Fig 2: Lung metastases have prominent mesenchymal features and secrete CXCL2 and M2 macrophage infiltrates. (A) ECAD and VIM immunohistochemical staining of lung metastases and primary tumours. The scale is 200 µm. (B, C) The proportion of positive cells per 100 cells, statistical number of A, ***, P < 0.001. (D) Immunohistochemical depictions of CXCL2 and CD163 in lung metastases and primary tumours. The scale is 200 µm. (E, F) The proportion of positive cells per 100 cells, statistical number of D, ***, P < 0.001; **, P < 0.01.

Fig 3: SNAIL expression is closely related to macrophage promotion of tumour cell migration and metastasis. (A) Immunohistochemical staining of SNAIL in primary tumours with or without CRC lung metastases, and lung metastases. The scale is 200 µm. (B) The proportion of positive cells per 100 cells, statistical number of A, ***, P < 0.001; **, P < 0.01; *, P < 0.05. (C) SNAIL overexpression and M2 macrophage coculture for cell proliferation and migration. (D-G) C Statistics of colony number and migrating cell number, **, P < 0.01; *, P < 0.05. (H-I) Effect of SNAIL overexpression on VIM and ECAD expression in CRC cells, **, P < 0.01. (J) Effect of SNAIL overexpression on CXCL2 expression in CRC cells, ***, P < 0.001. (K) Effect of SNAIL overexpression on CXCL2 expression in CRC cells co-cultured with TAMs, ***, P < 0.001.

Fig 4: High SNAIL expression promotes the secretion of CXCL2 by tumour cells after the tumour stroma activated M2 macrophages and induced the migration and proliferation of tumour cells. (A) SNAIL overexpression in BALB/c nude mice and NOD-SCID mice. (B) Statistical comparison of tumour volume after tumour formation, **, P < 0.01; *, P < 0.05. (C) Representative diagram of ECAD, VIM, CXCL2 and CD163 immunohistochemical staining in different treatment groups. (D-G) The proportion of positive cells per 100 cells, statistical number of C, ***, P < 0.001; **, P < 0.01.

Fig 5: SNAIL-dominated EMT and CXCL2-dominated macrophage infiltration are important features of lung metastases. (A) Colorectal carcinoma in situ and lung metastases heatmap of the 30 DEGs with |log2fold change | > 2, P < .05. Red: higher expression; blue: lower expression. (B) Colorectal carcinoma in situ and lung metastases module-trait associations. Each row corresponds to a module eigengene, and each column corresponds to a trait. Each cell contains the corresponding correlation and p value. The table is colour-coded by correlation according to the colour legend. (C) A scatter plot of gene significance (GS) and module members (MM) of relapses in the SteelBlue module. (D) Modular analysis of PPI networks in lung metastases. (E) Module analysis and statistical node number of the PPI network of lung metastatic tumours. (F) Pathways enriched by lung metastases, including the Intestinal Immune Network For IGA Production, Cytokine Receptor Interaction, Chemokine Signalling Pathway, and JAK STAT Signalling Pathway. (G) Heatmap of immune cell infiltration in lung metastases versus primary tumours. (H) Violin diagram of immune cell infiltration in lung metastases versus primary tumours.