M2 Tumor-Associated Macrophages-Derived Exosomal MALAT1 Promotes Glycolysis and Gastric Cancer Progression

Abstract

M2-polarized tumor-associated macrophages (M2 TAMs) promote cancer progression. Exosomes mediate cellular communication in the tumor microenvironment (TME). However, the roles of exosomes from M2 TAMs in gastric cancer progression are unclear. Herein, it is reported that M2 TAMs-derived exosomes induced aerobic glycolysis in gastric cancer cells and enhanced their proliferation, metastasis, and chemoresistance in a glycolysis-dependent manner. It is identified that MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is enriched in M2 TAM exosomes and confirmed that MALAT1 transfer from M2 TAMs to gastric cancer cells via exosomes mediates this effect. Mechanistically, MALAT1 interacted with the δ-catenin protein and suppressed its ubiquitination and degradation by β-TRCP. In addition, MALAT1 upregulated HIF-1α expression by acting as a sponge for miR-217-5p. The activation of β-catenin and HIF-1α signaling pathways by M2 TAM exosomes collectively led to enhanced aerobic glycolysis in gastric cancer cells. Finally, a dual-targeted inhibition of MALAT1 in both gastric cancer cells and macrophages by exosome-mediated delivery of siRNA remarkably suppressed gastric cancer growth and improved chemosensitivity in mouse tumor models. Taken together, these results suggest that M2 TAMs-derived exosomes promote gastric cancer progression via MALAT1-mediated regulation of glycolysis. The findings offer a potential target for gastric cancer therapy.

Keywords: MALAT1; exosomes; gastric cancer; glycolysis; tumor‐associated macrophages.

Figure 1

M2‐polarized macrophages induce aerobic glycolysis in gastric cancer cells and promote their proliferation, migration, invasion, and chemoresistance. A,B) QRT‐PCR A) and western blot B) analyses of glycolysis‐related gene expression in gastric cancer cells treated with conditioned medium from M2 macrophages (M2‐CM). C) Glucose uptake, lactate production, ATP level, and LDH activity assays for gastric cancer cells treated with M2‐CM. D,E) Cell counting and colony formation D), transwell migration, and matrigel invasion E) assays for gastric cancer cells treated with M2‐CM in the presence or absence of 2‐DG. Scale bar: 200 µm. F) Flow cytometric analyses on oxaliplatin‐induced apoptosis in gastric cancer cells treated with M2‐CM in the presence or absence of 2‐DG. G) CCK8 assay for IC50 of oxaliplatin in M2‐CM‐treated gastric cancer cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Exosomes from M2‐polarized macrophages induce aerobic glycolysis and promote gastric cancer cell proliferation, migration, and invasion. A) TEM analysis of exosomes from M2 macrophages (M2‐EX). Scale bar: 100 nm. B) Western blot analyses of exosomal proteins in M2‐EX. C) Cellular uptake assay for Dio‐labeled M2‐EX in gastric cancer cells. Scale bar: 25 µm. D,E) QRT‐PCR D) and western blot E) analyses of glycolysis‐related gene expression in gastric cancer cells treated with M2‐EX. F) Glucose uptake, lactate production, ATP level, and LDH activity assays in gastric cancer cells treated with M2‐EX. G,H) Cell counting and colony formation G), transwell migration, and matrigel invasion H) assays for gastric cancer cells treated with M2‐EX in the presence or absence of 2‐DG. Scale bar: 200 µm. I) Flow cytometric analyses of oxaliplatin‐induced apoptosis in gastric cancer cells treated with M2‐EX in the presence or absence of 2‐DG. J) CCK8 assay for IC50 of oxaliplatin in gastric cancer cells treated with M2‐EX. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Exosomal MALAT1 from M2‐polarized macrophages induces aerobic glycolysis and gastric cancer cell proliferation, migration, and invasion. A) Heatmap of RNA‐sequencing analyses of M0‐EX and M2‐EX. B) QRT‐PCR analyses on the top 6 enriched lncRNAs in M2‐polarized macrophages and M2‐EX. C) QRT‐PCR analyses on the top 6 enriched lncRNAs in gastric cancer cells treated with M2‐EX. D) Fluorescent imaging of gastric cancer cells incubated with Cy3‐labeled MALAT1‐enriched M2‐EX. Scale bar: 25 µm. E) Glucose uptake, lactate production, ATP level, and LDH activity assays in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. F,G) Cell counting and colony formation F), transwell migration, and matrigel invasion G) assays for gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. Scale bar: 200 µm. H) Flow cytometric analyses of oxaliplatin‐induced apoptosis in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. I) CCK8 assay for IC50 of oxaliplatin in control or MALAT1‐depleted M2‐EX‐treated gastric cancer cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Exosomal MALAT1 from M2‐polarized macrophages stabilizes δ‐catenin protein. A) RIP assay for the interaction between δ‐catenin protein and MALAT1. B) Fluorescent imaging of the co‐localization of Cy3‐labeled MALAT1 transferred by M2‐EX and δ‐catenin protein in gastric cancer cells. Scale bar: 25 µm. C,D) Western blot assay for δ‐catenin C) and β‐catenin signaling pathway D) proteins in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. E) MG‐132 assay for δ‐catenin protein levels in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. F) CHX assay for the half‐life of δ‐catenin protein in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. G) Western blot assay for the ubiquitination of δ‐catenin protein in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. H) Co‐IP assay for the interaction between δ‐catenin and β‐TRCP in gastric cancer cells. I) Co‐IP assay for the impact of control or MALAT1‐depleted M2‐EX on δ‐catenin and β‐TRCP protein interaction. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Exosomal MALAT1 from M2‐polarized macrophages upregulates HIF‐1α by sponging miR‐217‐5p. A) Bioinformatic analyses of potential MALAT1‐interacting miRNAs. B) RIP assay for the interaction between AGO2 protein and MALAT1. C) QRT‐PCR analyses of miR‐217‐5p expression in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. D) Luciferase assay for the activities of WT and MUT MALAT1 reporters. E) Bioinformatic analyses of potential target genes of miR‐217‐5p. F) QRT‐PCR analyses of HIF‐1α expression in gastric cancer cells transfected with miR‐217‐5p mimics or inhibitors. G) Luciferase assay for the activities of WT and MUT HIF‐1α reporters. H) Western blot analyses of HIF‐1α protein expression in gastric cancer cells treated with miR‐217‐5p mimics and inhibitors. I) Luciferase assay for the activities of WT and MUT HIF‐1α reporters in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. J,K) QRT‐PCR J) and western blot K) analyses of HIF‐1α protein expression in gastric cancer cells treated with control or MALAT1‐depleted M2‐EX. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Exosomes from M2‐polarized macrophages activate β‐catenin and HIF‐1α signaling pathways to induce aerobic glycolysis and gastric cancer progression. A,B) Western blot A) and qRT‐PCR B) analyses of glycolysis‐related gene expression in gastric cancer cells treated with M2‐EX in the presence or absence of ICG‐001 and PX‐478. C) Glucose uptake, lactate production, ATP level, and LDH activity assays for gastric cancer cells treated with M2‐EX in the presence or absence of ICG‐001 and PX‐478. D,E) Cell counting and colony formation D), transwell migration, and matrigel invasion E) assays for gastric cancer cells treated with M2‐EX in the presence or absence of ICG‐001 and PX‐478. Scale bar: 200 µm. F) Flow cytometric analyses of oxaliplatin‐induced apoptosis in gastric cancer cells treated with M2‐EX in the presence or absence of ICG001 and PX‐478. G) CCK8 assay for IC50 of oxaliplatin in M2‐EX‐treated gastric cancer cells with the presence or absence of ICG001 and PX‐478. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Targeted inhibition of MALAT1 suppresses the promoting effects of M2‐polarized macrophages on gastric cancer progression and improves chemotherapy efficacy. A) Representative images of tumors in different groups of mouse models as indicated. B) Tumor volumes and growth curves in different groups of mouse models as indicated. C,D) Western blot analyses of the expression of glycolysis‐related proteins C), δ‐catenin, and HIF‐1α D) in different groups of tumors as indicated. E) Immunohistochemical staining of δ‐catenin and HIF‐1α protein expression in different groups of tumors as indicated. Scale bar: 50 µm. F) Ki‐67 staining and TUNEL staining for tumors in different groups of mouse models as indicated. Scale bar: 50 µm. G) Flow cytometric analyses of M2peps‐293T‐EX and RGD‐293T‐EX by macrophages and gastric cancer cells. H) QRT‐PCR analyses of MALAT1 expression in macrophages and gastric cancer cells treated with MALAT1 siRNA‐loaded M2peps‐293T‐EX and RGD‐293T‐EX. I,J) Tumor images I), volumes, and growth curves J) in different groups of mouse models as indicated. K) Western blot analyses of δ‐catenin and HIF‐1α protein expression in different groups of tumors as indicated. L) QRT‐PCR analyses of gene expression in different groups of tumors as indicated. M) Immunohistochemical staining of protein expression in different groups of tumors as indicated. Scale bar: 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

The proposed model for the roles of M2 TAMs in promoting glycolysis and gastric cancer progression.