Fig 1: Loss of CES2 attenuates PDAC growth in vitro and in vivo. A) Cell confluency of CFPAC-1 and AsPC-1 was checked every 12 h starting 48 h after the cells were seeded. B) Colony formation in soft agar or anchorage-independent growth in 96-well round bottom ultralow attachment (ULA) plate of CFPAC-1 and AsPC-1 cells. C) Growth of AsPC1 and CFPAC1 was measured by MTS assay following 96-h treatment with either DMSO or the indicated concentrations of fenofibrate. D) The growth of AsPC-1 cells expressing high (control) or low (shCES2) level of CES2 in nude mice was monitored every week. The effect of CES2 knockdown in xenograft tumor tissue was confirmed by immunohistochemistry using a-CES2 antibody. E) Luciferase-based noninvasive bioluminescence imaging of nude mice inoculated with control and shCES2 AsPC-1 cells at day 28 and day 35.
Fig 2: Detecting hCES2 activity in orthotopic SU.86.86 xenografts. A few drops of probe 2 (20 µM) was added to a SU.86.86 frozen tissue slice (10 µm in thickness) on a microscope slide and imaged. (A) Single channel red fluorescence as a function of time whereby probe 2 added to SU.86.86 CES2 OE tissues (top row) led to larger time dependent increase in fluorescence, compared to control vector SU.86.86 tissues (bottom row). (B) Ratiometric images obtained after 3 hour incubation. Tissues were washed 3 × with PBS to obtain images in the yellow channel. A higher red/yellow ratio was observed for CES2 OE orthotopic xenografts compared to the respective control. Average fluorescence was collected at 12 different regions of the tissues at 3 hours to construct a box plot (far right). Magnification is 20×. Scale bars = 50 µm. Statistical significance is calculated using the Mann–Whitney–Wilcoxon test compared to CES2 OE (****p < 0.0001).
Fig 3: CES2 expression predicts poor prognosis in PDAC patients. Kaplan–Meier survival curves depict the relationship between tumoral mRNA expression of CES2 > or = an optimal cutoff value and overall survival in the TCGA-PDAC transcriptomic dataset. The cutoff value was determined using log rank statistics-based methods (see Materials and methods). Results of the univariable and multivariable Cox proportional hazard model analyses are provided. For multivariable analyses, age (stratified by median), sex, and stage were included as co-variables.
Fig 4: Diagram of reciprocal regulation between CES2 and HNF4a. Proposed regulatory pathway wherein CES2 promotes the catabolism of choline-containing phospholipids to yield lipid species that sustain HNF4a protein expression and activation in a soluble epoxide hydrolase (sEH)-dependent manner; activated HNF4a in turn increases the transcription of CES2. Elevated CES2 enables tumor progression and maintains the progenitor phenotype.
Fig 5: Measuring hCES2 activity in pancreatic cancer cells. Probe 2 (20 µM) was incubated for 3 hours with the pancreatic cancer cell line, SU.86.86. Top row, probe 2 with SU.86.86 cells containing a vector overexpressing CES2 (CES2 OE) displayed strong red over yellow fluorescence, indicating high conversion of probe 2 to product 3. Bottom row, probe 2 with parental SU.86.86 showed weak red fluorescence but high yellow fluorescence indicative of poor conversion of probe 2 to 3. Far top right, Western blot showing hCES2 expression levels. Far bottom right, bar graph quantifying the ratio of red over yellow fluorescence for CES2 OE and parental. Light gray bars indicate pretreatment with the CES2 inhibitor, LPA, dark gray bars are no pretreatment (images in Fig. S9†). Ratios are an average of 3 regions in an 8-chambered well plate. Magnification is 20×. Scale bars = 50 µm. Statistical significance is calculated using unpaired t test compared with CES2 (***p < 0.001).
Supplier Page from MilliporeSigma for Anti-CES2 antibody produced in rabbit