The Study Of 3D Co-Culture System For Mimicking Tumor Microenvironment

The manifestation of the tumor is a complex process based upon a sequence of interactions between microenvironment and the tumor cells.The tumor microenvironment plays a vital role in tumor occurrence,growth,drug resistance,and metastasis.Therefore identifying the interaction between tumor microenvironment and tumor cells exhibit the main solution for cancer therapy.The macrophage and angiogenesis have an important role in the tumor microenvironment.Macrophage cells play a vital role in providing pro-inflammatory and anti-inflammatory signaling in tumor sites.The hydrogel has widely used in three-dimensional cell culture due to its unique properties.This thesis was used in the 3D co-culture system for breast cancer cell(4T1)and human umbilical vein endothelial cells(HUVECs)and THP-1 cells.This project was used biomaterials including sodium alginate,gelatin,chitosan and hyaluronic acid,polyethylene glycol(PEG)to build hydrogels.Cancer stem cells and normal stem cells have many similar signaling pathways,so we have mimicked stem cells microenvironment to select tumor stem cells differentiation.This project was used hydrogels for simulating the microenvironment of stem cells to select and enrich cancer stem cells.We have explored the screening efficiency of immobilized cytokine,elastic modulus,and hyaluronic concentration.We have obtained the optimal system by evaluating the colony numbers and diameters.In this project we have investigated the effects of the different biomaterials characteristics on the THP-1 cell growth and select the suitable biomaterials combination for the THP-1 growth,we have detected the mechanical properties of different biomaterials combination,and have selected the suitable HA molecular weight and concentration,according to the results HA 4.8 molecular weight and 0.1% concentration were selected for THP-1 growth.According to the results as a suitable alginate stiffness 190 Pa(1%)concentration was selected and Chitosan presence effect for hydrogel stiffness was evaluated.In this study,we have considered many factors,including cell percentage,the total number,different cultivation methods and the environment in a co-culture system.Analyzing of tumor cell growth,we have created a suitable co-culture system for 4T1 with HUVECs and THP1 in the three-dimensional environment.We have mainly studied the reaction of tumor cells under the action of the microenvironment in the aspects of stem cell properties(CD44,CD24),and invasive migration ability(MMP9)and multi-drug resistance(MDR1).Co-culture THP-1 with 4T1 normal cancer cells were expressed high growth kinetics,cell proliferation,and upregulated gene expression in the contact co-culture system,and stem cells were expressed in high growth kinetics,cell proliferation,and upregulated gene expression in non-contact co-culture group.Co-culture HUVECs with 4T1 also normal cancer cells were expressed high growth kinetics,cell proliferation,and upregulated gene expression in the contact co-culture system,and stem cells were expressed in high growth kinetics,cell proliferation,and upregulated gene expression in non-contact co-culture group.In drug analysis we have found breast cancer normal 4T1 cells co-culture groups have more drug resistance than control groups,also contact co-culture group has more resistance in normal 4T1 group.Also in the cancer stem cell group has more drug resistance in co-culture groups than control group same as the normal group,but non-contact group has high drug resistance than contact co-culture group.We have found CSCs are indeed the major culprits of tumor development and responsible for therapeutic resistance and malignant progression in human patients,treatment approaches that target CSCs could potentially increase the efficacy of currently available treatment regimens and reduce the risk of tumor relapse and metastasis.