Gastrointestinal stromal tumors regulate macrophage M2 polarization through the MIF/CXCR4 axis to immune escape

Abstract

Purpose: The infiltration of immune cells and their roles of the infiltrating-immune cells in gastrointestinal stromal tumor (GIST) is still unclear. We aimed to discover the infiltration cell types and the relationship between the infiltrating-immune cells and the progression of GIST.

Experimental design: Single-cell RNA sequencing were performed to discover types of the infiltrating-immune cells and to analyze CellChat between cells. Immunohistochemistry of 80 GIST samples were used to clarify the relation between macrophages and recurrence risk. In vitro, flow cytometry and Real-time PCR were performed to uncover a potential mechanism of tumor cell regulation of macrophages.

Results: Tumor cells, macrophages, and T-cells were the predominant cell types. The MIF/CXCR4 axis was the most common ligand-receptor interaction between macrophages and tumor cells. As the risk increased, expression levels of CD68, CD206, MIF, and CXCR4 gradually increased. In vitro, we found that GIST882 was able to secrete MIF and GIST882 cell supernatant upregulated M2 polarization. Real-time PCR showed that expression levels of IL-10 mRNA and Arginase-1 mRNA were also the highest in the GIST882 cell supernatant group.

Conclusions: These findings identify that macrophages are the most abundant infiltrating cells in GIST. The MIF/CXCR4 axis is the most common ligand-receptor interaction between macrophages and tumor cells. GIST cells can regulate macrophage M2 polarization through the MIF/CXCR4 axis.

Keywords: M2 polarization; gastrointestinal stromal tumor; macrophage; single-cell RNA sequencing; tumor microenvironment.

Figure 1

Cell clusters were visualized by uniform manifold approximation and projection (UMAP) in these two tumors.

Figure 2

Distribution of infiltration cells in two tumors. G01: high risk GIST; G02: very low risk GIST.

Figure 3

Interactions numbers and interaction weights of cell-to-cell. (A) Numbers of interactions in G01. (B) Weights of interactions in G01. (C) Numbers of interactions in G02. (D) Weights of interactions in G2. G01, high risk GIST; G02, very low risk GIST.

Figure 4

Outgoing and incoming signaling patterns of Cell-Chat in G01.

Figure 5

Outgoing and incoming signaling patterns of Cell-Chat in G02.

Figure 6

MIF/CXCR4 axis was the most crosstalk ligand-receptor between tumor cells and macrophages in G01 (A) and G02 (B).

Figure 7

Immunohistochemical characterization of CD8, CD68, MIF, CD206 and CXCR4 in GIST samples.

Figure 8

(A) Immunofluorescence showed that co-expression of CD68 and CXCR4 in GIST sample. (B) Immunofluorescence showed that MIF expression is present both inside and outside GIST cells.

Figure 9

(A) (A) There were differences in CD8+T cell infiltration among different groups (0.35 ± 0.15 vs 0.27 ± 0.10 vs 0.09 ± 0.02, P=0.003); (B) There were differences in the infiltration of CD68+macrophage cells among different groups (0.38 ± 0.09 vs 0.64 ± 0.17 vs 0.98 ± 0.19, P=0.03). (B) (A) As the risk increases, the expression of CXCR4 gradually increases, and the difference is statistically significant (0.43 ± 0.24 vs 0.90 ± 0.68 vs 1.64 ± 0.53, P<0.001); (B) As the risk increases, the expression of MIF gradually increases, and the difference is statistically significant (0.009 ± 0.003 vs 0.70 ± 0.02 vs 0.12 ± 0.03, P<0.001); (C) As the risk increases, the expression of CD206 gradually increases, and the difference is statistically significant (0.04 ± 0.01 vs 0.07 ± 0.0.02 vs 0.15 ± 0.03, P<0.001); The statistical method is non-parametric test, n=80, *P<0.05; **P<0.01.

Figure 10

(A) GIST882 cells could express CD117; (B) GIST-882 cells could secrete MIF and MIF increased gradually along with the time. ISO-1 could inhibit the secretion of MIF.

Figure 11

(A) (A) GIST882 CM group; (B) GIST882 CM+ISO-1 group; Ctrl group vs GIST882 CM group, 1.6% vs 27.3%, P=0.000; GIST882 CM group vs GIST882 CM+ISO-1 group,27.3% vs 11.9%, P=0.006; ISO-1:MIF inhibitor; The statistical method is chi square test, **:P<0.01. (B) (A) GIST882CM group; (B) GIST882CM+WZ811 group. Ctrl vs GIST882CM group:1.6% vs 24.8%, P=0.000; GIST882CM group vs GIST882CM+WZ811group:24.8% vs 9.3%, P=0.004; WZ811:CXCR4 antagonists; The statistical method is chi square test, **:P<0.01.

Figure 12

(A) Ctrl vs GIST882CM vs GIST882CM +ISO-1 vs GIST882CM +WZ811:1.17 ± 0.43 vs 5.40 ± 1.21 vs 1.17 ± 0.23 vs 1.57 ± 0.57; (B) Ctrl vs GIST882CM vs GIST882CM +ISO-1 vs GIST882CM +WZ811:1.05 ± 0.18 vs 1.83 ± 0.18 vs 0.84 ± 0.30 vs 0.80 ± 0.17; ISO-1: MIF inhibitor; WZ811: CXCR4 antagonists; The statistical method is T-test, mean ± SEM, n=3, *P<0.05; **P<0.01.