Research On The Related Effects Of Long-pentraxin3 In Milky Spots Metastasis Of Gastric Cancer

Gastric cancer(GC)is a life-threatening malignant tumor,which is the second leading cause of cancer-related mortality.The regeneration and metastasis of tumors have brought huge challenges to the prevention and treatment of gastric cancer.The tumor microenvironment is formed by the interaction between tumor cells and the host,which can regulate the crosstalk between cancer cells and other cytokines.Tumor-associated macrophages(TAMs)play an important leading role in tumor-related inflammation.As an important regulator of tumorigenesis,these cells have dual tumor and anti-tumor activities.TAMs promote the progression and metastasis of solid tumors by releasing a variety of cytokines,including chemokines,inflammatory factors and growth factors.At the same time,studies have proved that TAMs could affect the stemness of tumors.TAMs are mainly expressed in two subtypes,namely M1(classical activated macrophages),which are polarized by lipopolysaccharides(LPs)and interferon gamma(IFN-?)and M2(alternatively activated macrophages).Polarization in the presence of IL-4,IL-10 or IL-13,these two cell types usually have opposite effects on tumor progression.In malignant tumors,macrophages mainly express M2-like phenotypes,which can improve the growth and survival of tumor cells,and stimulate angiogenesis and tumor metastasis.Peritoneal metastasis is still one of the most common metastatic modes of gastric cancer,which has a significant impact on the survival rate of gastric cancer patients.Since milky spots are the main implantation sites of tumor cells in the peritoneal spread,the research on the mechanism of peritoneal metastasis mainly focuses on milky spots.Milky spots are primitive lymphoid tissues in the abdominal cavity of humans and animals,which are mainly distributed in the greater omentum,mesenteric and pelvic floor.It is a tiny anatomical structure,mainly composed of a large number of macrophages and lymphocytes,which participate in the removal of abdominal particles,bacteria and tumor cells.In addition,milky spot is a common metastatic site of ovarian cancer,colon cancer and gastric cancer.More importantly,milky spot is the first line of defense of the peritoneum.During the process of gastric cancer peritoneal metastasis,milky spot is preferentially and selectively invaded to form micrometastasis.Long-pentraxin3(PTX3)is a prototype member of the long-pentraxin subfamily,which is produced by innate immune cells and plays a vital role in inflammation regulation,innate immunity and tumor-related inflammation(including tumorigenesis,angiogenesis,metastasis and tumor immune regulation).Many studies have reported that PTX3 can play a biological role in various types of malignant tumors(breast cancer,lung cancer,skin cancer,melanoma,etc.),and can promote or inhibit tumor cell invasion and metastasis.The biological effects of PTX3 on progression,milky spot metastasis,and stemness in gastric cancer have not been revealed.Therefore,it is important to clarify the biological characteristics and mechanisms of PTX3 in gastric cancer cells.In this study,through a series of experiments in vitro and in vivo,we focused on the link between PTX3 and gastric cancer stemness and other characteristics,and further revealed the biological role of PTX3 in the environment of gastric cancer milky spot metastasis.Part ?Expression of PTX3 in gastric cancer tissues and cells and expression relevance with tumor-associated macrophagesObjective:To investigate the expression of PTX3 in gastric cancer tissues and cell lines and expression relevance with macrophages.Methods:Firstly,we analyzed the expression level of PTX3 in gastric cancer tissues and normal tissues through the TCGA database,and then used immunohistochemistry(IHC),western-blot and real-time quantitative PCR(qRT-PCR)experiments to detect the expression of PTX3.Furthermore,the differences in expression of PTX3 in gastric cancer cell lines were verified by western-blot and qRT-PCR.In addition,we performed IHC experiments in gastric cancer tissues with different expression levels of PTX3 to detect the protein expression of CD86 and CD206,so as to explore the correlation between PTX3 and the expression of M1 macrophages and M2 macrophages.Results:Analysis of the TCGA database indicated that the expression of PTX3 mRNA in gastric cancer tissues(n=375)was lower than that in adj acent tissues(n=32)(*p<0.05).IHC scores,tissue western-blot and tissue qRT-PCR results indicated that the expression of PTX3 in gastric cancer tissues was significantly lower than that in normal tissues(**p<0.01,***p<0.001).In addition,PTX3 expressions in gastric cancer cell lines BGC-823 and SGC-7901 and gastric mucosal epithelial cells GES-1 were detected by western-blotting and qRT-PCR and found that PTX3 was low expressed in BGC-823 and SGC-7901 cells while high expressed in GES-1 cells(***p<0.001).Subsequently,we analyzed the correlation between PTX3 and macrophages through IHC tests.In order to quantify and distinguish the phenotype of macrophages,the expression of CD86(M1 marker)and CD206(M2 marker)were detected.The results showed that there was significantly more infiltration of CD86+cells in highly PTX3 expressed tissues,while in the low PTX3 expression tissues,CD206+macrophages showed their significantly increased infiltration(*p<0.05,**p<0.01)Conclusions:1.The mRNA expression and protein expression of PTX3 are significantly reduced in gastric cancer tissues and gastric cancer cell lines.2.The expression of PTX3 is positively correlated with the expression of M1 macrophages CD86,and negatively correlated with the expression of M2 macrophages CD206 in gastric cancer tissues.Part ?Effects of PTX3 on epithelial-mesenchymal transition and stemness in gastric cancerObjective:To explore whether PTX3 is involved in the regulation of gastric cancer migration,invasion,cell stemness,epithelial-mesenchymal transition in vitro and vivo.Methods:The gastric cancer cell lines BGC-823 and SGC-7901 were transiently transfected with over-expressing PTX3 plasmid.The effects of up-regulated PTX3 on the migration,invasion and stemness of gastric cancer cells were investigated by transwell experiments and sphere forming assay and western blot.In addition,the PTX3 overexpression lentiviral vector was transfected into gastric cancer cell BGC-823 and then selected the stable cells.The negative control group(Scramble)and LV5-PTX3-transfected BGC-823 cells(3×106)in 100?l PBS were subcutaneously inoculated into the left flank of the animals.The tumor sizes were quantified by means of a vernier caliper and documented on alternate days.Tumor volume was calculated by following formula:tumor volume[mm]3=(length[mm])(width[mm])2×0.5.Following the injection for 25 days,the xenograft tumors were removed from the animals and investigated by immunohistochemistry.Results:Compared with the control group(CTL),the migration and invasion ability of gastric cancer cells in the experimental group(PTX3)decreased significantly(**p<0.01)from the transwell experiment.Moreover,the sphere formation ability in the PTX3-overexpressed group was weaker than that in the control group via the sphere forming assay(**p<0.01).Furthermore,western-blot experiments were carried out after transfection of gastric cancer cells PTX3 overexpression plasmids for 48 hours,which indicated that PTX3 reduced protein expressions of MMP2 and MMP9,which represent tumor cell invasiveness.In addition,in the detection of epithelial-mesenchymal transition(EMT)related indexes,overexpression PTX3 decreased the protein expression levels of Snail,N-cadherin and Vemintin while increased the E-cadherin expression.More importantly,we found that PTX3 inhibited the expression of SOX2,ALDH1,CD44,CD133 and LGR5 in gastric cancer by western-blotting.In vivo experiment,the growth rate and weight of subcutaneous tumor in PTX3-overexpression group were significantly lower than those in the control group.Meanwhile,we also found that the E-cadherin expression level increased while N-cadherin decreased in the group of up-regulated PTX3 compared with the control group through the IHC detection of tumor tissues.Conclusions:1.Up-regulation of PTX3 could reduce the ability of migration and invasion in gastric cancer cells,further suppress the epithelial-mesenchymal transition process of gastric cancer cells.2.Overexpression of PTX3 is involved in the attenuation of stemness and inhibited the ability of forming spheres in the gastric cancer cells.3.Up-regulation of PTX3 could depress the ability of subcutaneous tumorigenesis and inhibit the process of epithelial-mesenchymal transition in gastric cancer cells.Part ?Effects of PTX3 on macrophage polarization in the milky spots metastasis environment of gastric cancer cells and related mechanisms research in vitroObjective:To delve the effect of PTX3 on the polarization of macrophages under mimic milky spots metastasis environment in vitro and related mechanisms researchMethods:First,we applied recombinant human Pentraxin3(rhPTX3)to test the viability assay of rhPTX3 on the THP-1 cells.Cells were exposed to serial concentrations of rhPTX3 from 0 to 60ng/ml for 48h or 72h.The cell inhibitory activities were measured using CCK8 assays.Subsequently,we analyzed the effect of rhPTX3 on the M2 macrophages polarization induced by IL13 and IL4 in vitro.THP-1 cells were treated with 320 nM PMA for 24h,followed by cultured by the additional IL4 and IL13 without(Untreated)or with rhPTX3(40ng/ml)for another 48h.The expression of CD206 was detected by flow cytometry.To further demonstrate the effect of PTX3 on the polarization of M2 macrophages,the transcriptional changes of specific M2 marker genes were tested by qRT-PCR.The mRNA levels of M2 marker,Argl,IL10,TGF-? and CCL22 were tested.To investigate the impact of PTX3 on the polarization of macrophages in the environment of GC milky spot metastasis,we co-cultured gastric cancer cell lines with macrophages to simply simulate the environment of GC metastasis into milky spots.We divided the co-culture systems into two groups in each gastric cancer cells line,one of which we overexpressed PTX3.After 48 hours,the expression of CD86 and CD206 were detected at the protein level by flow cytometry and immunofluorescence.At the same time,the gene expressions of M1 markers IL1?,TNF-?,CXCL9,CCR7 and M2 markers Arg1,IL10,TGF-? and CCL22 were observed by qRT-PCR experiment.In addition,transfected the gastric cancer cells with PTX3 overexpression plasmid,the gene expression of IL4 and IL10 was detected by qRT-PCR after 24h later,checked the protein levels of IL4,IL10,JNK,and p-JNK by western-blotting after transfection 48h.Results:1.CCK8 assays found that there was no significant growth inhibition of recombinant PTX3 on THP-1 cells and cells survival was highest at 40ng/ml.2.Exogenous PTX3 reduced the protein expression of CD206 as well as gene expression of Arg1,IL10,TGF-? and CCL22 in M2 macrophages through flow cytometry and qRT-PCR experiments(*p<0.05,**p<0.01).3.Under the mimic milky spots metastasis environment in vitro,overexpression of PTX3 of gastric cancer cells decreased the protein level of CD206 of macrophages while increased CD86 protein level from flow cytometry and immunofluorescence experiments.Moreover,The Arg1,IL10,TGF-?and CCL22 gene expression of macrophages decreased while the IL1?,TNF-?,CXCL9,CCR7 gene expression increased in the experimental group(*p<0.05,**p<0.01).4.Overexpression of PTX3 in gastric cancer cells decreased the expression of IL4 and IL10 and the expression of p-JNK1/2.Conclusions:1.Exogenous PTX3 inhibits the polarization of M2 macrophages by inhibiting the protein level and transcription level of M2 macrophages.2.Under the condition of simulated milky spots metastasis in vitro,up-expression of PTX3 in gastric cancer cells suppresses the polarization of M2 macrophages and promotes the M1 polarization.3.Up-regulation PTX3 negatively regulates IL4 and IL10 expression in gastric cancer cells by inhibiting JNK1/2 phosphorylation,thereby inhibiting the M2 macrophages polarization.Part ?Implication of PTX3 in the milky spots metastasis of gastric cancer in vivoObjective:To investigate the effects of PTX3 on milky spots metastasis of gastric cancer in vivo.Methods:10 six-week-old strain 615 mice were randomly separated into two groups(five mice in each group),which were raised under standard laboratory conditions.LV-PTX3-transfected MFC cells(PTX3)and negative control cells(Scramble)(5×105)in 100?l PBS were inoculated into the abdominal cavity of the animals.We euthanized the mice on the 14th days respectively,then extracted the omenta of each mouse,compared and evaluated the tumor metastasis degree of the milky spots,and investigated by immunohistochemistry to observe the expression of LGR5,CD86 and CD206.Results:1.On the 14th days after intraperitoneal injection of MFCs,the number of tumor colonization of gastric cancer cells in the peritoneal milky spots of the experimental group overexpressing PTX3 were lower than those of the control group.2.In the experimental group,the expression of M2 macrophage marker CD206 in the milky spots decreased,while the expression of M1 macrophage marker CD86 increased.3.The expression level of LGR5 protein in milky spots in the group that PTX3 overexpression was significantly lower than that in the control group.Conclusions:1.Overexpression of PTX3 inhibits the colonization and metastasis of gastric cancer cells in the milky spots.2.Overexpression of PTX3 suppresses the polarization of M2 macrophages while promotes M1 macrophages polarization in milky spots.3.Up-regulation of PTX3 reduces the stemness of gastric cancer cells in the milky spot metastasis.