High Throughput Screening And Identification Of Growth Factors And Chemokines Derived By Gastric Cancer Associated Fibroblast

Recently, a lot of new treament and measurment have been applied to gastric cancer, such as recognition of tumor microenvironment and rapid development of proteomeology which can improve the investigation of pathogenesis and treatment of gastric cancer. The tumor microenvironment is consisted of three parts:functional endepidermis, extracellular matrix and mesenchymal cells. The mesenchymal cells include fibroblasts,various immunocytes and vascular/lympgatic vessel endotheliocytes.Tumour cells can also synthesize and secrete new matrix contents into extracellular space. Tumor microenvironment plays a very important role in the process of the tumor genesis and development:(1)Gene instability caused by microenvironment may induce gene mutation and DNA damage; furthermore, it can also lead to disorder of DNA repair pathways.(2)Many physical and chemical factors play a significant role in the carcinogenicity of functional cells by damaging tissue structure of microenvironment, alterating mesenchymal cytokine expression spectra and remodeling extracellular matrix.(3) The oxygen, nutrients and growth stimulation signal which are need by tumor cells for growth, invasion and metastasis are considered to come from surrounding stromal cells.(4)At the appropriate microenvironment, malignant tumor cells can be induced back to the differential state. (5)A few anti-tumor components in the ECM,such as tumstatin and endostatin, have been abstracted successfully in clinical trials.Fibroblasts are widely used as model cells in many researches, which may be used as tissue engineering seed cells for clinical research in the future. Some former studies have shown that fibroblasts in the tumor microenvironment are very important and prospectful.Fibroblasts are main stromal cells of tissue microenvironment. They are also the main cells which produce ECM and cathepsin, occupying "maternal cell matrix" status. Fibroblasts are thought as "guardian" of normal tissue structure because they can inhibit periphery cellula epithelialis from being canceration. What is more, malignant tumor cells in normal fibroblasts environment can differentiate into normal cells.Fibroblasts are the "controlers" of epithelial cell proliferation and differentiation. Transformed fibroblasts can express a variety of cytokines, adhesion molecules, and then the genesis, growth, angiogenesis, invasion and metastasis of the epithelial and tumor cells will be enhanced. Accordingly, furhter study of fibroblasts in the tumor microenvironment will contribute to understanding biological behavior of the tumorigenesis mechanism and tumor invasion and metastasis to discovery new therapeutic target.For fibroblasts have tissue specificity, does any tissue specific excreted factors or signal protein work during the procedure of gastric tumor carcinogenesis? Whether there are any unknown fibroblast growth factors that have close relation with gastric cancer? There are few cytological studies involving establishment and analysis of gastric cancer stromal fibroblasts model or special analysis of gastric cancer microenvironment and the important stromal cells. To invesgate the microenvironment of gastric cancer, we are going to begin with investigating gastric cancer stromal fibroblasts. Firstly, gastric cancer-asociated fibroblast (GCAF) and their couterpart normal human gastric mucosa fibroblast (HGMF) were isolated from 10 resected gastric cancer samples and primary cultured. We observed the diffrences of morphology, bioloagical feature and impact on gastirc cancer cell growth between GCAF and HGMF. Moreover, using Human chemokine antibody array, we compared the relative expression levels of cytokines in their conditioned medium. Some high expressed and significantly different growth factors and chemokines were determined as key different mediators secreted by GCAF and HGMF. Finally, we confirmed the different expression levels of these key cytokines in serum of gastric cancer patient and health controls by enzyme-linked immunosorbent assay (ELISA). Our findings indicate that GCAF and HGMF have different expression spectrums of cytokines, some of which may contribute to their different impact on gastric cancer cell growth and involve in promotion of gastric cancer progression.Part 1 Primary culture and identification of HGMF and optimization of culture conditionObjective Primary culture and identification of HGMF, and then optimize the culture condition in order to improve the success culture rate to make it meet the needs of research.Methods1. HGMF Primary separate and culture:obtained normal gastric mucosa tissue from resected gastric cancer specimens, using outgrowth method primary culture HGMF.2. Identification of HGMF:Immunohistochemical and FCM method were used to identifica primary cultured HGMF.3. Optimize culture conditions:compared original generation rate of HGMF under various culture conditions,such as culture surface, types and PH value of culture medium.Results1. We primarily separate and culture HGMF and found out ways and specific conditions of taking clinical specimens, transportationas as well as the culture time.2. Optimized culture conditions:RPMI-1640 mixes with 20% FCS, plastic culture surface and PH=7.4 were ideal culture conditions.3. Immunohistochemical staining and FCM method indicated primarily cultured HGMF negatively expressed keratin and desmin and positively expressed vimentin.Conclusion1. Primary culturation of HGMF had special requirement;optimized conditions may improve the repeatability and successful rate of primary culture.2. Immunohistochemical staining and FCM method proved that the primarily cultured cells come from mesoderm.Part 2 Differences of morphology, biological characteristic and function between human gastric mucosa fibroblasts and gastric cancer associated fibroblastsObjectivePairs of primary cultured GCAF and HGMF from the morphology, cell proliferation, total protein content, cell cycle and cell culture supernatant effect on gastric cancer cells and note the differences between the two kinds of cells. And to provide experimental basis for further research.Methods1.Observed HE staining cells under light microscope and transmission electron microscopy observation morphological differences between HGMF and GCAF.2.CCK-8 method were used to detected their proliferative activity and draw a growth curve.3.BCA was used to assay total protein of HGMF and GCAF.4.Cell cycle of HGMF and GCAF were detected by flow cytometry.5.CCK-8 method, tanswell small chamber testing and flow cytometry were used to conditioned media of GCAF effect on proliferation, invasion capacity and cell cycle of human gastric cancer cell line BGC-823,respectively.Results1.Morphology contrast:HGMF had long shuttle-shaped, consistent-size, showing a small amount of double-nucleated cells under light microscope;otherwise.GCAF had larger cell body, different sizes and irregular cell shape.And large cells, dual-core or multi-core cell were more common.2.Contrast of cell proliferation capability:conpared with HGMF,GCAF had stronger proliferative activity.3.Protein concentration of 1×106 GCAF and HGMF was 1.0018±0.0940mg/ml and 0.7688±0.0441mg/ml,respectively.GCAF's protein concentration significantly higher than that of HGMF's,4.Proliferation index of GCAF and HGMF was 42.0200±2.7314%和35.2520±4.4368%,respectively.And SPF of the two was 19.5040±2.7944%和9.3180±2.2068%,respectively.5.GCAF-CM enhanced the proliferation and invasive ability of gatric cancer cell line BGC-823.Conclusion There were significant differences of morphology, biological features and functions between primary cultured GCAF and HGMF.Part 3 Cytokine microarray of culture supernatant between human gastric mucosa fibroblasts and gastric cancer associated fibroblasts and ELISA authenticationObjectiveTo explore expression levels of specific cytokines in culture supernatant of GCAF and HGMF,and then select some relevant different factors.Investigated their expression laws in large sample of GCAF-HGMF culture supernatant and gastric cancer-normal serum.To screening clinical diagnosis and treatment fibroblast derived targets of gastric cancerMethods1.Preparation of cell culture supernatant:1×106 well growing GCAF and HGMF of the third-generation,cultued with RPMI1640, culture supernatant were collected back after 48 hours later.2. RayBio(?) Cytokine microarray was used to detect expression of 120 factors in GCAF and HGMF supernatant,and then images and data were collected and standard values expression of every antibody were calculated.3.According to the results of cytokine microarray screening,we chose some high expressed and significantly different growth factors and chemokines. Study their expression laws in large sample of GCAF-HGMF culture supernatant and gastric cancer-normal serum by use of ELISA method.Results1.There wre a total of 67 high expression cytokines in the two groups,including 23 cytokines,8 growth factors,20 decapentaplegics and 16 chemotactic factors.Compared with the HGMF-CM,there are 22 cytokines in GCAF-CM higher expression in the value of≥1.3.2. The expression levels of HGF in GCAF and HGMF supernatant is 448.32± 100.13pg/ml and 101.78±16.35pg/ml (P=0.0000),TGF-(31 is 191.41±21.04pg/ml and 109.45±23.84pg/ml (P=0.0000),Angiogenin is 67.35±17.17pg/ml and 43.53±16.01 pg/ml (P=0.0088),IGF-1 is 16.24±3.54ng/ml and 12.30±2.62ng/ml (P=0.0111),CCL5/RANTES is 487.15±117.54pg/ml and 271.05±78.94pg/ml (P=0.0002),MCP-1/CXCL2 is 63.47±15.24pg/ml and 51.46±11.33pg/ml (P=0.0524),CCL16/HCC-4 is 16.19±5.51pg/ml and 8.66±3.43pg/ml (P=0.0018), CXCL9/MIG is 278.36±59.45pg/ml and 171.58±58.48pg/ml (P=0.0008), MEC/CCL28 is 219.51±64.61pg/ml and 114.32±22.13pg/ml (P=0.0000), GROa/CXCL1 is 16.98±6.78pg/ml and 10.91±3.58pg/ml (P=0.0221).The expression levels of HGF in gastric cancer and normal serum is 687.52±127.37pg/ml and 211.02±38.36pg/ml,TGF-β1 is 219.51±64.61pg/ml and 114.32±22.13pg/ml (P=0.0000),Angiogenin is 63.76±15.22ng/ml and 38.42±9.80ng/ml (P=0.0000), ICAM-1 is 3.44±0.65ng/ml and 2.75±0.64ng/ml (P=0.0000),IGF-1 is 59.14±14.52ng/ml and 41.56±12.72ng/ml (P=0.0000), CCL5/RANTES is 5.21±0.97ng/ml and 3.88±0.83ng/ml (P=0.0000),MCP-1/CXCL2 is 188.07±45.23pg/ml and 128.76±30.68pg/ml (P=0.0000),CCL16/HCC-4 is 35.78±10.36pg/ml and 11.48±4.01pg/ml (P=0.0000),CXCL9/MIG is 758.67±184.41pg/ml and 480.14±149.88pg/ml (P=0.0000),MEC/CCL28 is 886.46±255.51pg/ml and 432.45±171.63pg/ml (P=0.0000),GROa/CXCL1 is 142.47±8.41pg/ml and 18.54±8.47 (P=0.0000)ConclusionThe ability to secrete cytokines between GCAF and HGMF were significantly different.The expression level of HGF,Angiogenin,TGF-β1,IGF-1,CCL5/ RANTES,MCP-1/CXCL2,CCL16/HCC-4,CXCL9/MIG,MEC/CCL28 and GROα/CXCL1 of gastric cancer serum was significantly higher than that of normal serum. Differenct factors secreted by fibroblasts may serve as indicators of of diagnosis and prognosis in gastric cancer.