Cisplatin-based chemotherapy is frequently used to treat advanced gastric cancer (GC). However, the resistance often occurs with the mechanisms being not well understood. Recently, emerging evidence indicates that tumor-associated macrophages (TAMs) play an important role in chemoresistance of cancer. As the important mediators in intercellular communications, exosomes secreted by host cells mediate the exchange of genetic materials and proteins to be involved in tumor aggressiveness. The aim of the study was to investigate whether exosomes derived from TAMs mediate cisplatin resistance in gastric cancer. M2 polarized macrophages were obtained from mouse bone marrow or human PBMCs stimulated with IL-4 and IL-13. Exosomes isolated from M2 macrophages culture medium were characterized, and miRNA expression profiles of M2 derived exosomes (M2-exos) were analyzed using miRNA microarray. In vitro cell coculture was further conducted to investigate M2-exos mediated crosstalk between TAMs and tumor cells. Moreover, the in vivo experiments were performed using a subcutaneous transplantation tumor model in athymic nude mice. In this study, we showed that M2 polarized macrophages promoted cisplatin (DDP) resistance in gastric cancer cells and exosomes derived from M2 macrophages (M2-exos) are involved in mediating the resistance to DDP. Using miRNA profiles assay, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and cell lysate isolated from M2 polarized macrophage. Functional studies revealed that exosomal miR-21 can be directly transferred from macrophages to the gastric cancer cells, where it suppresses cell apoptosis and enhances activation of PI3K/AKT signaling pathway by down-regulation of PTEN. Our findings suggest that exosomal transfer of tumor-associated macrophages derived miR-21 confer DDP resistance in gastric cancer, and targeting exosome communication may be a promising new therapeutic strategy for gastric cancer patients.

Cancer immunotherapy has revolutionized cancer treatment, and it relies heavily on the comprehensive understanding of the immune landscape of the tumor microenvironment (TME). Here, we obtain a detailed immune cell atlas of esophageal squamous cell carcinoma (ESCC) at single-cell resolution. Exhausted T and NK cells, regulatory T cells (Tregs), alternatively activated macrophages and tolerogenic dendritic cells are dominant in the TME. Transcriptional profiling coupled with T cell receptor (TCR) sequencing reveal lineage connections in T cell populations. CD8 T cells show continuous progression from pre-exhausted to exhausted T cells. While exhausted CD4, CD8 T and NK cells are major proliferative cell components in the TME, the crosstalk between macrophages and Tregs contributes to potential immunosuppression in the TME. Our results indicate several immunosuppressive mechanisms that may be simultaneously responsible for the failure of immuno-surveillance. Specific targeting of these immunosuppressive pathways may reactivate anti-tumor immune responses in ESCC.

Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment and have been shown to contribute to tumor aggressiveness. However, the detailed mechanisms underlying the pro-metastatic effect of TAMs on gastric cancer are not clearly defined. Here, we show that TAMs are enriched in gastric cancer. TAMs are characterized by M2-polarized phenotype and promote migration of gastric cancer cells in vitro and in vivo. Furthermore, we find that M2-derived exosomes determine the TAMs-mediated pro-migratory activity. Using mass spectrometry, we identify that apolipoprotein E (ApoE) is highly specific and effective protein in M2 macrophages-derived exosomes. Moreover, TAMs are uniquely immune cells population expressed ApoE in gastric cancer microenvironment. However, exosomes derived from M2 macrophages of Apoe ^−/− mice have no significant effect on the migration of gastric cancer cells in vitro and in vivo. Mechanistically, M2 macrophage-derived exosomes mediate an intercellular transfer of ApoE-activating PI3K-Akt signaling pathway in recipient gastric cancer cells to remodel the cytoskeleton-supporting migration. Collectively, our findings signify that the exosome-mediated transfer of functional ApoE protein from TAMs to the tumor cells promotes the migration of gastric cancer cells.

Regulatory B cells (Bregs) play a critical role in inflammation and autoimmune disease. We characterized the role of Bregs in the progression of gastric cancer. We detected an increase in Bregs producing IL-10 both in peripheral blood mononuclear cells (PBMCs) and in gastric tumors. Multicolor flow cytometry analysis revealed that a subset of CD19+CD24hiCD38hi B cells produces IL-10. Functional studies indicated that increased Bregs do not inhibit the proliferation of CD3+T cells or CD4+ helper T cells (Th cells). However, Bregs do suppress the secretion of IFN-γ and TNF-α by CD4+Th cells. CD19+CD24hiCD38hiBregs were also found to correlate positively with CD4+FoxP3+ regulatory T cells (Tregs). Neutralization experiments showed that Bregs convert CD4+CD25- effector T cells to CD4+FoxP3+Tregs via TGF-β1. Collectively, these findings demonstrate that increased Bregs play a immunosuppressive role in gastric cancer by inhibiting T cells cytokines as well as conversion to Tregs. These results may provide new clues about the underlying mechanisms of immune escape in gastric cancer.

Heterotypic interactions between tumor cells and macrophages can enable tumor progression and hold potential for the development of therapeutic interventions. However, the communication between tumors and macrophages and its mechanism are poorly understood. Here, we find that tumor‐associated macrophages (TAM) from tumor‐bearing mice have high amounts of lipid as compared to macrophages from tumor‐free mice. TAM also present high lipid content in clinical human gastric cancer patients. Functionally, TAM with high lipid levels are characterized by polarized M2‐like profiling, and exhibit decreased phagocytic potency and upregulated programmed death ligand 1 (PD‐L1) expression, blocking anti–tumor T cell responses to support their immunosuppressive function. Mechanistically, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identifies the specific PI3K pathway enriched within lipid‐laid TAM. Lipid accumulation in TAM is mainly caused by increased uptake of extracellular lipids from tumor cells, which leads to the upregulated expression of gamma isoform of phosphoinositide 3‐kinase (PI3K‐γ) polarizing TAM to M2‐like profiling. Correspondingly, a preclinical gastric cancer model is used to show pharmacological targeting of PI3K‐γ in high‐lipid TAM with a selective inhibitor, IPI549. IPI549 restores the functional activity of macrophages and substantially enhances the phagocytosis activity and promotes cytotoxic‐T‐cell‐mediated tumor regression. Collectively, this symbiotic tumor‐macrophage interplay provides a potential therapeutic target for gastric cancer patients through targeting PI3K‐γ in lipid‐laden TAM.

Immune checkpoint blockade (ICB) immunotherapy increases antitumor immunity by blocking cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and displays robust clinical responses in various cancers. However, ICB immunotherapy also triggers severe inflammatory side effects, known as immune-related adverse effects (irAEs). One of the most common toxicities is immune checkpoint blockade-associated colitis (ICB associated colitis). The exact mechanism of ICB associated colitis remains to be explored. Here, we combined ICB (anti–CTLA-4 and anti-PD-1) treatment with a standard colitis model, in which a more severe form of colitis is induced in mice, to recapitulate the clinical observations in patients receiving combined ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) therapy, during which colitis is the most frequent complication encountered. We found that the composition of the gut microbiota changed in ICB associated colitis. Principal component analysis of the gut microbiome showed an obvious reduction in the abundance of Lactobacillus in severe ICB associated colitis. Lactobacillus depletion completely by vancomycin augmented the immunopathology of ICB. Furthermore, we found that the ICB toxicity could be totally eliminated via the administration of a widely available probiotic Lactobacillus reuteri (L.reuteri). Oral administration of L. reuteri therapeutically inhibited the development and progression of colitis, thus ameliorating the loss of body weight and inflammatory status induced by ICB treatment. Mechanistically, the protective effect of L. reuteri was associated with a decrease in the distribution of group 3 innate lymphocytes (ILC3s) induced by ICB associated colitis. In conclusion, our study highlights the immunomodulatory mechanism of the gut microbiota and suggests that manipulating the gut microbiota by administrating L. reuteri can mitigate the autoimmunity induced by ICB, thus allowing ICB immunotherapy to stimulate the desired immune response without an apparent immunopathology.