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. 2022 Apr;13(2):1302-1313.
doi: 10.1002/jcsm.12917. Epub 2022 Feb 11.

Human pancreatic tumour organoid-derived factors enhance myogenic differentiation

Rianne D W Vaes  1 , David P J van Dijk  1 , Elham Aïda Farshadi  2 , Steven W M Olde Damink  1   3 , Sander S Rensen  1 , Ramon C Langen  4
Affiliations

Human pancreatic tumour organoid-derived factors enhance myogenic differentiation

Rianne D W Vaes et al. J Cachexia Sarcopenia Muscle. 2022 Apr.

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: 3.1-fold, 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: -2.1-fold, P < 0.001; PANCO-12a: -2.0-fold, P < 0.001) and myogenic factor 5 (MYF5) (PANCO-9a: -2.1-fold, P < 0.001; PANCO-12a: -1.8-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.

Keywords: Cachexia; E3 ubiquitin ligases; Myogenesis; Organoids; Skeletal muscle atrophy.

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Conflict of interest statement

The authors have nothing to disclose.

Figures

Figure 1
Figure 1
Effect of tumour‐derived factors from established 2D pancreatic cancer cell lines on C2C12 myotubes. Mature C2C12 myotubes were treated with CM from PK‐45H, PANC‐1, PK‐1, and KLM‐1. (A) After 4 h, NF‐κB luciferase activity luciferase was assessed. (B) mRNA expression of Atrogin‐1/MAFbx and MuRF1 were determined after 24 h. Data were normalized to CYPA, B2M, and RPLP0 reference genes and obtained from three independent experiments. Data are presented as mean ± SEM.
Figure 2
Figure 2
Pancreatic tumour organoid‐derived factors do not induce C2C12 muscle atrophy signalling. Mature C2C12 myotubes were treated with CM from PANCO‐9a and PANCO‐12a organoid cultures. (A) After 4 h, NF‐κB‐induced luciferase activity was assessed. (B) mRNA expression of Atrogin‐1/MAFbx, MuRF1 and (C) Redd1 was determined after 24 h. Data were normalized to CYPA, B2M, and RPLP0 reference genes and obtained from three independent experiments. Data are presented as mean ± SEM.
Figure 3
Figure 3
Tumour organoid‐derived CM transiently stimulates proliferation and increases fusion of myoblasts during differentiation. (A) Representative phase‐contrast images of C2C12 myoblasts differentiated for 5 days in DM control medium [DM, 50% (v/v) DMEM/F12] (control CM) or DM containing 50% (v/v) (tumour) organoid CM (NP; normal pancreas). Scale bar = 400 μm. (B) C2C12 myoblasts were differentiated in DM control medium [DM, 50% (v/v) DMEM/F12], DM containing 10 nM IGF‐1, or DM containing 50% (v/v) tumour organoid CM. NucLight rapid red was used to stain nuclei. Representative phase‐contrast images overlaid with red‐fluorescence images are presented. Scale bar = 400 μm. (C) The number of red stained nuclei on each individual image (12 h time interval) was quantified and plotted against time. (D) Bar graph showing the number of nuclei after 48 h of differentiation. Nuclei counts were obtained from three independent experiments. Data are presented as mean ± SEM. (E) mRNA expression of CCND1 was determined after 48 h. Data were normalized to CYPA, B2M, and RPLP0 reference genes and obtained from three independent experiments. Data are presented as mean ± SEM.
Figure 4
Figure 4
Enhanced myogenic differentiation induced by tumour organoid‐derived CM is accompanied by increased MyHC‐IIb expression and suppression of self‐renewal markers. (A) Graphical representation of the myogenic process. Myoblasts have the ability to proliferate, self‐renew, differentiate, and fuse into myotubes. During proliferation, myoblasts express the paired‐box 7 (PAX7) transcription factor. Upon myogenic commitment, a decrease in Pax7 expression results in cell cycle arrest, which is accompanied by increased expression of myogenic factor 5 (MYF5) and myoblast determination (MYOD) transcription factors. Myogenin (MYOG) is a transcription factor that is highly expressed during the fusion of myoblasts into myotubes. This protein results in the transcription of genes required for the fusion of myoblasts into myotubes, including myomaker (MYMK). mRNA expression of (B) satellite cell marker PAX7, (C) key‐myogenic regulators and muscle specific genes (MYF5, MYOD, MYOG, MCK, MYMK), and (D) Ca2+ flux regulating signalling channels (RYR1, SERCA2, CACNB1, DHPR) were determined after 48 h. (E) Expression of myosin heavy chain isoforms (MYH1, MYH2, MYH4, MYH7) was determined after 72 h. Data were normalized to CYPA, B2M, and RPLP0 reference genes and obtained from three independent experiments. Data are presented as mean ± SEM. CACNB1, calcium voltage‐gated channel auxiliary subunit beta1; DHPR, dihydropteridine reductase; MCK, muscle creatine kinase; MYH1, myosin heavy chain 1, MyHC‐IIx; MYH2, myosin heavy chain 2, MyHC‐IIa; MYH4, myosin heavy chain 4, MyHC‐IIb; MYH7, myosin heavy chain 7, MyHC‐I; RYR1, ryanodine receptor 1; SERCA2, sarcoplasmic/endoplasmic reticulum calcium ATPase 2.
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