Human pancreatic tumour organoid‐derived factors enhance myogenic differentiation

Abstract

Background Most patients with pancreatic cancer develop cachexia, which is characterized by progressive muscle loss. The mechanisms underlying muscle loss in cancer cachexia remain elusive. Pancreatic tumour organoids are 3D cell culture models that retain key characteristics of the parent tumour. We aimed to investigate the effect of pancreatic tumour organoid-derived factors on processes that determine skeletal muscle mass, including the regulation of muscle protein turnover and myogenesis. Methods Conditioned medium (CM) was collected from human pancreatic cancer cell lines (PK-45H, PANC-1, PK-1, and KLM-1), pancreatic tumour organoid cultures from a severely cachectic (PANCO-9a) and a non-cachectic patient (PANCO-12a), and a normal pancreas organoid culture. Differentiating C2C12 myoblasts and mature C2C12 myotubes were exposed to CM for 24 h or maintained in control medium. In myotubes, NF-kB activation was monitored using a NF-κB luciferase reporter construct, and mRNA expression of E3-ubiquitin ligases and REDD1 was analysed by RT-qPCR. C2C12 myoblast proliferation and differentiation were monitored by live cell imaging and myogenic markers and myosin heavy chain (MyHC) isoforms were assessed by RT-qPCR. Results Whereas CM from PK-1 and KLM-1 cells significantly induced NF-κB activation in C2C12 myotubes (PK-1: 3.1fold, P < 0.001; KLM-1: 2.1-fold, P = 0.01), Atrogin-1/MAFbx and MuRF1 mRNA were only minimally and inconsistently upregulated by the CM of pancreatic cancer cell lines. Similarly, E3-ubiquitin ligases and REDD1 mRNA expression in myotubes were not altered by exposure to pancreatic tumour organoid CM. Compared with the control condition, CM from both PANCO-9a and PANCO-12a tumour organoids increased proliferation of myoblasts, which was accompanied by significant downregulation of the satellite cell marker paired-box 7 (PAX7) (PANCO-9a: À2.1-fold, P < 0.001; PANCO-12a: À2.0-fold, P < 0.001) and myogenic factor 5 (MYF5) (PANCO-9a: À2.1-fold, P < 0.001; PANCO-12a: À1.8-fold, P < 0.001) after 48 h of differentiation. Live cell imaging revealed accelerated alignment and fusion of myoblasts exposed to CM from PANCO-9a and PANCO-12a, which was in line with significantly increased Myomaker mRNA expression levels (PANCO-9a: 2.4-fold, P = 0.001; PANCO-12a: 2.2-fold, P = 0.004). These morphological and transcriptional alterations were accompanied by increased expression of muscle differentiation markers such as MyHC-IIB (PANCO-9a: 2.5-fold, P = 0.04; PANCO-12a: 3.1-fold, P = 0.006). Although the impact of organoid CM on myogenesis was not associated with the cachexia phenotype of the donor patients, it was specific for tumour organoids, as CM of control pancreas organoids did not modulate myogenic fusion. Conclusions These data show that pancreatic tumour organoid-derived factors alter the kinetics of myogenesis, which may eventually contribute to impaired muscle mass maintenance in cancer cachexia.

Extracellular Vesicles Inhibit the Response of Pancreatic Ductal Adenocarcinoma Cells to Gemcitabine and TRAIL Treatment

“…There have been several studies considering the effects of PDAC-cell-derived CM on other stromal components such as fibroblasts [ 11 ] and vice versa [ 20 , 21 ]. This work, however, is one of the first to consider the effects of PDAC-cell-derived CM on PC cells directly and specifically during gemcitabine and TRAIL treatment.…”

“…CM broadly contains two fractions: the soluble fraction, comprising cytokines and nutrients, and an insoluble fraction, comprising extracellular vesicles. Some work has been carried out considering the effects of PDAC-derived CM on other cell types, in terms of proteomic changes in the recipient cells [ 10 ] and cellular differentiation [ 11 ]. Little to no work has been completed to investigate the role of PDAC-cell-derived CM in PDAC cells in vitro.…”

DUSP1 promotes muscle atrophy by inhibiting myocyte differentiation in cachectic patients

“…Progressive impairment and dysfunction of multiple organs are caused by cachexia that is responsible for approximately 20% of all cancer mortalities ( 2 ). Cancer cachexia remains unclear in terms of its pathophysiology, but the consensus is that there is a complex interaction between tumor and patient-derived factors which result in a negative energy balance ( 3 ). Accordingly, identifying crucial molecules that contribute to the pathogenic process of cancer cachexia could facilitate the formulation of therapeutic strategies for preventing or alleviating cachexia.…”

Patient‐derived pancreatic tumour organoid implantation establishes novel pre‐cachexia mouse models

“…Combining detailed cachexia phenotyping of patients with generation of organoids from their pancreatic tumours creates unique opportunities to identify fundamental molecular mechanisms underlying clinical cachexia phenotypes. Therefore, our group has recently generated a human pancreatic cancer organoid biobank that encompasses the whole cachexia spectrum as observed in clinical practice 22 and has shown that pancreatic cancer organoid cultures produce known cachexia‐related factors that affect skeletal muscle cell differentiation 23 …”

“…Therefore, our group has recently generated a human pancreatic cancer organoid biobank that encompasses the whole cachexia spectrum as observed in clinical practice 22 and has shown that pancreatic cancer organoid cultures produce known cachexia-related factors that affect skeletal muscle cell differentiation. 23 This study aimed to investigate the impact of implantation of pancreatic tumour organoids from patients with and without cachexia on body weight and skeletal muscle characteristics in mice.…”