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The cells or xenografts were fixed with 4% PFA, embedded in paraffin, and incubated at 4C overnight with primary antibodies against TLR5 (ab13876, Abcam, UK) and Ki67 (ab9260, MILLIPORE, USA). The samples were then washed, incubated with secondary antibody for 30min at room temperature, and stained with diaminobenzidine. Positive reactions were defined as those showing brown signals in the cell cytoplasm. The intensity of staining was scored as follows: grade 0=negative; grade 1=weak; grade 2=moderate; grade 3=strong. The percentage ranking of positive cells: 0=less than 5%; 1=525%; 2=2650%; 3=5175%; 4=greater than 75%. A final score was calculated by multiplying the two scores. In terms of the IF assay, cells or xenografts were fixed with 4% PFA, washed three times with PBS, and treated with Triton X-100. The samples were then incubated overnight with CD11b (ab184308, Abcam, USA), iNOS (178945, Abcam, USA) or Arg-1 (96183, Abcam, USA) antibody (1:100) at 4C. The next day, they were washed with Tris-buffered saline and Tween 20 and stained with secondary antibodies (TRITC for CD11b, KGAA99, KeyGEN BioTECH, China and FITC for iNOS and Arg-1, Jackson ImmunoResearch 111-095-003, USA). A biological fluorescence microscope (Olympus BX43, Japan) was used to detect fluorescence. The image analysis software ImageJ (1.52o) was utilized for staining quantification by calculating the value of integral optical density (IOD).

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