Developing 3D In Vitro Tumor Models: Extracellular Matrix Remodeling by Collagenases to Increase Drug Efficacy

Background: Solid tumors display a highly complex biological microenvironment, named Tumor Microenvironment (TME), characterized by a dense Extracellular Matrix (ECM), abnormal vasculature, and dynamic tumor–stroma interactions that collectively limit drug penetration and therapeutic efficacy. Traditional two-dimensional (2D) culture systems fail to adequately reproduce these features, underscoring the need for advanced three-dimensional (3D) models that better reflect tumor architecture and resistance mechanisms. Hypothesis and Aim of the Study:The research project aims 1) to develop a two-step, synergistic strategy based on the controlled remodeling of the collagen-rich tumor ECM using ultrapure recombinant collagenases, which enhances drug penetration and antitumor efficacy of the anticancer drug Doxorubicin (DOX) in solid tumors and 2) to conjugate collagenases and DOX to biocompatible and stimuli-responsive nanoparticles to improve intratumoral drug bioavailability while reducing off-target effects. To test these hypotheses, the project has been focused on the 1) evaluation and comparation of the therapeutic performance of DOX-loaded bovine serum albumin (BSA) nanoparticles and glutathione-responsive poly(vinyl pyrrolidone) (PVP) nanoparticles (NPs) in advanced 3D tumor models and 2) validation of a vascularized tumor model incorporating microvascular fragments to better recapitulate the tumor microenvironment and improve the predictive value of in vitro drug testing. Methods: Primary tumor cells (PTCs) were isolated from a breast tumor chemically induced in Wistar rats, using ultrapure recombinant collagenases. PTCs were used to generate 3D tumor spheroids capable of producing an endogenous type I collagen–rich matrix, as confirmed by immunofluorescence staining. ECM remodeling was achieved using ultrapure recombinant collagenases (class I and II). Doxorubicin (DOX) was administered in PTC spheroids as a free drug, conjugated to pH-sensitive BSA NPs, or incorporated into glutathione-responsive PVP NPs, and their efficacy was evaluated through a cell viability assay. BSA–DOX NPs were synthesized using the desolvation method and crosslinked with glutaraldehyde, whereas enzyme-loaded BSA nanoparticles were thermally crosslinked. The resulting constructs were characterized by Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and spectrophotometry. In addition, Microvascular Fragments (MVFs) isolated from rat adipose tissue were co-cultured with PTCs to generate so-called “Angiotumoroids” that recapitulate the tumor–endothelial cell crosstalk. Co-cultures were characterized by Scanning Electron Microscopy (SEM) and immunofluorescence, while protein expression was evaluated by proteomic analysis. Across all 3D models investigated in this study, drug uptake was assessed by confocal microscopy image analysis, and cell viability was assessed using an ATP-based luminescent assay.Results and discussion: Tumor spheroids developed a dense, collagen-rich ECM that markedly limited doxorubicin penetration. Pre-treatment with recombinant collagenases reduced ECM density and synergistically enhanced intratumoral DOX accumulation and antitumor efficacy, both for free DOX and nanoparticle-based formulations (PVP NPs and BSA NPs). It is well known that tumor cells present higher levels of intracellular glutathione compared to normal ones. Therefore, DOX was conjugated to OPVP NPs through a redox-responsive linker to obtain a controlled drug release mechanism. On the other hand, tumor cells and microenvironment present low pH, and, therefore, BSA–DOX nanoparticles were formulated to exhibit pH-dependent aggregation behavior, suggesting a potential acidity mediated retention within the tumor microenvironment in vivo. To improve the immunocompatibility of collagenases, the enzymes were conjugated to BSA nanoparticles via thermal crosslinking, a strategy that preserved their catalytic activity more effectively than conventional chemical crosslinking approaches. Finally, advanced three-dimensional tumor models were generated by co-culturing Microvascular Fragments (MVFs) with PTCs, giving rise to structures termed Angiotumoroids. These models displayed a homogeneous endothelial organization throughout the spheroid architecture and demonstrated pronounced neoangiogenic potential, mediated both by direct tumor– endothelial cell interactions and by soluble pro-angiogenic factors. Moreover, Angiotumoroids exhibited marked overexpression of efflux transporters, such as ATP Binding Cassette Subfamily B Member 1 (ABCB1), which conferred a drug-resistant phenotype, as confirmed by cytotoxicity assays. Taken together, these characteristics support the use of angiotumoroids as robust and physiologically relevant in vitro platforms for studying mechanisms of tumor drug resistance and assessing treatment efficacy. Conclusions: The combined targeting of the tumor ECM and the use of responsive nanoparticles significantly improve chemotherapeutic delivery in solid tumor models. Advanced 3D and vascularized tumor systems represent powerful and predictive platforms for investigating tumor resistance mechanisms and optimizing innovative drug-delivery strategies.