Microenvironmental modulation of breast cancer stem cells through biological matrix.

摘要

Breast cancer is the second leading cause of death among American women and almost 12.5% of the women are diagnosed with invasive breast cancer in the course of her lifetime. Recent advancement in breast cancer research has identified a rare, highly tumorigenic population of cells within tumors known as cancer stem cells (CSCs) or tumor-initiating cells (TICs). These cells evidenced to share very similar properties to normal adult stem cells are hypothesized to be main regulators in tumor growth, metastasis and relapse. Evidence also suggests that tumor microenvironment plays a critical role in the development and progression of cancer, constantly by modulating cell-matrix interactions. Scientists have tried to characterize and identify the TIC population but the actual extracellular cues in deciphering the fate of TICs have not been explored. The basic unanswered question is the phenotypic stability of this TIC population in tissue extracellular matrix setting. This basic understanding of the TICs with its surrounding extracellular matrix will help us design novel material to culture and maintain these cells for clinical studies.;The in vivo complexity makes it difficult to identify parameters in a diverse milieu that affect TICs behavior. In this thesis work we aim to create an in vitro culture substrate combining extracellular matrix proteins and topography to mimic the original tumor environment. Such matrix will help to maintain, propagate and expand TIC population for scientific study. The results presented in this work that the microenvironmental cues play a considerable role in tumor relapse and progression by altering the cancer stem cell behavior and thus this knowledge could be used to design novel cancer therapeutics.;In chapter one, we define the cancer stem cell hypothesis, its biological implications and the significance of our work. We discuss the various phenomena taking place in the tumor microenvironment that triggers tumor relapse and distant metastasis. This chapter also elaborates on the various processes like self-renewal, differentiation and epithelial to mesenchymal transitions that take place in the tumor, triggered by certain microenvironmental cues. We detail various strategies to isolate and test cancer stem cells from a Her2/neu-induced mouse mammary tumor model. Finally we describe our cell model and validate its use as a well characterized cancer stem cell model for our further studies.;In chapter two, we fabricated 3D fibrous scaffold utilizing electrospinning technique. Polymer scaffold with varying composition and orientation were created by this method. Wavy microfeatures were fabricated by oxygen plasma treatment on polydimethylsiloxane (PDMS) films. 2D thin films with extracellular matrix proteins using dip coating and layer by layer method was also prepared. All matrices were visualized using SEM or AFM techniques and optimized for cell culture studies. Initial adhesion and change in morphology of tumor cells to such matrices were also evaluated.;In chapter three, we investigated the response of mouse mammary breast cancer H6O5 cells' response to topographical effect of electrospun fibrous scaffolds with random and aligned fiber orientations. Breast cancer cells were cultured on these fibrous scaffolds for 3-5 days. The cells showed elongated spindle-like morphology in the aligned fibers whereas kept mostly flat stellar shape in the random fibers. Gene expression profiling of these cells post seeding, showed up-regulation of transforming growth factor beta-1 (TGFbeta-1) along with other mesenchymal biomarkers, suggesting that these cells are undergoing epithelial-mesenchymal transitions in response to the polymer scaffold. The results of this study indicate that the topographical cue may play a significant role in tumor progression.;In chapter four, we demonstrated how TIC population would respond when subjected to a unique microenvironment composed of different extracellular proteins. The inherent characteristics of these cells to undergo differentiation and self-renewal under the influence of different ECM proteins were evaluated. The TIC-enriched population isolated from Her2/neu-induced mouse mammary tumors was cultured on collagen, fibronectin and laminin coated substrates for one to two weeks. Our observations indicate that laminin substrate can maintain majority of the self-renewing and tumorigenic TIC population, whereas collagen induced a more differentiated phenotype of the cells. Also interestingly, fibronectin substrates dictated an invasive phenotype of TICs as evidenced from the EMT-related gene expression pattern. We also extended this study with human breast cancer cells and observed similar responses.