Study On The Role Of Esophageal Stromal Fibroblast And Its Mechanism In Esophageal Carcinogenesis

Squamous cell carcinoma of the esophagus is one of the most commonmalignant tumors in China and the cancer patients have higher mortality ratesand5-year survival rate is lower. Invasion and metastasis is an importantfactor affecting mortality and prognosis in patients.Precise mechanism ofesophageal cancer remains unclear. Previous studies on esophageal cancermainly paid much attention to malignant transformation of squamousepithelial cells, but paid little attention to changes in the stromamicroenvironment surrounding epithelial cells. Tumor microenvironmentcontained matrix protein, immune cells, activated fibroblasts and adjacentunique vasculature. Activated cancer-associated fibroblasts are the maineffecting cells. Compared with normal fibroblasts, activated cancer-associatedfibroblasts has undergone significant changes on morphological structure,growth pattern,athletic ability and proliferation activity. They can also secretemore cytokines to promote tumor cell proliferation. Paracrine signalings andextracellular matrix components could mediate epithelial-mesenchymalinteractions.Objective：The process of esophageal carcinoma is moderately advanced. Atypicalhyperplasia squamous epithelia is intermediate stage between normalsquamous epithelia and carcinoma epithelia. Atypical hyperplasia squamousepithelia is different from normal squamous epithelia and carcinoma epitheliain structure and function. We don’t know if there is atypical hyperplasiafibroblasts in stroma of precancerous lesion which is intermediate stagebetween normal fibroblasts and cancer-associated fibroblast. Compared withnormal fibroblasts and cancer-associated fibroblasts, what features is itsstructure and function? What is atypical hyperplasia fibroblasts’role and its mechanism in esophageal carcinogenesis? Studies on the different stagefibroblasts may be helpful for early diagnosis and treatment for esophagealprecancerous lesion and provide targets of drug action.Method:In this study,we study phenotypic transformation of fibroblast andchanges of expression of proteins(including α-SMA、TGFβ1、HGF、MMP2、MMP9and VEGF)secreted by interstitial fibroblasts and changes ofexpression of proteins(including c-met and smad4) in the epithelia in theesophageal carcinogenesis process. Detection of α-SMA, TGF-β1andSMAD4, HGF, HGF, VEGF and MMP2and MMP9expression changes inesophageal normal group and low grade intraepithelial neoplasia group, highgrade intraepithelial neoplasia group, early in the cancer and advanced cancergroups via immunohistochemical staining. Counting microvessel density ineach group and statistically analyze the correlations between indicators andlesions. Analyze correlations between α-SMA and TGFβ1， correlationsbetween α-SMA and HGF. Analyze correlations between TGFβ1and smad4，correlations between HGF and c-met;In vitro, interstitial fibroblasts successfully were isolated and culturedfrom different stages of lesion of esophagus, including NFs, AFs and CAFs.Observation of morphological characteristics of three types of stromalfibroblasts and detection of immunological and biological characteristics andexpression of related genes in them were carried out. To analyze and comparethe results of interaction of three kinds of stromal fibroblasts and cancer cells.Comprehensively analyze esophageal stromal fibroblasts function in thesetting and progression of esophageal squamous cell carcinoma.Threeesophageal interstitial fibroblasts were cultured by explants adherent methodand enzymatic digestion method in vitro. To observe cell morphology andinternal structure using inverted microscope, transmission electronmicroscope.Using immuno-cell staing method to detect the expression ofα-SMA, CK and Vimentin. Using tetrazolium blue colorimetric method drawsa7days growth curve of NFs, AFs and CAFs. Detection of NFs, AFs and CAFs cell cycle using flow cytometry. detecting intracellular level of α-SMA、TGFβ1、HGF、MMP2、MMP9and VEGF-A mRNA using RT-PCR method.Using transwell technique to detect gene expression changes of each cell aftercancer cells indirectly contacted with three types of stromalfibroblasts.Detection of cancer cells impact on invasiveness of stromalfibroblasts. Cancer cell lines were mixed with three kinds of esophagealstromal fibroblasts respectively when injected into nude mice subcutaneouslyand observe the growth of the tumors. Immunohistochemical detection ofproliferation of cancer cells and tumor microvessel formation was carried out.Result:The first part: In esophageal carcinogenesis, esophageal stromalfibroblasts changed from α-SMA-phenotype to α-SMA+phenotype.Expression of TGFβ1、HGF、c-met、MMP2、MMP9and VEGF proteinincreased. Inversely, smad4protein decreased. Alpha-SMA correlated withTGFβ1and HGF positively. MVD increased with progression of esophagealcarcinogenesis. MVD correlated with TGF-beta1, HGF and VEGF expressionpositively.The second part: NFs, AFs and CAFs could be cultured in vitrosuccessfully with purity approaching to95%. In light microscope three kindsof stromal fibroblasts show to be long flat spindle. No differences in size. TheSynapse was abundant. There was synaptic inhibition and contact inhibition inNFs, otherwise in AFs and CAFs. Under electron microscopy, three kinds ofesophageal fibroblasts were rich in rough endoplasmic reticulum, Ribosome,Golgi. Microwires were visible in esophageal normal fibroblasts, abundant inAFs and CAFs. Local micro-filaments formed dense patches. Negativeexpression of CK and positive expression of vimentin could be observed inthem. Alpha-SMA was negative expression in NFs, but positive in AFs andCAFs. NFs, AFs and CAFs growth curves were s-shaped. Logarithmic growthstage was from3rd to5th day. CAFs were activated fibroblast. Its proliferationcapacity is significantly stronger than NFs and AFs. The ability of AFsproliferation is between NFs and CAFs. Percentage of s-phase cells in NFs, AFs and CAFs group is23.7%,32.8%,33.4%respectively.The third part: From NFs→AFs→CAFs, there were no difference in theexpression of vimentin mRNA, whereas the the expression of α-SMA mRNAincreased in three kinds of fibroblasts. The expression of VEGF-A, TGFβ1,HGF, MMP2and MMP9mRNA was higher in CAFs than that in NFs and AFs.VEGF-A、MMP2、MMP9didn’t expressed in NFs and AFs, but expressedprominently in CAFs.Weak expression of TGFβ1and HGF was observed inAFs. Overexpression of TGFβ1and HGF was in CAFs. After esophagealcarcinoma cells ECa109indirectly was co-cultured with three kinds ofesophageal stromal fibroblasts, TGFβ1and HGF expression enchanced in NFs.VEGF-A,TGFβ1,HGF, MMP2and MMP9expression enchanced in AFs andCAFs. NFs didn’t obviously promote proliferation of ECa109line. AFs andCAFs will significantly promote the proliferation of ECa109line. PCNAexpression in esophageal cancer cell line ECa109increased significantly.Invasive experiments found that only a small amount of esophageal cancercells in the control group penetrate through Matrigel with cell number for(18.0±5.4), NFs group with cell number for (35.0±4), AFs group with cellnumber for (125.2±7.1), CAFs group with cell number for (135.5±8). Animalmodels showed that esophageal stromal fibroblasts and cancer cell strain ofmixed injection can shortened the time for tumor formation, accelerate thegrowth of tumors. it is not confirmed that they can stimulate the proliferationof tumor cells, but can increase tumor microvascular visibly.Conclusions:1In the course of esophageal carcinogenesis, immunophenotype ofesophageal stromal fibroblasts transformed and interstitial fibroblasts wereprogressively activated.there are intermediate stages.With their activation,esophageal stromal fibroblasts’ structures and biological characteristicsgradually changed, secretory function enhanced.2Three esophageal interstitial fibroblasts could be successfully culturedin vitro.3Cancer cells can indirectly stimulate esophageal stromal fibroblasts through paracrine cytokine and change their function. Esophageal stromalfibroblasts can indirectly stimulate the proliferation and invasiveness of cancercells through cytokine secretion.4After Esophageal normal stromal fibroblasts direct contact with cancercells,its phenotype transformed to the phenotype of cancer-associatedcarcinomas。 Activated esophageal stromal fibroblasts cancer cells’ directcontact with cancer cells may accelerate the building and growth of tumors.