Sexual Dimorphism In Liver Metastasis And Its Underlying Mechanism

Tumor metastasis is a leading cause of tumor-associated patients death,approximately accounts for 90% of deaths.Liver is the primary target organ of tumor metastasis,and its morbidity is increasing.Early liver metastasis is not easy to detect,and prognosis is poor.Thus investigating the pathogenesis of liver metastasis and the preventing target are important to protect people's health.Tumor metastasis is the main cause of death in patients with malignant tumors.Approximately 90% of cancer patients die due to the tumor metastasis.The liver is one of the most important target organs for tumor metastasis.The incidence of metastatic liver cancer(tumor with liver metastasis)keeps increasing year by year,the onset of which is hidden while the prognosis is poor.Therefore,the in-depth study of the pathogenesis of liver metastasis and the search for potential prevention and treatment targets are of great significance to protect people's health.Gender is a key factor affecting cancer development,treatment response and prognosis.For most non-gender-specific tumors,the morbidity and mortality of males are higher than female patients,but the mechanism that causes this difference is not yet clear.Amount of research uncover that tumor can be influenced by gender,but its conclusion is controversial.Meanwhile,the study about gender how to affect the tumor microenvironment is known less.The importance of gender in tumorigenesis has attracted accumulating attention in recent years,but it how to influence tumor metastasis,especially metastatic liver cancer remain far from clear.The sex difference for metastatic liver cancer and liver microenvironment need to be further confirmed.Gender is a key factor influencing tumorigenesis and development,treatment responsiveness and prognosis.For most non-gender-specific tumors,the morbidity and mortality of males are higher than female patients,but the mechanism that causes this difference is not yet clear.Many studies have revealed the influence of gender factors on tumor cells themselves,but the specific conclusion is still controversial.At the same time,the influence of gender factors on the tumor microenvironment is still rarely reported.Unlikely to the large amount of attention on the gender difference in tumor occurrence,there are relatively few studies on the gender difference of tumor metastasis,especially liver metastasis.The gender difference of metastatic liver cancer needs further research to confirm.The influence of gender factors on the "soil" of the liver metastasis,the liver microenvironment,is still unclear.Tumor liver metastasis is a complex and inefficient process.Extensive research confirms that the key of tumor successful colonization is that the interaction of circulating tumor cells and various components within liver microenvironment.Liver cells,such as parenchymal cells immune cells and hepatic stellate cells are implied.They can promote or inhabit tumor progression,which dependent on the relation of immune cells and tumor.Sex hormones can directly affect the differentiation and function of immune cells through its corresponding receptors.Thus,sex hormones may influence metastatic liver cancer by influencing immune cells within liver microenvironment.But this hypothesis and related mechanisms remain to further investigate.Tumor liver metastasis is a complex and inefficient process.Extensive researches have confirmed that the interaction of circulating tumor cells with various components of the liver microenvironment after entering the liver is the key to successful metastasis.The parenchymal cells,immune cells,and hepatic stellate cells in the liver microenvironment are all involved in the response to tumor cells,and promote or inhibit the process of tumor metastasis at different stages of liver metastasis.In this process,the interaction of immune cells and tumor cells is the key to tumor liver metastasis.Many studies have found that sex hormones can directly affect the differentiation and function of immune cells through their corresponding receptors.Therefore,sex hormones may affect tumor liver metastasis by affecting the function of immune cells in the liver microenvironment,but the hypothesis needs to be further verified,and related mechanisms need to be further studied.Neutrophils are the first line of defense against the invasion of exogenous pathogens,and play an important role in the tumor metastasis.It is involved in multiple stages of tumor metastasis,such as inhibiting the killing of circulating tumor cells by NK,promoting the penetration of blood vessels and promoting angiogenesis,etc.The removal of neutrophils can significantly inhibit tumor metastasis.Previous studies have found that the development of neutrophils is regulated by androgen/androgen receptor(AR)signaling pathways.However,the specific regulatory mechanism and the effect of this regulation on gender differences in metastatic liver cancer still need to be further studied.Neutrophils are the first defense against the invasion of foreign pathogens in body,and play an important role in the process of tumor metastasis.It is involved in multiple stages of tumor metastasis,such as inhibiting the killing of circulating tumor cells by NK cells,promoting the penetration of blood vessels and promoting angiogenesis,etc.Clearing neutrophils can significantly inhibit tumor metastasis.Previous studies have found that the development of neutrophils is regulated by androgen/androgen receptor(AR)signaling pathways.However,the specific regulatory mechanism and the effect of this regulation on gender differences in metastatic liver cancer need further study.In our study,the gender differences in metastatic liver cancer were investigated by using clinical data,experimental liver metastasis model and molecular biology methods,and the molecular mechanism of gender differences was also discussed.This study not only helps to elucidate the new mechanism of metastatic liver cancer development,but more importantly,this study can also provide a basis for gender-based treatment strategies for patients with metastatic liver cancer.In our study,the patient's clinical data,experimental liver metastasis model and molecular biology are comprehensively used to deeply study the gender difference of metastatic liver cancer,and explored the molecular mechanism that caused this gender difference.This study not only helps to elucidate the new mechanism of metastatic liver cancer development and provides potential targets for clinical prevention and treatment,but more importantly,this study can also provide a basis for gender-based treatment strategies for patients with metastatic liver cancer,thus provide a theoretical basis for basic research results to be applied.Methods:1.The statistical analysis of cancer cases with various common tumors in the SEER database was performed to compare the proportion and the overall survival of male and female cancer patients with liver metastasis for single-factor analysis.The statistical analysis of cases with various common tumors in the SEER database was performed to analyze the proportion of male and female cancer patients with liver metastasis and the impact of gender on the overall survival of patients with metastatic liver cancer.2.To establish an animal model of liver metastasis by splenic injection compared the effect of gender on tumor load.Inoculate tumor cells through the spleen to establish an experimental liver metastasis animal model,and analyze the effect of gender on experimental liver metastasis.3.Subcutaneous tumors of male mice grew more rapidly than those of female mice;But there was no difference in the pulmonary metastasis model.Establish subcutaneous tumor-bearing and lung metastasis animal models,and compare whether there are gender differences in tumor growth in other parts.4.The effect of male hormone or female hormone on liver metastasis of tumor was preliminarily discussed by castration of male mice and female mice.The effect of androgen supplementation on liver metastasis in castrated male mice was preliminarily determined.AR knockout of androgen receptor further clarified the effect of tumor liver metastasis through androgen /AR receptor signaling pathway.Preliminary study of male and female mice castrated on what kind of sex hormones affect tumor metastasis.Then supplement male hormones in castrated males to preliminarily determine the effect of male hormones on tumor liver metastasis.Male hormone receptor(AR)knockouts mice to establish a liver metastasis model to further clarify that androgens affect tumor liver metastasis through the androgen/AR receptor signaling pathway.5.q-PCR was used to detect the expression levels of androgen receptors and estrogen receptors in different tumor cells,and the effects of androgen on the proliferation of the above tumor cells were detected in vitro,so as to analyze whether androgen directly acted on tumor cells to promote their proliferation.Expression of Ki67 and subcutaneous tumor CD31 in liver sections were defined by immunohistochemical staining.Q-PCR was used to detect the expression levels of androgen receptors and estrogen receptors in different tumor cells,vitro detection is applied on the effect of androgen on the proliferation of tumor cells.Analyze whether androgen directly acts on tumor cells to promote their proliferation.Immunohistochemistry staining was used to detect the expression of Ki67 in liver slices and CD31 in subcutaneous tumors.6.Tumor cells were inoculated to the spleen with CFSE staining to observe the early retention of tumor cells in the liver;Tumor cells with stable expression of Luciferase were constructed.After spleen inoculation,early in vivo imaging was used to detect bioluminescence and count the total flux in the liver.Tumor cells were inoculated to the spleen with CFSE staining and tumor cells stably expressing luciferase were constructed.Immunofluorescence staining and live imaging were used to observe the early retention of tumor cells in the liver.7.Flow cytometry was used to detect the changes of NK cells,NKT cells,neutrophils,mononuclear cells and macrophages in the liver of male mice,castrated male mice and female mice without tumor and under tumor bearing conditions.Flow cytometry was used to detect changes in NK cells,NKT cells,neutrophils,mononuclear cells and macrophages in the liver of male,castrated male mice and female mice under tumor-free and tumor-bearing conditions.8.After the corresponding immune cells of male mice and female mice were cleared by immunodeficient mice and neutralizing antibodies,the liver metastasis model was established to explore which immune cells would be affected by androgens to promote liver metastasis of tumor.Use immunodeficiency mice and immune cell neutralizing antibodies to clear the corresponding immune cells of male and female rats,and then inoculate tumor cells through the spleen to explore which immune cells affect male tumors to promote liver metastasis.9.q-PCR was used to detect the expression of chemokines related to recruitment of neutrophils in the liver.Flow cytometry was used to detect the apoptosis of neutrophils and the content of reactive oxygen species in neutrophils in the liver of tumor-bearing mice.The peripheral blood cell count was measured by a hematology analyzer.10.The effect of androgens on the precursor cells of neutrophils was detected by flow cytometry.q-PCR was used to detect the expression of aging related molecules CXCR2 and CXCR4 in bone marrow cells of non-tumor bearing mice and tumor bearing mice.Flow cytometry was used to detect the expression of CXCR2 and CXCR4 in bone marrow by neutrophils.Results:1.The proportion of male patients with liver metastasis is significantly higher than that of female patients,and the overall survival time of male patients with cancer is lower than that of female patients.Among the patients with liver metastasis of colon cancer,lung cancer or pancreatic cancer,the overall survival time of male patients is also significantly lower than that of female patients.2.In the animal model of liver metastasis,the tumor load of male mice is significantly higher than that of female mice.By observing the liver metastasis in liver HE,the liver area occupied by liver metastasis was significantly higher than that of female mice and was not related to the tumor growth pattern.3.Subcutaneous tumors of male mice grew faster than those of female mice.However,there was no difference in the pulmonary metastasis model.4.After castration,the load of tumor metastasis in male mice decreased obviously,and the load level was close to that of female mice.After adding androgens to castrated male mice,the tumor load level returned to that of non-castrated male mice.In male androgen receptor knockout mice(Ar-/-),tumor load was reduced compared to wild-type male mice.When compared with wild-type male mice,it shows a lower tumor burden in male androgen receptor knockout mice(Ar-/-).5.The expression levels of androgen receptor and estrogen receptor in tumor cells were low by quantitative PCR;CCK8 showed no difference in the growth rate of tumor cells co-culturing with different concentrations of androgen.There was no difference in the expression of Ki67 in liver metastasis and subcutaneous tumor proliferation in mice of different genders.Tumor angiogenesis was higher in males than in females.6.Immunofluorescence detection showed that more tumor cells remained in the liver of male mice 24 hours after splenic inoculation.Twenty-four hours after spleen inoculation,tumor cells in the liver of male mice were significantly more than those of female mice.7.The different immune cells in the liver detected by flow cytometry showed that the ratio of neutrophils in male mice was significantly higher than that in female and castrated male mice under the two conditions of liver tumor-free and tumor-bearing.8.After T cells were cleared via T cell deficient mice and neutralizing antibody,gender differences still existed in the level of liver tumor bearing after spleen inoculation.However,the gender differences in metastatic HCC are eliminated after neutrophils were cleared by anti-Ly6 G neutralizing antibody.9.Androgens can significantly increase the number of neutrophils in peripheral blood and promote the expression of neutrophils chemokines in the liver,thereby promoting the accumulation of neutrophils in the liver.10.Androgens promoted the release of neutrophils by inhibiting the expression of CXCR4 in bone marrow neutrophils.Conclusion:This study demonstrated from clinical data and zoological level that males had a significant predisposition to liver metastasis and a worse prognosis.Further research revealed that the sex difference was caused by a neutrophil-dependent pathway through the androgen/androgen receptor signaling pathway.Androgens on the one hand promoted the proliferation of neutrophils in the bone marrow,on the other hand promoted neutrophil to release into the blood by inhibiting the expression of CXCR4,leading to the neutrophilic granulocyte aggregation in the liver and the increasing engraftment in the liver tumor cells and angiogenesis.Finally,the cancer spread to the liver.Targeting the androgen/androgen receptor signaling pathway or clearing neutrophils in male mice can effectively inhibit tumor liver metastasis.This study not only provides a new target for the treatment of metastatic liver cancer,but also provides a basis for the clinical strategy of treating patients according to gender.