Investigating the role of the extracellular matrix on CAR-T cell function against triple-negative breast cancer in 3D

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic cancer type with a low overall survival with limited therapeutic options. Chimeric antigen receptor T (CAR-T) cell therapy emerged as a promising treatment for solid tumours, eliciting targeted immune responses in 2D monolayer cultures. However, its efficacy against TNBC solid tumours has shown incomplete immune responses in early-phase clinical trials. This highlights the need to develop 3D models to study CAR-T cell interaction and response against solid tumours, bridging the gap between oversimplified 2D monolayer cultures and not fully human physiologically relevant animal models. The aim of this thesis was to design and develop a 3D hydrogel model to study CAR-T cell and TNBC spheroid interaction where the effect of the extracellular matrix (ECM) is investigated. Gelatine-alginate hydrogels were selected and used as an ECM mimicking biomaterial as they combine the cell adhesion molecules (e.g., RGD motifs)
and bioactivity of gelatine with the tuneable mechanical properties provided by the addition of alginate in the mixture. Gelatine-alginate hydrogels were formulated by manipulating both polymer concentrations (10% w/v, 7% w/v, and 5% w/v gelatine, and 1% w/v, 2% w/v and 3% w/v alginate) and characterised to be used as proxy for the ECM of TNBC. T cell infiltration, motility and migration towards a chemotactic gradient was higher in the formulation containing 1% w/v alginate and 5% w/v gelatine (5G1A). Large and compact MDA-MB-231 spheroids (700-900 µm in diameter) were generated. MDA MB-231 spheroids exhibited a well-defined 3D structure comprised of an outer layer of proliferating cells surrounded by a necrotic core which was larger in the absence of the 5G1A hydrogel. Metabolic assays confirmed a higher glycolytic state in spheroids in the hydrogel. CAR-T cells targeting folate receptor alpha (FRA-CAR) displayed a moderate lytic effect against spheroids in the hydrogel via reporter assays, while
modular image analysis revealed a null cytotoxic effect in the presence of the 5G1A hydrogel and a moderate effect that halted after 12 hours in the hydrogel’s absence. Our research implied that the fibrous ECM may play a more important role on CAR-T cell-TNBC interactions and its therapeutic response than previously reported.

Date of Award	17 Jun 2025
Original language	English
Awarding Institution	University of Bristol
Sponsors	CONACyT & Cytoseek Limited
Supervisor	Adam W Perriman (Supervisor), James Armstrong (Supervisor) & Ben Carter (Supervisor)