Extracellular vesicles derived from tumour cells as a trigger of energy crisis in the skeletal muscle

Abstract

Background: Cachexia, a syndrome frequently occurring in cancer patients, is characterized by muscle wasting, altered energy and protein metabolism and impaired myogenesis. Tumour-derived microvesicles (TMVs) containing proteins, messenger RNAs (mRNAs), and non-coding RNAs could contribute to cancer-induced muscle wasting.

Methods: Differential ultracentrifugation was used to isolate TMVs from the conditioned medium of Lewis lung carcinoma and C26 colon carcinoma cell cultures. TMVs were added to the culture medium of C2C12 myoblasts and myotubes for 24-48-72 h, and the effects on protein and energy metabolism were assessed. TMVs were also isolated from the blood of C26-bearing mice. MicroRNA (miR) profile of TMVs was obtained by RNA-seq and validated by digital drop PCR. Selected miRs were overexpressed in C2C12 myoblasts to assess the effects on myogenic differentiation.

Results: Differentiation was delayed in C2C12 myoblasts exposed to TMVs, according to reduced expression of myosin heavy chain (MyHC; about 62% of controls at Day 4) and myogenin (about 68% of controls at Day 4). As for myotubes, TMVs did not affect the expression of MyHC, while revealed able to modulate mitochondria and oxidative metabolism. Indeed, reduced mRNA levels of PGC-1α (C = 1 ± 0.2, TMV = 0.57 ± 0.06, normalized fold change, P < 0.05) and Cytochrome C (C = 1 ± 0.2, TMV = 0.65 ± 0.04, normalized fold change, P < 0.05), associated with increased BNIP3 expression (C = 1 ± 0.1, TMV = 1.29 ± 0.2, normalized fold change, P < 0.05), were observed, suggesting reduced mitochondrial biogenesis/amount and enhanced mitophagy. These changes were paralleled by decreased oxygen consumption (C = 686.9 ± 44 pmol/min, TMV = 552.25 ± 24 pmol/min, P < 0.01) and increased lactate levels (C = 0.0063 ± 0.00045 nmol/μL, TMV = 0.0094 ± 0.00087 nmol/μL, P < 0.01). A total of 118 miRs were found in MVs derived from the plasma of the C26 hosts; however, only three of them were down-regulated (RNA-seq): miR-181a-5p (-1.46 fold change), miR-375-3p (-2.52 fold change), and miR-455-5p (-3.87 fold change). No correlation could be observed among miRs in the MVs obtained from the blood of the C26 host and those released by C26 cells in the culture medium. Overexpression of miR-148a-3p and miR-181a-5p in C2C12 myoblasts revealed the ability to impinge on the mRNA levels of Myf5, Myog, and MyHC (Myh4 and Myh7).

Conclusions: These results show that in C2C12 cultures, TMVs are able to affect both differentiation and the mitochondrial system. Such effects could be related to TMV-contained miRs.

Keywords: Cancer cachexia; MicroRNAs; Microvesicles; Mitochondria; Muscle wasting; Myogenesis.

Figure 1

Effect of tumour‐derived microvesicles on C2C12 myoblast differentiation. (A) Phase‐contrast microscopy of C2C12 myoblast cultures during differentiation in the presence or in the absence of C26‐MV or LLC‐MV (scale bar: 100 μm); (B) heat map representing changes in protein expression. Data are expressed as arbitrary units (AI); n = 3 for each experimental condition, statistical significance is set at P < 0.05; (C) representative western blotting pattern of protein expression reported in panel (B); (D) western blotting showing caspase‐3 expression; (E) representative blots and quantification of Sunset analysis for the assessment of protein synthesis 4 and 24 h after exposure to C26‐MV or LLC‐MV. Data are expressed as % of controls, n = 3. Significance of the differences: *P < 0.05, **P < 0.01 vs. controls. LLC, Lewis lung carcinoma; MV, microvesicles.

Figure 2

Effect of tumour‐derived microvesicles on C2C12 myotubes. (A) Immunofluorescence analysis of MyHC expression after 24 h of tumour‐derived microvesicle exposure; (B) heat map (ΔΔCt; n = 3) showing the expression of mitochondria‐related genes. (C–D) Mitochondrial respiration and oxygen consumption rate (OCR) in C2C12 myotubes exposed to LLC and C26‐MVs for 24 h (n = 3). ATP, adenosine triphosphate; BR, basal respiration; MR, maximal respiration; PL, proton leak; SRC, spare respiratory capacity. Data expressed as picomoles per minute. (E) Lactate release from C2C12 myotubes exposed to LLC‐MVs or C26‐MVs. Data are expressed as means ± SD, n = 3. Significance of the difference: *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.

Figure 3

Tumour‐derived microvesicles infused into healthy mice partially recapitulate cachexia. Changes in (A, C) gastrocnemius and (B, D) tibialis anterior mass in healthy mice infused for 48, 72, and 96 h with C26‐MV or AH130‐MV. Data (mg/10 g initial body weight, means ± SD) are expressed as % of controls, n = 5. Significance of the differences: *P < 0.05, **P < 0.01 vs. controls. MV, microvesicles.

igure 4

F miR expression profile in circulating MVs of C26‐bearing mice. (A) PCA plot (PC1 vs. PC2) representing sample variability in the library; (B) raw read abundance and molecular diversity of each sncRNA biotype; (C) top 20 miRs detected in circulating MVs; (D) volcano plot reporting all detected miRNAs in the gastrocnemius, according to P value (−log10) and fold change (log2) distribution (controls: n = 4; C26: n = 5). Significance of the difference set at P < 0.05; (E) expression of selected myomiRs in the gastrocnemius of controls and C26 hosts (means ± SEM); (F) panel representing the changes in the levels of the snoRNA Gm23011 in the gastrocnemius of the C26 hosts. For all ‘box and whisker’ plots, data are represented as: line = median, whiskers = min to max (controls: n = 4; C26: n = 5; P < 0.05). miR, microRNA; MV, microvesicles.

Figure 5

miR expression profile in MVs isolated from the C26 CM. (A) Top 20 miRs detected in MVs isolated from the C26 CM; (B) molecular diversity of each sncRNA biotype; (C) top 5 rRNAs and (D) top 5 snoRNAs in MVs isolated from the C26 CM. For all ‘box and whisker; plots, data are represented as: line = median, whiskers = min to max (n = 3). miR, microRNA; MV, microvesicles.

Figure 6

Effect of miRs contained into C26‐MVs on the expression of genes involved in myogenesis. (A) Heat map (ΔΔCt; n = 3) showing the expression of genes involved in C2C12 overexpressing specific miRs at Days 2 and 4 of differentiation. Significance of the differences: *P < 0.05, **P < 0.01 vs. SCR (scramble) for each time point. SCR, scrambled sequence.