The Mechanism Of Shuangshen Granules To Regulate Lung Pre-metastasis Niche By Myeloid Derived Suppressor Cells

Research background:Pre-metastatic niche is the novel target of tumor-prevention,and it was described as a important direction that could lead to a great change of tumor treatment mode.The concept of pre-metastatic niche was raised by Kaplan in Nature magazine.And a more clear definition was put forward in 2016 in Cancer cell:"a supportive and receptive tissue microenvironment to form the metastatic designated sites,or fertile soil in preparation for metastatic tumor cell seed colonizatino,thus suporting tumor settlement in the distant organ and promoting tumor metastasis.The intervene of pre-metastatic niche becomes a important therapeutic measure to prevent/reduce distant organ metastasis in patients who can not be resected the primary tumor timely or completely.It has been a hot research topic to prevent tumor cell metastasis through intervening pre-metastatic niche,and this treatment mode was hailed as a novel mode to reform tumor therapy.Myeloid derived suppressor cells(MDSCs)with slprl-stat3 signaling activation play a important support role in pre-metastatic formation and lung metastasis.It is a complicated process from tumor cell extravasation,infiltrate into vessles and arrived at target organ,any step failure will stop the metastatic process of tumor cells.Research showed that accumulation of MDSCs in lung pre-metastatic niche is a key process in fomation of pre-metastatic niche.The crosstalk between MDSCs and tumor cells is a key rate-limiting step of circulative tumor cells seed in pre-metastatic niche.The activation of slprl-stat3 signaling in MDSCs caused by its activation in tumor cells is a guarantee of persistent recruit and survive of MDSCs.It’s a reliable means to repress cancer metastasis through disrupt lung pre-metastatic niche by depressing slprl-stat3 signaling in MDSCs.Choosing effective Chinese medicinal formulae of anti tumor metastasis,exploiting the advantages of intervening multi target,disrupting the formation of pre-metastatic niche would be a novel research direction of traditional Chinese medicine treatment of tumor.The tumor-treatment model of traditional Chinese medicine is to help the human healthy as a whole,so as to inhibit tumor metastasis.Supplemeting Qi and activating blood circulation is a effective method to anti-tumor metastasis based on clinical practice.Shuangshen granules(SSG)(American ginseng,Cordyceps sinensis and pseudo-ginseng)formulated by professor Hua baoj in is based on the above method and qi upbearing-downbearing theory.Preliminary experiment had proved that SSG could reduce the rate of MDSCs in lung.The main content is whether SSG could bate the formation of lung pre-metastatic niche through inhibit slprl-stat3 signal pathway in MDSCs.Revealing the mechanism of anti-tumor metastasis effect of SSG based on pre-metastatic niche model had important significance in research on improving the efficiency of anti-tumor metastasis through compatibilities.And this study will provide experiment and scientific basis to theory of "preventive treatment of disease" and "caring the healthy part firstly" of TCM,also it has realistic significance in transform the tumor treatment model in TCM.Aim of the study:Theoretical study:The efficacy of Supplemeting Qi and activating blood circulation prescription based on qi upbearing-downbearing theory on anti-tumor metastasis,and it value in theory of "caring the healthy part firstly".Experimental study:Analysing SSG based on the theory of "qi upbearing-downbearing" and the method of "Supplemeting Qi and activating blood circulation",investigating the efficacy of SSG in anti-tumor metastasis through intervene pre-metastatic niche,proceeding the following experiments:(1)in vivo experiment:①investigate the efficacy of SSG on private tumor and lung metastasis through intervene pre-metastatic niche formation.②experiment on effect of SSG on specific tumor derived secreted factors(TDSFs)of lung pre-metastatic niche.③experiment on effect of SSG on recruitment suppression of MDSCs into lung pre-metastatic niche.④experiment on effect of SSG on biomarker(Fibronectin,Lox,Versican,MMP9)of pre-metastatic niche.(2)in vitro experiment:①experiment on effect of SSG on tumor secretion of TDSFs.②after making the activation of slprl-stat3 signal pathway on MDSCs differentiation clear and definite,Efficacy of SSG on this process could be investigated.③building co-culture model simulating the pre-metastatic niche,effect of SSG on biomarker of pre-metastatic niche will be tested.In general,the study will discuss effect of SSG on lung "healthy part" and anti-tumor metastasis under the theory of "qi upbearing-downbearing" and the method of "Supplemeting Qi and activating blood circulation".Methods:(1)construct tumor model of Lewis lung cancer in C57BL/6 mice,small animal in vivo imaging technology and periodic sampling(lung and tumor)methods were used to evaluate the effect of SSG on primary tumor and lung metastasis.(2)three methods including fluorescence microscope,immunohistochemical and fluorescent quantitation by microplate Reader were used to building animal model of pre-metastatic nice.(3)Enzyme-linked immunosorbent assay(ELIS A)was used to test lung specific TDSFs(VEGFA、TGF-β、GM-CSF and TNF-α)in different time(14d and 28d).(4)Flow cytometry was used to quantitative analyse the rate and phenotype of MDSCs in lung pre-metastatic niche model.(5)Western blot and Immunohistochemical staining were used to evaluate the specific biomarker of lung pre-metastatic niche.(6)Co culture technique was used to build the in vitro model of pre-metastatic niche,TDSFs and biomarker of pre-metastatic niche under the action of SSG was analysed.(7)In the co culture model,effect of slprl-stat3 signal pathway activation of Lewis cance cell on differentiation from myeloid cell to MDSCs was evaluted,intervention effect of SSG on this process was also analysed.Results:(1)The anti-tumor effect of SSG:results of small animal in vivo imaging technology shows that both SSG and 5-Fu have a effect on inhibition of tumor growth.SSG have a notable anti-tumor effect and inhibit the primary tumor growth statistically significant p/sec/cm2/sr in 14d(P<0.05),however,in 28d after injection of Lewis cells,the weight of primary tumor in SSG group was similar to the controls(P>0.05);5-Fu still have a tumor-growth inhibition effect on 28d compare to control group(P<0.05).(2)Tumor inhibition rate:mice were regularly sacrificed on the 14d、28d after having an inoculation of Lewis cells,tumor were peeled to calculate the tumor inhibition rate.On the 14d and 28d,tumor inhibition rates of SSG and 5-Fu group were 38.2%、4.2%;41.8%、20.1%,respectively。(3)Lung metastasis:mice were sacrificed on 14d、17d、20d、23d、26d、29d、32d、35d after Lewis cells inoculation.4 methods were used to evaluate lung metastasis,including:①Lungs were resected and covered with Bulin solutino for perusal and take count of metastasis.②frozen sections observed by fluorescence microscope;③ lungs were grinding and Microplate Reader was used for its Fluorescent quantitative analysis.④paraffin embedding and HE staining were also used for observe the lung metastasis.Before 26d,fewer lung metastasis were observed in SSG and 5-Fu group compare to controls(P<0.05).After 29d,no significance lung metastasis were found between SSG group and controls(P>0.05).(4)rate of MDSCs in lung:compare to controls,SSG could significantly reduced the rate of lung MDSCs(33.9%vs.9.9%,P<0.05)。(5)tumor derived secreted factors in blood:ELISA was used to quantitative analyse VEGF、TGF-β、GM-CSF and TNF-αlevels in mice blood after inoculation of tumor cells on 14d and 28d.Compared to control group,SSG could normalize the GM-CSF and TNF-a(P<0.05),and have no effect on VEGF and TGF-β on 14d;while on 28d,levels of TGF-β and GM-CSF of SSG group were significantly lowed than controls(P<0.05),the levels of VEGF and TNF-α were similar between SSG group and control group.(6)specific biomarker of lung pre-metastatic niche:Versican、Fibronectin、Lox、MMP9 were considered to be the specific biomarker of lung pre-metastatic niche.Their expression levels were evaluated by qualitative and quantitative methods:immunohistochemical and Western blot.Similar results were found in two methods,that is SSG can decrease the four biomarker levels significantly compare to control(P<0.05).5-Fu was no better than the SSG group in decreasing biomarker levels(P>0.05).(7)Effect of SSG on TDSFs in vitro pre-metastatic niche model:in co-culture model,TDSFs in liquid supernatant were analyzed in 12h and 24h.In 12h after co-culture,all TDSFs levels had similar change as the controls(P>0.05).In 24h,results showed that SSG can significantly decrease levels of TGF-β and GM-CSF(P<0.05),no significant change was seen in VEGF and TNF-a.What is more,5-Fu had no significantly effect on the expression of all TDSFs in this study.(8)Efficacy of SSG on slprl-stat3 signaling pathway.Real time quantitative fluorescence polymerase chain reaction(PCR)was used to detect the expression level of slprl and stat3 gene in different tissue and cells(lung,bone marrow and tumor),western blot was used to verify the PCR results.We found that SSG could significantly inhibited both gene and protein levels of slprl and stat3 in tumor,bone marrow and lung tissues(P<0.05).5-Fu could only decrease the gene and protein of s1pr1 and stat3 in tumor(P<0.05),have no significance impact on the levels of two genes and proteins in bone marrow and lung tissue(P>0.05).(9)Effect of SSG on process of myeloid cells differentiate into MDSCs:firstly,we make slprl-stat3 highly expressed Lewis cells by sew2871(a agnoist of slprl).Then we co-culture these active cells with normal myeloid cells to observe the differentiation of myeloid cells by flow cytometry.We found that in this model,the differentiation rate of MDSCs is significantly high than controls(43%vs.5%,P<0.05).Secondly,we evaluated the effect of SSG on MDSCs differentiation.Wild type Lewis cells have no effect on the differentiation of MDSCs,the inhibit effect of SSG is not significantly either.However,a significantly supression of slprl-stat3 signaling in Lewis cells was found in SSG alcohol extract group,a supression in differentiation from myeloid cells to MDSCs seems secondary to this phenomenon.5-Fu have similar effect as SSG,both of them reached the statistical difference(P<0.05).(9)Effect of SSG on specific lung pre-metastatic niche biomarker in vitro pre-metastatic niche model:western blot was used to quantify the protein levels of biomarker in mixture of lung cells and MDSCs.Compare to controls with pure lung cells or MDSCs,SSG has a significantly inhibition on expression levels of Version、Fibronectin、Lox and MMP9 in pre-metastatic niche model(P<0.05).Conclusions:(1)Shuangshen granules,a Supplemeting Qi and activating blood circulation prescription under qi upbearing-downbearing theory could not only inhibit primary tumor,but also prevent tumor cells colonized and metastasize to distant target organ through reconfigure the pre-metastatic niche.(2)Shuangshen granules could reduce the rate of MDSCs in lung pre-metastatic niche and inhibit of some TDSFs secreted by tumor cells.(3)there is a close relationship between slprl-stat3 signaling activation in Lewis lung cancer cells and differentiation from myeloid cells to MDSCs,also the slprl-stat3 signaling activation in lung.Shuanshen granules could inhibit the differentiation from myeloid cells to MDSCs and lead to a decrease rate of MDSCs in lung pre-metastatic niche because of its inhibition effect on activation of slprl-stat3 signaling in tumor cells.Shuanshen granules could reduce the expression level of biomarker of pre-metastatic niche,such as Version、Fibronectin、Lox and MMP9,reconstructs the lung pre-metastatic niche,reduce lung metastasis.