020 | A three-dimensional vascularized cancer model mimicking the tumor microenvironment for drug resistance studies

Alessandra Lo Cicero¹, Gabriele Lo Buglio¹, Francesco La Monica¹, Simona Campora¹, Margot Lo Pinto², Simone Dario Scilabra², Giulio Ghersi^1|3 | ¹Department of Biological, Chemical and Pharmaceutical Sciences and Technologies STEBICEF, University of Palermo, Palermo, Italy; ²Proteomics Group of RiMED Foundation, Research Department IRCCS ISMETT Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione, Palermo, Italy; ³Abiel srl, Palermo, Italy.

In solid tumors, the organization of the extracellular matrix (ECM), stromal components, and functional vasculature critically regulate tumor progression and therapeutic response. Conventional 2D culture systems fail to recapitulate these features, while most 3D spheroid models lack a mature and functional vascular compartment, which limits their translational relevance, particularly in drug resistance studies. We developed a vascularized 3D breast cancer model (Angiotumoroids) by combining primary tumor cells (PTCs) from rat mammary carcinoma with adipose-derived microvascular fragments (MVFs), which retain the structural and cellular complexity of native blood vessels. By modulating MVF density within the constructs, angiotumoroids exhibited progressive integration of MVFs, confirmed by increased CD31 expression, and developed vascular-like networks characterized by active angiogenic sprouting. Tumor-vascular crosstalk influenced ECM remodeling, as demonstrated by a marked increase in active MMP9 in vascularized constructs, while MMP2 levels remained largely unchanged. Embedding angiotumoroids in collagen matrices further revealed robust and branched neovascularization, particularly at intermediate MVF densities, highlighting the synergistic contribution of juxtacrine and paracrine signaling between tumor cells and MVFs. Importantly, proteomic profiling revealed that angiotumoroids possess a distinct molecular signature compared to non-vascularized PTC spheroids, with enrichment of angiogenesis-related proteins (including VEGFA), ECM components (collagens I and IV), and pathways associated with matrix remodeling. Notably, proteins linked to multidrug resistance were significantly upregulated, including the ABC transporter ABCB1 (P-glycoprotein). Consistent with these findings, vascularized angiotumoroids displayed altered doxorubicin distribution, with increased and spatially localized drug accumulation but reduced cytotoxic efficacy. Despite enhanced uptake, tumor cells exhibited greater survival and recovery over time, supporting a mechanism of drug sequestration and efflux within vascularized compartments that ultimately limits therapeutic effectiveness. Overall, the angiotumoroid model represents a robust and scalable platform for studying tumor angiogenesis, ECM remodeling, and vascular-associated drug resistance. By integrating structural, functional, and molecular features of the tumor microenvironment, it provides a powerful tool for evaluating anticancer drug delivery and for developing strategies aimed at overcoming microenvironment-mediated chemoresistance.