Optimizing a five-factor cocktail to prepare reparative macrophages for wound healing.

The treatment of non-healing wounds, such as diabetic ulcers, remains a critical clinical challenge. Recent breakthroughs in cell therapy have shown great promise, with one primary focus on preparing cells with comprehensive reparative functions and foreseeable safety. In our previous study, we recapitulated the pro-regenerative and immunosuppressive functions of tumor-associated macrophages (TAMs) in non-tumor-derived macrophages, endowing the latter with characteristics for promoting diabetic wound healing - termed TAMs-educated macrophages (TAMEMs). To eliminate the use of tumor-derived sources and devise a more controllable method to prepare TAMEM-like cells, in this study, we identify a cocktail comprising five recombinant proteins as an essential condition to induce non-polarized macrophages (termed TAMEMs5) into therapeutic cells with pro-healing functions. The screened five factors are osteopontin (OPN), macrophage inflammatory protein (MIP)-2, chemokine (C-C motif) ligand 8 (CCL8), vascular endothelial growth factor (VEGF)-B, and macrophage colony-stimulating factor (M-CSF). We demonstrate the rationale for screening these factors and the phenotype of TAMEMs5 prepared from murine bone marrow-derived macrophages, which exhibit angiogenic and immunomodulatory effects in vitro. Then, we induce primary human monocytes from periphery blood into TAMEMs5, which show pro-healing effects in a human primary cell-based ex vivo model (T-SkinTM). Our study demonstrates a simple, effective, and controllable approach to induce primary macrophages to possess repairing activities, which may provide insights for developing cell-based therapeutics for non-healing wounds clinically.

Tumor-associated macrophages-educated reparative macrophages promote diabetic wound healing.

Nonhealing diabetic wounds, with persistent inflammation and damaged vasculature, have failed conventional treatments and require comprehensive interference. Here, inspired by tumor-associated macrophages (TAMs) that produce abundant immunosuppressive and proliferative factors in tumor development, we generate macrophages to recapitulate TAMs' reparative functions, by culturing normal macrophages with TAMs' conditional medium (TAMs-CM). These TAMs-educated macrophages (TAMEMs) outperform major macrophage phenotypes (M0, M1, or M2) in suppressing inflammation, stimulating angiogenesis, and activating fibroblasts in vitro. When delivered to skin wounds in diabetic mice, TAMEMs efficiently promote healing. Based on TAMs-CM's composition, we further reconstitute a nine-factor cocktail to train human primary monocytes into TAMEMsC-h , which fully resemble TAMEMs' functions without using tumor components, thereby having increased safety and enabling the preparation of autologous cells. Our study demonstrates that recapitulating TAMs' unique reparative activities in nontumor cells can lead to an effective cell therapeutic approach with high translational potential for regenerative medicine.

Smectite promotes probiotic biofilm formation in the gut for cancer immunotherapy.

Administration of probiotics to regulate the immune system is a potential anti-tumor strategy. However, oral administration of probiotics is ineffective because of the poor inhabitation of exogenous bacteria in host intestines. Here we report that smectite, a type of mineral clay and established anti-diarrhea drug, promotes expansion of probiotics (especially Lactobacillus) in the murine gut and subsequently elicits anti-tumor immune responses. The ion-exchangeable microstructure of smectite preferentially promotes lactic acid bacteria (LABs) to form biofilms on smectite in vitro and in vivo. In mouse models, smectite laden with LAB biofilms (Lactobacillus and Bifidobacterium) inhibits tumor growth (when used alone) and enhances the efficacy of chemotherapy or immunotherapy (when used in combination with either of them) by activating dendritic cells (DCs) via Toll-like receptor 2 (TLR2) signaling. Our findings suggest oral administration of smectite as a promising strategy to enrich probiotics in vivo for cancer immunotherapy.

High-salt diet inhibits tumour growth in mice via regulating myeloid-derived suppressor cell differentiation.

High-salt diets are associated with an elevated risk of autoimmune diseases, and immune dysregulation plays a key role in cancer development. However, the correlation between high-salt diets (HSD) and cancer development remains unclear. Here, we report that HSD increases the local concentration of sodium chloride in tumour tissue, inducing high osmotic stress that decreases both the production of cytokines required for myeloid-derived suppressor cells (MDSCs) expansion and MDSCs accumulation in the blood, spleen, and tumour. Consequently, the two major types of MDSCs change their phenotypes: monocytic-MDSCs differentiate into antitumour macrophages, and granulocytic-MDSCs adopt pro-inflammatory functions, thereby reactivating the antitumour actions of T cells. In addition, the expression of p38 mitogen-activated protein kinase-dependent nuclear factor of activated T cells 5 is enhanced in HSD-induced M-MDSC differentiation. Collectively, our study indicates that high-salt intake inhibits tumour growth in mice by activating antitumour immune surveillance through modulating the activities of MDSCs.

A toll-like receptor agonist mimicking microbial signal to generate tumor-suppressive macrophages.

Switching macrophages from a pro-tumor type to an anti-tumor state is a promising strategy for cancer immunotherapy. Existing agents, many derived from bacterial components, have safety or specificity concerns. Here, we postulate that the structures of the bacterial signals can be mimicked by using non-toxic biomolecules of simple design. Based on bioactivity screening, we devise a glucomannan polysaccharide with acetyl modification at a degree of 1.8 (acGM-1.8), which specifically activates toll-like receptor 2 (TLR2) signaling and consequently induces macrophages into an anti-tumor phenotype. For acGM-1.8, the degree of acetyl modification, glucomannan pattern, and acetylation-induced assembly are three crucial factors for its bioactivity. In mice, intratumoral injection of acGM-1.8 suppresses the growth of two tumor models, and this polysaccharide demonstrates higher safety than four classical TLR agonists. In summary, we report the design of a new, safe, and specific TLR2 agonist that can generate macrophages with strong anti-tumor potential in mice.

A Jak2-selective inhibitor potently reverses the immune suppression by modulating the tumor microenvironment for cancer immunotherapy.

Small molecule therapeutics can be potent tools for cancer immunotherapy. They may be devised to target the tumor associated macrophages (TAMs) and regulatory T cells (Treg), which are major immunosuppressive cells in the tumor microenvironment. The infiltration and functionalization of these cells, which essentially promote tumor development, are mediated by the hyper-activation of the Jak-STAT3 signaling pathway. Here, we demonstrated that compound 9#, a novel inhibitor of Jak2, could suppress Jak2-STAT3 signaling in macrophages (peritoneal macrophages and THP-1 cells) and direct the macrophages toward the pro-inflammatory (M1-like) phenotype. When tested in ex vivo TAM culture and in vivo tumor models, compound 9# could reverse the phenotype of TAM from M2- to M1-type by promoting IL-12 expression. Further study suggested that compound 9# also inhibited the induction of Treg both in vitro and in vivo via blockage of Jak2 signaling. Finally, compound 9# potently increased the frequency and anti-tumor activity of CD4+ and CD8+ T lymphocytes, leading to effective suppression of tumor growth. Taken together, our findings indicated that compound 9# could be a potential candidate of small molecule therapeutics for cancer immunotherapy.