| IntroductionGastric cancer is globally the fourth most common malignancy,and it ranks the third leading cause of cancer-related death.Although the diagnosis and treatment of gastric cancer have greatly improved in recent decades,its prognosis remains very poor,with a 5-year survival rate of only 29.6%.Prognosis evaluation is critical when determining treatment strategies for gastric cancer patients.For patients who will have a poor outcome,aggressive treatments should be given to eliminate cancer cells to the utmost,thus patients' survival can be improved.For patients who potentially have a favorable prognosis,treatments should not be so aggressive as to avoid overtreatment.Presently,the prognosis of gastric cancer was evaluated with TNM staging system,which includes invasion depth(T stage),node metastasis(N stage)and remote metastasis(M stage).The TNM staging system is the most important prognostic factor for gastric cancer,and it has been well recognized and widely applied.However,even for patients with the same TNM stage,the long-term outcome may differ markedly from patient to patient.Therefore,it is necessary to explore new variables for predicting outcomes of gastric cancer patients.Traditional prognostic variables of gastric cancer,including the TNM staging system,mainly focused on the biological behavior of cancer cells.Histologically,cancer tissues are composed of parenchyma and stroma,and tumor stroma constitutes a permissive microenvironment for cancer cells.Besides cancer cells(tumor parenchyma),tumor stroma also contributes to the growth,invasion and metastasis of tumor.Thus,the characters of tumor microenvironment might be employed as useful markers for prognosis evaluation.Recently,tumor stroma percentage(TSP)and Klintrup–M?kinen grade(KM grade),two makers of tumor microenvironment,have emerged as prognostic variables for some cancer tpyes.TSP reflects stroma to parenchyma ratio in tumor,whereas KM grade is a semiquantitative evaluation for immune cells at the invasive front of the tumor,and a higher KM grade reflects a more prominent inflammatory reaction in tumor microenvironment.The prognostic value of TSP and KM grade has been validated in previous studies,and these two markers was combined to provide the Glasgow microenvironment score(GMS),a new variable of tumor microenvironment.In GMS system,high KM grade is defined as 0 score,low KM grade/low TSP as 1 score,and low KM grade/high TSP as 2 score.The prognostic value of GMS was superior to that of single TSP or KM grade.However,it remains unclear whether GMS can serve as a prognostic variable for gastric cancer.In the present study,we investigated the association between GMS and survival of gastric cancer patients,and we found the in tumor microenvironment gastric cancer may be partially classified as inflammatory and desmoplastic microenvironment.Gastric cancer with the inflammatory microenvironment showed a favorable prognosis,whereas the desmoplastic microenvironment was closely associated with rapid progression and poor outcomes.One prominent feature of the desmoplastic microenvironment is the enrichment of collagen fibers,but how collagen fibers are changed and the influences of collagen fibers on gastric cancer remain to be elucidated.We then focused on collagen fibers to establish their association with the prognosis of gastric cancer,and we carried out qualitative and quantitative analysis of collagen reorganization in gastric cancer.Furthermore,the effects of collagen fibers on the proliferation and invasion of gastric cancer cells were investigated.As collagen fibers located in extracellular space,its influences ought to be transmitted by membrane receptors on cancer cells.DDR2(discoidin domain receptor 2)is an important receptor for type I collagen,but its role in gastric cancer is still unknown.Employing a three-dimensional culture system to mimic the tumor microenvironment with abundant type I collagen,we investigated how DDR2 affects the proliferation and invasion of cancer cells to clarify the role of DDR2 in the progression of gastric cancer.Methods and results1.GMS were evaluated based on histopathology.Of all the 225 cases of gastric cancer,32 cases were 0 score,111 cases were 1 score,and 82 cases 2 score.Gastric cancer with GMS 0 score showed increased survival than that with GMS 1 score(5-year OS: 78.1% vs 54.1%;5-year DFS: 62.5% vs 45.9%).Patients with GMS 2 score had the worst prognosis,and their 5-year OS and 5-year DFS were both 24.4%.Using Cox multivariate analysis,we found GMS was an independent prognostic factor for gastric cancer.2.Immunohistochemistry showed that GMS 0 score was significantly associated with deficiency of mismatch repair gene(P = 0.042)and a higher expression of PD-L1(P = 0.030).Chromogenic in situ hybridization showed that patients with GMS 0 score were more likely to be positive for Epstein-Barr virus(P = 0.037).3.Immunohistochemistry showed that there existed more cancer-associated fibroblasts in gastric cancer with GMS 2 score(P< 0.001).Using picrosirius red staining and two-photon microscopy,we found more collagen fibers in gastric cancer with GMS 2 score(P< 0.001),and the maturity of these collagen fibers was reduced.4.A total of 155 cases of gastric cancer from The Cancer Genome Atlas project that had undergone mRNA profiling were analyzed.Gastric cancers with GMS 0 score exhibited high expression of 282 genes,and the most enriched genes were associated with inflammation pathways.Meanwhile,1589 genes were expressed at the highest abundance in gastric cancers with GMS 2 score,and the most enriched genes were associated with cell-cell interactions or cell-ECM interactions.5.Using open-source data from Internet(Kaplan-Meier Plotter),we found mRNA expression of type I collagen was closely associated with reduced overall survival.In a cohort containing 225 cases of gastric cancer,we also found that the cases enriched with collagen fibers had a significantly lower rate of 5-year OS and 5-year DFS.In vitro assays showed that when concentration of type I collagen was increased in microenvironment,gastric cancer cells exhibited an enhanced capability of invasion.When gastric cancers were implanted to nude mice with increased concentration of type I collagen,the volume of the xenografts could also be significantly increased.6.In gastric cancer,collagen reorganization is prominent,including increased collagen deposition and reduced maturity.Furthermore,the morphology of collagen fibers could be dramatically changed: they exhibit a thicker,straighter and less eosinophilic appearance,and the density was usually increased.7.The parameters for collagen fibers,not only structural parameters(length,width and straightness)for individual collagen fibers,but also organizational parameters(alignment,density)for all collagen fibers,were significantly elevated in gastric cancer.Moreover,the length,width and density of collagen fibers were all correlated with patients' prognosis,and the width showed the maximum value in prognosis evaluation.8.DDR2 was expressed in gastric cancer cells,and lower expression of DDR2 was correlated with a poorer outcome.However,DDR2 expression was not an independent prognostic factor for gastric cancer.Weak expression of DDR2 in cancer cells was associated with increased collagen deposition in stroma of gastric cancer.9.Gastric cancer cells were cultured in 3D gels formed by type I collagen.Downregulation of DDR2 expression in cancer cells led to an increased cloning efficiency,rapid proliferation and enhanced invasion,whereas overexpressing DDR2 resulted in attenuated proliferation and impaired invasion.10.When gastric cancer cells were subcutaneously implanted in nude mice,reduced DDR2 expression caused increased tumorigenesis,and the volume of xenografts was increased significantly.However,DDR2 expression in cancer cells led to decreased growth of xenografts.Conclusions1.GMS could serve as a useful prognostic indicator for gastric cancer.The tumor microenvironment in gastric cancer may be partially classified as inflammatory or desmoplastic microenvironment that manifests different pathobiological features.The inflammatory microenvironment was a manifestation of better prognosis,and it was related to elevated PD-L1 expression,increased rate of EBV infection and deficient expression of mismatch repair genes.In contrast,the desmoplastic microenvironment was linked to a poorer survival,and there existed markedly increased CAFs and collagen deposition in tumor stroma.2.Type I collagen in microenvironment promoted growth and invasion of gastric cancer.In the stroma of gastric cancer,collagen fibers were strikingly reorganized,displaying an increased content(the quantitative change)and reduced maturity(the qualitative change).Moreover,the morphology and structure of collagen fibers were also changed,and the structural features could serve as prognostic factors for gastric cancer.3.DDR2 was expressed in gastric cancer cells,and in the microenvironment with abundant type I collagen,DDR2 expression resulted in attenuated proliferation and invasion of cancer cells.In gastric cancer,collagen content was negatively correlated with the DDR2 expression of cancer cells. |
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