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Introduction: Dacarbazine (DTIC)-based chemotherapy remains the first-line treat-ment for melanoma. However, its low response rate and evident side effects limit its clinical application. The combined use of chitosan oligosaccharide (COS) and other drugs has been shown to have good anti-tumor effects. Therefore, this study aimed to investigate whether COS can be combined with DTIC for the treatment of melanoma. Methods: By constructing a tumor-bearing model, statistical analysis of tumor size, TUNEL staining, and immunohistochemical detection of CD8, F4/80, and other indicators were con-ducted on tumor tissue to clarify the synergistic effect and molecular mechanism of the com-bination therapy. results: 3.1. COS-2 inhibited melanoma cell growth and induced apoptosis COS used in this study was prepared in our laboratory according to a previously described method (Lyu et al., 2014). Both TLC and HPLC showed that the synthesized COS, named COS-2, was mainly composed of 67% dimers and 33% trimers (Supplementary 1). COS has been shown to inhibit tumor progression in various cancers, including lung (Shen et al., 2009) and cervical (Huang et al., 2006) cancers; however, its therapeutic effects against melanoma remain unclear. Therefore, to examine the role of COS in the development of melanoma, we investigated the effects of COS-2 on the melanoma cell line B16 in vitro. MTT assay showed that COS-2 significantly inhibited the proliferation of B16 cells in a dose-dependent manner (Figure 1a). Transwell migration assay and wound healing assay showed that COS-2 suppressed the invasive and migratory abilities of B16 cells (Figures 1b, c). Consistent with the results of MTT assay, Ki67 staining showed that COS-2 inhibited the proliferation of B16 cells (Figure 1d). In addition, live/dead cell staining showed that COS-2 induced B16 cell necrosis. These results collectively indicated that COS-2 inhibited the proliferative, invasive, and metastatic abilities of B16 cells and promoted their death in vitro. COS has been reported to have similar inhibitory effects on the gastric cancer cell line SGC-7901(Luo et al., 2014). To assess whether COS-2 can enhance the anti-tumor effects of DTIC, the combination index (CI) of COS-2 and DTIC was calculated for B16 cells (CI values of <1.0 indicated synergism) (Wang et al., 2019). The results showed that COS-2 and DTIC had synergistic anti-tumor effects, with the tumor inhibition rate being increased by 22.88%. Similarly, a previous study demonstrated that chitotriose enhanced the inhibitory effects of doxorubicin on the proliferation of MDA-MB-231 cells, with a 16.49% increase in the inhibition rate (Li et al., 2023). 3.2. COS-2 combined with DTIC exerted synergistic therapeutic effects against melanoma To validate the synergistic anti-tumor effects of COS-2 and DTIC in vivo, we induced melanoma in C57BL/6 mice using B16 cells (Keller et al., 2017) (Figure 2a). Tumor-bearing mice were treated with varying concentrations of COS-2 and DTIC. The results indicated that 40-mg/kg DTIC combined with 200-mg/kg COS-2 resulted in a tumor inhibition rate of 82.28%. On the contrary, the tumor inhibition rate of 40-mg/kg DTIC was only 49.04% (Figures 2b, c). Tumor volume was significantly lower in the COS-2, DTIC, and combined treatment groups than in the control group. As shown in Figures 2d and 2e, the number of TUNEL-positive cells was higher in all treatment groups than in the control group, indicating that COS-2 and DTIC enhanced cell apoptosis. Notably, the apoptosis rate of the combined treatment group was significantly higher than that of the DTIC group. These results collectively indicated that COS-2 remarkably enhanced the therapeutic efficacy of DTIC. We speculate that COS-2 may enhance DTIC-induced apoptosis by regulating the expression of apoptosis-related genes or influencing the apoptosis signaling pathway. Meanwhile, DTIC itself, as a chemotherapeutic drug, may indirectly promote cell apoptosis by interfering with DNA replication or damaging DNA structure. Similarly, a study on pancreatic cancer showed that high- and medium-dose celastrol-conjugated COS (Cel-COS) had tumor inhibition rates of 67.83 % and 55.69%, respectively (Zeng et al., 2022), both of which are lower than the tumor inhibition rate of COS-2 combined with DTIC (82.28%), suggesting that the anti-tumor effects of COS-2 combined with DTIC are superior to those of Cel-CSO. 3.3. COS-2 alleviated DTIC-induced intestinal side effects and liver damage To examine the effects of COS-2 on intestinal inflammation induced by high-dose DTIC, we evaluated the levels of TNF-α and IL-6 in intestinal tissues of mice with melanoma (Figure 3h). The results showed that the levels of TNF-α and IL-6 were significantly higher in the DTIC group than in the control group, indicating that high-dose DTIC caused intestinal inflammation in mice with melanoma. However, combined treatment with COS-2 and DTIC decreased the levels of TNF-α and IL-6 by approximately 39.44% and 27.1% when compared with the DTIC group, respectively (Figures 3f, g). These results indicate that COS-2 can alleviate intestinal inflammation caused by high-dose DTIC. 3.4. COS-2 improved immune function in mice with melanoma The abovementioned results suggest that COS-2 can enhance the anti-tumor efficacy of DTIC and alleviate liver injury and intestinal inflammation caused by high-dose DTIC. Inflammation is an important part of the immune response (Blair & Cook, 2008). Previous studies have shown that COS as an immunopotentiator can inhibit tumor growth (Zhai et al., 2018). In addition, it can decrease the interferon-gamma (IFN-γ)-induced expression of PD-L1 by activating APMK and inhibiting STAT1, consequently enhancing the ability of the immune system to eliminate tumor cells (Chen et al., 2022). Based on these findings, we speculated that COS-2 might suppress the growth of melanoma by regulating the immune system and attenuating inflammatory responses. Immunity is classified as specific and non-specific. We initially investigated the effects of COS-2 on specific immunity in mice with melanoma. ConA and LPS were used to induce the proliferation and differentiation of T lymphocytes and B lymphocytes in vitro (Yousef et al., 2012). Compared with normal mice (Figure 4a), mice with melanoma had a significantly lower proliferation rate of splenic lymphocytes. However, combined treatment with COS-2 and DTIC significantly increased the proliferation rate of splenic lymphocytes and enhanced specific immunity in mice with melanoma. To investigate the effects of COS-2 on non-specific immunity in mice with melanoma, the phagocytic ability of mouse monocytes was measured using the carbon clearance test. The results showed (Figure 4b) that the phagocytic index of the control group decreased by 46.64%, which was significantly lower than that of the normal group, indicating that the immune system of mice was inhibited during the development of melanoma. Combined treatment with COS-2 and DTIC restored the phagocytic index of mice with melanoma, suggesting that COS-2 enhanced non-specific immunity. The abovementioned results were validated via immunohistochemical analysis, with CD8 and F4/80 being used to characterize T cells and macrophages, respectively. As shown in Figures 4c and 4d, the number of CD8+-positive cells and F4/80-positive cells was approximately 181.28% and 150.83% higher in the combined treatment group than in the control group, respectively. Additionally, the immunohistochemical analysis of M1 type macrophage marker (CD86) was performed to further demonstrate that COS-2 was able to improved immune function in mice with melanoma. As shown in Figure 4e, the expression of CD86 was significantly increased. The results indicate that COS-2 not only significantly promotes the polarization of macrophages towards the M1 type, which is achieved by enhancing the expression of specific genes and the activity of signaling pathways within macrophages, but also that COS-2 has other effects on macrophages, such as upregulating the secretion of pro-inflammatory factors (such as TNF-α) while inhibiting the production of anti-inflammatory factors, thereby endowing macrophages with enhanced antigen presentation and tumor killing capabilities, but also exhibits direct anti-tumor activity, which may be related to COS-2-induced apoptosis or inhibition of tumor cell proliferation. These results suggested that COS-2 restored anti-tumor immunity in mice with melanoma. COS has been shown to exhibit immunostimulatory activity in various tumors; however, its immunoregulatory role in melanoma remains unclear. For example, COS-conjugated selenium (COS-Se) has been shown to improve immune function by increasing the phagocytic, spleen, and thymus indices in gastric cancer (Jiang et al., 2021). To the best of our knowledge, this study demonstrate that COS-2 enhances immune function by promoting the proliferation of T lymphocytes and the phagocytic activity of monocytes in melanoma. 3.5. COS-2 stimulated macrophages and increased the release of TNF-α To investigate the immunoregulatory effects of COS-2 in a controlled environment, we used RAW 264.7 macrophages as a model to examine the immune process in vitro (Zhang et al., 2014). MTT assay was performed to determine whether RAW 264.7 cells exerted inhibitive effects on B16 cells. The results showed that the culture supernatant of RAW 264.7 cells had no significant effect on the viability of B16 cells, which had a proliferation rate of 96% (Figure 5a). However, the