STING-IRG1 inhibits liver metastasis of colorectal cancer by regulating the polarization of tumor-associated macrophages

Abstract

The liver is the main site of colorectal cancer (CRC) metastasis. Tumor-associated macrophages (TAMs) play a key role in tumor metastasis. Therefore, modulating the function of tumor-associated macrophages is a potential therapeutic strategy to control tumor metastasis. We found in vivo experiments that the activation of STING inhibited CRC liver metastasis in model mice and affected the macrophage phenotype in the tumor microenvironment. Mechanistically, STING affects TAM polarization and regulates macrophage function through IRG1. And STING activates IRG1 to promote the nuclear translocation of TFEB, affecting the ability of macrophages to suppress tumor metastasis.Therefore, this study highlights the critical role of the STING-IRG1 axis on TAM reprogramming and its role in the process of tumor liver metastasis, which may provide a promising therapeutic strategy for CRC liver metastasis.

Keywords: Biological sciences; Immunity; cancer; molecular biology.

Graphical abstract

Figure 1

Expression of STING in TAMs correlates with liver metastasis of colorectal cancer (A) Representative IHC stained images of STING protein in primary liver metastatic and paracancerous tissue of human colorectal cancers. (B) Visualization of t-SNE in macrophage clusters based on single-cell transcriptomes. (C) Expression levels of STING in different macrophage subtypes. (D) Analysis of STING expression in primary CRC with metastasis tissues in CRC microarray profile. (E) Boxplots representing the distribution of M1 (CCL5⁺) or M2 (CCL18⁺) cells in primary and metastatic tumors. (F) Correlation between the mRNA expression of STING and M1/M2 macrophage obtained from Tumor Immune Estimation Resource (TIMER2.0, http://timer.cistrome.org/).

Figure 2

Activation of STING in vivo inhibits tumor metastasis (A) MC38-luciferase cells were injected to construct a transfer model and treated with DMXAA (i.p.) (10 mg/kg/3 days) or PBS (i.p.) for 3 weeks. Bioluminescence signal imaging of representative mice (left) and quantitation analyses of bioluminescence (right) were done following treatments,n ≥ 5. (B) Survival curves of liver metastatic mouse model after DMXAA and PBS treatment compared to the non-operated group (mock). (C) Representative macroscopic photograph of livers of the control model (top panel) and DMXAA (10 mg/kg/3 days)-treated mice (lower panel) with metastatic cancer nodules. (D) H&E staining of liver from control and DMXAA-treated liver metastasis mouse model. (E) Levels of ALT and AST in peripheral blood of mice after 3 weeks of DMXAA or PBS treatment. (F) Western blot to detect the protein levels of STING, TBK1, p-TBK1, IRF3, and p-IRF3 in mouse liver using GAPDH as the loading control. (G) Flow cytometric analysis of M1 (CD11c⁺) and M2(CD206⁺) ratios in F4/80⁺/CD11b⁺ macrophage populations isolated from liver metastases.

Figure 3

Inhibition of tumor growth in metastatic liver lesions accompanied by an increase in the proportion of M1-like macrophages after STING agonist treatment (A) MC38-luciferase cells were injected into WT or STING^gt/gt mice to generate a transfer model, followed by treatments with DMXAA (i.p.) (10 mg/kg/3days) or PBS for three weeks. Bioluminescence signal imaging of representative mice (left) and quantitation analyses of bioluminescence (right) were done after treatments, n ≥ 5. (B) H&E staining of liver metastatic lesions after the intrasplenic injection of MC38 cells to WT or STING^gt/gt mice. (C) Immunofluorescence staining of liver tissues with CD206⁺ (green), CD86⁺ (red) and DAPI (blue). (D) Flow cytometry analysis of CD11c⁺ and CD206⁺ macrophage subpopulations in tumor tissues of WT or STING ^gt/gt mice.

Figure 4

STING induces a pro-inflammatory phenotype in macrophages (A) Flow cytometric analysis of CD11c or CD206 in WT or STING^gt/gt BMDM. The BMDM was induced into M2 phenotype for treatment with DMXAA, followed by flow cytometry. (B) Bone marrow macrophages were isolated from WT and STING^gt/gt mice, and induced into M2-like macrophages using IL4. The cells were treated with or without DMXAA, and qPCR was performed following total RNA isolation. Each gene was normalized with the housekeeping gene HPRT.

Figure 5

STING activation affects colon cancer cell metastasis via macrophages (A) Determination of migration and invasion ability of MC38 after co-culture with M1 macrophages (RAW264.7) or M2 macrophages (RAW264.7) for 48 h with or without DMXAA or cGAMP treatment. (B) Determination of migration and invasion ability of DLD1 after co-culture with M1 macrophages (THP1) or M2 macrophages (THP1) for 48 h with or without cGAMP treatment. (C) With or without DMXAA or cGAMP treatment, MC38 cells were co-cultured with M1 or M2 macrophages and cell migration was determined using a wound healing assay.

Figure 6

STING reprograms TAMs to be tumoricidal (A) RAW264.7 cells were induced to TAM by tumor cell conditioned medium. Tumor cells (MC38 or CT26) and TAM were then labeled with fluorescence, and the number of tumor cells was counted after co-culture for 48 h. (B) With or without DMXAA, RAW264.7 cells were induced to TAM using tumor cell-conditioned media. MC38 cells were co-cultured with TAM and qPCR was done for the analysis of the mRNA levels of markers of TAM in RAW264.7. (C) WT or STING, bone marrow macrophages, were induced to TAM using tumor cell-conditioned media. Tumor cells (MC38 or CT26) and TAM were labeled with fluorescence, and the number of tumor cells was counted after co-culture for 48 h.

Figure 7

STING induces macrophage polarization via IRG1 (A) Mass spectrometry analysis revealed that STING interacts with IRG1. (B) Co-IP assay confirmed the interaction between STING and IRG1. (C) RAW264.7 cells were induced to become M2 macrophages using IL4 and then treated at different time points in the presence or absence of DMXAA. qPCR was performed to detect the mRNA level of the indicated markers CXCL10, IFNβ, IRG1 and iNOS. (D) Flow cytometry analysis of RAW264.7 cells after knockdown of IRG1 in IL4 or LPS/IFN-induced macrophages. (E) mRNA levels of M1-like macrophages-associated genes measured by qPCR in IRG1 knockdown RAW264.7 cells.

Figure 8

Activation of IRG1 in macrophages affects colon cancer cell metastasis (A) THP1 cells were silenced with shRNA against IRG1 or a non-targeting control. Macrophages were seeded into the transwell lower chamber to induce M2 polarization and tumor cells were seeded into transwell upper chambers (size:8 μM). 250 nM cGAMP was added to the transwell lower chamber. Migration and invasion assay of DLD1 after co-culture with M2 macrophages (THP1) for 48 h. (B) RAW264.7 cells were silenced with shRNA against IRG1 or a non-targeting control. Macrophages were seeded into the transwell lower chamber to induce M2 polarization and tumor cells were seeded into transwell upper chambers (size:8 μM). 50 μg/mL DMXAA was added to the transwell lower chamber. Migration and invasion assay of MC38 after co-culture with M2 macrophages (RAW264.7) for 48 h.

Figure 9

STING induces TFEB into the nucleus after activating IRG1 (A) Western Blot analysis of BMDM treated with different concentrations of DMXAA (0 μg,25 μg,50 μg,75 μg), the expression difference of TFEB in the cytoplasm and nucleus (β-Actin is a cytoplasmic internal reference protein, LaminB1 is a nuclear internal reference protein). (B) Western Blot analysis of TFEB expression differences in cytoplasm and nucleus after RAW264.7 and BMDM cells were treated with 150 μM OI (4-Octyl Itaconate) for 4 h (β-Actin is a cytoplasmic internal reference protein, LaminB1 is a nuclear internal reference protein), Whole: total cell protein, Cyto: cytoplasmic protein, Nuc: nuclear protein. (C) Western Blot analysis of TFEB protein expression difference in cytoplasm and nucleus after knockdown of IRG1 in RAW264.7 cells after 50 μg DMXAA treatment. (D) Western Blot detection of TFEB protein expression in RAW264.7 cells after IRG1 knockdown. (E) Immunofluorescence detection of nuclear translocation of TFEB protein after RAW264 cells were treated with 50 μg/mL DMXAA or RAW264.7 cells were treated with 150 μM OI (4-Octyl Itaconate). (F) After RAW264.7 knocked down IRG1, polarization was induced by IL4, and the migration and invasion of MC38 cells were detected after being treated with OI for 4 h and co-cultured with MC38 for 48 h in a transwell chamber. (G) After spleen injection of MC38-Luc cells to construct a colorectal cancer liver metastasis model, the liver metastasis model mice were treated with OI (i.p.) (50 mg/kg/2 day) or PBS for 3 weeks. Bioluminescence signal imaging (left) and quantitative analysis of bioluminescence (right) in representative mice, n ≥ 5. (H) ALT and AST levels in peripheral blood of mice with colorectal cancer liver metastases after 3 weeks of OI or PBS treatment.