Hypoxia at 3D organoid establishment selects essential subclones within heterogenous pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is especially hypoxic and composed of heterogeneous cell populations containing hypoxia-adapted cells. Hypoxia as a microenvironment of PDAC is known to cause epithelial-mesenchymal transition (EMT) and resistance to therapy. Therefore, cells adapted to hypoxia possess malignant traits that should be targeted for therapy. However, current 3D organoid culture systems are usually cultured under normoxia, losing hypoxia-adapted cells due to selectivity bias at the time of organoid establishment. To overcome any potential selection bias, we focused on oxygen concentration during the establishment of 3D organoids. We subjected identical PDAC surgical samples to normoxia (O2 20%) or hypoxia (O2 1%), yielding glandular and solid organoid morphology, respectively. Pancreatic cancer organoids established under hypoxia displayed higher expression of EMT-related proteins, a Moffitt basal-like subtype transcriptome, and higher 5-FU resistance in contrast to organoids established under normoxia. We suggest that hypoxia during organoid establishment efficiently selects for hypoxia-adapted cells possibly responsible for PDAC malignant traits, facilitating a fundamental source for elucidating and developing new treatment strategies against PDAC.

Growing Role of 3D In Vitro Cell Cultures in the Study of Cellular and Molecular Mechanisms: Short Focus on Breast Cancer, Endometriosis, Liver and Infectious Diseases

“…Currently, there is an ever-growing plethora of scientific articles reporting on the use of 3D cultures in oncological research of both so-called ‘solid’ [ 14 , 41 ] and ‘liquid’ tumors [ 42 , 43 ]. The shift from 2D in vitro systems to 3D cultures is explained by the fact that it is possible to tune these to more closely mimic in vivo tumor characteristics, including the heterogeneity of the tumor microenvironment (TME) [ 44 ], cell–cell (i.e., tumor cell–immune cell) [ 45 ] and cell–extracellular matrix contacts [ 46 ], hypoxia [ 47 ], nutrient and pH gradients [ 48 ] and biomechanical properties (such as extracellular matrix (ECM) stiffness) [ 49 ] ( Figure 1 a). This cancer-mimicking approach facilitates the study of mechanisms of cancer initiation, progression, resistance recurrence and tumour–stroma interaction, which can identify markers for early diagnosis and therapies [ 41 , 50 ].…”

“…BC cells shift from glycolysis to OXPHOS and FA metabolism, responding to a stiff microenvironment [66] Co The great potential of 3D models (like spheroids and organoids) and linked methods/technologies is to recreate important features of the tumor, such as the organization of the multicellular layer and the environment in which micro-metastases develop, as nutrients and oxygen are limited in these large structures [24,47]. In addition, they make it possible to represent and preserve the cellular heterogeneity present in tumors: combining different cell types in the same spheroid, i.e., tumor cells, monocytes and CAFs, allows for studying the role of these cells and the cell-cell interaction in tumour initiation and progression as well as all the variations in the signalling, gene expression and protein production pathways involved [14].…”