Francesco Pampaloni

Breast cancer (BC) is the most common type of cancer among women worldwide and underlies relapse, disease progression, and metastasis. Resistance to chemotherapy and programmed cell death (PCD), including apoptosis, strongly affects BC therapy success and remains a major challenge. Although necroptosis, a lytic, and via damage-associated molecular patterns (DAMP) release, highly immunogenic mode of PCD, might overcome apoptosis resistance, there is an urgent need for physiological and translational human models to model necroptosis and PCD resistance in BC. Here, we apply 3D patient-derived, metastatic human mammary organoids (hMOs) to model apoptosis resistance, necroptosis, and inflammatory signaling using single-cell CITE-sequencing, time-lapse live cell brightfield, and immunofluorescent confocal microscopy, as well as biochemical approaches. Smac mimetic-triggered apoptosis could be confirmed in a panel of BC hMOs. Upon inducing apoptosis resistance with caspase inhibition, BC hMOs rapidly undergo necroptosis with profound MLKL phosphorylation. Necroptotic cell death was preceded by prominent transcriptional upregulation of inflammatory cyto- and chemokines, including interferons, that activate natural killer cells. Finally, necroptosis and the expression and release of inflammatory messengers in metastatic BC hMOs could be attenuated upon the inhibition of linear ubiquitination. We describe a novel experimental platform to model PCD, inflammation, and necroptosis that allows therapeutic screening to overcome chemotherapy resistance in patient-derived metastatic BC hMOs. With this platform, we identified necroptosis-induced interferon signaling, suggesting Smac mimetics and necroptosis as a potential immunotherapy strategy against metastatic BC.

Self-organised three-dimensional (3D) cell cultures, collectively called 3D-oids, include spheroids, organoids and other co-culture models. Systematic evaluation of these models forms a critical new generation of high-content screening (HCS) systems for patient-specific drug analysis and cancer research. However, the standardisation of working with 3D-oids remains challenging and lacks convincing implementation. This study develops and tests HCS-3DX, a next-generation system for HCS analysis in 3D imaging and image evaluation. HCS-3DX is based on three main components: an automated Artificial Intelligence (AI)-driven micromanipulator for 3D-oid selection, an HCS foil multiwell plate for optimised imaging, and image-based AI software for single-cell data analysis. We validated HCS-3DX directly on 3D tumour models, including tumour-stroma co-cultures. Our data demonstrate that HCS-3DX achieves a resolution that overcomes the limitations of current systems and reliably and effectively performs 3D HCS at the single-cell level. Its application will enhance the accuracy and efficiency of drug screening processes, support personalised medicine approaches, and facilitate more detailed investigations into cellular behaviour within 3D structures.

Glioblastoma (GBM), the most malignant primary brain tumor in adults, has poor prognosis irrespective of therapeutic advances due to its radio-resistance and infiltrative growth into brain tissue. The present study assessed functions and putative druggability of BRCA1-associated ATM activator 1 (BRAT1) as a crucial factor driving key aspects of GBM, including enhanced DNA damage response and tumor migration. By a stable depletion of BRAT1 in GBM and glioma stem-like (GSC) cell lines, we observed a delay in DNA double-strand break repair and increased sensitivity to radiation treatment, corroborated by in vitro and in vivo studies demonstrating impaired tumor growth and invasion. Proteomic and phosphoproteomic analyses further emphasize the role of BRAT1’s cell migration and invasion capacity, with a notable proportion of downregulated proteins associated with these processes. In line with the genetic manipulation, we found that treatment with the BRAT1 inhibitor Curcusone D (CurD) significantly reduced GSC migration and invasion in an ex vivo slice culture model, particularly when combined with irradiation, resulting in a synergistic inhibition of tumor growth and infiltration. Our results reveal that BRAT1 contributes to GBM growth and invasion and suggest that therapeutic inhibition of BRAT1 with CurD or similar compounds might constitute a novel approach for anti-GBM directed treatments.

We introduce a platform for the fabrication of customizable wound healing dressing. The platform integrates electrospun nanofibers, bioprinted hydrogels, and cellular spheroids into hierarchical, fiber-reinforced hybrid constructs. The construct leverages the mechanical strength of polycaprolactone (PCL) nanofibers and the ECM-like properties of GelMA/PEGDA hydrogel. These materials support the incorporation of bone marrow-derived mesenchymal stem cell (BM-hMSC) spheroids, which act as a supportive “cell niche,” enhancing the viability of the hMSC during and after bioprinting, and facilitating their spreading across the construct during the maturation phase. The characterization of the hybrid constructs demonstrated strong structural integrity and enhanced mechanical properties, making them well-suited for clinical wound dressing applications. In vitro assays, including live/dead staining, MTT assays, and scratch assays, revealed increased cell attachment, proliferation, and migration. The spheroids maintained their viability over extended periods, significantly contributing to wound closure in the scratch assay. This innovative approach, which combines electrospinning and light-based bioprinting, offers a promising strategy for the development of customizable wound dressings that closely adapt to the complex architecture of human skin. The bioprinting approach allows for the creation of tailored geometries for specific clinical requirements. Future research will focus on optimizing scaffold design and conducting long-term in vivo studies to validate the platform’s clinical potential.

Breast cancer (BC) is the most common type of cancer among women worldwide and underlies relapse, disease progression and metastasis. Resistance to chemotherapy and programmed cell death (PCD), including apoptosis, strongly affects therapy success and remains a major challenge. Representative and translational models to understand, manipulate and cultivate advanced BC and to model PCD resistance are therefore urgently required. Smac mimetics are promising compounds to circumvent apoptosis resistance and are able to induce caspase-independent necroptosis, a lytic and inflammatory mode of PCD. Here, we apply primary, patient-derived human mammary organoids (hMOs) to investigate alternative forms of PCD to overcome apoptosis resistance. Using time lapse brightfield with immunofluorescent confocal microscopy, biochemistry and gene expression analysis, we demonstrate that Smac mimetics induce apoptosis in primary hMOs. By mimicking apoptosis resistance via caspase inhibition, hMOs undergo necroptosis, associated with expression and secretion of inflammatory mediators. Inhibition of linear ubiquitination by the LUBAC inhibitor HOIPIN-8 prevents necroptosis, as well as the expression and release of inflammatory mediators in hMOs. Our findings demonstrate that primary hMOs are effective models to model, study and manipulate PCD responses and inflammation in in primary BC organoids and open new therapeutic screening options for chemotherapy-resistant BC.

Light sheet fluorescence microscopy (LSFM) is a powerful tool for imaging large three-dimensional biological samples. However, the design and fabrication of specimen chambers for these systems present significant challenges, particularly in maintaining water-tight seals, preventing contamination, and ensuring the flexibility needed for precise positioning of the objective and sample. This study introduces open-hardware solutions to address these challenges, utilising a combination of 3D printing, silicone injection moulding, and FEP-foil thermoforming. We describe the development of custom, highly flexible silicone seals and connectors through a laboratory-scale injection moulding process. These components enable precise, low-resistance movement of imaging objectives and specimen holders, which is crucial for maintaining imaging accuracy. Additionally, we introduce a novel “optical window” design that isolates the objective lens from the immersion medium, significantly reducing the risk of contamination and facilitating easy exchange of chambers and lenses without compromising sterility. The practicality of these designs is demonstrated through their application in long-term live imaging of Tribolium castaneum embryos, honey bee embryos, and human mesenchymal stem cell spheroids. By providing open-source CAD and 3D printing files, this work promotes accessibility and customization in microscopy, enabling researchers to easily replicate and adapt these solutions to their specific needs.

Self-organized three-dimensional (3D) cell cultures, collectively called 3D-oids, include spheroids, organoids and other co-culture models. Systematic evaluation of these models forms a critical new generation of high-content screening (HCS) systems for patient-specific drug analysis and cancer research. However, the standardisation of working with 3D-oids remains challenging and lacks convincing implementation. This study develops and tests HCS-3D X , a next-generation system revolutionising HCS analysis in 3D imaging and image evaluation. HCS-3D X is based on three main components: an automated Artificial Intelligence (AI)-driven micromanipulator for 3D-oid selection, an HCS foil multiwell plate for optimised imaging, and image-based AI software for single-cell data analysis. We validated HCS-3D X directly on 3D tumour models, including tumour-stroma co-cultures. Our data demonstrate that HCS-3D X achieves a resolution that overcomes the limitations of current systems and reliably and effectively performs 3D HCS at the single-cell level. Its application will enhance the accuracy and efficiency of drug screening processes, support personalised medicine approaches, and facilitate more detailed investigations into cellular behaviour within 3D structures.