(A) Tumor sizes of control (CON) and melittin (MEL) groups were measured and calculated. Tumor-bearing mice were administered the melittin peptide (0.5 mg/kg) every 3 days (an overall total of five times) starting on day 5 after tumor inoculation. N=5 animals per group. (B) Mantel-Cox survival curve. N=8 animals per group. (C) Hematological profiling. Blood was collected and parameters for myeloid function were analyzed. (D) Percentage of neutrophils and lymphocytes in blood leukocytes from tumor bearing mice (left). Neutrophil/lymphocyte ratio (N/L ratio) was used to verify whether acute cytotoxicity occurred as a result of melittin treatment (right). All of the data are presented as the mean ± SEM. *P < 0.05, **P < 0.01.

(A-B) The cell cycle was detected by PI staining and plotted as a histogram after gating on single cells. Representative figures of three replicates for the cell cycle are shown. The peaks correspond to the G1/G0, S, and G2/M phases. The results are given as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

(A) Splenocytes from tumor bearing mice were stained for detecting CD4 T cells (CD3⁺CD4⁺CD8^-), CD8 T cells (CD3⁺CD8⁺CD4^-), regulatory T cells (TREG; CD4⁺CD25⁺Foxp3⁺), B cells (B220⁺CD19⁺CD25^-), regulatory B cells (BREG; B220⁺CD19⁺CD25⁺), dendritic cells (DC; CD45⁺CD11b⁺CD11c⁺), and macrophages (MAC; CD45⁺F4/80⁺). Ratios of each immune cell in splenocytes were determined by flow cytometric analysis. (B) TAMs from tumor tissue were marked as CD11b⁺F4/80⁺ and presented as a contoured line (left) after gating on CD45⁺ cells from total live gated cells. The percentages of CD11b⁺F4/80⁺ cells in CD45⁺ cells are shown as a bar graph (right). (C) Binding of melittin to CD4⁺, CD8⁺ and CD11b⁺ cells in a mixed population of splenocytes was detected. (D) Percentages of melittin⁺CD11b⁺ cells in total CD11b⁺ cells treated with either DMSO or cytochalasin D (Cyto D) were measured to verify whether the co-staining was associated with phagocytosis. (E) Melittin binding subsets of CD11b⁺ cells were identified as F4/80⁺ macrophages, CD11c⁺ dendritic cells, and Gr-1⁺ neutrophils following a gating strategy. (F) Subpopulations of melittin-bound macrophages were identified as CD86⁺ (M1) and CD206⁺ (M2). All plots are representative of three replicates. (G) Tumor size was monitored after macrophage depletion. Clodronate liposome (Clo) or vehicle liposome (Con) was injected by i.p. 3 days before tumor inoculation, followed by treatment every 4 days. 0.5mg/kg of melittin was co-treated with Clo (Clo+Mel). N=3-4 animals per group. All of the values are shown as the mean ± SEM. **P <0.01, ***P < 0.001.

(A-B) M1-like macrophages that infiltrated tumor cells were marked as F4/80⁺CD86⁺ (upper panel), and M2-like TAMs were marked as F4/80⁺CD206⁺ (lower panel). (C-D) F4/80⁺CD86⁺ and F4/80⁺CD206⁺ macrophages in splenocytes are shown. The M1/M2 ratios were calculated based on the dot plots of CD86⁺ (M1) and CD206⁺ (M2) cells in F4/80⁺ macrophages. All plots were gated on CD45⁺ cells from total live gated cells. The values are presented as the mean ± SEM; ** P <0.01.

(A) qPCR of M2 phenotypic markers (Vegf, Mrc1/CD206, Il-10, and Tgf-β) and a M1 phenotypic marker (Tnf-α) in bone marrow-derived macrophages (BMDM). The cells were stimulated with LPS or IL-4 for 24 h and incubated in the presence of PBS or melittin for another 24 h. Fold increase of each gene was normalized against the level of the unstimulated group (indexed as NO TREAT). (B) Relative mRNA levels of Vegf and flt1/VEGFR1 from melittin-treated tumor cells were compared with the PBS-treated control group and the results are shown as fold-difference. (C) TNF-α, IL-10, and TGF-β production was measured in the supernatant of the BMDM cultures using an ELISA. (D-E) VEGF and CD206 expressions were determined by Western blot analysis. β-actin was used as a loading control. Representative blots of 4-5 experiments are shown. Values are the mean and error bars represent SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 represent a significant difference between the LPS or IL-4 treated CON group and the NO TREAT group. #P < 0.05, ##P < 0.01, and ###P < 0.001 represent a significant difference between the melittin treated group and the CON group. The non-marked data showed no significant (ns) differences when compared to the CON group. The data are presented as the mean ± SEM.

(A-B) Intracellular ROS productions of unstimulated cells (NO TREAT) or M1-polarized macrophages, treated with PBS (CON) or melittin (MEL) were measured by H₂DCFDA staining respectively. (C) Relative phagocytosis index of melittin or cytochalashin D (cytoD)-treated BMDMs compared with control was estimated by measuring internalized latex-bead fluorescence intensity. The mean ± SEM for three replicates are shown. *P < 0.05, ***P < 0.0001.

(A) VEGF (red) and (B) CD31 (green) positive cells were visualized with immunofluorescence staining in a paraffin sectioned tumor. The nuclei were counterstained with DAPI (blue). (C-D) The intensity of VEGF (C) and CD31 (D) were quantified by image J. The data are presented as the mean ± SEM; **P < 0.01, *** P < 0.001, versus the corresponding control group. Total magnification, 400X. Scale bars, 50 μm.