Development of 3D collagen-based scaffolds for the investigation of extracellular matrix changes on breast cancer progression and evaluation of anti-cancer therapeutics

<p dir="ltr">Advancements in the treatment of triple-negative breast cancer (TNBC), the most aggressive form of breast cancer (BC), is hindered by the use of 2D cancer models that fail to replicate the physiological tumour microenvironment, which plays a significant role in the regulation of cell behaviour and cancer progression. Changes to the breast extracellular matrix (ECM) such as an increase in ECM stiffness and increased deposition of ECM components including hyaluronic acid (HyA) and chondroitin sulfate (CS) have been identified as contributors to TNBC progression, however, their role in the tumour microenvironment can not be accurately investigated with 2D cancer models. In addition, the use of 2D cancer models at the initial stages of drug discovery negatively impacts clinical approval of new treatments with large discrepancies between 2D studies and clinical trials. Thus, the work presented in this thesis aimed to develop a physiologically relevant 3D in vitro model using collagen-based scaffolds, modified to mimic the breast ECM stiffness and the ECM changes associated with TNBC progression such as increasing concentrations of HyA and CS, for the investigation of TNBC progression, and to evaluate anti-cancer therapeutic testing in the form of chemotherapy and gene therapy, for the advancement of TNBC treatment.</p><p dir="ltr">Chapter 2 of this thesis outlined the fabrication and characterisation of four collagen-based scaffolds (CHyA low, CHyA high, CCS low and CCS high) composed of native breast tissue components and stiffness to mimic healthy and cancerous breast ECM (0.5kPa and 1kPa) respectively. The scaffolds were found to be highly biocompatible, support the attachment and viability of two TNBC cell lines, MDA-MB-231 and MDA-MB-436, which exhibited the expression of characteristics associated with the two cell lines, deeming the scaffolds suitable for TNBC research.</p><p dir="ltr">In Chapter 3, the effect of ECM stiffness on TNBC progression was investigated by utilising the healthy and cancerous scaffolds developed in Chapter 2 and assessing the effect of stiffness on cell metabolic activity, proliferation, migration, morphology and epithelial-mesenchymal transition (EMT) markers. An increase in ECM stiffness was found to increase the aggressiveness of TNBC by promoting collective amoeboid migration, the elevation of pro-inflammatory cytokines and a hybrid EMT phenotype. </p><p dir="ltr">In Chapter 4, the effect of increasing HyA and CS concentrations in the ECM on TNBC progression was investigated utilising the cancerous scaffolds developed in Chapter 2. HyA and CS were found to exert different effects on the behaviour of TNBC; an increase in HyA was considered to enhance collective amoeboid migration of TNBC cells, increase the expression of pro-inflammatory cytokines and promote a hybrid EMT phenotype while an increase in CS was found to also encourage collective amoeboid migration of TNBC but reduced the expression of pro-inflammatory cytokines.</p><p dir="ltr">Finally, in Chapter 5 our data highlighted the vast potential of 3D collagen-based scaffolds to be utilised as models for pre-clinical screening of chemotherapeutics, the exploration of microRNA (miR) delivery for targeted TNBC treatment and the combination of chemotherapy with gene therapy. Our results highlighted the ability to assess the impact of the ECM on chemotherapeutic efficacy. Furthermore, our scaffolds supported the known expression of miR-23a, a miRNA overexpressed in cancers and associated with poor patient prognosis, in TNBC cells and we were able to demonstrate knockdown when miR-23a inhibitor was delivered via gene therapy using modified glycosaminoglycan enhanced transduction (GET) cell penetrating peptides. Knockdown of miR-23a resulted in the decrease of cell migration, cell viability, angiogenesis and chemoresistance in TNBC cells, therefore uncovering some of the mechanisms in which miR-23a may be promoting TNBC progression, confirming its potential as a target for TNBC gene therapy. </p><p dir="ltr">Collectively, this thesis describes the fabrication and characterisation of physiologically relevant collagen-based scaffolds that mimic the ECM changes that take place as TNBC progresses. These scaffolds can be utilised to investigate the impact these ECM changes have on TNBC progression and therefore advance the understanding of TNBC which can lead to the identification of better treatment targets. In summary, these collagen-based scaffolds have vast potential as platforms for investigating ECM effects and the assessment of novel treatments in a wide variety of cancers.</p>