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. 2021 Sep 24;13(19):4781.
doi: 10.3390/cancers13194781.

Heterogeneity of Melanoma Cell Responses to Sleep Apnea-Derived Plasma Exosomes and to Intermittent Hypoxia

Abdelnaby Khalyfa  1 , Wojciech Trzepizur  2 , Alex Gileles-Hillel  3   4 , Zhuanhong Qiao  1 , David Sanz-Rubio  5 , José M Marin  5   6 , Miguel A Martinez-Garcia  6   7 , Francisco Campos-Rodriguez  6   8 , Isaac Almendros  6   9 , Ramon Farre  6   9 , Manuel Sanchez-de-la-Torre  6   10 , Francisco García-Río  6   11 , David Gozal  1
Affiliations

Heterogeneity of Melanoma Cell Responses to Sleep Apnea-Derived Plasma Exosomes and to Intermittent Hypoxia

Abdelnaby Khalyfa et al. Cancers (Basel). .

Abstract

Obstructive sleep apnea (OSA) is associated with increased cutaneous melanoma incidence and adverse outcomes. Exosomes are secreted by most cells, and play a role in OSA-associated tumor progression and metastasis. We aimed to study the effects of plasma exosomes from OSA patients before and after adherent treatment with continuous positive airway pressure (CPAP) on melanoma cells lines, and also to identify exosomal miRNAs from melanoma cells exposed to intermittent hypoxia (IH) or normoxia. Plasma-derived exosomes were isolated from moderate-to-severe OSA patients before (V1) and after (V2) adherent CPAP treatment for one year. Exosomes were co-incubated with three3 different melanoma cell lines (CRL 1424; CRL 1619; CRL 1675) that are characterized by genotypes involving different mutations in BRAF, STK11, CDKN2A, and PTEN genes to assess the effect of exosomes on cell proliferation and migration, as well as on pAMK activity in the presence or absence of a chemical activator. Subsequently, CRL-1424 and CRL-1675 cells were exposed to intermittent hypoxia (IH) and normoxia, and exosomal miRNAs were identified followed by GO and KEG pathways and gene networks. The exosomes from these IH-exposed melanoma cells were also administered to THP1 macrophages to examine changes in M1 and M2 polarity markers. Plasma exosomes from V1 increased CRL-1424 melanoma cell proliferation and migration compared to V2, but not the other two cell lines. Exposure to CRL-1424 exosomes reduced pAMPK/tAMPK in V1 compared to V2, and treatment with AMPK activator reversed the effects. Unique exosomal miRNAs profiles were identified for CRL-1424 and CRL-1675 in IH compared to normoxia, with six miRNAs being regulated and several KEGG pathways were identified. Two M1 markers (CXCL10 and IL6) were significantly increased in monocytes when treated with exosomes from IH-exposed CRL-1424 and CRL-1625 cells. Our findings suggest that exosomes from untreated OSA patients increase CRL-1424 melanoma malignant properties, an effect that is not observed in two other melanoma cell lines. Exosomal cargo from CRL-1424 cells showed a unique miRNA signature compared to CRL-1675 cells after IH exposures, suggesting that melanoma cells are differentially susceptible to IH, even if they retain similar effects on immune cell polarity. It is postulated that mutations in STK-11 gene encoding for the serine/threonine kinase family that acts as a tumor suppressor may underlie susceptibility to IH-induced metabolic dysfunction, as illustrated by CRL-1424 cells.

Keywords: AMPK; CPAP; CRL-1424; CRL-1619; CRL-1675; exosomes; intermittent hypoxia; melanoma; miRNAs; positive airway pressure; sleep apnea.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schema illustrating the experimental design and data analyses. (A) Plasma exosomes were isolated and purified from obstructive sleep apnea (OSA) patients either before CPAP treatment (OSA-V1) or after treatment (OSA-V2). (B) Diagram illustrating cell culture exposed to normoxia and IH for 48 h, and exosomes characterization, exosomal miRNAs microarrays profiling, Gene Ontology and KEGG pathways.
Figure 2
Figure 2
Effects of plasma exosomes-derived from OSA patients before CPAP treatment (OSA-V1) and after adherent CPAP treatment for 1 year (OSA-V2) on melanoma cells proliferation. Exosomes-derived from OSA patients were incubated in 96-wells plates for 24 h. The cells were treated with CyQUANT GR dye/cell lysis buffer, and the fluorescence was measured using a microplate reader (the excitation maximum was 485 nm, and the emission maximum was 530 nm). (A) represents the average of fluorescence intensity of cell proliferation for CRL-1424 cells, (B) CRL-1619 cells, and (C) CRL-1675 cells with statistical significance set at p < 0.05 (n = 20/condition).
Figure 3
Figure 3
Effects of plasma exosomes-derived from OSA patients before CPAP treatment (OSA-V1) and after adherent CPAP treatment for 1 year (OSA-V2) on melanoma cell migration. Cells were seeded in 24-well trans-well inserts with 8 μm pore size. Cells were allowed to migrate for 40 h, and then were stained with 0.2% crystal violet solution followed by phase contrast imaging. The migration rate was determined by counting the number of cells contained in the photos of 5 different fields at original magnification. (A) represents average of different fields for each subject for each group using CRL-1424 cells, (B) CRL-1629, and (C) CRL-1675, (n = 20/group), with statistical significance set at p < 0.05.
Figure 4
Figure 4
Effects of plasma exosomes-derived from OSA patients before CPAP treatment (OSA-V1) and after adherent CPAP treatment for 1 year (OSA-V2) on the presence or absence of AMPK activation using naïve melanoma cells (CRL-1424). Cells were grown on 12-well plates and treated with exosomes for 24 h in the presence or absence of AMPK activator. In the presence of AMPK activator, cells were also grown in 12-well plates and treated with AMPK activator for 4 h, and media were then removed and replaced with new media followed by adding exosomes for 24 h. Panel (A) shows a representative image of Western blots for no exosomes, OSA-V1 and OSA-V2 on CRL-1424 cells. Panel (B) shows a histogram for the densitometric quantification of Western blots bands (n = 8/condition) with statistical significance set at p < 0.05. Panel (C) shows a representative Western blot for CRL-1424 cells (no exosomes, OSA-V1 and OSA-V2). Panel (D) shows a histogram for the densitometric quantification of Western blots bands (n = 8/condition) with significance set at p < 0.05.
Figure 5
Figure 5
Differentially expressed exosomal miRNAs derived from the supernatants of melanoma cells (CRL-1424 and CRL-1675) after exposures to normoxia (RA) and IH conditions. Panel (A) shows a principal component analysis (PCA) plot of exosomal miRNAs for CRL-1424 cells. PCA clustering of miRNA expression by subject group is denoted by different colors and reveals complete and accurate separation of miRNA expression across the subjects from the two groups. Panel (B) shows a heatmap illustrating miRNA expression patterns (dark red, increased miRNA expression, light blue, reduced miRNA expression in exosomes. Panel (C) shows the PCA of exosomal-miRNA. Panel (D) shows a heatmap illustrating miRNA expression patterns for CRL-1675 cells. The dendrograms show hierarchical clustering representing the similarities and dissimilarities in expression profiles among individuals and miRNAs. In silico target gene predictions were used for GO, KEGG pathways, and gene network interactions.
Figure 5
Figure 5
Differentially expressed exosomal miRNAs derived from the supernatants of melanoma cells (CRL-1424 and CRL-1675) after exposures to normoxia (RA) and IH conditions. Panel (A) shows a principal component analysis (PCA) plot of exosomal miRNAs for CRL-1424 cells. PCA clustering of miRNA expression by subject group is denoted by different colors and reveals complete and accurate separation of miRNA expression across the subjects from the two groups. Panel (B) shows a heatmap illustrating miRNA expression patterns (dark red, increased miRNA expression, light blue, reduced miRNA expression in exosomes. Panel (C) shows the PCA of exosomal-miRNA. Panel (D) shows a heatmap illustrating miRNA expression patterns for CRL-1675 cells. The dendrograms show hierarchical clustering representing the similarities and dissimilarities in expression profiles among individuals and miRNAs. In silico target gene predictions were used for GO, KEGG pathways, and gene network interactions.
Figure 6
Figure 6
Gene network representation of interactions between gene target predictions of exosomal-miRNAs derived from melanoma cells (CRL-1424) exposed to normoxia or IH conditions. Three gene networks were generated from gene lists presented in Table 1. Panel (A) represents the gene network for the AMPK pathway, Panel (B) MAPK pathway, and Panel (C) cAMP pathway.
Figure 6
Figure 6
Gene network representation of interactions between gene target predictions of exosomal-miRNAs derived from melanoma cells (CRL-1424) exposed to normoxia or IH conditions. Three gene networks were generated from gene lists presented in Table 1. Panel (A) represents the gene network for the AMPK pathway, Panel (B) MAPK pathway, and Panel (C) cAMP pathway.
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