Total glucosides of paeony inhibit breast cancer growth by inhibiting TAMs infiltration through NF-κB/CCL2 signaling

Abstract

Purpose: The high density of tumor-associated macrophages (TAMs) and inflammatory factors are crucial elements leading to tumor immune tolerance. Previously, we found that total glucosides of paeony (TGP) have strong inhibitory effects on the release of various inflammatory factors; however, it is unclear whether the inhibitory effects can improve the inflammatory microenvironment of tumors. Therefore, in the present study, we investigated the mechanism via which TGP depresses tumor growth and metastasis via modulation of TAM infiltration in breast cancer.

Methods: We assessed the effects of TGP on various mouse models of tumor. Lung metastasis was detected using hematoxylin and eosin staining. T cell (CD3⁺CD4⁺ and CD3⁺CD8⁺) effector and memory subsets, and TAM (CD45⁺CD11b⁺F4/80⁺) populations in the tumor microenvironment (TME) were examined using flow cytometry. Lipopolysaccharide (LPS)-stimulated macrophage experiments were used to investigate the TGP anti-inflammatory effects in vitro. Furthermore, conditional medium (CM) was added to detect 4T1 breast cancer cell growth using a Real-Time Cell Analyzer (RTCA) xCELLigence system. Inflammatory cytokine and chemokine levels were measured using cytometric bead array (CBA) kits and quantitative polymerase chain reaction (qPCR). NF-κB expression in the nucleus was examined by immunofluorescence and Western blot analysis.

Results: TGP suppressed tumor growth and lung metastasis, decreased CD45⁺CD11b⁺F4/80⁺ (TAMs) population obviously, and increased CD44^LowCD62L^Hi (T memory stem cells) and CD44^HiCD62L^Hi (central memory cells) populations in the tumor-infiltrating CD4⁺ and CD8⁺ T cells. In addition, TGP reduced inflammatory factor levels in tumors, thus inhibiting the infiltration of TAMs to improve the inflammation immunosuppressive microenvironment. In the in vitro experiment, TGP inhibited IL-10 and C-C Motif Chemokine Ligand 2 (CCL2) secretion and mRNA expression in LPS-stimulated macrophages to inhibit 4T1 cell growth and restrain macrophages M2 polarization. In addition, TGP can directly inhibit 4T1 cell proliferation by restraining autocrine CCL2 and IL-10. Further mechanistic studies reavealed that TGP inhibited CCL2 secretion by inhibiting NF-κB accumulation in the nucleus in macrophages.

Conclusion: TGP reduced TAM recruitment mainly through the NF-κB/CCL2 signaling pathway, thereby promoting T cell infiltration in the TME. TGP has a unique advantage in balancing the inflammatory response. Furthermore, our results present novel insights on the mechanisms underlying TAM infiltration that were inhibited by TGP, with potential application in development of novel therapies targeting CCL2 pathways.

Keywords: Breast cancer; CCL2; NF-κB; Total glucosides of paeoniae; Tumor-associated macrophages.