Fig. 5: Effect of hypoxia and hypoxia-mimicking compounds on PDAC response to treatment.

A PDAC organoids were grown in monoculture and co-culture with PSCs under normoxic and hypoxic conditions. The cells were grown for 4 days on the OrganoPlate® and subjected to 1 µM Gemcitabine and 1 µM Roxadustat for 72 h, before their survival was analyzed with Cell Titer Glo 3D viability assay. The graph shows the survival of the organoids normalized to a no-treatment control (=medium only). 0.1% DMSO was used as a vehicle control and showed no effect on the cells. The data was compared with Ordinary one-way ANOVA and Tukey’s multiple comparison test and shown are mean and SD (****p ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05). B Diagram showing key regulators of HIF1α in hypoxia and normoxia. During normoxia HIF1α is constantly hydroxylated by prolyl hydroxylases (PHDs), that use oxygen (O2) and iron (Fe2+), thus leading to subsequent ubiquitination and proteasomal degradation of HIF1α. In contrast, under hypoxia or due to hypoxia mimicking factors (such as Roxadustat treatment), PHDs are inhibited, thus enabling the HIF1α subunit to bind to the HIF1β subunit. This HIF-complex can migrate to the nucleus, bind a hypoxia responsive element (HRE), and lead to the activation of several genes involved in angiogenesis, glycolysis, proliferation, and survival [42]. C Caspase 3/7 staining of chips in hypoxia was compared to chips in normoxia to show respective areas of apoptosis. D Quantification of the Caspase 3/7 assay staining depicting mono-, and co-cultured cells in normoxia and hypoxia when treated with Gemcitabine or Gemcitabine with Roxadustat. The data was compared with Ordinary one-way ANOVA and Tukey’s multiple comparison test and shown are mean and SD (****p ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05), (n = 3, N = 2). E Reactive Oxygen Species (ROS) staining with ROS marker, Hoechst for nuclei staining and Propidium Iodide (PI) for dead cell staining in PDAC monoculture. F Quantification of ROS intensity to the total cell count in hypoxia samples was normalized to normoxia samples comparing untreated samples with samples treated with Gemcitabine with or without Roxadustat. G Monocultures treated with 500 nM Gemcitabine (G), 1 uM Roxadustat (R), 1 nM Echinomycin (E) and 10 uM Kc7f2 (K). H Co-cultures treated with 500 nM Gemcitabine (G), 1 uM Roxadustat (R), 1 nM Echinomycin (E) and 10 uM Kc7f2 (K) to determine the effect of HIF-inhibitors on PDAC treatment. Shown are mean and SD (****p ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05), (N = 3, n = 3). The data was analyzed with two-way ANOVA and Sidak’s multiple comparison test. Shown are maximum projections (10× magnification) of PDAC monoculture for all images, imaged on the ImageXpress Micro Confocal (Molecular Devices, US). Scale Bar = 200 um.