Biology subjects

Giustarini, G.

Publications and source records attributed to Giustarini, G..

3 recordsLinked to original sources

bioRxiv2026

Immune reprogramming of 3D tumor models via optoporation-mediatedtargeted gene delivery to macrophages

The dynamics of the tumor microenvironment (TME) are a key determinant of cancer progression and therapeutic resistance through complex interactions between tumor, stromal and immune cell populations. Among these, tumor-associated macrophages (TAMs) play a central role in promoting tumor growth and immune suppression. However, the specific contributions of TAMs remain poorly understood due to the lack of tools enabling selective genetic manipulation in three-dimensional (3D) tumor models. Here, we present a gold nanoparticle-assisted optoporation approach that enables spatially selective plasmid-based gene delivery to TAMs within intact heterocellular 3D pancreatic ductal adenocarcinoma (PDAC) spheroids, thereby modulating the TME. In two-dimensional (2D) TAM cultures, conventional transfection of IRF5- and IKBKB-encoding plasmids validated their capacity to induce TAM repolarization, as evidenced by activation of interferon signaling. Extending this approach to 3D PDAC spheroids, nanoparticle-assisted optoporation achieved selective transfection of TAMs with IRF5- and IKBKB-encoding plasmids by transiently generating nanoscale membrane pores in illuminated cells. TAMs transfection elicited a robust interferon response, marked by transcriptional upregulation of IFNA, IFNB1, and CXCL10, and increased protein levels of IFNB1, IFNL1, and CXCL13, together with downregulation of pro-tumorigenic markers CEACAM5, IL19, and IL32. These coordinated changes indicate a shift towards an anti-tumorigenic TME. By enabling minimally invasive, TAM-specific gene delivery in complex multicellular 3D spheroids, this strategy allows precise modulation of the TME and opens new avenues for modeling its dynamics in cancer progression and therapeutic response.

bioengineering↗

bioRxiv2026

Multi-omics and functional analysis of a bioengineered vascularized pancreatic cancer model reveal an immunosuppressive and therapy-resistant niche

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by therapy resistance and an immunosuppressive tumor microenvironment. To comprehensively characterize the complex stromal-immune cell interactions that drive PDAC aggressiveness, we applied an integrated multi-modal approach combining single-cell RNA sequencing, spatial transcriptomics, proteomics, immunofluorescence, and microfluidic-based functional assays to bioengineered spheroid models with increasing cellular complexity (up to four cell types) integrating human pancreatic cancer cells, pancreatic stellate cells, endothelial cells, and monocyte-derived macrophages. By incorporating vascularization within the OrganiX microfluidic platform, we enable studies of immune cell trafficking in vascularized tumors. Multi-omics phenotyping revealed coordinated molecular programs in our four-cell organotypic spheroid models, including enhanced hypoxic and glycolytic pathways, NF-{kappa}B activation, and ECM remodeling. Stromal and immune cells acquired tumor-associated phenotypes mirroring patient heterogeneity, including IL-1{beta}+ macrophages, inflammatory cancer-associated fibroblasts (iCAFs), and antigen-presenting CAFs (apCAFs). The four-cell model exhibited superior clinical relevance, with gene expression signatures that correlated more closely with poor-prognosis patient cohorts and cancer hallmarks that were functionally validated through microfluidic-based assays demonstrating enhanced tumor invasion, angiogenesis, and therapeutic resistance. Finally, live imaging combined with transcriptomic readouts captures dynamic interactions between neutrophils and cancer cells in the vascularized microtumor, enabling direct observation of intravascular events relevant to metastatic dissemination. This integrated analysis demonstrated the recreation of a human-relevant aggressive PDAC niche, establishing a framework that bridges in vitro cellular crosstalk studies with patient-relevant therapeutic responses, offering a powerful translational tool for therapy development in PDAC.

cancer biology↗

bioRxiv2025

Microplastics cross the murine intestine and induce inflammatory cell death after phagocytosis by human monocytes and neutrophils

Microplastics are contaminating the environment but also our food and drinking products. In a crucial study, microplastics have been detected in circulating human blood, urging the investigation of the effects of microplastics on human health. Here we aimed to determine the distribution of microplastics after oral exposure in mice and their interactions with and effects on mouse and human innate immune cells. We established that both 1 and 10m polystyrene (PS) particles penetrated the intestinal epithelium after oral administration and could be detected in blood and liver of mice after ten days of oral administration. Using intravital microscopy we captured the in vivo phagocytosis of 1m PS by mouse neutrophils in the liver. Pristine PS were barely phagocytosed by primary human phagocytes, however, 1 and 10{micro}m PS pre-incubated with plasma were readily phagocytosed by human neutrophils and monocytes. Plasma-coated 1 and 10m PS both increased human neutrophil and monocyte cell death but only after phagocytosis. Importantly, neutrophil cell death occurred a few hours after phagocytosing a single coated 10{micro}m PS while PS of 1{micro}m needed to be administered at a ratio of 27 particles per cell to induce significant neutrophil cell death. Neutrophil cell death upon microplastic exposure was characterized by extracellular DNA, which together with other released DAMPs can potentially trigger inflammation. Our findings suggest that microplastics could negatively impact the immune system and human health.

immunology↗