Legumain Promotes Gastric Cancer Progression Through Tumor-associated Macrophages In vitro and In vivo

Abstract

Tumor-associated macrophages (TAMs) play a crucial role in the tumor microenvironment. Legumain (LGMN) has been shown to be a tumor-promoting protein, but the effect of LGMN on TAMs in the progression of gastric cancer (GC) is under exploration. Our studies included the construction of LGMN-knockdown and LGMN-overexpressing TAMs induced from the human cell line THP-1 (PMA/IL-4/IL-13) and murine cell line Raw264.7 (IL-4/IL-13). A CCK-8 assay and transwell migration assay indicated that upregulation of LGMN expression in TAMs stimulated cell proliferation, migration and invasion in vitro, while downregulation of LGMN expression reduced cell proliferation, migration and invasion. In vivo experiments revealed slower growth, less angiogenesis, and less Ki67 expression in LGMN-knockdown TAMs injected with gastric cancer cells compared to control TAMs injected with GC cells. Together, these study results suggested that LGMN⁺ TAMs, which may serve as a potential target for GC treatment, promoted gastric cancer cell proliferation and angiogenesis in vitro and in vivo.

Keywords: Legumain; angiogenesis; gastric cancer; tumor-associated macrophages.

Figure 1

Efficient knockdown or overexpression of LGMN in monocytes/macrophages. (A) Expression of LGMN and CD68 in gastric cancer tissue samples and adjacent normal tissue samples. (B and C) Protein bands for LGMN and β-actin in THP-1 (PMA/IL-4/IL-13) cells and Raw264.7 (IL-4/IL-13) cells with or without LGMN knockdown. (D and E) Protein bands for FLAG-LGMN and β-actin in THP-1 (PMA/IL-4/IL-13) cells and Raw264.7 (IL-4/IL-13) cells with or without LGMN overexpression.

Figure 2

Knocking down LGMN expression in TAMs reduced their activity and migration. (A and B) Growth curves for THP-1 and Raw264.7 cells with or without LGMN knockdown. (C and D) Crystal violet staining after a transwell migration assay and statistical analysis of the cells/field for THP-1 and Raw264.7 cells with or without LGMN knockdown. (E) Growth curves for MKN28 cells cocultured with THP-1 cells with or without LGMN knockdown. (F) Transwell migration assay of MKN28 cells cocultured with THP-1 cells with or without LGMN knockdown.

Figure 3

Overexpression of LGMN in TAMs enhanced their activity and migration. (A and B) Growth curves for THP-1 and Raw264.7 cells with or without LGMN knockdown. (C and D) Crystal violet staining after a transwell assay and statistical analysis of the cells/field for THP-1 and Raw264.7 cells with or without LGMN overexpression. (E) Growth curves for MKN28 cells cocultured with THP-1 cells with or without LGMN overexpression. (F) Transwell migration assay of MKN28 cells cocultured with THP-1 cells with or without LGMN overexpression.

Figure 4

LGMN-KD TAMs reduced tumor development in vivo. (A) Tumor formation after subcutaneous injection of SGC7901 gastric cancer cells mixed with TAMs induced from THP-1 cells (PMA/IL-4/IL-13) with or without LGMN KD. (B) Dissected tumors. (C) Tumor weights for the NC and KD groups. (D) H&E staining for the NC and KD groups.

Figure 5

LGMN-KD TAMs reduced tumor cell proliferation and angiogenesis in vivo. (A) Green fluorescence-labeled CD68, red fluorescence-labeled AEP and DAPI-labeled nuclei in LGMN-NC and LGMN-KD tissue samples. (B) Red fluorescence-labeled AEP, DAPI-labeled nuclei and merged images of LGMN-NC and LGMN-KD tissue samples. (C) Immunohistochemical staining for Ki67 in LGMN-NC and LGMN-KD tissue samples.

Figure 6

LGMN-KD TAMs reduced tumor cell proliferation and angiogenesis. Diagram of TAMs promoting GC proliferation and angiogenesis.