Functions And Mechanisms Of Eosinophil-derived Chemokine CCL6 In Tumor Metastasis

Background:Metastasis is the most critical aspect of tumorigenesis and causes nearly 90%of cancer mortality.Current anti-metastatic therapies often have limited success.A large number of studies showed that interplays between primary cancer and immune cell responses,acting together to control metastatic progression.The functions of various immune cells and local inflammatory components in tumor metastasis have been well explored,such as neutrophils,NK cells,regulatory T cells,monocytes/macrophages,platelets,which play indispensable roles in carcinogenesis,invasion,progression,and metastasis.Exploring the role and mechanism of immune cells and related microenvironments in tumor metastasis is essential for better preventing and treating metastatic diseases.Although eosinophils and their mediators in the inflammatory environment have been largely appreciated in many recent researches,they are thought to play controversial roles in cancer development.Eosinophils are a group of multifunctional immune cells that are involved in innate and adaptive immune responses through the expression of various receptors and mediators.They contribute to different diseases,such as parasitic infection and allergic asthma,and appear to be associated with cancer biology.Clinically,tumor-associated tissue eosinophilia（TATE）has been well described in a range of tumor types and sites,and it has been associated with clinical prognoses.Recent studies showed that activated tumor-homing eosinophils play an important role in orchestrating antitumor immune responses.It is reported that eosinophils promote tumor cell death apoptosis as well as regulate the CD8⁺T cell immune response.However,as multi-functional leucocytes,eosinophils are thought to play an opposing role in cancer development depending on the stimuli.Although some studies have suggested the disadvantages of eosinophils in primary tumors,there is little information available about the effects of eosinophils in the context of inflammation on tumor metastasis.Purpose:We investigated the function of eosinophils in the context of eosinophilic inflammation and eosinophilia by testing whether they can modulate experimental tumor cell metastasis and explored the underlying mechanisms.Methods:1.We established a melanoma model of lung metastasis following the classic OVA-induced asthma model in mice,and a DSS-induced colitis model with eosinophilia in mice,followed by tumor cell injection.These models were used to explore the relevance of eosinophilic inflammation and metastasis.2.Cd3δ-Il-5 transgenic（Il-5 Tg）mice in which a very high level of eosinophils is produced in the bone marrow and eosinophil-null（Eos-null）mice were used to establish bone metastatic models and clarify the effect of eosinophilia on metastasis.3.We isolated and purified eosinophils to study the effects on tumor cell growth,apoptosis,proliferation,and migration.Adoptive transfer of eosinophils was also performed in the metastasis model to test the direct effect of eosinophils on the colonization of tumor cells.4.We further characterized immune cell populations in the metastatic microenvironment at 24 h post-transfer of eosinophils.5.The effective factor of eosinophils promoting tumor cell migration and colonization were identified.We generated C-C motif chemokine ligand 6（Ccl6）knockout(Ccl6^-/-)mice to explore eosinophil-derived CCL6 in promoting metastasis.6.We established stable knockdown of CC Chemokine receptor 1（Ccr1,the receptor of CCL6）in B16-F10 cells and administrated BX471（antagonist of CCR1）in B16-F10cells to study the function of CCL6-CCR1 signaling in metastasis in vitro and in vivo.7.Eosinophils in malignant pleural effusion（MPE）of patients with cancer were detected,as well as the expression of CCL23（the human orthologue of murine CCL6）.Results:1.We show that airway inflammatory eosinophilia and colonic inflammation with eosinophil infiltration are both associated with increased metastasis in mice.2.Eosinophilia is responsible for increased bone metastasis in eosinophil-enriched Il-5 Tg mice.3.Eosinophils promote tumor cell migration in vitro and colonization in vivo.4.There were no significant changes in the number of alveolar macrophages,CD4⁺T and CD8⁺T lymphocytes,NK cells,DCs,neutrophils,or monocytes,except increased eosinophils following eosinophils transfer.5.Eosinophils promote tumor cell migration and metastasis formation through secreting CCL6.Genetic knockout of Ccl6 in Il-5 Tg mice remarkably attenuates bone metastasis.6.Inhibition of CCR1 in tumor cells reduces tumor cell migration and colonization in vitro and in vivo.7.We also observe increased eosinophils in the malignant pleural effusion（MPE）of cancer patients with elevated expression of CCL23.Conclusion:Our study identifies a CCL6-dependent pro-metastatic activity of eosinophils in the context of eosinophilic inflammation or eosinophilia,which can inhibit by targeting CCR1 and represent an approach to prevent metastatic disease.