Introduction: Tumor cell interactions with the microenvironment, especially those of bone-marrow-derived myeloid cells, are important in various aspects of tumor metastasis. Myeloid-derived suppressor cells (MDSCs) have been suggested to constitute tumor-favoring microenvironments. In this study, we elucidated a novel mechanism by which the MDSCs can mediate spontaneous distant metastasis of breast cancer cells. Methods: Murine breast cancer cells, 4T1 and EMT6, were orthotopically grafted into the mammary fat pads of syngeneic BALB/c mice. CD11b⁺Gr-1⁺ MDSCs in the spleen, liver, lung and primary tumor mass were analyzed. To evaluate the role of MDSCs in the distant metastasis, MDSCs were depleted or reconstituted in tumor-bearing mice. To evaluate whether MDSCs in the metastasizing tumor microenvironment affect breast cancer cell behavior, MDSCs and cancer cells were co-cultivated. To investigate the role of MDSCs in in vivo metastasis, we blocked the interactions between MDSCs and cancer cells. Results: Using a murine breast cancer cell model, we showed that murine breast cancer cells with high IL-6 expression recruited more MDSCs and that the metastasizing capacity of cancer cells paralleled MDSC recruitment in tumor-bearing mice. Metastasizing, but not non-metastasizing, tumor-derived factors induced MDSCs to increase IL-6 production and full activation of recruited MDSCs occurred in the primary tumor site and metastatic organ in the vicinity of metastasizing cancer cells, but not in lymphoid organs. In addition, tumor-expanded MDSCs expressed Adam-family proteases, which facilitated shedding of IL-6 receptor, thereby contributing to breast cancer cell invasiveness and distant metastasis through IL-6 trans-signaling. The critical role of IL-6 trans-signaling was confirmed in both the afferent and efferent pathways of metastasis. Conclusion: In this study, we showed that metastasizing cancer cells induced higher MDSCs infiltration and prompted them to secret exaggerated IL-6 as well as soluble IL-6Rα, which, in turn, triggered a persistent increase of pSTAT3 in tumor cells. This potential tumor-MDSC axis involving IL-6 trans-signaling directly affected breast cancer cell aggressiveness, leading to spontaneous metastasis.

Pulmonary fibrosis is a potentially life-threatening disease that may be caused by overt or asymptomatic inflammatory responses. However, the precise mechanisms by which tissue injury is translated into inflammation and consequent fibrosis remain to be established. Here, we show that in a lung injury model, bleomycin induced the secretion of IL-6 by epithelial cells in a transglutaminase 2 (TG2)–dependent manner. This response represents a key step in the differentiation of IL-17–producing T cells and subsequent inflammatory amplification in the lung. The essential role of epithelial cells, but not inflammatory cells, TG2 was confirmed in bone marrow chimeras; chimeras made in TG2-deficient recipients showed reduced inflammation and fibrosis, compared with those in wild-type mice, regardless of the bone marrow cell phenotype. Epithelial TG2 thus appears to be a critical inducer of inflammation after noninfectious pulmonary injury. We further demonstrated that fibroblast-derived TG2, acting downstream of transforming growth factor-β, is also important in the effector phase of fibrogenesis. Therefore, TG2 represents an interesting potential target for therapeutic intervention.

We previously reported that IL-6 and transglutaminase 2 (TG2) were expressed in more aggressive basal-like breast cancer cells, and TG2 and IL-6 expression gave these cells stem-cell-like phenotypes, increased invasive ability, and increased metastatic potential. In the present study, the underlying mechanism by which IL-6 production is induced in luminal-type breast cancer cells was evaluated, and TG2 overexpression, IL-1β stimulation, and IL-6 expression were found to give cancerous cells a hormone-independent phenotype. Luminal-type breast cancer cells (MCF7 cells) were stably transfected with TG2. To evaluate the requirement for IL-6 neogenesis, MCF7 cells were stimulated with various cytokines. To evaluate tumorigenesis, cancer cells were grown in a three-dimensional culture system and grafted into the mammary fat pads of NOD/scid/IL-2Rγ−/− mice. IL-1β induced IL-6 production in TG2-expressing MCF7 cells through an NF-kB-, PI3K-, and JNK-dependent mechanism. IL-1β increased stem-cell-like phenotypes, invasiveness, survival in a three-dimensional culture model, and estrogen-independent tumor growth of TG2-expressing MCF7 cells, which was attenuated by either anti-IL-6 or anti-IL-1β antibody treatment. Within the inflammatory tumor microenvironment, IL-1β increases luminal-type breast cancer cell aggressiveness by stimulating IL-6 production through a TG2-dependent mechanism.

Despite the capability of extracellular vesicles (EVs) derived from Gram‐negative and Gram‐positive bacteria to induce potent anti‐tumour responses, large‐scale production of bacterial EVs remains as a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs, namely, outer membrane vesicles (OMVs). By combining metal precipitation and size‐exclusion chromatography, we isolated 357 mg in total protein amount of E. coli OMVs, which was equivalent to 3.93 × 10¹⁵ particles (1.10 × 10¹⁰ particles/μg in total protein amounts of OMVs) from 160 L of the conditioned medium. We show that these mass‐produced E. coli OMVs led to complete remission of two mouse syngeneic tumour models. Further analysis of tumour microenvironment in neoantigen‐expressing tumour models revealed that E. coli OMV treatment causes increased infiltration and activation of CD8⁺ T cells, especially those of cancer antigen‐specific CD8⁺ T cells with high expression of TCF‐1 and PD‐1. Furthermore, E. coli OMVs showed synergistic anti‐tumour activity with anti‐PD‐1 antibody immunotherapy, inducing substantial tumour growth inhibition and infiltration of activated cancer antigen‐specific stem‐like CD8⁺ T cells into the tumour microenvironment. These data highlight the potent anti‐tumour activities of mass‐produced E. coli OMVs as a novel candidate for developing next‐generation cancer immunotherapeutic agents.