Role Of Lung Mesenchymal Stromal Cells In Establishing The Pre-metastatic Niches

Metastasis is the major cause of cancer-related death and a key factor in the failure of cancer treatment.Disseminated tumor cells face many obstacles and likely,only few of them can colonize in the distant organs.Therefore,colonization of distant organs is a rate-limiting step of metastasis.The local microenvironment in the distant organ,also known as pre-metastatic niche,determines whether or not colonization of cancer cells can be established.Tumor-derived growth factors,inflammatory cytokines,chemokines,and exosomes are shown to be critical for the initiation and evolution of pre-metastatic niches.Immune cells,such as neutrophils,macrophages,MDSCs,and T cells,are believed to be the recipients of the tumor-secreted factors and represent critical constituents of premetastatic niches.Mesenchymal stromal cells(MSCs)exist in almost every type of tissues and are critical for maintaining tissue homeostasis and regeneration.During tumor development,bone marrow and tissue resident MSCs can be mobilized and modulate tumor cells directly or indirectly.Various growth factors and chemokines can be produced by tumor MSCs to control tumor growth and metastasis.However,our knowledge of how local MSCs contribute to the pre-metastatic niches and how they interact with immune cells in forming the pre-metastatic niches is very limited.By analyzing the components of the lungs at different stages during tumor development,the changes of the expression profile of MSCs and the changes of the immune cell population were determined.Furthermore,we explored the specific role of MSCs involved in pre-metastatic niche remodeling,and revealed the underlying mechanism.In this study,lung MSCs were identified from different stages of the MMTVPy MT spontaneous breast cancer mouse model,and their roles in metastasis were elucidated by a combination of bioinformatics,molecular and cell biology analyses.Our main findings are as follows:1.Mesenchymal stromal cells(MSCs)are crucial components of PMNs.We isolated and expanded lung local mesenchymal stromal cells(LMSCs)from female MMTV-Py MT mice at different stages of their tumor development.We found that MSCs from all stages possess similar surface markers,proliferation capabilities,and differentiation potentials.When administered intravenously to MMTV-Py MT mice,those MSCs from mice with late pre-metastatic and metastatic lung tumor exhibited a strong promotion of lung metastasis.The same was seen with murine breast cancer cell line 4T1 implanted into the mammary gland fat pad of wild type mice.2.The pro-metastatic property of C3-producing MSCs is endowed by type 2 cytokines.RNA-seq revealed that there is a high expression of complement C3 in late pre-metastatic and metastatic LMSCs.The metastasis promotion effect was not seen in mice deficient in C3 a receptor.We also found that IL-4 and IL-13 were up-regulated in mice bearing pre-metastatic and metastatic lung tumor.Furthermore,we also showed that IL-4 and IL-13 could increase C3 expression through Stat6.Significantly,in Stat6 deficient mice,there was no LMSCs-mediated metastasis of 4T1 cells.In MMTV-Py MT mice and 4T1-inoculated mice,anti-IL-4 blocked LMSCs promoted lung metastasis.3.C3 prepares PMNs by recruiting neutrophils and driving the formation of neutrophil extracellular traps.The effect of C3 a was found to be exerted through recruiting neutrophils and the subsequent neutrophil extracellular traps(NETs),as administration of DNAase abolished the LMSCs-induced metastasis promoting effect.4.C3 expression levels are associated with lung metastasis in breast cancer patients.To address the clinical relevance of these findings we determined C3 a levels in serum from 41 breast cancer patients and 7 healthy controls by ELISA.The patients with metastasis had the highest levels of C3.Furthermore,we examined the levels of C3 in lung metastatic specimens obtained from patients with breast cancers and in noncancerous lung tissues from patients with primary lung cancers.C3 expression was found to be elevated in the lung metastasis sites.Additionally,LMSCs-marked by Nestin and C3 were co-localized in pulmonary metastasis sample,suggesting that C3 may also be produced by LMSCs in cancer patients.These data indicate that C3 was probably also involved in lung metastasis of breast cancer in human patients.Taking together,our study demonstrated that under the influence of type 2 inflammation,LMSCs participate in the formation of the metastasis destination of breast cancer cells through the previous unappreciated Stat6-MSC-C3-neutrophil axis.Our studies establish a novel link between mesenchymal stromal cells and immune cells during the formation of PMNs.Th2 cells,MSCs and neutrophils are functionally connected and sequentially act to prepare the PMNs for the colonization of cancer cells.These findings suggest that targeting the Th2 cytokines-Stat6-C3-NETs axis may represent a potential strategy for controlling breast cancer metastasis to the lungs.