The development of anticancer therapies has increasingly relied on advanced 3D in vitro models, which more accurately mimic the tumor microenvironment compared to traditional 2D cultures. This review describes the evolution of these 3D models, highlighting significant advancements and their impact on cancer research. We discuss the integration of machine learning (ML) and artificial intelligence (AI) in enhancing the predictive power and efficiency of these models, potentially reducing the dependence on animal testing. ML and AI offer innovative approaches for analyzing complex data, optimizing experimental conditions, and predicting therapeutic outcomes with higher accuracy. By leveraging these technologies, the next generation of 3D in vitro models could revolutionize anticancer drug development, offering effective alternatives to animal experiments.

Several limitations, including dark toxicity, reduced tumor tissue selectivity, low photostability and poor biocompatibility hamper the clinical use of Photodynamic therapy (PDT) in cancer treatment. To overcome these limitations, new PSs have been synthetized, and often combined with drug delivery systems, to improve selectivity and reduce toxicity. In this context, BODIPYs (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) have recently emerged as promising and easy-to-handle scaffolds for the preparation of effective PDT antitumor agents. In this study, the anticancer photodynamic effect of newly prepared negatively charged polymethyl methacrylate (nPMMA)-bounded BODIPYs (3@nPMMA and 6@nPMMA) was evaluated on a panel of 2D- and 3D-cultured cancer cell lines and compared with free BODIPYs. In particular, the effect on cell viability was evaluated, along with their ability to accumulate into the cells, induce apoptotic and/or necrotic cell death, and inhibit cellular migration. Our results indicated that 3@nPMMA and 6@nPMMA reduce cancer cell viability in 3D models of HC116 and MCF7 cells more effectively than the corresponding free compounds. Importantly, we demonstrated that MDA-MB231 and SKOV3 cell migration ability was significantly impaired by the PDT treatment mediated by 3@nPMMA and 6@nPMMA nanoparticles, likely indicating the capability of this approach to reduce metastatic tumor potential.

A promising approach enhancing osteosarcoma (OS) prognosis involves the combination of various techniques, such as chemo‐ and photodynamic therapy, delivered through nanocarriers for synergistic cell death. Among the potential candidates for improving drug accumulation at the tumor site, mesenchymal stromal cells (MSCs) exhibit a significant advantage due to their tumor‐homing ability and intracellular drug retention. This study evaluates the efficacy of chemo‐releasing and photoactive bimodal nanoparticles, kPCe6 NPs, delivered via MSCs. In vitro analyses show that cells internalize and retain kPCe6 NPs in a dose‐dependent manner and that kPCe6‐loaded cells induce massive tumor cell death in a tridimensional tumor model. Results from an in vivo orthotopic OS murine model show negligible tumor cell death upon peritumoral administration of two doses containing 10⁶ loaded cells. To gain insight into this observation, this work investigates the role of cell dose in treatment efficacy. The results indicate that achieving a tumor reduction higher than 90% requires a substantial number of loaded cells, approximately 35% of the entire tumor mass, highlighting the criticality of the cell dose for the success of this therapeutic approach and its potential impact on clinical translation in OS patients, particularly when the number of tumor cells is limited.

Beatrice Costa was not included as a first co-author in the original publication

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel had been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3-dioxygenase 1 (IDO1), whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetic, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies. This work describes the development of novel biomimetic and carrier-free nanobinders (NB) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive prodrugs, and capable of hijacking human serum albumin (HSA). A fine tuning of the preparation conditions allowed to identify NB@5 as the best-performing prodrugs-based nanoformulation. Our data show that NB@5 effectively binds with HSA, demonstrating its protective role in the controlled release of drugs in vitro and suggesting that NB could exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully demonstrates that the drugs encapsulated within the nanobinders are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1. This study highlights the potential of prodrugs-based nanobinders as a promising avenue for the targeted chemoimmunotherapy of TNBC.