Neutrophils Enhance Tumor Cell Invasiveness Via FAM3C-mediated Epithelial-to-mesenchymal Transition In Gastric Cancer

Background:Lymph node metastasis(LNM)is the major metastasis route of gastric cancer(GC)and serves as the most important hallmark of tumor progression dismal prognosis.Lymphatic invasion(LI)is a precursor step of LNM.Tumor microenvironment(TME)plays an important role in carcinogenesis,tumor progression,treatment response and prognosis.Our previous studies suggest that TANs is significantly correlated with LNM in gastric cancer,but its mechanism is unknown.Neutrophils distributed in TME are called tumor-associated neutrophils(TANs),which play multiple roles in tumor development.TCM(traditional Chinese medicine)treats patients as a whole,treats them according to syndrome differentiation,and combines righting and dispelling evil.Tumor microenvironment has gradually become an important target of TCM for anti-tumor.To explore the correlation between TANs and syndrome differentiation of gastric cancer,as well as the role of TANs in lymphatic invasion and LNM in gastric cancer and its mechanism,in order to provide new ideas for the mechanism of lymph node metastasis and clinical treatment of GC.Part Ⅰ:The correlation between tumor-associated neutrophils and syndrome differentiation of early gastric cancerObjective:To explore the TCM syndrome differentiation and clinicopathological features(including TANs)of early gastric cancer.Methods:Patients with T1b early gastric cancer(tumor infiltrating submucosa)were collected from The Affiliated Hospital of Nanjing University of Chinese Medicine(Jiangsu Province Hospital of Chinese Medicine)from January 2011 to December 2020,and the preoperative TCM syndrome differentiation,clinicopathological features including gender,age,tumor location,tumor size,macroscopic type,depth of tumor infiltration,Lauren classification,histological classification,the presence of lymphatic invasion,perineural invasion and Helicobacter pylori(H.pylori)infection,and the status of lymph node metastasis(LNM)were collected.Analysis of the tumor-associated neutrophils(TANs)was determined by hematoxylin&eosin(H&E)staining or immunohistochemistry(IHC)with CD66b,and based on the median TAN number of 10 per non-overlapping high-power field(HPF)in primary tumor tissues,these patients were divided into high-and low-TANs groups.The neutrophil and lymphocyte count in the blood routine of patients at admission were collected,and the neutrophil to lymphocyte ratio(NLR)was calculated.Taking the median of NLR as the critical value,these patients were divided into high-and low-level NLR groups.To explore the correlation between TCM syndrome differentiation and clinicopathological features,TANs and NLR.Results:A total of 257 cases of T1b early gastric cancer were included in this study.The preoperative TCM classification of 257 patients were qi stagnation and blood stasis syndrome(194 cases,75.5%),liver Qi invading the stomach syndrome(9 cases,3.5%),stomach heat injuring Yin Syndrome(6 cases,2.3%),phlegm dampness coagulation syndrome(14 cases,5.4%),spleen stomach deficiency cold syndrome(8 cases,3,1%)and Qi and blood deficiency syndrome(26 cases,10.1%).And there was no correlation between gender,age,tumor primary site,tumor size,gross appearance,depth of invasion(SM1 vs SM2),Lauren classification,histological differentiation,nerve invasion,lymphatic invasion,helicobacter pylori infection and lymph node metastasis(P>0.05).There was no correlation between TANs and NLR in these six gastric cancer syndrome differentiation types(P>0.05).Conclusion:Syndrome differentiation types of early gastric cancer can not correspond to clinicopathological features effectively.The internal relationship between the two evaluation systems of Chinese and Western medicine needs to be further studied.Part Ⅱ:Clinicopathological study of tumor associated neutrophils promoting lymph node metastasis of early gastric cancerObjective:To investigate the clinicopathological significance of tumor-associated neutrophils in early gastric cancer,especially the correlation with lymphatic invasion and lymph node metastasis.Methods:The consecutive cases of early gastric cancer(infiltrating mucosa or submucosa)from January 2011 to December 2020 in The Affiliated Hospital of Nanjing University of Chinese Medicine(Jiangsu Province Hospital of Chinese Medicine)were retrospectively analyzed.The clinicopathological features including gender,age,tumor location,tumor size,macroscopic type,depth of tumor infiltration,Lauren classification,histological classification,the presence of lymphatic invasion,perineural invasion and Helicobacter pylori(H.pylori)infection,and the status of lymph node metastasis(LNM)were collected.Analysis of the tumor-associated neutrophils(TANs)was determined by hematoxylin&eosin(H&E)staining or immunohistochemistry(IHC)with CD66b,and based on the median TAN number of 10 per non-overlapping high-power field(HPF)in primary tumor tissues,these patients were divided into high-and low-TANs groups.Lymphatic invasion was determined by H&E staining,and IHC was used to differentiate lymphatic invasion and vascular invasion.The correlation between TANs,clinicopathological features and LI was analyzed.Results:A total of 413 cases of early gastric cancer from 2011 to 2020 were included in this study,including 156 cases of T1a(2011-2017)and 257 cases of T1b(2011-2020).There were 70 cases had LNM,of which the LNM in T1a patients was 5.8%(9/156)and that in T1b patients was 23.7%(61/257).Univariate analysis showed that tumor size(P=0.003),depth of invasion(P=0.000),Lauren classification(P=0.000),histological grade(P=0.001),LI(P=0.000),Nerve invasion(P=0.011)and TANs(P=0.000)were significantly correlated with LNM,among which Histological classification(P=0.026),LI(P=0.000)and TANs(P=0.013)were independent risk factors for LNM in 322 cases of EGC.According to the layered analysis of infiltration depth:univariate analysis revealed that depth of invasion(P=0.014),Lauren classification(P=0.000),histological classification(P=0.000),lymphatic invasion(P=0.000),and high TANs(P=0.000)were significantly associated with LNM,among which Lauren classification(P=0.007,LI(P=0.000)and high TANs group(P=0.002)were independent risk factors for LNM in 257 cases of T1b EGC.In T1b-SM2 tumors,the risk factors included tumor diameter(P=0.049),Lauren classification(P=0.000),histological classification(P=0.017),lymphatic invasion(P=0.000)and high TANs(P=0.000),among which Lauren type(P=0.025),LI group(P=0.000)and high TANs group(P=0.003)were independent factors of LNM in T1b-SM2 tumors.However,TANs was not an independent risk factor for LNM of T1a EGC and T1b-SM1 EGC.There is almost no LI in T1 a tumors,and LI mainly occurs in T1b tumors.Further analysis showed that TANs was an independent risk factor for lymphatic invasion in T1b,SM1,and SM2 tumors.Neutrophils could be detected in about half of lymphatic cancer emboli in T1b tumors,presenting tumor cell-neutrophil clusters in lymphatic vessels.The prevalence of neutrophils in these cancer emboli was 46.97%(31/66)in T1b tumors and 45.61%(26/57)in SM2 tumors,respectively.Importantly,the incidence of LNM in patients with lymphatic tumor cell-neutrophil clusters was increased to 77.42%(24/31)(P=0.006)in T1b tumors and 80.77%(21/26)(P=0.003)in SM2 tumors,respectively.However,the incidence of LNM in patients with neutrophil-free cancer emboli was 42.86%(15/35)in T1b tumors and 41.94%(13/31)in SM2 tumors.Conclusion:TANs is an independent risk factor for lymph node metastasis in early gastric cancer,especially in T1b-SM2 stage.TANs can promote lymph node metastasis of early gastric cancer,and its mechanism needs to be further studied.Part Ⅲ:Molecular mechanism of tumor associated neutrophils promoting invasiveness of early gastric cancerObjective:To investigate the molecular mechanism of how tumor-associated neutrophils promote lymph node metastasis.Methods:(1)The isolation and culture of peripheral blood neutrophils were treated with the following two groups:① the peripheral neutrophils from healthy donors or advanced gastric cancer patients were isolated and cultured for certain time to generate condition media(CMs).② the neutrophils isolated from healthy donors were cultured with MKN28 or MKN45 tumor cells for 6 h and then purified,and the CMs from the tumor-educated neutrophils(Edu-Neu)were collected after additional 12-hour culture.(2)Wound-healing assay and Trans well assay were used to observe effect on migration and invasion of tumor cells.(3)RNA-seq was used to detect gene expression profiles of Edu-Neu,and bioinformatics analysis was performed.(4)Western blot,immunofluorescence assay,immunohistochemistry(IHC),enzyme-linked immunosorbent assay(ELISA)and real-time fluorescence quantitative PCR(RT-PCR)were used to detect the expression of related proteins and mRNA,which includes epithelial to mesenchymal transition(EMT)related proteins E-cadherin(E-cad),ZO-1,claudin-1,vimentin(Vim),N-cadherin(N-cad)and related transcription factors(ZEB1,snail,slug,Twist and βCatenin),TANs marker CD66b,pathway related proteins(JNK pathway,Smad pathway),related proteins of RNA-seq(FAM3C,integrin),etc.(5)STRING website predicts protein-protein interaction of integrins.The combination was confirmed by co-IP and double immunohistochemistry staining.(6)Establishment of subcutaneous or abdominal allograft tumor model of gastric cancer in C57BL/6 mice:four-week-old male C57BL/6 mice were randomly divided into two groups(subcutaneous allograft group and abdominal allograft group),and each contained 12 mice.After digestion and washing,MFC cells(1.0 ×106 cells per mouse)were inoculated into the hypochondrium of mice subcutaneously or into abdominal cavity.Each group was further divided into two subgroups,neutrophil deletion group and control group,each containing 6 mice.For neutrophil depletion,mouse anti-Ly6G antibody was daily used intraperitoneally after inoculation(25 μg per day),while mice in the control group received equivalent dose of IgG2a isotype.Two weeks later,these mice were euthanized and these subcutaneous or abdominal tumors were collected.Results:(1)CMs of Edu-Neu,rather than CMs of isolated neutrophils,is required to enhance tumor cell migration and invasiveness.(2)7531 differential expressed genes(DEGs)overlapped in the three donors as showed in the Venn Diagram,including 6698 up-regulated genes and 833 down-regulated genes.Gene Ontology(GO)analysis and KEGG pathway analysis,indicated that co-culture with gastric cancer cell results in profound molecular alterations in neutrophils.Forty-seven DEGs were obtained among "cytokine activity" category in "molecular function",and the growth differentiation factor 15(GDF15),macrophage migration inhibitory factor(MIF),Bone morphogenetic protein 4(BMP4)and family with sequence similarity 3 member C(FAM3C)were the four most up-regulated genes.(3)CMs of Edu-Neu promote EMT status of MKN45 or MKN28 cells:the expression of E-cadherin(E-cad),ZO-1 and Claudin-1 is decreased,while the expression of vimentin(Vim)and N-cdaherin(N-cad)was increased.Tumor tissue studies showed,E-cad expression in tumor tissues or lymphatic cancer emboli was decreased in high-TANs group with regard to that in low-TNAs group(P<0.01),and E-cad levels in lymphatic cancer emboli were further decreased than in tumor tissues in high-TANs group(P<0.01).(4)FAM3C is identified to be involved in tumor cell EMT medicated by the educated-neutrophils:FAM3C treatment decreased E-cad expression and increased Vim expression of MKN45 and MKN28 cells in a dose-dependent manner.GDF15,MIF and BMP4 had no similar effect.Blockage of FAM3C with neutralizing antibody reversed the enhanced invasiveness or induced EMT of tumor cells by the Edu-Neu.FAM3C was detected in TANs in human gastric tumor tissues and cancer emboli with IHC,but not in neutrophils in normal stomach tissues.TGCA database analysis showed:FAM3C mRNA levels in gastric tumor tissues are much higher than that in corresponding normal stomach tissues(P<0.001).FAM3C level in tumors is related to TNM staging of gastric cancer although it does not reach remarkable significance(P=0.063).FAM3C level in tumors is negative associated with cumulative survival of gastric cancer patients(P=0.022).Mechanically,FAM3C promotes gastric cancer cell EMT via JNK-ZEB1/Snail signaling activation.(5)Gastric cancer cells educate neutrophils FAM3C expression via TGFβ1:RNA-seq showed that transforming growth factor beta receptor associated protein 1(TGFBRAP1)expression increased in the educated-neutrophils(P=0.000).TGCA database analysis showed:TGFβ1 mRNA level in tumor tissues was associated with TNM staging positively(P=0.0043)and overall survival negatively(P=0.022).TGFβ1 m RNA level correlates with FAM3C level in gastric cancer(P=0.0023).FAM3C was up-regulated in a dose-dependent manner by exogenous TGFβ1.Treatment with neutralizing anti-TGFβ1 antibody Disitertide or anti-TGFβ1R antibody LY-364947 inhibited FAM3C expression of Edu-Neu and reversed EMT markers in tumor cells.Mechanically,tumor cells increases FAM3C levels through TGFβ1-Smad2/3 pathway in neutrophils.(6)The RNA-seq assay has shown that several integrins including α2,α3,α6,αE,αv,β1,β4,β5,β6 and β8 were up-regulated in the educated-neutrophils by tumor cells,and the subunitsα6(ITGA6),β1(ITGB1)and β4(ITGB4)were the most significantly increased ones.Co-culture with MKN45 cells up-regulated mRNA expression of the three subunits,and treatment with TGFβ1 inhibitor,Disitertide,or TGFβ1R inhibitor,LY-364947,could attenuate these effects.Furthermore,exogenous TGFβ1 also increased the mRNA levels of these subunits in neutrophils.(7)Prediction analysis of protein-protein interaction indicated that the subunits α6,β1 andβ4 can interact with CD151.Edu-Neu or FAM3C treatment could increase CD 151 levels in MKN45 cells.TCGA data analysis indicated that CD 151 mRNA levels correlate positively with FAM3C levels in gastric tumor tissues(P=0.000),and CD151 mRNA level in gastric tumor tissues is negatively associated with overall survival(OS)of gastric cancer patients(P=0.023).Co-IP assays showed that ITGA6,ITGB1 and ITGB4 can bind with CD151.Subunit α6 was detected in neutrophils and CD151 was found in tumor cells using dual-color IHC assays in gastric cancer tissue and lymphatic cancer emboli.(8)Subcutaneous allograft tumor model of gastric cancer showed:after neutrophils depletion with anti-Ly6G antibody,the tumor volumes were less than in control group(P<0.01).IHC assays revealed that E-cad was up-regulated while Vim was down-regulated in anti-Ly6G-treated group with control group(P<0.01).The abdominal allograft models also showed E-cad was up-regulated while Vim was down-regulated in anti-Ly6G-treated group,and tumor cells were spindle in control IgG group,and tumors invaded into liver.Whereas,tumor cells were polygonal in anti-Ly6G group,and tumor was not found to invade into liver and the liver envelope was intact.Conclusions:Crosstalk between neutrophils and tumor cells is prerequisite for tumor invasiveness,endowing neutrophils to promote tumor EMT and LNM.This study clearly identifies the functional roles of neutrophils in tumor invasiveness,and is expected to provide a new strategy for the prevention and treatment of lymph node metastasis of gastric cancer.