The Role Of Eosinophils In Tumor Initiation Progression And Metastasis

IntroductionCancer is one of the diseases that is seriously harmful to people's health,and the incidence rate increases year by year.Although the mechanism of cancer initiation,progression and metastasis has not been clearly explored,a large number of studies have shown that cancer is a disease resulted from external environmental courses and internal causes that includes mutation of genes or tumor suppressor gene inactivation,abnormal function of immune cells and so on.The past decade of cancer research has been marked by a growing appreciation of the role of immunity,especially the immune-related cells and cytokines,such as Neutrophils,Th17 cells,Treg cells,DC cells and the like.Some of these studies suggested that Eosinophils(Eos)may also have a regulatory role in the development and metastasis of cancer.However,the molecular mechanism of Eos remains largely unclear.Eos is a class of white blood cells mentioned and named by Paul Ehrlich in 1879 for the first time,accounting for a very low proportion of white blood cells in healthy individuals.Eos plays a role in the:innate immune and adaptive immune regulation to maintain the stability of the environment,such as mediating tissue damage repair.In addition,as multifunctional leukocytes,the latest research showed that Eos has several"new functions".Eos is essential for development and proliferation of B cells,as well as promoting the mobilization of stem cells.Moreover,The number of Eosinophils in peripheral blood and tissues is significantly increased in diseases such as parasite infection or allergic disorders,especially asthma.In recent years,"tumor-associated Eosinophilia" was frequently reported in patients with colorectal cancer,breast cancer,ovarian cancer,cervical cancer,oral squamous cell carcinoma,Hodgkin's lymphoma and prostate cancer and so on.While its presence has been associated with a good prognosis in some studies,suggesting that Eos may play a protective role against tumors.On the other hand,an association between allergic conditions and a trend of decreased risk in numerous malignances was reported in clinic research,while there were evidences from animal experiments that allergic inflammation can promote tumor progression and metastasis.Cytokines are necessary in sculpting the tumor microenvironment,involving regulation of tumor growth and progression.Eos can produce and secrete variety of cytokines.To this end,the role of some cytokines associated with Eosinophils were increasingly appreciated in recent years,such as IL-5.IL-5 is produced and secreted by lymphocytes,mast cells,type II innate lymphoid(ILC2)cells and Eosinophils.IL-5 is one of the most important factor in the differentiation and survival of Eosinophils.Eosinophil expressing IL-5 receptor on surface.The lack of IL-5 directly leads to a significant decrease in Eosinophil levels,whereas IL-5 over expression in CD3+ T Cells leads to the development of a massive Eosinophilia.Many studies suggest that IL-5 involves the pathological process of the tumor,but the results remain controversial.Therefore,Eos may have a regular role in cancer initiation,progression and metastasis,which have the potential to become an important target for cancer treatment.However,current research of the role that Eos involed in cancer were mostly on the basis of epidemiological investigation,relative mouse studies are ralely reported.This experiment was divided into two parts:(1).The first project will use the Eos-related gene knockout and transgenic mice(such as Phil,NJ1638)and lung cancer-induced model mice(KrasG12D P53f/f)to study the role of Eos in lung cancer initiation and progression.(2)Eos-related gene knockout and transgenic mice(eg,Phil,NJ1638)were used to explore the mechanism of Eos in tumor lung metastasis in experimental tumor metastasis models.Part I The role of Eosinophils in KrasG12D induced lung tumor initiation and progressionObjectiveTo determine whether Eosinophils play a potential role on anti-tumor response in tumor initiation and progression and the possible underlying mechanisms.MethodsHere Phil mice(genetic deletion of Eos)and NJ mice(profound Eos expansion)were back-crossed onto a P53f/f KrasG12D background.Mice were induced of lung cancer by receiving adenoviral Cre recombinase.Mouse lungs were collected for histological e-valuation by staining with Haemotoxylin and Eosin(H&E).Tumor infiltrating leukocyte subpopulations upon depletion of Eosinophils in Phil/P53f/f fKrasG12D mice were also assessed by Flow cytometry.ResultsTumor from NJ1638/P53f/fKrasG12D mice exhibited large numbers of Eosinophils infiltrating in tumor foci compared with P53f/fKrasGl2D tumor bearded mice.However,tumor number in early time point and tumor burden in following t:ime course have no significant differences,as well as the result in Phil/P53f/fKrasG12D mice.Flow cytometry showed that CD4+/CD8+ Treg cells infiltration were the same between Phil/P53f/fKrasG12D and P53f/fKrasG12Dmice.Conclusions Our data indicated that the filtration and depletion of Eosinophils have not significantly effect in KrasG12D induced lung cancer model.The role of Eosinophils in lung tumor initiation and progression needs further study.Part ? Regulation of Eos in tumor lung metastasisObjectiveTo investigate the regulation roles and possible mechanisms of Eos in tumor lung metastasis.MethodsWe established a widely used B16-F10 mouse melanoma pulmonary metastatic model and Lewis lung adenocarcinoma(LLC)cells with NJ1638 mice and their littermates We counted the surface tumor foci numbers 12 days(B16-F10)or 21 days(LLC)after tail vein injection of tumor cells.tumor proliferation by Ki67 staining as well as apoptosis by the TUNEL assay was performed.Short-term lung colonization assay was also used to monitor tumor cell extravasation in vivo.Tumor cells were labeled with CMFDA in vitro and then injected into the tail vein of NJJ638 mice and their littermates.The lungs were then isolated at 3,6,and 24 h after the injection.Then we analyzed the vascular permeability in the process of metastasis using Evans blue.ResultsThe lungs of the NJ1638 mice showed dramatically diminished tumors compared with the wild type(WT)controls.There were significantly fewer metastatic foci on the lung surfaces of the transgenic mice than on those of the WT littermates,but the size distributions of individual metastatic foci were not significantly different between these mice.And much fewer cells were detected in NJ1638 mice in short-term time of 6h and 24h.After injection with tumor cells,the lungs of WT mice took up the Evans blue,whereas the lungs of Il-5 Tg mice remained clear.Eos depletion in WT mice did not affect metastasis,and loss of Eos in the Il-5 Tg mice produce a very mild but significant increase in the number of metastatic foci.ConclusionHigh levels of IL-5 in vivo might protect from cancer metastasis,by blocking tumor cell metastasis at a very early stage of permeating vascular barriers,as well as maintain the stability of vascular barriers,and the inhibition of metastasis in the NJ1638 mice was only partially dependent on Eos.