Whereas splenic M1 macrophage numbers/percentages remained unchanged, a significant decrease in numbers and a reductive trend in percentages of M2 macrophages was detected in the spleen of the 4T1 tumor-bearing mice upon chitin + anti-PD-1 combination treatment (Fig. 8C, Additional file 15: Fig. S13C), resulting in a significantly increased M1/M2 macrophage ratio compared to the other treatment groups (Fig. 8D). This evidence concerns the gene PDCD1 and neoplasm.