Effects of Matrix Stiffness and Fibrous Architecture on Doxorubicin Response in Engineered 3D Prostate Cancer Scaffolds

Abstract

Two-dimensional cell culture has been considered the gold standard for studying human biology in vitro for a long time. In this approach, cells are grown as flat monolayers and are expected to replicate the behavior of cells inside the human body. However, organs, cells, and particularly relevant to this study, tumors, do not grow in two-dimensional conditions. Instead, they continuously interact with their three-dimensional surrounding environment. This three-dimensional environment can be replicated in a biological laboratory through various approaches, including three-dimensional bioprinting, tumor spheroids, and three-dimensional hydrogel models. The goal of all these systems is to reproduce the conditions of the human body as faithfully as possible. This three-dimensional environment has distinct mechanical, chemical, and structural properties that influence the tumor microenvironment, cell-cell interactions, and cell-extracellular matrix interactions. In this project, we focus specifically on the mechanical properties and fibrous architecture of three-dimensional scaffold models. As cancer progresses, the extracellular matrix surrounding the tumor undergoes extensive structural remodeling. Cancer-associated fibroblasts and tumor cells deposit increasing quantities of collagen, which assembles into a dense fibrous network throughout the tumor stroma. Crosslinking enzymes further stabilize these collagen fibers, progressively stiffening the matrix and creating a physically restrictive environment around the tumor. This has consequences that extend beyond mechanics alone: the fibrous network has been reported to limit drug penetration to cancer cells deep inside the tumor, activate signaling pathways that promote cancer cell survival and invasion, and create physical barriers that protect certain cancer cell populations from chemotherapy, all of which may contribute to treatment resistance. In this study, short electrospun poly(ε-caprolactone) (PCL) fibers, a biocompatible synthetic polyester, were incorporated into gelatin-based hydrogel scaffolds to introduce this fibrous architectural element into the hydrogel matrix in a controlled and tunable manner. The mechanical stiffness of the tumor microenvironment is a recognized regulator of cancer cell behavior, drug delivery, and phenotypic identity. Prostate cancer was selected as the model disease for this study because it is one of the most diagnosed malignancies in men worldwide, and treatment of its advanced, castration-resistant form remains a major clinical challenge. In prostate cancer, the extracellular matrix stiffens progressively as disease advances, from approximately 3 to 4 kPa in normal prostate tissue to values exceeding 7 kPa in cancerous tissue. This mechanical change has been shown to promote more aggressive cancer cell behavior through mechanotransduction pathways, and to restrict the transport of chemotherapeutic drugs into the tumor interior. However, the combined effects of matrix stiffness and fibrous architecture on drug distribution and cancer cell response have not been investigated simultaneously in a single engineered prostate cancer model system. This thesis presents an investigation using three groups of three-dimensional scaffolds: gelatin methacryloyl (GelMA), a photocrosslinkable gelatin-based hydrogel, fabricated at approximately 10 kPa and approximately 20 kPa, and GelMA reinforced with short electrospun PCL fiber particles, fabricated at approximately 20 kPa. The 20 kPa GelMA-only group was included to match the composite's stiffness, allowing stiffness-driven and fiber-driven contributions to be distinguished from one another. The 10 kPa and 20 kPa scaffolds were used to model the soft and stiffer prostate tumor microenvironments, respectively. All experiments were conducted using PC3 human prostate cancer cells, an androgen receptor-negative cell line representative of castration-resistant, therapy-refractory disease. Scaffold fabrication and characterization confirmed GelMA synthesis by proton NMR spectroscopy, with a degree of methacrylation of approximately 40% across all batches, and compression testing and oscillatory rheology confirmed moduli within the target stiffness ranges for each of the three scaffold groups. Doxorubicin distribution and cancer cell response were then investigated using doxorubicin, a clinically used chemotherapy agent with intrinsic red fluorescence that allows direct confocal imaging of its spatial distribution without additional labeling. Confocal z-stack imaging demonstrated rapid doxorubicin penetration throughout all three scaffold groups, with no detectable concentration gradient between the outer surface and scaffold core in any group. Within the GelMA-PCL composite scaffolds, however, doxorubicin fluorescence was locally concentrated at structures consistent with fiber locations, despite bulk penetration being complete and comparable across all three groups. Despite this comparable bulk drug penetration, PC3 cell viability after doxorubicin exposure differed substantially between groups: cells in GelMA-only scaffolds at 20 kPa (stiffness-matched to the composite) showed the highest relative viability, cells in GelMA-only scaffolds at 10 kPa showed intermediate viability, and cells in GelMA-PCL composite scaffolds showed the lowest relative viability of the three groups. Live-cell imaging suggested that this reduced viability in the composite group may be linked to localized doxorubicin binding at PCL fiber surfaces, producing regions of concentrated drug exposure despite comparable bulk distribution throughout the construct. These findings suggest that matrix stiffness and fibrous architecture act through distinct, and in this case competing, mechanisms in regulating chemotherapy response: higher stiffness was associated with greater cancer cell resistance to doxorubicin, which may reflect stiffness-driven signaling, the smaller mesh size of the more crosslinked network, or both, while fibrous architecture, independent of bulk drug delivery, appears to sensitize cells locally through fiber-associated drug accumulation. If confirmed, this would provide a mechanistic link between the physical tumor microenvironment and chemotherapy response that is more complex than a simple barrier-and-protection model. Beyond prostate cancer, the scaffold platform developed in this work offers a reproducible and tunable three-dimensional model applicable to other solid tumors, with direct relevance to understanding how the physical composition, not just the stiffness, of the tumor microenvironment shapes chemotherapy efficacy.