Engineering the tumour microenvironment in vitro using self-assembling peptide hydrogels

Abstract

Solid tumours comprise of a variety of cell types and components, collectively known as the tumour microenvironment (TME). The TME includes: vasculature, immune cells, extracellular matrix (ECM), hypoxia and lymphatic vessels. The TME is dynamic and complex, but essential for tumours to grow, metastasise and withstand anti-cancer drugs. To create effective anti-cancer drugs that target the TME, representative models are needed. Traditional two-dimensional (2D) in vitro models lack translatability to in vivo, due to the absence of a three-dimensional (3D) environment and physical components such as the ECM. The ECM is a 3D network of proteins and macromolecules and provides structural and biochemical support for cells. The development and use of 3D in vitro models can augment and expand the quality of data collected in in vitro studies, by culturing cells within a 3D environment that mimic the physical and chemical barriers observed within the TME. Hydrogels, 3D networks of hydrophilic chains that can retain large quantities of water, are popular biomaterials used for disease modelling purposes, due to their biocompatibility and ability to mimic the 3D environment. However, naturally derived hydrogels are limited by their batch-to-batch variability, rendering them inappropriate for pharmaceutical use. A class of synthetic hydrogels, self-assembling peptide hydrogels (SAPHs), are gaining popularity in tissue engineering applications due to their chemical definition, biocompatibility and tuneable properties. A SAPH system has not been used previously for modelling breast cancer and vasculature; this would allow more representative in vitro modelling of the TME using a synthetic platform. This thesis aims to explore the use of a commercially available SAPH for modelling breast cancer and vasculature in vitro, thereby better recapitulating the TME in addition to using a synthetic hydrogel system. Features of the TME studied were: hypoxia, epithelial to mesenchymal transition (EMT), cell invasion, anti-cancer drug resistance and vasculature formation. The breast cancer cell lines MCF-7 and MDA-MB-231, representing early-stage and metastatic breast cancer respectively were studied, in parallel to using human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs), to create tubes resembling vasculature. Compared with collagen I and Matrigel, the SAPH PeptiGelAlpha1 exhibited superior mechanical properties and was formed from uniformly sized nanofibres. MCF-7 and MDA-MB-231 cells were viable and proliferated within PeptiGelAlpha1 over a 14-day period. MCF-7 cells formed spheroids within SAPH, similar to that of collagen I, whilst MDA-MB-231 cells tended to remain dispersed. PeptiGelAlpha1 appeared to be more appropriate for in vitro modelling of early breast cancer, such as with MCF-7 cells, as shown by ECM deposition, their highly invasive potential, and evidence of remodelling the peptide matrix, which were not observed with MDA-MB-231 cells. Preliminary data showed that encapsulation in PeptiGelAlpha1 resulted in a greater percentage of viable cells when treated with tamoxifen, compared with 2D culture. HUVECs and MSCs were not able to form tube-like structures within the hydrogel, although RGD functionalisation did positively influence cell morphology and proliferation. This benefit was concentration-dependent. The work in this thesis shows that a commercially available SAPH has potential to be a suitable candidate for in vitro modelling of breast cancer, and has important implications for studying tumour biology and tissue engineering vasculature in vitro.

Date of Award	1 Aug 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alberto Saiani (Supervisor) & Olga Tsigkou (Supervisor)