Figure 1 from PPARδ Orchestrates a Prometastatic Metabolic Response to Microenvironmental Cues in Pancreatic Cancer

Induction of EMT-like phenotype and metabolic switch in PDAC cells upon starvation. A, Representative images illustrating morphologic changes for PDAC-354 cells in response to treatment for 72 hours with the complex I inhibitor metformin (3 mmol/L), the β-oxidation inhibitor etomoxir (20 μmol/L), the complex II inhibitor malonate (5 mmol/L), the pyruvate carrier inhibitor UK5099 (100 μmol/L), or tumor-like conditions low pH (HCl 50 μmol/L) + low glucose (1 mmol/L) + 3%O2. Scale bar, 50 μm. B, Expression of EMT-associated genes (ZEB1, SNAI1, SNAI2, LOXL2, and VIM) was determined by RT-qPCR after cells were treated for 48 hours as indicated in A or with MCM. Pooled data for PDAC-185, A6L, 215, 253, and 354 (n ≥ 4 for each cell type). Data are normalized to HPRT1. Eto, etomoxier; Mal, malonate; Met, metformin. C, PDAC-215, 253, and 354 cells were treated with MCM or 20 μmol/L etomoxir for 48 hours and seeded in modified Boyden invasion chambers containing 20% FBS in the lower compartment. The number of invasive cells was analyzed after 16 hours (n = 7–11). D, GFP+-Luciferase+-PDAC-354 cells were treated with control, MCM, or 20 μmol/L etomoxir for 48 hours and then injected intrasplenically to assess their metastatic capacity (n = 9 mice/group). Representative photographs of liver metastasis (top) and subsequent hematoxylin and eosin staining (bottom). Scale bar, 200 μm. E, Representative OCR profile for PDAC-253 cells treated for 48 hours as indicated (mitochondrial stress test). O, ATP synthase inhibitor oligomycin; F, mitochondrial OXPHOS uncoupler FCCP carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; A+R, complex III inhibitor antimycin A + electron transport change inhibitor rotenone. F, Maximal and ATP-linked respiration (resp) in non-CSC vs. CSC cells. Bars represent pooled data from PDAC-215, 253, and 354, showing individual data points corresponding to each PDX (n = 5–7). G, Representative extracellular acidification rate (ECAR) profile for PDAC-253 cells treated for 48 hours as indicated (glycolysis test). G, glucose; O, ATP synthase inhibitor oligomycin; 2DG, glycolysis inhibitor 2-deoxy-glucose. H, Glycolysis, glycolytic capacity, and glycolytic reserve in adherent (non-CSC) vs. sphere-derived cells (CSC). Bars represent pooled data from PDAC-215, 253, and 354, showing individual data points corresponding to each PDX (n = 4–5). All data are represented as the mean ± SEM. *, P P P