cancers Article 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 and David Gozal 1, * 1 Department of Child Health and the Child Health Research Institute, University of Missouri School of Medicine, Columbia, MO 65201, USA; khalyfaa@health.missouri.edu (A.K.); qiaoz@health.missouri.edu (Z.Q.) 2 Department of Respiratory and Sleep Medicine, INSERM Unit 1063, Angers University Hospital, 49000 Angers, France; wotrzepizur@chu-angers.fr 3 Pediatric Pulmonology and Sleep Unit, Hadassah Medical Center, Jerusalem 9987500, Israel; alex.gileles@mail.huji.ac.il 4 Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9987500, Israel 5 Translational Research Unit, Hospital Universitario Miguel Servet, IISAragon, Department of Medicine, University of Zaragoza, 50002 Zaragoza, Spain; dsanz@iisaragon.es (D.S.-R.); jmmarint@unizar.es (J.M.M.) 6 CIBER de Enfermedades Respiratorias, ISCIII, 28012 Madrid, Spain; martinez_miggar@gva.es (M.A.M.-G.);  franciscoj.campos.sspa@juntadeandalucia.es (F.C.-R.); isaac.almendros@ub.edu (I.A.); rfarre@ub.edu (R.F.);  manuel.sanchez@udl.cat (M.S.-d.-l.-T.); francisco.garcia@uam.es (F.G.-R.) Citation: Khalyfa, A.; Trzepizur, W.; 7 Pneumology Department, University and Polytechnic la Fe Hospital, 46026 Valencia, Spain 8 Respiratory Department, Hospital Universitario de Valme, 41014 Sevilla, Spain Gileles-Hillel, A.; Qiao, Z.; 9 Unitat de Biofísica i Bioenginyeria, Facultat de Medicina i Ciencies de la Salut, Sanz-Rubio, D.; Marin, J.M.; Martinez-Garcia, M.A.; Universitat de Barcelona-Institut Investigacions Biomediques August Pi Sunyer, 08036 Barcelona, Spain 10 Group of Precision Medicine in Chronic Diseases, Hospital Arnau de Vilanova-Santa Maria, IRBLleida, Campos-Rodriguez, F.; Almendros, I.; Department of Nursing and Physiotherapy, Faculty of Nursing and Physiotherapy, University of Lleida, Farre, R.; et al. Heterogeneity of 25198 Lleida, Spain Melanoma Cell Responses to Sleep 11 Respiratory Department, Hospital Universitario La Paz-IdiPAZ, Universidad Autónoma de Madrid, Apnea-Derived Plasma Exosomes 28046 Madrid, Spain and to Intermittent Hypoxia. Cancers * Correspondence: gozald@health.missouri.edu 2021, 13, 4781. https://doi.org/ 10.3390/cancers13194781 Simple Summary: Obstructive sleep apnea has been implicated in deleterious effects on cancer incidence and outcomes. Among the cancers identified as affected by sleep apnea, melanoma has Academic Editors: Jozsef Tímar and consistently emerged as particularly susceptible. Furthermore, evidence suggests that exosomes, Andrea Ladányi microparticles that originate from all cells and contain a variety of biologically active molecules such as microRNAs, can modify cancer cell properties. Here, we examined whether exosomes from sleep Received: 1 September 2021 Accepted: 21 September 2021 apnea patients can alter melanoma cell properties, and whether such effects were attributable to Published: 24 September 2021 selected microRNA candidates, and affected melanoma cells harboring different mutations differently. In these experiments, the divergent responses of three melanoma cell lines to exosomes from sleep Publisher’s Note: MDPI stays neutral apnea patients before and after treatment indicated that STK11 gene mutations in melanoma cells with regard to jurisdictional claims in make them particularly responsive to sleep apnea by altering cellular metabolism, and that specific published maps and institutional affil- microRNAs appear to underlie such effects. iations. 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 Copyright: © 2021 by the authors. patients before and after adherent treatment with continuous positive airway pressure (CPAP) on Licensee MDPI, Basel, Switzerland. melanoma cells lines, and also to identify exosomal miRNAs from melanoma cells exposed to This article is an open access article intermittent hypoxia (IH) or normoxia. Plasma-derived exosomes were isolated from moderate-to- distributed under the terms and severe OSA patients before (V1) and after (V2) adherent CPAP treatment for one year. Exosomes conditions of the Creative Commons were co-incubated with three3 different melanoma cell lines (CRL 1424; CRL 1619; CRL 1675) that Attribution (CC BY) license (https:// are characterized by genotypes involving different mutations in BRAF, STK11, CDKN2A, and PTEN creativecommons.org/licenses/by/ genes to assess the effect of exosomes on cell proliferation and migration, as well as on pAMK activity 4.0/). Cancers 2021, 13, 4781. https://doi.org/10.3390/cancers13194781 https://www.mdpi.com/journal/cancers Cancers 2021, 13, 4781 2 of 25 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: sleep apnea; positive airway pressure; CPAP; exosomes; melanoma; miRNAs; AMPK; CRL-1424; CRL-1619; CRL-1675; intermittent hypoxia 1. Introduction Obstructive sleep apnea (OSA) is a frequent disease that occurs among individuals of all ages, and has recently been associated with an increased risk of cancer incidence and mortality [1,2]. Among the potential tumors exhibiting significant associations with OSA related to incidence and outcomes [3–8], cutaneous melanoma has emerged as one of the malignancies that most consistently emerges in epidemiological studies [9], while such association may not be as reliably present for other tumor types [8,10,11]. Previously, in murine models we have collected evidence supporting the conceptual framework that sleep apnea, i.e., intermittent hypoxia (IH) and sleep fragmentation (SF) could causally modify cancer-related biological processes. These observations prompted us to hypothesize that selected types of solid cancers may be particularly responsive to the underlying presence of OSA, and that their incidence or outcomes may be adversely affected in OSA patients [12,13]. In the United States, cutaneous melanoma rates (the most aggressive skin tumor type) have continuously increased over the last two decades, and continue to increase. In the framework of OSA, in addition to the aforementioned epidemiological evidence, several studies using animal models to study melanoma tumor growth [14,15], metastasis [12] and potential interactions with obesity [16] confirmed that intermittent hypoxia mimicking the oscillations in oxygen tension that characterize OSA induce significant changes in the biological properties of melanoma cells, thereby promoting accelerated proliferation, invasion and resistance to therapy [12,16–21]. However, it has also become apparent that there is considerable variability in the response to hypoxia by any type of malignant tumor, and that such heterogeneity may account for the conflicting findings in epidemiological studies attempting to link between OSA and cancer [22]. Thus, improved understanding of the molecular and cellular processes underlying melanoma plasticity and heterogeneity is of paramount importance. Tumor cells and adjoining cells within the tumor microenvironment can communi- cate by a variety of processes involving cell–cell interactions as well as paracrine mecha- nisms [23,24]. More recently, exosomes have been suggested as one of the key mechanisms of intercellular communication in the tumor microenvironment, in light of their ubiquitous Cancers 2021, 13, 4781 3 of 25 presence in bodily fluids, as well as their ability to reliably deliver genetic messages be- tween cells, ultimately altering the phenotype of the target cells [25–34]. In previous studies, we showed that both exosomes derived from plasma of mice exposed to IH or SF emulating the perturbations of OSA, and exosomes from plasma samples of patients with OSA altered tumor growth and enhanced specific properties of cancer tumor cell cultures [27,28]. In this study, we hypothesized that circulating exosomes from patients with OSA will induce changes in naïve human melanoma cells in vitro, but that such effects may vary among melanoma cell lines, and that such differences will also be reflected in the exosomal miRNA cargo originating from melanoma cells exposed to IH in vitro. Furthermore, we aimed to assess the effect of exosomes derived from IH-exposed melanoma cells on THP1 monocytes or THP1 differentiated macrophages. 2. Materials and Methods 2.1. Human Subjects Human studies were carried out in the Sleep Clinic of the Hospital Universitario Miguel Servet, a large teaching university hospital in Zaragoza, Spain, as part of the EPIOSA study (NCT02131610) [35] and in a multicenter trial conducted by the Spanish Sleep Net- work to assess the efficacy of CPAP in women with OSA (NCT02047071; [36,37]. All studies were approved by the appropriate human subject bioethics committees and all partici- pants provided signed informed consents. Adult patients (mean age: 48.5 ± 6.7 years; male:female: 1:1; range: 20–65 years) with polysomnographically or polygraphically diag- nosed OSA at baseline (V1) and after 12 months of adherent CPAP treatment (6.0 ± 2.1 h/night throughout the duration) (V2) were selected into this study (n = 26). Data from all sleep studies were scored using AASM guidelines [38] by trained personnel that were blinded to the aims or nature of the current study. Optimal titration of CPAP was implemented by using auto-CPAP (Autoset-T; ResMed, Sydney, Australia), according to the guidelines of the Spanish Sleep and Breathing Group [39]. Fasting blood samples were drawn at V1 and V2 using a 21G butterfly needle into ethylenediaminetetraacetic acid (EDTA) (PreAnalytix GmbH, Bene, Hombrechtikon, Switzerland). Plasma was separated by centrifugation and stored at −80 ◦ C until analy- ses. A detailed description of Materials and Methods is provided in the Supplementary Materials. These data include exosome isolation, characterization from plasma and cell culture conditions, in vitro proliferation and migration, Western blotting, semi-quantitative reverse transcription–PCR (qRT-PCR), and miRNA isolation from exosomes derived melanoma cells. 2.2. Cell Lines and Culture Reagents Three human primary melanoma cells, namely CRL-1424, CRL-1619 and CRL-1675 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human skin malignant melanoma derived from adult male (31 years old) were grown in McCoy’s 5a Medium Modified (CRL-1424, Cat. # 302007). Human skin malignant melanoma derived from adult female (54 years old) were grown in Dulbecco’s Modified Eagle’s Medium (CRL-1619, Ca. # 302002). Human malignant melanoma cells derived from metastatic site of subcutaneous tissue derived from an adult male (29 years old) were grown in McCoy’s 5a Medium Modified (HTB-63, Cat. # 302007). The three cell lines were selected based on the fact that they have different known gene mutations: CRL-1424 is mutant in BRAF (+/+), CDKN2A (+/+), and STK11 (+/+); CRL-1619 is mutant in BRAF (+/−), CDKN2A (+/+); CRL-1675 cell is mutant in CDKN2A (+/+), not STK11, but also mutant in PTEN (+/+). Cells and culture media were obtained from ATCC (Manassas, VA, USA). Cells were grown in the recommended medium supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), and an antibiotic/antimycotic solution at final concentrations of 100 U/mL penicillin, 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in T-25 tissue culture flasks (Techno Plastic Cancers 2021, 13, 4781 4 of 25 Products AG, Trasadingen, Switzerland) in a standard humidified incubator with 5% CO2 balanced-air at 37 ◦ C. 2.3. Exosome Isolation 2.3.1. Plasma Exosomes Exosomes were isolated from plasma samples using the Total Exosome Isolation Kit (TEIR) according to the manufacturer’s protocol instructions (Life Technologies, Carls- bad, CA, USA). After isolation, exosomes were carefully characterized as previously reported [32,40–42] based on approaches that strictly adhere to MISEV2018 guidelines [43]. Briefly, plasma was centrifuged at 2000× g for 20 min to remove cell/debris. The super- natants were collected. Plasma was then pre-filtered using 0.22 µm centrifuge tube filter (Sigma-Aldrich, St. Louis, MO, USA), and for 120 µl of plasma, 60 µl 1× PBS was added followed by 6 µl of proteinase K (Life Technologies, Carlsbad, CA, USA). After incubation at 37 ◦ C for 12 min, 36 µL of TEIR buffer was added and incubated immediately at 4 ◦ C for 35 min. This was followed by centrifugation at 10,000× g at 22 ◦ C for 6 min, and pellets were solubilized in 60 µL of 1× PBS. Plasma samples were filtered again using 0.22 µm centrifuge tube filter (Sigma-Aldrich, St. Louis, MO, USA) to remove any large molecules. Exosomes samples were stored at −20 ◦ C until use. 2.3.2. Cell Culture Exosomes Exosomes derived from melanoma cell supernatants after exposures to normoxia or IH were isolated using TEIR as per the manufacturer’s protocol. Briefly, supernatants were centrifuged at 2000× g for 20 min followed by filtration using 0.22 µM filter. The reagent (500 µL) was added to 1000 µL ml of cell media sample, and the solution was incubated overnight at 4 ◦ C. The precipitated exosomes were recovered by standard centrifugation at 10,000 × g for 75 min, and the pellet was then re-suspended in PBS and stored at −20 ◦ C. 2.4. Exosome Uptake by Melanoma Cells Human plasma exosomes were labeled with the fluorescent linker PKH67-Green (Sigma-Aldrich, St. Louis, MO, USA). Second, exosomes derived from human melanoma cells exposed to either IH or normoxia for 48 h were labeled with fluorescent linker PKH26- Red (Sigma-Aldrich, St. Louis, MO, USA). For details about IH exposures, please see IH section below. Labeled exosomes were further incubated at 37 ◦ C for 10 min. Samples were filtered through a microspin column G-25 (Sigma-Aldrich, St. Louis, MO, USA) to remove unbound dye. The reaction of all samples was stopped by adding ExoQuick-TC reagent (System Biosciences, Palo Alto, CA, USA), followed by placing the labeled exosome samples at 4 ◦ C for 40 min following by centrifugation for 3 min at 14,500× g. The pellets were suspended in 1× PBS, and the labeled exosomes were added to human melanoma cells for 24 h in a cell culture incubator at 37 ◦ C. The cell membranes were permeabilized by incubation with 0.25% (v/v) Triton-X-100 in PBS for 10 min. The labeled exosomes were monitored for delivery into the intended cells using a Leica SP5 Tandem Scanner Spectral 2-photon confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA) with a 63× oil-immersion lens. As negative control, PKH67-Green was prepared and added to each cell plate with all reagents, but without any exosomes, to monitor background unincorporated dyes. Cell nuclei were imaged by staining with DAPI at a concentration of 1 µg/mL in PBS (Life Technologies, Carlsbad, CA, USA) at room temperature for 5 min [44–46]. 2.5. Exosome Size Determination and Quantification Morphological evaluation of exosomes was performed using electron microscopy as previously described [32,40]. Exosomes derived from patient plasma or melanoma cell cultures’ sizes distribution were assessed using electron microscopy (model Tecnai F30 at 300 KV; Thermo-Fisher, Hillsboro, OR, USA). Exosomes were placed on Formvar-carbon coated electron microscopy grids and allowed to stand for 5–10 min for exosome adsorption. Cancers 2021, 13, 4781 5 of 25 Grids with adherent exosomes were washed three times with 25 µL drops of DPBS, then were fixed with 2% paraformaldehyde in DPBS for 7 min, followed by incubation with 25 µL drops of 2% uranyl acetate and then examined by electron microscopy. Size distribution of exosomes was inspected and quantified as previously described [32]. Exosomes, either from plasma or cell cultures, were quantified using either enzymatic fluorescent assays (FluoroCet #FCET96A quantitation kit; System Biosciences, Mountain View, CA, USA) according to the manufacturer’s protocol, or with the Spectradyne nCS1 nanoparticle analyzer (Spectradyne, Cherry Hills, CA, USA) which employs resistive pulse sensing technology, and further analyzed for selective subpopulation surface markers using FACS analysis (FACSCalibur, BD Biosciences), whereby purified exosomes were incubated with magnetic beads 9.1 µm with exosomes markers under gentle agitation at 4 ◦ C overnight according to manufacturer’s recommendations. Control experiments were also performed with all the reagents and beads, but in the absence of exosomes. Twenty- five thousand events were computed, and data were analyzed using FlowJo Software (Tree Star, Inc, Ashland, OR, USA). 2.6. Proliferation and Migration Assays All three human melanoma cell lines were used for experiments with OSA-derived plasma exosomes. Cells (2 × 104) were grown as described above, and a similar number of exosomes derived from either OSA-V1 or OSA-V2 were added for 24 h. Medium was removed from the plates 24 h after adding the exosomes, and the monolayers were rinsed with cold PBS and 200 µL of CyQUANT GR dye/cell lysis buffer (included in the CyQUANT kit, Invitrogen, Eugene, OR, USA) was added to each well. Fluorescence was measured using a microplate reader. The excitation maximum was 485 nm, and the emission maximum was 530 nm. Migration assays were performed using human melanoma cells in 24-well trans-well inserts with 8 µm pore size (Corning, NY, USA). Cells (3.0 × 104) were cultured in the top in serum free medium in single culture, with and without exosomes isolated from OSA-V1 or OSA-V2 conditions. The cells were allowed to migrate for 40 h from the starved medium side to the lower compartment while using 10% FBS as chemoattractant. Cells were stained with 0.2% crystal violet solution (Sigma-Aldrich, St. Louis, MO, USA) and phase contrast images of the migrated cells were imaged using NIS-Elements AR vs. 3.10 imaging software. Sections were visualized with a Nikon Elipse Ti microscope using 4× objectives and photographed with a Cool SNAP EZ camera. The migration rate was determined by counting the number of cells contained in the photos of 5 random-selected different fields at original magnification by operators who were blinded to the experimental conditions. 2.7. p-AMPK and p-AMPK Activator Western Blots Human melanoma cells (CRL-1424) were grown as described above in 12-well plates in 10% FBS for 24 hr. Culture media were then replaced with 10% depleted FBS, and the cells were used under two different conditions. First, cells were treated with exosomes derived from OSA-V1, OSA-V2 (equal number of exosomes) or without exosomes for 24 h. Second, cells were treated with 15 µM of p-AMPK activator (PF06409577, Sigma- Aldrich (St-Louis, MO, USA) for 4 h, and equal numbers of exosomes were added to each individual well-plate for 24 h. Protein cell lysed concentrations were quantified using the BCA kit (Life Technologies, Rand Island, NY, USA). Equal amounts of total protein were electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE, [10% and transferred into nitrocellulose membrane (Millipore, Billerica, MA, USA)]. The membranes were blotted with 5% skimmed milk and subsequently probed overnight at 4 ◦ C with primary antibodies. The membranes were incubated with polyclonal p-AMPK ((Phospho-AMPKα (Thr172) (40H9), Cell Signaling, Danvers, MA, USA)) and AMPK polyclonal antibody (AMPKα, D5A2, Cell Signaling, Danvers, MA, USA) overnight at 4 ◦ C, then washed three times for 10 min each with 25 mM Tris-HCL, pH 7.4, 3.0 mM KCl, Cancers 2021, 13, 4781 6 of 25 140 mM NaCl, and 0.05% Tween-20 (TBST), incubated with anti-rabbit immunoglobulin G:HRP-linked antibody (Cell Signaling Technology; 1:2000 dilution in blocking buffer with gentle agitation for 1 h at room temperature), and finally immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Chemidoc XRS+; Bio- Rad, Hercules, CA, USA), and quantified by the Image Lab software (Bio-Rad, Hercules, CA, USA). The intensity of p-AMPK was adjusted by using total AMPK as a control. All the whole western blot figures can be found in the supplementary materials. 2.8. Cell Cultures and Intermittent Hypoxia Melanoma CRL-1424 cells or CRL-1675 cells were grown in T25 flasks in recom- mended media as described above, supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), and an antibiotic/antimycotic solution at final concentrations of 100 U/mL penicillin, 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Subsequently, cell media were replaced with 10% depleted FBS (System Biosciences, LLC, Palo Alto, CA, USA) and cells were exposed to normoxia or IH for 48 h. Cells were placed in a cell culture incubator at 37 ◦ C and 100% humidity, and connected to a gas source providing a gas mixture with either 21% O2 (normoxia) or 1% O2 (hypoxia) for 48 h alternating every 30 min. This profile generated nadir partial pressure of oxygen in the 3–7 mmHg range [47]. Cell culture supernatants were collected, and exosomes were isolated as described above. 2.9. qRT-PCR Total RNA was extracted using RNeasy Kit (Qiagen, MD, USA) following the manu- facturer’s instructions. Quantitative polymerase chain reaction analysis (qRT-PCR) was performed for selected messenger RNAs (mRNAs) using ABI PRISM 7500 System (Applied Biosystems, Foster City, CA, USA). For RNA, cDNA was synthesized using 200 ng of total RNA using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). The thermal cycling conditions were 95 ◦ C for 10 min, followed by 40 cycles at 94 ◦ C for 15 s, 60 ◦ C for 30 s, and 72 ◦ C for 30 s. Human 18S gene was used as a reference gene to normalize the expression ratios. The cycle number (Ct) values were averaged and the difference between the housekeeping gene Ct and the gene of interest Ct was calculated to determine the relative expression of the gene of interest using the 2−∆∆Ct method. 2.10. Exosomal miRNA Isolation from Melanoma Cells Two different melanoma cells CRL-1424 and CRL-1675 were used and grown at 37 ◦ C for 48 h supplement with 10% depleted FBS. Supernatants were collected and centrifuged at 2000× g for 35 min. Exosomes were isolated using TEIR for cell culture media (Life Technologies, Grand Island, NY, USA) as described by the manufacture protocol. The supernatant was transferred into a new centrifuge tube and 500 µL of TEIR was added for 1 mL of cell culture media, and further incubated at 4 ◦ C overnight for 16 h. Following incubation, the samples were centrifuged at 10,000× g for 75 min at 4 ◦ C. The supernatants were discarded, and the pellet was re-suspended in 1X PBS. 2.10.1. miRNAs Microarray Processing and Analysis Total RNA including miRNA was isolated from purified exosomes obtained from cells either exposed to normoxia or IH for 48 h using miRNeasy Mini Kit-column-based system (Cat. # 217004, Qiagen, Germantown, MD, USA) following the manufacturer’s instructions (Qiagen, Turnberry Lane, Valencia, CA, USA) [48]. Exosomes were homogenized in QIAzol Lysis Reagent. After addition of chloroform, the homogenate was separated into aqueous and organic phases by centrifugation using the miRNeasy Mini Kit (Qiagen® ) which combines phenol/guanidine-based lysis of samples and a silica membrane column-based purification as described by the manufacturer protocol [47]. The RNA quality and integrity were determined using the Eukaryote Total RNA Nano 6000 LabChip assay (Agilent Technologies, Santa Clara, CA, USA) on the Agilent 2100 Bioanalyzer [47]. Cancers 2021, 13, 4781 7 of 25 Each sample was prepared according to Agilent’s miRNA recommended approach using the one-color technique and profiled on the Agilent human miRNA microarray (Agilent Technologies, Santa Clara, CA, USA) [47]. Each array consisted of 60-mer DNA probes synthesized in situ that represent 2006 human miRNAs. The labeled RNAs were hybridized to custom human miRNAs microarrays 8 × 60 K (Agilent Technologies, Santa Clara, CA, USA). Following hybridization and washing, the arrays were scanned with an Agilent microarray scanner using high dynamic-range settings as specified by the man- ufacturer. Microarray results were extracted using Agilent Feature Extraction software (v12.0) to quantify signal intensities [32,47]. The quality control for each miRNA was evaluated based on their Agilent Feature Extraction quality control report. miRNA mi- croarray data were log-base 2 transformed and quantile normalized. The normalized data were expressed as the difference of log of g processed signal (Agilent Feature Extraction). Undetected probes were excluded from further analysis. Background-subtracted intensities were normalized for detected miRNA probes using the quantile method across all miRNA microarray experiments as described previously. 2.10.2. Target Gene Prediction and Function Enrichment Analysis To identify the underlying target genes of DE miRNAs, the miRWalk2.0 database ( zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/ accessed on 16 July 2021) was used. This database contained a total of 12 bioinformatic algorithms, including DIANA microT (version 4.0), DIANA microT CDS (version 5.0), miRanda rel (version 1.0), mirBridge (version 1.0), miRDB (version 4.0), miRmap (version 1.0), miRNAMap (version 1.0), PicTar (version 2.0), PITA (version 6.0), RNA22 (version 2.0), RNAhybrid (version 2.1) and Tar- getscan (version 6.2). The Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool (version 6.8; http://david.abcc.ncifcrf.gov, accessed on 16 July 2021) was used for Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of all target genes of DE miRNAs. A p value of <0.05 was set as the cut off value. 2.11. THP-1 Monocytes and Differentiation to Macrophages THP-1 cells were used in two different conditions. First, THP-1 monocytes (0.2 × 106) cells were seeded in 12-well plates for 48 h. Second, THP-1 (0.2 × 106) cells were differ- entiated into macrophages in 12-well plates containing 2 mL of the RPMI 1640 medium containing 150 µM phorbol 12-myristate 13-acetate (PMA) for 48 h at 5% CO2 at 37 ◦ C. Media were replaced with depleted FBS and exosomes derived from IH or normoxia were added for 24 h. Cells were collected and used for either flow cytometry to evaluate cell polarity using specific antibodies or for total RNA isolation to evaluate macrophage-related gene expression. For immunofluorescence analysis, cells were stained with different antibodies such as APC/Cy7 anti-human CD11b (Cat. # 301341), antibody. Cells were stained with 1µg antibody in 100 µL. Cell Staining Buffer was then added and further incubated at 4 ◦ C for 20 min in the dark. Samples were washed twice with Cell Staining Buffer and pellets were resuspended in 0.5 mL of Cell Staining Buffer. Flow cytometric measurements were performed using a 4-color FACS Calibur (Becton Dickinson, San Jose, CA, USA). Following incubation, samples were washed and resuspended in PBS and 10,000 events were recorded. 2.12. Data Analyses Results are presented as means ± SD, unless stated otherwise. Comparisons of all quantitative data groups were performed using unpaired Student t-tests, non-parametric tests, or ANOVA procedures as appropriate depending on the variable distributions. Statistical analyses were performed using SPSS version 21.0 (SPPS Inc., Chicago, IL, USA). For all comparisons, a two-tailed p < 0.05 was considered to define statistical significance. 2.12. Data Analyses Results are presented as means ± SD, unless stated otherwise. Comparisons of all quantitative data groups were performed using unpaired Student t-tests, non-parametric tests, or ANOVA procedures as appropriate depending on the variable distributions. Sta- Cancers 2021, 13, 4781 tistical analyses were performed using SPSS version 21.0 (SPPS Inc., Chicago, IL, USA).8 of 25 For all comparisons, a two-tailed p < 0.05 was considered to define statistical significance. 3. Results 3. Results 3.1. Human 3.1. Human Subjects Subjects Subjectssuffering Subjects sufferingfrom frommoderate moderateto tosevere severeOSA OSAwerewereassessed assessed at at two two different different times: times: (A) untreated (A) untreated (OSA-V1; (OSA-V1; nn == 26; 26; 20 20males), males),and and(B) (B)treated treatedwith withCPAP CPAPforfor a period a periodof of 12 months 12 months(OSA-V2, (OSA-V2,n n= = 26), 26),asasshown shownininFigure Figure1A. 1A.There Therewere were no no significant differencesin significant differences in subject BMI between the V1 and V2 conditions (31.7 ± 3.7 2 kg/m2). Twelve subject BMI between the V1 and V2 conditions (31.7 ± 3.7 kg/m ). Twelve of the subjectsof the subjects werehypertensive were hypertensiveandand1616reported reportedexcessive excessivedaytime daytimesleepiness sleepiness(Epworth (Epworthscores scores>>12). 12). (A) Human subjects OSA-V2 (Post-CPAP OSA-V1 treatment for 12 months) Exosomes Exosomes Exosome uptake p-AMPK/total-AMPK pAMPK activation Melanoma cell - Proliferation - Migration (B) Melanoma cells 1424 and 1675 cells Normoxia Intermittent hypoxia Exosomes Exosomal miRNAs THP-1 THP-1 Monocytes Macrophages Gene ontology qRT-PCR qRT-PCR Markers Markers KEGG pathways M1 and M2 M1 and M2 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. However, the apnea–hypopnea index (AHI), a measure of OSA severity was markedly higher among OSA-V1 subjects (70.13 ± 16.78 events/h) compared to OSA-V2 (2.71 ± 1.32 events/h; p = 0.0001). In addition, significant differences emerged in diastolic blood pressure between OSA-V1 (82.31 ± 8.59 mmHg) and OSA-V2 (73.15 ± 10.12 mmHg; p = 0.01). In parallel, three commercially available melanoma cell lines (CRL-1424, CRL- 1619, and CRL-1675) were exposed to IH or normoxia as indicated in Figure 1B. Cancers 2021, 13, 4781 9 of 25 3.2. Exosome Characterization and Uptake Exosomes derived from OSA subjects or from the supernatants of the cell lines were characterized as mentioned. Exosome counts were determined in OSA-V1 (4.53 × 108 /mL) or OSA-V2 (4.48 × 108 /mL; p = 0.24, n = 20/group). The purity of the plasma-derived exosomes was also confirmed by TEM (Figure S1A). Flow cytometry analyses for exosome surface markers including tetraspanin (CD63) in the presence or absence of exosomes were performed as shown in Figure S1B. In plasma exosomes labeled with PKH67, we observed exosome internalization in melanoma cells as fluorescent membrane dye (Figure S2), il- lustrating that exosomes were functionally intact. Similar procedures were applied for exosomes derived from melanoma cell cultures, including Spectradyne nCS1, flow cytome- try (CD63), and TEM (University of Missouri Microscopy Core, Columbia, MO, USA) as described in Methods. Exosome counts in CRL-1424 cell culture supernatants exposed to IH (3.46 ± 0.23 × 108 /mL) or normoxia (3.46 ± 0.22 × 108 /mL; n = 8/group; p = 0.16), and in CRL-1675 cell culture supernatants (3.31 ± 0.21 × 108 /mL in IH; 3.18 ± 0.19 × 108 /mL in normoxia; n = 8/group; p = 0.23) were similar and unaltered by the type of exposure. 3.3. Cell Proliferation and Migration The effect on proliferation and migration of plasma-derived exosomes on melanoma cells was assessed in CRL-1424, CRL-1619, and CRL-1675 cell lines. As shown in Figure 2, exosomes derived from OSA-V1 increased proliferation of CRL-1424 cells when com- pared to OSA-V2 (average of fluorescence signal OSA-V1: 270,303.88 ± 44,237.86 ×103 vs. OSA-V2: 204,727.75 ± 21,704.25 × 103 , p = 0.001, n = 12; Figure 2A); however, no Cancers 2021, 13, x FOR PEER REVIEW 10 of significant differences in proliferation emerged for either CRL-1619 or CRL-1675 25 lines cell after treatment with exosomes (Figure 2B,C). Figure 2. Effects of plasma exosomes-derived from OSA patients before CPAP treatment (OSA-V1) and after adherent CPAP Figure 2. Effects of plasma exosomes-derived from OSA patients before CPAP treatment (OSA-V1) and after adherent treatment CPAP for 1 yearfor treatment (OSA-V2) on melanoma 1 year (OSA-V2) cells proliferation. on melanoma Exosomes-derived cells proliferation. fromfrom Exosomes-derived OSAOSA patients were patients incubated were incu- in 96-wells batedplates for 24plates in 96-wells h. Theforcells 24 h.were The treated with cells were CyQUANT treated GR dye/cell with CyQUANT lysis buffer, GR dye/cell and the lysis buffer, fluorescence and was measured the fluorescence was usingmeasured using reader a microplate a microplate reader (themaximum (the excitation excitation maximum was 485 nm,wasand 485 the nm,emission and the emission maximum maximum was 530 was 530(A) nm). nm). (A) represents represents the average of fluorescence intensity of cell proliferation for CRL-1424 cells, (B) CRL-1619 cells, and (C) CRL- the average of fluorescence intensity of cell proliferation for CRL-1424 cells, (B) CRL-1619 cells, and (C) CRL-1675 cells with 1675 cells with statistical significance set at p < 0.05 (n = 20/condition). statistical significance set at p < 0.05 (n = 20/condition). Cancers 2021, 13, 4781 10 of 25 In migration assays, the three melanoma cell types were seeded in trans-wells indi- vidually and treated with exosomes derived from OSA-V1 and OSA-V2, cells were then counted (Figure 3A–C). We found that only CRL-1424 cells showed a significant increase in migration when cells were treated with exosomes from OSA-V1 (1000.74 ± 112.62) com- pared to the same patients treated with CPAP OSA-V2 (700.10 ± 103.18; p = 0.008; n = 20) (Figure 3A). However, there were no significant differences in melanoma cell migration in Cancers 2021, 13, x FOR PEER REVIEW 11 of 25 either CRL-1619 or CRL-1675 cells after exposure to exosomes from OSA-V1 compared to OSA-V2, as shown in Figure 3B,C. FigureFigure 3. Effects 3. Effects of plasma of plasma exosomes-derived exosomes-derived fromfrom OSAOSA patients patients before before CPAPCPAP treatment treatment (OSA-V1)and (OSA-V1) andafter afteradherent adherent CPAP CPAP treatment for 1 year (OSA-V2) on melanoma cell migration. Cells were seeded in 24-well trans-well inserts with 8 μm pore pore treatment for 1 year (OSA-V2) on melanoma cell migration. Cells were seeded in 24-well trans-well inserts with 8 µm size. size. allowed Cells were Cells wereto allowed to migrate migrate for for then 40 h, and 40 h,were and then were stained stained with 0.2%with 0.2% crystal crystal violet violet followed solution solution followed by phaseby phase contrast contrast imaging. imaging. The The migration ratemigration rate wasby was determined determined counting bythecounting number the number of cells of cells in contained contained in the the photos of photos of 5 different 5 different fields at original magnification. (A) represents average of different fields for each subject for each group using CRL-1424 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) cells, CRL-1675, (B) CRL-1629,(nand= 20/group), with (n (C) CRL-1675, statistical significance = 20/group), set at p