The Study of 2d and 3d Hypoxic Microenviroment on the Label-Free Selection and Regulation of Pancreatic Cancer Stem Cells for Tumor Model Establishment and the Application of Therapeutic Target

Cancer stem cells (CSCs) represent a subpopulation of tumor cells that exhibit capacities for self-renewal, tumor initiation, metastasis, and resistance to chemotherapy and radiotherapy. However, the major obstacle associated with the use of CSCs is the difficulty in their isolation and enrichment. Hyaluronic acid (HA) is found at high levels in the stem cell niche and malignant tumor. Collagen-rich fibrotic extracellular matrix in pancreatic adenocarcinoma has been identified to contribute to cancer progression and chemoresistance. Therefore, this proposal will establish a novel and label-free 3D model system to select and enrich pancreatic CSCs by using niche mimetic collagen/HA multilayer film system with hypoxic niches. It is considered that this platform may approach pathologically relevant culture conditions and advance our understanding of tumor formation, invasion and identify the routes toward more effective therapy. During the first year, series of biomimetic substrate such as collagen/ HA will be fabricated and the surface properties include the surface morphology, surface charge, stiffness, roughness, hydration, and thickness will be characterized. We will combine sequential measurements by Quartz Crystal Microbalance with Dissipation (QCM-D), atomic force microscopy (AFM), and zeta potential to characterize the surface properties variation. In addition, colony number and size distribution of CSCs on these substrates will be counted and analyzed. Furthermore, the cell viability, proliferation rate, and cytotoxicity of colonies selected from different substrates and normal population cancer cells will be analysis. Besides, the constitutive expression of cancer stem cell markers including the CD133, CD44, EpCAM etc, will be determined by flow cytometry. During the second year, the further bio-effects of pancreatic cancer cells on 2D versus 3D colony selection system under ambient and hypoxic niche will be compared. The HA niches and collagen microenviroment related genes, drug resistance related gene, and stem cell related gene will be analyzed with reverse transcription-polymerase chain reaction (RT-PCR). In addition, the colonies isolated from series substrates will be measured with drug sensitivity. During the third year, invasion ability, telomere repeat length and self-renewal ability of the cells isolated from 2D and 3D hypoxic microenviroment will be characterized to realize the hypoxia effect on tumor dimensionality and CSCs responses. Eventually, in order to make sure the colony selected from 3D hypoxic microenviroment will display the CSC properties and tumor formation ability, the in vivo tumorigenic assays will also be proceeded to investigate the ability of tumor formation. We believe the 3D colony selection and culture system may provide new insights regarding the role of tumor dimensionality in guiding hypoxia-related tumor and CSCs responses. This system may help us to investigate CSC behavior and this technique may use on the application of drug testing and cancer therapeutic target.

Project ID:PB10507-2927
External Project ID:MOST105-2221-E182-017