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

该项目使用光穿孔在胰腺导管腺癌（PDAC）的血管化体外器官型模型中的精确位置控制单个巨噬细胞的表型。这种细胞表型的靶向调节改善了对肿瘤微环境的控制，提供了更接近体内病理生理条件的肿瘤-免疫相互作用模型。PDAC是世界上癌症相关死亡的第四大原因，因为它对治疗的反应有限。肿瘤相关巨噬细胞（TAM）被认为是良好的治疗靶点。TAM参与癌细胞增殖、侵袭和血管生成，但巨噬细胞极化维持肿瘤存活的机制知之甚少。高质量的肿瘤免疫微环境模型对于阐明TAM的作用和评估候选药物是必要的。体外器官型模型是特别有吸引力的，因为它们成本低，快速和简单的建立和修改，并易于测定。Adriani博士的血管化PDAC类器官模型与人PDAC的组织结构非常相似，为肿瘤-免疫相互作用的高含量成像（HCI）、流式细胞术和转录组学研究提供了平台[]。该模型显示TAM呈现升高的糖酵解基因转录物水平（例如HK 2、GPI、TPI 1和PGK 1），并且药物处理对TAM中的糖酵解抑制破坏了它们的促转移表型。然而，对巨噬细胞的协同作用及其与肿瘤微环境在促进肿瘤生长中的相互作用的理解有限。光穿孔提供了一种改变类器官中选定细胞表型的途径，具有以高空间和时间分辨率剖析复杂细胞相互作用的潜力。因此，该研究项目将开发一个用于体外血管化PDAC模型的3D光致发光的平台，以对单个巨噬细胞进行局部基因编辑，以抑制其糖酵解开关并逆转其促肿瘤功能。mRNA将被加载到纳米颗粒中并通过光致发光选择性地递送到细胞内。光穿孔是选择性递送至细胞的有力方法。使用近红外扫描激光器与光响应纳米颗粒耦合，它局部破坏靠近细胞-纳米颗粒界面的细胞膜，将有效载荷从溶液传递到细胞胞质溶胶。Chiappini博士使用多孔硅纳米粒子将核酸光致发光到许多细胞类型，效率高达80%。Chiappini实验室还证明了肿瘤球体内单个细胞的光致发光。血管化肿瘤的光穿孔将为整个肿瘤块提供更好的上级通道，从而提高调节微环境相互作用的能力。免疫染色后进行HCI成像将允许评估靶细胞光致发光后的表型变化，这归因于特征性巨噬细胞标志物，如CD 68、CD 206和CD 163。流式细胞术将使我们能够对从设备中检索到的不同细胞类型进行更深入的表型表征，并且可以运行scRNA-seq来评估基因编辑前后复杂的细胞相互作用引起的基因表达变化。

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.