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Hindawi Journal of Oncology Volume 2020, Article ID 4291827, 11 pages https://doi.org/10.1155/2020/4291827 Research Article Role of the Exosome Secretion Machinery in Ovarian Carcinoma: In Vitro and In Vivo Models 1 1 2 3,4 Esther Channah Broner, Hadil Onallah, Tali Tavor Re’em, Ben Davidson , and Reuven Reich Institute of Drug Research, School of Pharmacy, Faculty of Medicine, e Hebrew University of Jerusalem, Jerusalem 91120, Israel Department of Pharmaceutical Engineering, Azrieli College of Engineering, Jerusalem, Israel Department of Pathology, Oslo University Hospital, Norwegian Radium Hospital, Oslo N-0310, Norway University of Oslo, Faculty of Medicine, Institute of Clinical Medicine, Oslo N-0316, Norway Correspondence should be addressed to Ben Davidson; email@example.com and Reuven Reich; firstname.lastname@example.org Received 6 October 2019; Revised 3 March 2020; Accepted 15 April 2020; Published 30 May 2020 Academic Editor: Vincenzo Coppola Copyright © 2020 Esther Channah Broner et al. ,is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. We recently reported on the expression and clinical role of molecules that mediate exosome secretion in high-grade serous carcinoma. In the present study, the biological role of these molecules was analyzed. Methods. OVCAR8 and ES-2 ovarian carcinoma cells were studied using a combination of CRISPR/Cas9 technology and two 3D in vitro models—spheroids emulating eﬀusions and alginate scaﬀolds representing solid lesions. Isolation of exosomes was validated by electron microscopy. TSAP6, NSMASE2, RAB27A, and RAB27B mRNA and protein levels were analyzed using qRT-PCR and Western blotting, respectively. Tumor aggressiveness was studied in vitro using scratch assay, invasion assay, and matrix metalloproteinase (MMP) activity assay and in vivo using a mouse model. Results. In OVCAR8 cells, mRNA expression of TSAP6 and RAB27A was signiﬁcantly higher in spheroids compared to scaﬀolds, whereas the opposite was true for NSMASE2 mRNA. In ES-2 cells, TSAP6 and RAB27B mRNA expression was signiﬁcantly higher in spheroids versus scaﬀolds. In addition, nSMase2 and TSAP6 protein expression was signiﬁcantly higher in scaﬀolds compared to spheroids. CRISPR-edited cells with silencing of NSMASE2, TSAP6, and RAB27A/B had reduced migration, invasion, and MMP activity. Additionally, knockout (KO) of these molecules resulted in signiﬁcantly diminished exosome secretion. In vivo assay showed that when injected to mice, OVCAR8 RAB27A/B KO cells, as opposed to control OVCAR8 cells, did not form ascites or visible tumor lesions and had reduced MMP expression. Conclusion. ,e present study provides evidence that diﬀerent models for culturing ovarian carcinoma cells aﬀect the expression of molecules mediating exosome secretion and that these molecules have a tumor-promoting role. Silencing these molecules may consequently have therapeutic relevance in this cancer. dismal for the majority of patients, particularly when the 1. Introduction disease is diagnosed at advanced stage . Ovarian cancer, the most aggressive gynecologic cancer, was Exosomes are endogenous nanovesicles, 30–120 nm in predicted to be diagnosed in 22,530 women and led to 13,980 diameter, that are secreted from cells and are found in bodily fatalities in the USA in 2019 . Global ﬁgures for 2018 are ﬂuids including plasma , urine , saliva , breast milk 295,414 new cases and 184,799 deaths, making this cancer , semen , and eﬀusions [9, 10]. ,ese nanovesicles th th the 8 most common and 8 most lethal cancer in women contain bioactive molecules including lipids, proteins, DNA, . While novel targeted therapies, such as PARP inhibitors, mRNA, miRs, and long noncoding RNA [11, 12]. have extended the survival of patients carrying mutations in Exosomes derived from tumor cells have been shown to BRCA and related DNA repair genes, prognosis remains have cancer-promoting eﬀects, including increased motility 2 Journal of Oncology through extracellular matrix (ECM) , increased degra- 2.2. Exosome Extraction and Quantitation. 4 ×10 cells were dation of ECM , and invasiveness . Cancer-derived seeded in 100 mm dishes and incubated overnight with exosomes can also coerce surrounding cells to contribute to supplemented medium of RPMI 5% FCS. On the next day, their cancer microenvironment: tumor exosomes can the medium was replaced with serum-free medium transform surrounding ﬁbroblasts into cancer-associated (EXCELL advanced CHO; Sigma-Aldrich, St. Louis MO) ﬁbroblasts. Exosomes can also cause increased vascular and cells were incubated for further 48 hours. ,e medium permeability , contributing to the metastatic capability of was collected and ﬁltered with Amicon Ultra-15 Centrifugal the tumor, as well as condition premetastatic tumor niches at 0.1μM PVDF Filter Units (Merck Millipore Ltd). Next, the various distant anatomic sites [17, 18]. medium was concentrated with 3000 MWCO vivaspin 20 A previous study by our group reported on anatomic (Sartorius, Gottingen, ¨ Germany). Exosomes were extracted site-dependent expression of components of exosomes se- from the supernatant according to the user’s manual of cretion machinery, i.e. neutral sphingomyelinase2 ExoQuick-TC (System Biosciences, Mountain View CA). (nSMase2), Tumor suppressor-activated pathway 6 ,e resulting pellet containing exosomes was resuspended in (TSAP6), Ras-related protein 27A/B (Rab27a/b) and ADP- 100μl of PBS and quantiﬁed for protein concentration by ribosylation factor 6 (ARF6) in high-grade serous carcinoma Bradford assay. Exosomes were detected and quantiﬁed (HGSC), the most common and clinically aggressive his- using the qNano Gold analyzer (IZON Science Ltd., tologic type of ovarian carcinoma (OC). TSAP6 and RAB27a Christchurch, New Zealand). levels in eﬀusion specimens were additionally signiﬁcantly related to overall survival . 2.3. Electron Microscopy (EM). Exosomes extracted from all Another previous study by our group, focusing on Ezrin genotypes examined were negatively stained with Sodium expression in OC, suggested that tumor growth in a spheroid phosphotungstate (PTA) and imaged by a transmission model closely resembles tumor growth in ascites and pleural electron microscope, JEM-1400 plus, available in the Inter- eﬀusion, whereas cells cultured on macroporous scaﬀold are Departmental Equipment Department at the Ein Karem a model for solid tumor growth, thereby creating an in- campus of the Hebrew University. ,is procedure was clusive three-dimensional (3D) in vitro model that emulates performed by the staﬀ of the department according to the diﬀerent anatomic sites aﬀected by OC . In the standard protocol. OVCAR8 control-derived exosomes were present study, we utilized this model together with CRISPR- diluted 1 :1000 in PBS. Cas9 Knockouts (KO) in OC cell lines. Our objective was to demonstrate that NSMASE2, TSAP6, and RAB27A/B-re- lated pathways are essential for exosome secretion in OC cell 2.4.qRT-PCR. RNA was extracted with Tri-Reagent (Sigma- lines and may consequently constitute a potential thera- Aldrich) and converted to cDNA with qScript cDNA syn- peutic target in OC. thesis kit (Quanta Biosciences, Gaithersburg MD). qPCR was performed with KAPA SYBERFAST Universal qPCR kit 2. Methods and Materials (Kapa Biosystems, Wilmington MA) on the CFX Connect Real-Time system (Bio-Rad Laboratories, Hercules CA). ,e 2.1. Cell Culture. ,e OVCAR8 and ES-2 OC cell lines were ﬁnal concentrations of template and primers were deter- purchased from ATCC (Manassas VA) and cultured at 37 C, mined individually for every assay, calibrated based on a 5% CO in their appropriate medium (RPMI + 5% FCS and standard curve. Speciﬁc primer sequences were designed DMEM + 10% FCS, resp.), supplemented with 1% nones- utilizing the NCBI Primer Blast tool (Table 1). Analysis was sential amino acids, 1% L-glutamine, 1% BME vitamins, 1% performed with BioRad CFX Manager Software. ,e ΔCT of sodium pyruvate and 1% penicillin, streptomycin, and genes of interest was calculated relative to the RPLP0 amphotericin. All reagents were purchased from Biological -ΔΔCT housekeeping gene and expressed as 2 versus average Industries (Beit HaEmek, Israel). OVCAR8 and ES-2 OC primary as a control, except for TSAP6 which was analyzed spheroids were formed by constant shaking of 400,000 cells as TSAP6/RPLP0. per well in 6-well plates for 48 hours on a vertical shaker. When necessary, standard PCR was run using the above Alginate scaﬀolds with a diameter of 5 mm and thickness of equipment and primers with 2G FAST KAPA master mix 2 mm were prepared as high guluronic acid (LVG) alginate (Kapa Biosystems). ,e products were run with electro- (NovaMatrix FMC Biopolymers, Drammen, Norway). ,e phoresis on 1.5% agarose gel stained with ethidium bromide, macroporous structure was obtained by employing a freeze- imaged, and then analyzed by ImageJ. drying technique: alginate was dissolved in DDW (1.2% w/v solution), homogenized and cross-linked with a solution of calcium gluconate (1.2% w/v) by homogenizer apparatus. 2.5. Immunoblotting. Samples were lysed with 1% NP-40, ,e ﬁnal concentration of cross-linked solution was 1.0% 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 0.5 mM EDTA, and 0.2% w/v for the polymer and cross-linker, respectively. 10% glycerol, 1% protease inhibitor cocktail (Sigma- ,e cross-linked solution was doled out into 96-well plates Aldrich), and 0.1% SDS and quantiﬁed using the Bradford (100μL/well) and then transferred to +4 C for 1 hour, assay. Twenty-ﬁve micrograms of protein was loaded on 10% subsequently to −20 C for 24 hours, and then lyophilized. SDS polyacrylamide gels, separated by electrophoresis, and 400,000 cells were seeded on each alginate scaﬀold and were transferred to PVDF membranes (Millipore, Billerica MA) cultured for 1-2 weeks. and blocked with 5% nonfat milk in TBST. Primary Journal of Oncology 3 Table 1: qPCR primers. ml, 10% SDS polyacrylamide gels, and separated by elec- trophoresis. ,e gels were incubated overnight at 37 C and Forward 5′-AGGACTGGCTGGCTGATTTC-3′ NSMASE2 Reverse 5′-TGTCGTCAGAGGAGCAGTTATC-3′ then washed in 2.5% triton, developed, dried, and analyzed with the ImageJ software. Forward 5′-GGGAGTTCAGCTTCGTTCAG-3′ TSAP6 Reverse 5′-TGGGAGGCAGGTAGAACTTG-3′ Forward 5′-GCTTTGGGAGACTCTGGTGTA-3′ RAB27A 2.9. Invasion Assay. Cells of all genotypes (n � 50,000) were Reverse 5′-TCAATGCCCACTGTTGTGATAA-3′ seeded on Matrigel-coated ﬁlters in Boyden chambers and 5′-TAGACTTTCGGGAAAAACGTGTG- Forward ° incubated for 6 hours at 37 C with conditioned medium RAB27B 3′ from the 3T3 ﬁbroblast cell line, used as chemoattractant. Reverse 5′-AGAAGCTCTGTTGACTGGTGA -3′ Invading cells were stained and quantiﬁed under an invert microscope. antibodies were as follows: nSMase2 (H00055512-D01; Abnova, Taipei City, Taiwan), TSAP6 (sc-20531; Santa Cruz Biotechnology, Santa Cruz CA), Rab27A (ab55667, Abcam, 2.10. In Vivo Tumor Aggressiveness Assay. OVCAR8, Cambridge, UK), and GAPDH (14C10, Cell Signaling, RAB27A-KO, and RAB27B-KO cells (2 ×10 ) were injected Danvers MA). Band density was measured using the Image J IP into female SCID mice (n � 12; 5 controls, 4 RAB27A-KO, software, divided by the loading control (GAPDH), and 3 RAB27B-KO). Mice were monitored for 21 days, until compared to control. symptoms of OC appeared. ,e tumors were removed, ﬁxed in formalin, and embedded in paraﬃn. H&E sections were assessed by an experienced surgical pathologist (BD). ,e 2.6. CRISPR-Cas9 Knockouts (KO). Clustered Regularly presence of ascites ﬂuid was noted. All experiments were Interspaced Short Palindromic Repeats- (CRISPR-) associ- authorized by the Animal Ethics Committee of the Hebrew ated 9 (Cas-9) KOs were generated in OVCAR8 cells using University of Jerusalem. the pSpCas9 (BB)-2A-Puro (PX459) plasmid obtained from Addgene (Watertown MA) according to the manufacturer’s protocol. Brieﬂy, speciﬁc single guide RNA (sgRNA) inserts 2.11.Immunohistochemistry(IHC). Formalin-ﬁxed, paraﬃn- were designed to target the beginning of the 5′ sequence of embedded sections from the in vivo mouse model were an- every gene (Table 2). alyzed for Ki-67, p53, MMP2, and MMP9 protein expression ,e inserts were 5′ phosphorylated and annealed on a using the Dako EnVision System (Dako, Glostrup, Denmark). ° ° ramp between 95 C and 25 C. ,e vector was digested by the ,e Ki-67 antibody was a rabbit polyclonal antibody pur- BBSI restriction enzyme. Ligation of the insert to the plasmid chased from Abcam (cat no. 15580; Cambridge, UK), applied was performed with T4 ligase. ,e plasmids were then at 1 :1000 dilution with antigen retrieval in HpH (pH 9.0) transformed to DHα5 E. coli cells. Plasmids were extracted solution (Dako). ,e p53 antibody was a rabbit polyclonal from the DHα5 E. coli cells with GeneJet Plasmid Miniprep antibody purchased from Novocastra (part of Leica Bio- (Fermentas Life Sciences) and were sent to the Center for systems; cat no. NCL-p53-CM1; Wetzlar, Germany), applied Genomic Technologies at the Hebrew University, Givat Ram, at 1 : 3000 dilution with antigen retrieval in citrate buﬀer (pH Jerusalem, for sequencing, in order to conﬁrm the insertion of 6.0). ,e MMP2 antibody was a rabbit polyclonal antibody the sgRNA insert. Plasmids were subsequently transfected purchased from LabVision (cat no. RB-9233-P1; Fremont into OVCAR8 cells with Lipofectamine 2000 (Invitrogen, CA), applied at 1 : 400 dilution with antigen retrieval in Tris- Carlsbad CA). Selection by Puromycin (A.G. Scientiﬁc Inc. EDTA buﬀer. ,e MMP9 antibody was a rabbit polyclonal San Diego CA) was performed, and the surviving resistant antibody purchased from NeoMarkers (part of LabVision; cat cells were seeded as single cell colonies, resulting in formation no. RB-1539-P1; Fremont CA), applied at 1 : 200 dilution with of single cell clones of-KO cells, which were then analyzed for no antigen retrieval. Positive controls consisted of normal KO with PCR or qPCR as described above. spleen (Ki-67), colon carcinoma (p53), placenta (MMP2), and lung carcinoma (MMP9). 2.7. Migration—Scratch Assay. OVCAR8 cells with NSMASE, TSAP6, and RAB27A/B KOs and OVCAR8 2.11.1. IHC Scoring. Nuclear staining was scored as positive control cells were seeded to conﬂuence in 6-well plates. Each for Ki-67 and p53 and cytoplasmic staining for MMP2 and well was scratched twice with a sterile tip. ,e scratch in MMP9. Scoring was performed by an experienced surgical NSMASE2 and TSAP6 KO cells was imaged at t � 0 and pathologist (BD), using a 0–4 scale as follows: 0 � no t � 18 hrs. RAB27A/B KOs were imaged at t � 0 and staining, 1 � 1–5%, 2 � 6–25%, 3 � 26–75%, 4 � 76–100% of t � 24 hrs. Wound closure was analyzed by T-scratch soft- tumor cells. ware . 2.8. Matrix Metalloproteinases (MMP) Activity Assay. KO 2.12. Statistical Analysis. Student’s t-test was employed, cells and OVCAR8 control cells (n = 500,000) were incu- signiﬁcance was determined as p< 0.05. High and low bated overnight in DMEM medium supplemented with 0.1% mRNA and protein expression was deﬁned relative to ex- BSA. ,e conditioned medium was loaded on 1 mg gelatin/ pression in control cells in each experiment. 4 Journal of Oncology Table 2: CRISPR-Cas9 sgRNA insert sequences. Sense CACCGTTCACCTCGGTGGGCCATG NSMASE2 Antisense AAACCATGGCCCACCGAGGTGAAC Sense CACCGCCATTGCAAACTCGCTCAAC TSAP6 Antisense AAACCGTTGAGCGAGTTTGCAATGG Sense CACCCAGTATTCATACCCACTCCG RAB27A Antisense AAACCGGAGTGGGTATGAATACTG Sense CACCGCACTCGCAGTCCTGACGGGGCAGGG RAB27B Antisense AAACCCCCTGCCCGTCAGGACTGCGAGTG signiﬁcantly less in the scratch assay (Figure 4(a)). After 24 3. Results hours, the wounds in OVCAR8 cells with KO mutation of 3.1. Growth Form in 3D Models Inﬂuences Gene and Protein NSMASE2 and TSAP6 were 73% and 55% open, respectively, Expression in OC. Our aim was to develop a 3D model that versus 27% of the OVCAR8 control (p< 0.001 and p< 0.03, emulates OC. To this end, we successfully introduced two resp.). separate 3D in vitro models utilizing the OVCAR8 and ES-2 RAB27A/B KO wounds analyzed after 18 hours were OC cell lines—a spheroid model and a scaﬀold model. 81% and 71% open versus 43% of the control (p< 0.0001 and Analysis of the genes involved in exosome secretion in p< 0.0005, respectively). OVCAR8 cells showed diﬀerent NSMASE2, TSAP6, and In MMP activity tests, MMP9 activity was signiﬁcantly RAB27A levels as function of the culture method reduced in OVCAR8 NSMASE2, TSAP6, and RAB27A KO (Figures 1(a)–1(d)). mRNA expression of TSAP6 and cells compared to OVCAR8 control cells (p< 0.05; RAB27A was higher in spheroids compared to scaﬀolds Figure 4(b)). (p< 0.01 and p< 0.001, respectively), whereas the opposite In the Boyden chamber invasion assay, OVCAR8 was true for NSMASE2 mRNA (p � 0.0001). No change was RAB27A/B KO cells inﬁltrated signiﬁcantly less than control observed in RAB27B mRNA levels. In ES-2 cells, TSAP6 and OVCAR8 cells: an average of 32 and 44 cells succeeded in RAB27B mRNA expression was higher in spheroids versus inﬁltrating, respectively, versus an average of 73 cells in the scaﬀolds (p< 0.03, p< 0.02, respectively) (Supplementary control (p< 0.0005 and p< 0.05, respectively). A trend was Figures 1(a) and 1(b)). noted for NSMASE2-KO cells where only 52 cells inﬁltrated nSMase2 and TSAP6 protein expression was higher in (p � 0.087; Figure 4(c)). scaﬀolds compared to spheroids (p< 0.006 and p< 0.0004, respectively). ,e growth form did not aﬀect RAB27A protein expression (Figures 2(a)–2(c), Supplementary 3.5. Suppression of Exosome Secretion Pathways Leads to Figure S2). RAB27B protein expression was not analyzed Absence of Ascites Fluid and Reduced Tumor Size In Vivo. due to lack of adequate antibody. Seven weeks following the injection of 2 ×10 cells of OVCAR8 control, RAB27A-KO and RAB27B-KO to SCID mice, only the mice injected with OVCAR8 control cells 3.2. CRISPR Cas9 KO in OC Cell Lines. In order to evaluate the distinct role of the genes putatively involved in exosome developed ascites, with a volume ranging from 0.75 to 1.5 ml. No ascites ascites was observed in the mice injected secretion, OVCAR8 cells were subjected to KO with the with KOs. ,e entire group of RAB27A-KO and RAB27B- CRISPR Cas9 protocol for the various genes in this study. KO mice exhibited no visible tumors by gross examina- KOs were validated by qPCR showing the absence of mRNA tion, whereas tumor nodules were detected in the and by Western Blot, proving that this gene was silenced and OVCAR8 control group. Microscopic assessment of the not expressed in this cell line. ,e percentages of KO abdominal organs from the 12 mice showed that in achieved and protein levels are detailed in Table 3. RAB27A-KO and RAB27B-KO mice, the majority of or- gans were tumor-free, with isolated tumor nodules 3.3. Exosome Secretion Pathway KO Leads to Decreased ranging from 0.5 mm to 4 mm and 0.5 to 2 mm, respec- Exosome Secretion in OC Cell Lines. EM visualization tively, whereas the OVCAR8 control group had multiple showed that under normal culture conditions, OVCAR8 lesions with a diameter of up to 7 mm (Figure 5, Sup- cells secrete exosomes (Figure 3(a)). However, when mea- plementary Table S1). ,e average diameter of metastasis, sured by the qNano Gold analyzer, exosome secretion was including tumor-negative organs, was 0.59 mm for the signiﬁcantly reduced in OVCAR8 KO cells compared to RAB27A-KO group (total diameter of all metastases control OVCAR8 cells (TSAP6 KO: p< 0.05; RAB27A KO: 13.5 mm in 23 organs), 0.35 mm for the RAB27B-KO p< 0.05; RAB27B KO: p< 0.01; Figure 3(b)). group (total diameter of all metastases 5.5 mm in 16 or- gans), and 1.53 mm for the control group (total diameter of all metastases 44.5 mm in 29 organs; p< 0.05). 3.4. Suppression of Exosome Secretion Pathways Leads to IHC analysis showed comparable expression of Ki-67 and Reduced Migration, MMP Activity, and Invasion in OC Cells. p53 in tumors from the RAB27A-KO and RAB27B-KO mice OVCAR8 cells with KO of any of the four genes, that is, compared to controls. However, MMP2 and MMP9 expression NSMASE2, TSAP6, RAB27A, and RAB27B, migrated Journal of Oncology 5 10 6 ∗∗ 0 0 OVCAR8 spheroid OVCAR8 scaﬀold OVCAR8 spheroid OVCAR8 scaﬀold (a) (b) 2.5 3 ∗∗ ns 2.0 1.5 1.0 0.5 0.0 0 OVCAR8 spheroid OVCAR8 scaﬀold OVCAR8 spheroid OVCAR8 scaﬀold (c) (d) Figure 1: (a–d) Gene expression dependent on 3D cell culture form: diﬀerential expression of mRNA expression in spheroids and scaﬀolds is seen for the following genes: (a) NSMASE2 (p � 0.0001), (b) TSAP6 (p< 0.01), (c) RAB27A (p< 0.001), (d) RAB27B (p< 0.55) in OVCAR8 cells. 0.8 6 ns ∗ ∗∗∗ 1.5 0.6 1.0 0.4 0.5 0.2 0.0 0.0 OVCAR8 OVCAR8 OVCAR8 OVCAR8 OVCAR8 OVCAR8 spheroid scaffold spheroid scaffold spheroid scaffold Spheroids Scaffolds Spheroids Scaffolds Spheroids Scaffolds nSMASE2 TSAP6 RAB27A GAPDH GAPDH GAPDH (a) (b) (c) Figure 2: (a–c) Diﬀerential protein expression depending on 3D growth form in OVCAR8 cell line: (a) nSMase2 and (b) TSAP6 protein expression is higher in scaﬀolds compared to spheroids (p< 0.006 and p< 0.0004, respectively). (c) RAB27A protein expression is not aﬀected by growth form; error bars indicate SEM. was signiﬁcantly lower in tumors from RAB27A-KO and exosome secretion machinery in HGSC cells and tumor- RAB27B-KO mice (Figure 6; Supplementary Table S1). derived exosomes . In the present study, we analyzed whether a novel 3D OC model representing OC progression can be relevant for studying exosome secretion pathways in 4. Discussion this cancer. We previously reported on the frequent expression and We propose that alginate scaﬀolds emulate solid tumors, clinical relevance of several molecules belonging to the while spheroids mimic tumor growth in eﬀusions. In the NSMASE2/GAPDH Fold change Fold change TSAP6/GAPDH Fold change Fold change RAB27A/GAPDH 6 Journal of Oncology Table 3: Percentage of KO generated by CRISPR Cas9 in OVCAR8 cells. KO A. qPCR NSMASE2 90% TSAP6 90% RAB27A 85% RAB27B 99% B. Western blot KO OV8 CONTROL NSMASE2 GAPDH TSAP6 GAPDH RAB27A GAPDH (A) OVCAR8 TSAP6 KO (B) OVCAR8 RAB27A KO 100 100 80 80 60 60 40 40 20 20 0 0 050 100 150 200 0 100 200 300 400 Particle diameter (nm) Particle diameter (nm) (C) OVCAR8 RAB27B KO (D) OVCAR8 100 100 80 80 60 60 40 40 20 20 OVACAR8-derived exosomes 0 0 0 50 100 150 200 250 0 50 100 150 200 250 Particle diameter (nm) Particle diameter (nm) (a) (b) Figure 3: (a) OVCAR8-derived exosomes, diameter 40–120 nm, as visualized by EM; PTA staining, X10K magniﬁcation (top), and X5K magniﬁcation (bottom). Scale bar indicates the size of the ﬁeld shown. (b) Exosome secretion in OVCAR8 KO cell lines: exosome secretion is reduced in (A) TSAP6 KO, (B) RAB27A KO, and (C) RAB27B KO compared to (D) OVCAR8 control cell lines. Frequency Frequency Frequency Frequency Journal of Oncology 7 Migration assay, KO cells ∗∗ ∗∗ ∗∗ OVCAR8 OVCAR8 OVCAR8 OVCAR8 OVCAR8 NSMASE2 KO TSAP6 KO RAB27A KO RAB27B KO ab c d e (a) MMP 9 activity ns MMP-9 ∗∗ (b) Figure 4: Continued. % MMP 9 activity % open wound OVCAR8 OVCAR8 NSMASE2 KO OVCAR8 TSAP6 KO OVCAR8 RAB27A KO OVCAR8 RAB27B KO OVCAR8 NSMASE2 KO TSAP6 KO RAB27A KO RAB27B KO 8 Journal of Oncology Invasion assay ∗∗ OVCAR8 NSMASE2 KO TSAP6 KO RAB27A KO RAB27B KO (c) Figure 4: (a) Migration assay. Below: extent of the wound open after 18 or 24 hours, respectively: (a) OVCAR8 control, (b) NSMASE2-KO (p< 0.001, n � 4), (c) TSAP6-KO (p< 0.03, n � 4), (d) RAB27A-KO (p< 0.0001, n � 8), (e) RAB27B-KO (p< 0.001, n � 8). Above: quantiﬁcation of experiment. Error bars represent SEM. (b) MMP activity assay. ,e width of the MMP9 gelatin consumed band is reduced in OVCAR8 NSMASE2 (p< 0.05), TSAP6 (p< 0.01), and RAB27A (p< 0.03) KO cells, but not in RAB27B KO cells. Error bars represent SEM. (c) Invasion assay: cells inﬁltrating through matrigel ﬁlters were quantiﬁed in this assay. RAB27A/B-KO cells inﬁltrated less than the OVCAR8 control cells. p< 0.0005 and p< 0.05, respectively. Error bars represent SEM. present study we show that the 3D form of growth inﬂuences Of note, there were notable diﬀerences between the both the mRNA and protein expression of the exosome mRNA and protein expression levels of Rab27A and TSAP6. secretion pathway in OC cell lines, as reﬂected by changes in We hypothesize this discrepancy to result from the action of NSMASE2, TSAP6, RAB27A, and RAB27B mRNA, as well as molecules regulating mRNA levels or due to epigenetic NSMASE2 and TSAP6 protein expression depending on the regulation of protein expression. 3D form applied. We utilized the CRISPR-Cas9 system and produced In OVCAR8 cells, TSAP6 and RAB27A mRNA levels NSMASE2, TSAP6, RAB27A, and RAB27B KOs in the were higher in spheroids than in scaﬀolds and RAB27B OVCAR8 cell line. EM showed that OVCAR8 cells produce mRNA expression was not inﬂuenced by the growth form. exosomes. ,e secretion of these exosomes was signiﬁcantly diminished in RAB27A-KO, RAB27B-KO, and TSAP6-KO ,is proﬁle partly concurs with our ﬁndings in our pre- vious study , where TSAP6 mRNA expression levels cells, well in agreement with the study of Bobrie et al., in were higher in HGSC eﬀusions compared to surgical which Rab27a mediated exosome secretion in breast car- specimens, while those of RAB27B expression did not diﬀer cinoma cells . Of note, in another study by the same between these anatomic locations. Data are discordant with group, Rab27a silencing reduced docking of multivesicular respect to RAB27A mRNA, which had comparable ex- endosomes (MVEs) and increased their size in Hela cells, pression levels in solid lesions and eﬀusions, despite the use whereas MVEs were redistributed towards the perinuclear of the same assay, presumably due to inherent diﬀerences region upon Rab27b silencing, documenting that these two between cell lines and clinical specimens. NSMASE2 and proteins have diﬀerent roles . TSAP6 protein levels were higher in scaﬀolds compared to Functional assays in the present study showed that spheroids, in agreement with the higher NSAMSE2 and molecules involved in the exosome machinery also regulate critical cancer-related processes. In migration assays, all KO TSAP6 expression in solid tumors versus eﬀusions in our previous study. ,ese data suggest that the diﬀerent culture cells migrated signiﬁcantly less, indicating that these genes, models faithfully emulate 3D growth in solid lesions and perhaps via exosome secretion, are needed for this metastatic eﬀusions in HGSC. feature. In the zymography assay that assesses the ability of Number of cells inﬁltrated OVCAR8 OVCAR8 NSMASE2 KO OVCAR8 TSAP6 KO OVCAR8 RAB27A KO OVCAR8 RAB27B KO Journal of Oncology 9 Intestine, RAB27A KO Liver, RAB27A KO Intestine, RAB27A KO (a) (b) (c) Kidney, RAB27B KO Liver, RAB27B KO Spleen, RAB27B KO (d) (e) (f) Skeletal muscle, control Pancreas, control Muscle, control (g) (h) (i) Fat, control Intestine, control (j) (k) Figure 5: Six sections from the liver, kidney, spleen, and intestine of mice in the RAB27A-KO and RAB27B-KO groups. No tumor nodules are seen. (g–k) Five sections of tumor nodules (marked with blue arrows) in the pancreas, intestine, muscle, and fat tissue in the OVACR control group (all ﬁgures at ×100 magniﬁcation). Diﬀerences in average tumor diameter between the KO groups and controls were signiﬁcant (p< 0.05; see text). cancer cells to degrade gelatin via MMPs, NSMASE2-KO, growth of the primary tumor, and reduced lung dissemi- RAB27A-KO, and TSAP6-KO cells showed reduced MMP9 nation following the injection of 4T1 metastatic carcinoma activity. In the Boyden Chamber invasion assay that mea- cells to mice. Overall, exosome pathway KO cells appear to sures another metastatic characteristic, that is, the ability to possess less malignant and less metastatic characteristics, degrade matrigel, RAB27A-KO and RAB27B- KO cells suggesting that the exosome machinery pathway is impor- inﬁltrated signiﬁcantly less. ,is ﬁnding is well in agreement tant for tumor progression. with the study of Geback ¨ et al. , in which Rab27A si- ,e development of a malignant eﬀusion in the peri- lencing resulted in decreased MMP9 activity, suppressed toneal and/or pleural cavities represents metastatic disease 10 Journal of Oncology MMP9, RAB27B KO Ki-67, RAB27A KO p53, RAB27A KO MMP2, RAB27B KO (a) (b) (c) (d) MMP9, control Ki-67, control p53, control MMP2, control (e) (f) (g) (h) Figure 6: Protein expression of Ki-67, p53, MMP2, and MMP9 by IHC. Ki-67 and p53 expression is comparable in RAB27A-KO cells and controls. In contrast, a RAB27B-KO shows no expression of MMP2 and MMP9, whereas control cells are diﬀusely positive for both proteins. Tumor areas are marked with blue arrows (all ﬁgures at ×100 magniﬁcation). that is not amenable to surgical removal, where tumor cells Conflicts of Interest are chemoresistant and possess cancer stem cell charac- ,e authors have no conﬂicts of interest. teristics [24, 25]. ,is form of tumor progression is therefore a highly lethal stage of OC, in addition to its detrimental eﬀect on the quality of life of OC patients. In Authors’ Contributions contrast to OVCAR8 controls, no development of ascites was observed following injection of RAB27A and RAB27B Esther Channah Broner performed the experiments and KO cells to mice, and solid tumor nodules were signiﬁ- wrote the manuscript. Hadil Onallah performed the ex- cantly smaller, indicating that these molecules may have periments and critically read the manuscript. Tali Tavor direct clinical relevance in HGSC. Tumors from mice in- Re’em prepared the 3D constructs and critically read the jected with RAB27A and RAB27B KO cells additionally had manuscript. Ben Davidson designed the study, microscop- lower MMP expression. Overall, the experimental data, ically assessed the tumors in the mice experiments, per- combined with the association between TSAP and poor formed and scored the immunostains, and co-wrote the outcome in our previous study , support a role as manuscript. Reuven Reich designed the study, supervised oncogenes rather than tumor suppressors for the exosome the experiments, and cowrote the manuscript. Esther machinery components. An association between Rab27a Channah Broner and Hadil Onallah contributed equally to protein and favorable outcome was nevertheless observed this work. for exosomal levels of this protein in our previous study , raising the possibility that extracellular Rab27a has Supplementary Materials other functions than its intracellular counterpart. In conclusion, this study presents a 3D in vitro model Figure 1: (a) RAB27B (p< 0.02) and (b) TSAP6 (p< 0.03) that may allow exploration of the molecular drivers of the mRNA expression in the ES-2 cell line. Figure 2: images of diﬀerent growth forms of OC. Our in vivo model shows that the actual Western blots of Figures 2(a)–2(c). Table S1: interfering with the exosome secretion pathway contributes Isolated tumor nodules ranges in mm in the various organs. to attenuating the aggressiveness of OC, a ﬁnding that may (Supplementary Materials) have therapeutic relevance. Analysis of the content of exosomes derived from the scaﬀold and spheroid model, References including proteins, mRNA, and noncoding RNA, may be of interest in future research.  R. L. Siegel, K. D. Miller, and A. Jemal, “Cancer statistics, 2019,” CA: A Cancer Journal for Clinicians, vol. 69, no. 1, Data Availability pp. 7–34, 2019.  F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. 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Journal of Oncology – Hindawi Publishing Corporation
Published: May 30, 2020
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