 6 h post-injection, the mice were irradiated with X-ray (5 Gy) on the tumor location for a total of three times within a week. The size of the tumor and weight of mice were measured daily. The formula: volume = 1/2×LW 2 (L and W represent the length and width of the tumor, respectively) was used to calculate the volume of the tumor. After 10 days from the first treatment, the mice were sacrificed. The weight of the tumors was measured. The main organs (heart, liver, spleen, lung, and kidney) and tumor were collected and used for hematoxylin and eosin (H & E)-stained, while the spleen and tumor were used for flow cytometry analysis.

Flow cytometry analysis
All tumor and spleen were harvested, the tumor was mechanically cut into small pieces and digested with collagenase and hyaluronidase at 37 °C. After 1 h, the tumor tissue suspension was ground and

Statistical analysis
All tests were carried out with GraphPad Prism 6 software (GraphPad Software, San Diego, USA).
The data were presented as mean ± SD. The statistical analyses were performed by unpaired two-tailed Student's t-tests. P < 0.05 was considered statistically significant.