The Mechanism Of CRIP1 Regulating Lymphatic Permeability In Gastric Cancer Through Polarization Of Tumor Associated Macrophage Mediated By CCL5

Objective: The high incidence and death rate of gastric cancer(GC)is a serious threat to the lives and health of people around the world.According to the latest cancer data of 185 countries worldwide,there are about 1 million new cases of GC each year,and nearly770,000 people die of GC each year.In China,one new case of GC is diagnosed every minute averagely,and about two people die from GC every three minutes,which brings a serious economic and social burden to China.The metastasis and spread of the primary site of GC is the main culprit of patients’ failed treatment outcome,and lymph node metastasis is the most common form of GC metastasis and spread.We found that the CRIP1 gene was closely related to GC lymph node metastasis through GC tissue microarray.As part of Lin11,Isl-1,Mec-3(LIM structure),namely,bis-zinc finger protein family,CRIP1(cysteine-rich intestinal protein 1)was first identified in the small intestine.The research of CRIP1 in GC is still in its infancy,and it was first mentioned in GC in a study by Benjamin,which introduced CRIP1 as a novel and independent factor in determining the prognosis of GC patients.Currently,CRIP1 is demonstrated to promote lymph node metastasis in many different tumors such as thyroid cancer and colorectal cancer.Lymph node metastasis refers to the spread of tumor cells of epithelial origin into the lymphatic vessels,where it follows the lymphatic circulation into the draining lymph nodes and colonizes them.Recently,the dynamics of lymphatic vessels in lymph node metastasis has attracted the attention of researchers.Both lymphangiogenesis and lymphatic permeability changes in tumors can affect lymph node metastasis,but most studies in this field have focused on lymphangiogenesis,while the mechanism of lymphatic permeability in lymph node metastasis is still unclear.The tumor microenvironment(TME)plays an important role in the process of lymph node metastasis,and its interaction with tumor cells is involved in the whole process of tumor metastasis.As an important component of the tumor microenvironment,tumorassociated macrophages(TAM)can influence tumor growth,angiogenesis,immune regulation and metastasis.In recent years,researchers have gradually discovered that M2-type TAM can promote tumor metastasis to lymph nodes in several ways.However,little has been reported about the interaction between M2 TAM and lymphatic permeability.Therefore,it deserves further investigation whether they can influence lymphatic permeability in the TME.Methods: We firstly screened CRIP1,a gene that may be related to lymph node metastasis,and compared its expression in different tumor tissues in the TCGA database.Then Realtime PCR assay and immunohistochemistry assay were applied to detect the expression level of CRIP1 in GC at the level of m RNA and protein in the tissue samples in our biological sample bank,further analyze the relationship between CRIP1 expression level and lymph node metastasis or survival based on the clinicopathological data.Later,a popliteal lymph node metastasis model was constructed using nude mice to determine the role of CRIP1 in lymph node metastasis.Next,we constructed an in vivo lymphatic permeability model with the help of Evans blue to detect the effect of CRIP1 on lymphatic permeability.Then we applied Luminex assay and ELISA assay to explore the cytokines that may affect lymphatic permeability,and employed neutralizing antibodies in the lymphatic permeability model to verify it.Furthermore,we applied mass spectrometry,GST pull down assay,PLA assay,Co-IP assay,Ch IP assay,dual-luciferase reporter gene assay and bioinformatics analysis to unfold the specific mechanism of CRIP1 regulation of this cytokine.Finally,we constructed a subcutaneous transplantation model in nude mice and applied immunofluorescence assay to investigate the effect of CRIP1 on TAM polarization in the TME.Later,we employed GC cells and THP-1 cells to construct a co-culture system in vitro to verify the effect of CRIP1 on TAM polarization,and applied ELISA assay to detect the changes of TAM secretion after polarization in the supernatant of the culture medium in co-culture system.The effect of TAM on lymphatic permeability was further verified by applying in vivo lymphatic permeability model and immunofluorescence staining.Results: Analysis of the TCGA database using the GEPIA website showed that CRIP1 was significantly highly expressed in a variety of malignancies(p<0.05)and that high CRIP1 expression was strongly associated with poor prognosis in ovarian cancer(p=0.033),lung cancer(p=0.042)and glioblastoma multiforme(p=0.034).Real-time PCR was applied to verify the results in GC tissues,which showed that CRIP1 was highly expressed in GC and correlated with lymph node metastasis(p=0.034).The immunohistochemical results of gastric cancer tissue microarrays also showed high expression of CRIP1 in GC tissues(p<0.001).The analysis of clinicopathological data showed that the expression of CRIP1 in GC was significantly correlated with Borrmann staging(p=0.003),T stage(p<0.001),N stage(p<0.001),TNM stage(p<0.001)and lymph node metastasis(p<0.001),and the results of survival analysis showed that patients with high CRIP1 expression had a significantly worse prognosis than those in the low CRIP1 expression group(p=0.021).The lymph node metastasis assay in nude mice showed that overexpression of CRIP1 promoted lymph node metastasis,increasing the percentage of metastatic lymph nodes from 40% to 80%(p=0.025),while the knockdown of CRIP1 inhibited lymph node metastasis,reducing the percentage of metastatic lymph nodes from 40% to 10%(p=0.031).In vivo lymphatic permeability model showed that overexpression of CRIP1 significantly increased the amount of Evans Blue that metastasized to axillary lymph nodes(p<0.001).Subsequent application of Luminex assay and ELISA assay to detect key factors regulating lymphatic permeability showed a significant increase in CCL5 in cell culture supernatant after stable overexpression of CRIP1(p=0.004),while the knockdown of CRIP1 decreased the expression level of CCL5(p<0.001).The further validation demonstrated that neutralizing antibodies of CCL5 could significantly reversed the elevation in Evans Blue caused by overexpression of CRIP1(p < 0.001).Mass spectrometry,GST pull down,PLA assay and Western blot assays showed that CRIP1 could bind to PKAα to promote CREB1 phosphorylation and thus enhance its transcriptional activity.ELISA results showed that overexpression of CREB1 increased CCL5 expression in cell culture supernatants(p<0.001),while the knockdown of CREB1 decreased its expression(p=0.001),and Real-time PCR similarly suggested that CREB1 could regulate the transcription of CCL5.Furthermore,Ch IP and dual-luciferase reporter gene assays confirmed that CREB1 could bind to the promoter region of CCL5 to regulate its transcription.Immunofluorescence experiments on sections of tumor-forming tissues demonstrated that overexpression of CRIP1 significantly increased the infiltration of M2 TAM(p=0.019),and the knockdown of CRIP1 decreased their proportion(p=0.003).The results of the coculture system showed that the supernatant of cell cultures overexpressing CREB1 induced THP-1 polarization toward M2(p=0.020),while neutralizing antibodies of CCL5 reversed this process(p=0.022).Meanwhile,the supernatant of cell cultures overexpressing CREB1 promoted TNF-α secretion by TAM(p=0.002),which was similarly reversed by neutralizing antibodies of CCL5(p<0.001).Subsequently,the lymphatic permeability model was applied again to confirm that TNF-α could affect lymphatic permeability(p<0.001).Finally,the application of immunofluorescence assays revealed that co-culture supernatants from the CREB1 overexpression group reduced VE-cadherin expression between lymphatic endothelial cells,while the addition of neutralizing antibodies of CCL5 or TNF-α restored VE-cadherin expression between lymphatic endothelial cells.Conclusion: CRIP1 is highly expressed in GC tissues and is closely associated with Borrmann staging,T stage,N stage,TNM stage,lymph node metastasis and poor prognosis of GC patients.CRIP1 can increase the permeability of lymphatic vessels and promote lymph node metastasis.The specific mechanism is that CRIP1 can bind to PKAα to promote the phosphorylation of CREB1 and enhance its transcriptional activity,thus promoting the expression of CCL5.CCL5 promotes M2-type polarization and secretion of TNF-α by TAM,and decreases the expression of VE-cadherin in lymphatic endothelial cells,thus increasing the permeability of lymphatic vessels and promoting lymph node metastasis.