Small molecular weight epigenetic inhibitors modulate the extracellular matrix during pancreatic acinar ductal metaplasia

Abstract

The pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment is distinguished by a high degree of fibrosis and inflammation, known as desmoplasia. Desmoplasia increases the stromal deposition and extracellular matrix (ECM) stiffness observed in the tumor microenvironment, contributing to the dampened penetration of pharmacological agents. The molecular and biophysical composition of the ECM during the earliest cellular changes in the development of PDAC, i.e. acinar ductal metaplasia (ADM), has not been extensively explored. We report that the mRNA expression of key protein components of the ECM increases during ADM in p48^Cre/+;LSL-Kras^G12D (KC) mouse acinar organoids cultured in Matrigel. Treatment of the organoids with small molecular weight epigenetic modulating compounds that inhibit or reverse ADM (largazole, FK228 and chaetocin) dramatically reduced the tissue mRNA expression of collagens, hyaluronan synthase, laminin and fibronectin. The storage moduli, determined by video tracking of fluorescent nanoparticles embedded into the Matrigel, increased during ADM and was reduced following treatment with the epigenetic modulating compounds. We report that the ECM of mouse organoids stiffens during ADM and is further enhanced by the presence of mutant Kras. Moreover, select HDAC and HMT inhibitors reduced the mRNA expression of ECM components and ECM stiffness during inhibition and reversal of ADM, suggesting that these compounds may be useful as adjuvants to enhance the tumor penetration of agents used to treat PDAC.

Keywords: Acinar ductal metaplasia; Biomechanics; Microrheology; Pancreas plasticity; Pancreatic cancer.

Fig. 1.. Expression of extracellular matrix components during ADM.

Pancreatic acinar cells from KC mice underwent ADM over 3 days of culture on Matrigel. Shown are the brightfield images for the (A) Day 0 (primarily acinar cell clusters) and (B) Day 3 (primarily ductal cells) cultures (4 × magnification). The mRNA expression of (C) collagens (D) hyaluronan synthase and (E) matrix metalloproteinases at Day 0 and Day 3 of ADM are presented. The mean data (biological replicates) ± SD are shown. *p < 0.05; **, p < 0.005 and ****, p < 0.0005.

Fig. 2.. Assay validation for video particle tracking analysis of shear modulus.

(A) Viscosity of glycerol solutions as determined by video particle tracking analysis compared to literature values. (B) Storage modulus (reported at 10 Hz) of Matrigel suspension of varying degrees of stiffness using video particle tracking. 1 × represents the Matrigel/media composition used in the cell culture experiments.

Fig. 3.. ECM stiffness increases in KC mouse organoids undergoing ADM.

(A) Storage moduli (reported at 10 Hz) during the progression of ADM for Cre mouse organoids. (B) Microscopic counting of acinar and ductal cells to obtain the percent of ADM for Cre (n = 3) and KC (n = 3) ADM organoids. (C) Storage moduli (reported at 10 Hz) during ADM progression for Cre and KC mouse organoids. Each data point is from an independent replicate; mean data (biological replicates) ± SD are shown. *p < 0.05.

Fig. 4.. Gene expression and ADM storage moduli changes by epigenetic compound treatment.

(A) ADM inhibition and reversal treatment schema in KC mouse organoid cultures. (B) Heatmap of the mRNA expression of ECM-related genes as determined by RNA sequencing in KC cultures treated with 1 μM chaetocin, 10 μM largazole (LZ) dimer and 1 μM FK228 following treatment in the ADM reversal mode. Storage moduli (reported at 10 Hz) of KC mouse organoids during ADM inhibition and reversal modes following treatment of (C) 1 μM TSA, (D) 10 μM largazole dimer, (E) 1 μM FK228 and (F) 1 μM chaetocin. The mean data (biological replicates) ± SD are shown.