Spatiotemporal modulation of tumour-immune interaction within organotypic models of pancreatic ductal adenocarcinoma through targeted gene editing del

This project uses optoporation to control the phenotype of individual macropahges at precise locations within a vascularised in vitro organotypic model of pancreatic ductal adenocarcinoma (PDAC). This targeted regulation of cell phenotype improves control over the tumour microenvironment, providing a model of tumour-immune interactions closer to the in vivo pathophysiological conditions.
PDAC is the fourth leading cause of cancer-related deaths in the world due to its limited response to treatment. Tumor-associated macrophages (TAMs) are considered a good therapeutic target. TAMs are involved in cancer cell proliferation, invasion and angiogenesis, but the mechanisms of macrophage polarization to sustain tumor survival are poorly understood.
High-quality models of the tumour-immune microenvironment are necessary to elucidate the role of TAMs and to evaluate candidate drugs. In vitro organotypic models are particularly appealing since they are low cost, rapid and simple to establish and modify, and easy to assay. Dr. Adriani' s organoid models of vascularised PDAC closely resemble the tissue architecture of human PDAC providing a platform for high-content imaging (HCI), flow cytometry and transcriptomics investigation of the tumour-immune interactions[]. This model showed that TAMs present elevated glycolytic gene transcript levels (e.g. HK2, GPI, TPI1 and PGK1) and that glycolysis inhibition with drug treatment in TAMs disrupts their pro-metastatic phenotype.
Yet there is a limited understanding of the cooperative role of macrophages and their interactions with the tumour microenvironment in promoting tumour growth. Optoporation provides a pathway to alter the phenotype of selected cells in organoids, with the potential to dissect complex cellular interactions with high spatial and temporal resolution.
Therefore, this research project will develop a platform for the 3D optoporation of in vitro vascularised PDAC models to locally perform gene editing on individual macrophages to inhibit their glycolytic switch and reverse their tumor-promoting functions. mRNA will be loaded into nanoparticles and selectively delivered intracellularly by optoporation.
Optoporation is a powerful approach for selective delivery to cells. Using a near-infrared scanning laser coupled with light-responsive nanoparticles it locally disrupts the cell membrane close to the cell-nanoparticle interface, delivering payloads from solution to the cell cytosol. Dr Chiappini uses porous silicon nanoparticles for optoporation of nucleic acids to many cell types with up to 80% efficiency. The Chiappini lab also demonstrated optoporation of individual cells within tumour spheroids. Optoporation of a vascularised tumour would provide superior access throughout the tumor mass, improving the ability to regulate microenvironmental interactions.
Immunostaining followed by HCI imaging will allow to assess phenotypic changes upon optoporation of the target cells thanks to characteristic macropahges markers such as CD68, CD206 and CD163. Flow cytometry will allow us a deeper phenotypical characterization of the different cell types retrieved from the device and scRNA-seq can be run to assess the changes in gene expression caused by the complex cellular interactions before and after the gene editing.