A Step-by-Step Methodological Guide for Developing Zonal Multicellular Scaffold-Based Pancreatic Cancer Models

The tumor microenvironment (TME), a complex heterogeneous mixture of various cellular, physical, and biochemical components and signals, is a major player in the process of tumor growth and its response to therapeutic methods. In vitro 2D monocellular cancer models are unable to mimic the complex in vivo characteristics of cancer TME involving cellular heterogeneity, presence of extracellular matrix (ECM) proteins, as well as spatial orientation and organization of different cell types forming the TME. In vivo animal-based studies have ethical concerns, are expensive and time-consuming, and involve models of non-human species. In vitro 3D models are capable of tiding over several issues associated with both 2D in vitro and in vivo animal models. We have recently developed a novel zonal multicellular 3D in vitro model for pancreatic cancer involving cancer cells, endothelial cells, and pancreatic stellate cells. Our model (i) can provide long-term culture (up to 4 weeks), (ii) can control the ECM biochemical configuration in a cell specific manner, (iii) shows large amounts of collagen secretion by the stellate cells mimicking desmoplasia, and (iv) expresses cell-specific markers throughout the whole culture period. This chapter describes the experimental methodology to form our hybrid multicellular 3D model for pancreatic ductal adenocarcinoma, including the immunofluorescence staining on the cell culture.