Research ArticleExperimental Studies

Effects of Cyclooxygenase-2 in Canine Melanoma-derived Extracellular Vesicles on Tumor Microenvironment In Vitro

NA-HYEONG KIM, JU-HYUN AN, JEONG-HWA LEE, SU-MIN PARK, KYEONG-BO KIM, TAE-HYUN KIM, YE-IN OH, KYEONG-WON SEO and HWA-YOUNG YOUN
Anticancer Research November 2022, 42 (11) 5397-5405; DOI: https://doi.org/10.21873/anticanres.16044

Abstract

Background/Aim: Tumor cell-derived extracellular vesicles (TEVs) promote tumor growth and metastasis; thus, they have drawn the attention of researchers. TEVs regulate the tumor microenvironment by facilitating crosstalk between immune and stromal cells. Macrophages are one of the key components involved in malignant behavior in melanomas. Generally, when activated, macrophages polarize into M1 (pro-inflammatory) or M2 (anti-inflammatory, pro-tumor) phenotypes. However, the role of canine melanoma-derived EVs in macrophage polarization is elusive. In this study, we aimed to analyze the pro- and anti-inflammatory cytokines that are common markers for M1 or M2 macrophages in vitro. Materials and Methods: The analysis was performed under coculture conditions of canine melanoma-derived (LMeC) EVs with canine macrophages (DH82). Quantitative reverse transcription polymerase chain reaction, western blotting, and immunofluorescence were used. Results: Canine melanoma-derived EVs polarized M1 macrophages (inducible nitric oxide synthase, tumor necrosis factor α) into M2 macrophages [cluster of differentiation (CD)206, interleukin-10] and cyclooxygenase-2 is a major factor in macrophage polarization in canine melanoma-derived EVs. Furthermore, we also found that melanoma-derived EVs induced the expression of angiogenic cytokines (vascular endothelial growth factor, transforming growth factor β) in endothelial cells. Conclusion: Melanoma-derived EVs perform an immunomodulatory function and can be used as targets in anti-inflammatory treatment.

Key Words:

Cancer cells affect the complex tumor microenvironment (TME) involving various surrounding cells (stromal cells, inflammatory cells, etc.), blood vessels, and extracellular matrix (1). Therefore, recently, the importance of studies on the microenvironment surrounding tumors is increasing to further understand the TME for anticancer drug development and utilization strategies (2).

Extracellular vesicles (EVs) are cell-derived membranous vesicles, ranging from 15 nm to several microns in diameter (3). EVs mimic the functions of the cell of origin by transferring bioactive cargoes [deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, and lipids] to the adjacent cells (4). Likewise, tumor-derived EVs (TEVs) can promote tumor-supporting processes, such as immunosuppression, invasion, angiogenesis, and metastasis, by modulating diverse immune cells in the TME. Specifically, TEVs suppress natural killer cells and cytotoxic cluster of differentiation (CD)8+ lymphocytes, induce myeloid-derived suppressor cells and M2-like macrophages, and stimulate regulatory T cell expansion (3, 5).

In addition, macrophages are a key component of the TME. Once differentiated, they are classified into two general polarized types (M1 and M2) based on their function. The M1 or pro-inflammatory phenotype exhibits enhanced phagocytosis and produces pro-inflammatory cytokines, whereas the M2 or anti-inflammatory phenotype resolves inflammation and promotes angiogenesis and tissue repair (6, 7). Tumor-associated macrophages (TAMs) are predominantly polarized to an M2-like phenotype, and this feature explains their ability to promote tumor growth and metastasis (8). Based on recent studies, TAMs serve as major metastasis promoters by releasing growth factors and various immunosuppressing proteins (9).

Malignant melanoma, the most aggressive tumor type in humans and dogs, is characterized by frequent relapse and metastases. In a recent study, melanoma-derived EVs (MEVs) were found to mediate immunosuppression and to support tumor growth via pro-angiogenic functions (10). Additionally, a recent investigation in humans detected cyclooxygenase-2 (COX-2) over-expression in oral and cutaneous melanomas, which correlated with malignancy and might be a marker of poor prognosis (11). Similarly, in canine melanoma, COX-2 expression was significantly greater in highly malignant cutaneous and oral melanomas (12). However, there have been few studies on TME-regulating key factors secreted from melanomas. Research on this aspect is necessary for the future development of personalized anticancer therapy.

In this study, we investigated the effects of canine MEVs on macrophage polarization and endothelial cells in the TME. The purpose of this study was to identify the major factors within MEVs, which affect the TME, through the analysis of anti-inflammatory cytokines and angiogenic factors.

Materials and Methods

Cell culture. The following cells were cultured in this study: LMeC, a canine melanoma cell line (Korean Cell Line Bank, Seoul, Republic of Korea); DH82, a canine macrophage-like cell line (Korean Cell Line Bank); and endothelial cells characterized in previous experiments (13). LMeCs and endothelial cells were cultured in Roswell Park Memorial Institute-1640 medium (RMPI; PAN Biotech, Aidenbach, Germany) with 10% heat-inactivated fetal bovine serum (FBS; PAN Biotech) and 1% penicillin-streptomycin (P/S; PAN Biotech) at 37°C in a 5% CO2 atmosphere. DH82 cells were cultured in Dulbecco’s modified Eagle’s medium (PAN Biotech) with 10% FBS and 1% P/S at 37°C in a 5% CO2 atmosphere. The culture medium was replaced every 2-3 days, and cells were sub-cultured when they reached 70-80% confluence.

Small interfering RNA (siRNA) transfection of LMeCs. When the confluency of LMeCs reached approximately 40%, they were transfected with COX-2 siRNA or control siRNA (sc-29279 and sc-37007, respectively; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 48 h using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturers’ instructions. COX-2 knockdown was confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting.

Isolation of canine melanoma cell line EVs. LMeCs were cultured for 48 h in RMPI with 5% exosome-depleted FBS (Thermo Fischer Scientific, San Jose, CA, USA) and 1% P/S (PAN Biotech). The media were collected and centrifuged at 100 × g for 5 min to remove cells and cell debris. Supernatants were transferred to a fresh tube, and an appropriate volume of ExoQuick-CG Exosome Precipitation Solution (System Biosciences, Palo Alto, CA, USA) was added. EVs were extracted according to the manufacturer’s instructions. The total protein concentration of EVs were measured using the bicinchoninic acid (BCA) assay.

Cell viability assay. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (D-Plus™ CCK Cell Viability Assay Kit; Dong-In Biotech, Seoul, Republic of Korea) to determine whether Lipofectamine RNAiMAX (Invitrogen) or GW4869 (Sigma–Aldrich, St Louis, MO, USA) had any influence on canine melanoma cell growth. The cells were seeded at a density of 3.3×104 cells/well in a 96-well plate. At 24 or 48 h after transfection or treatment with GW4869 (10 μM or 20 μM), a CCK-8 assay was conducted.

RNA extraction, complementary deoxyribonucleic acid (cDNA) synthesis, and real-time quantitative PCR. RNA was extracted using the Easy-Blue Total RNA Extraction kit (Intron Biotechnology, Sungnam, Republic of Korea). Total RNA concentration was measured at 260 nm using a NanoPhotometer (IMPLEN, Munich, Germany). Cell Script All-in-One 5x 1st cDNA Strand Synthesis Master Mix (Cell Safe, Seoul, Republic of Korea) was used to synthesize cDNA, and the samples were analyzed using AMPIGENE qPCR Green Mix Hi-ROX with SYBR Green dye (Enzo Life Sciences, Farmingdale, NY, USA) and forward and reverse primers (Bionics, Seoul, Republic of Korea). The expression levels of each gene were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequence of primers used in this study to amplify GAPDH, COX-2, interleukin 10 (IL-10), inducible nitric oxide synthase (iNOS), CD206, tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), and vascular endothelial growth factor (VEGF) of dogs are shown in Table I.

View this table:
Table I.

Sequences of PCR primers used in this study.

Coculture of LMeCs with DH82 cells. DH82 cells were seeded in six-well plates at a density of 5×105 cells/well and were incubated overnight. After adherence to the plates, lipopolysaccharide (LPS) (200 ng/ml) and/or GW4869 (10 μM) were added for 48 h. Next, the medium was removed, the cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS), and the cell culture medium was added. Using 0.4-μm pore size inserts, LMeCs were plated onto the macrophage cells at a density of 2×105 cells/well and ratio of 5:2. All cells were incubated for 48 h and then harvested for RNA extraction.

Western blot analysis. Total cellular proteins were extracted using the PRO-PREP Protein Extraction Kit (iNtRON Biotechnology, Seongnam, Republic of Korea) and quantified using the Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). A total of 20 μg of protein was analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with antibodies against CD81 and COX-2.

Statistical analysis. GraphPad Prism (version 6.01) software (GraphPad Software, La Jolla, CA, USA) was used to perform the statistical analysis. Student’s t-test and a one-way analysis of variance (ANOVA) were used to analyze the data, followed by the Bonferroni multiple comparison test. The data are presented as mean±standard deviation (SD). Differences with a p-value of <0.05 were considered statistically significant.

Results

Immunomodulatory effects of MEVs. To verify the innoxiousness of the treatment with 10 and 20 μM of GW4869 for 24 h and 48 h, a CCK-8 assay was performed; no significant difference was found between the control and experimental groups (Figure 1A). Treatment with the exosome inhibitor GW4869 resulted in a significant reduction in the levels of EV proteins produced by the melanoma cells (Figure 1B). To determine the effects of melanoma exosomes on M1 or M2 macrophage polarity the levels of certain cytokines were measured using RT-qPCR. The cytokines assayed were: TNF-α and iNOS for M1 macrophages or IL-10 and CD206 for M2 macrophages. LPS-treated DH82 canine macrophages cocultured with melanoma cells exhibited reduced levels of the messenger ribonucleic acid (mRNA) of TNF-α, a pro-inflammatory cytokine, and of iNOS, a marker of the M1 phenotype. By contrast, the levels of IL-10, an anti-inflammatory cytokine, and CD206, a marker of the M2 phenotype, were significantly increased when the LPS-treated DH82 cells were cocultured with melanoma cells. However, following pretreatment with GW4869 these effects were not observed (Figure 1C).

Figure 1.
Figure 1.

Expression of M1/M2 cytokines by canine macrophages (DH82) co-cultured with canine melanoma cell line (LMeC) and the effect of GW4869 treatment. (A) Cell viability assay using CCK-8 assays. Canine melanoma cells (LMeC) were incubated for 24 or 48 h with two different concentrations of GW4869 (10 and 20 μM). (B) Protein concentration of LMeC-derived EVs upon GW4869 treatment by BCA assays. (C) mRNA expression levels of common cytokines of M1/M2 macrophages co-cultured with LMeCs with or without GW4869 treatment using qRT-PCR. The results are shown as the mean±SD (*p<0.05, **p<0.01, and ***p<0.001, as analyzed by one-way ANOVA).

Identification of COX-2-depleted MEVs. CCK-8 analysis indicated that RNA transfection for 24 h and 48 h had no effect on the growth of melanoma cell (Figure 2A). Transfection with COX-2 siRNA- melanoma cells resulted in reduced expression of COX-2 mRNA, whereas transfection with control siRNA had no significant effect on the levels of COX-2 mRNA (Figure 2B). Protein levels changed accordingly (Figure 2C).

Figure 2.
Figure 2.

Identification of COX-2-depleted MEVs. (A) Cell viability analysis of LMeCs incubated for 24 and 48 h after transfection with COX-2 siRNA (siCOX-2) or scrambled siRNA(siRNA), or naïve EVs, using CCK-8 assays. COX-2 expression in LMeCs after 48 h of transfection with siCOX-2 or siRNA, or naïve EVs was determined by qRT-PCR (B) and western blot (C). The results are shown as the mean±SD (*p<0.05, as analyzed by one-way ANOVA).

COX-2 within MEVs: a major factor inducing M1 to M2 macrophage polarization in vitro. Based on the observation that coculture of melanoma cells with macrophages resulted in increased polarization towards the M2 phenotype, we hypothesized that COX-2 was the key MEV factor promoting M2 polarization. LPS-stimulated DH82 cells exhibited increased mRNA levels of M1 phenotype cytokines (iNOS, TNF-α). After the treatment with MEVs, M1 phenotype cytokine (iNOS, TNF-α) levels were decreased, but M2 phenotype cytokine (CD206, IL-10) levels were increased. These effects were not observed in the COX-2-depleted EV groups (Figure 3A). CD11c (M1 macrophage surface marker) and CD206 (M2 macrophage surface marker) were analyzed through immunofluorescence. LPS treatment increased the levels of CD11c+ macrophages. Following MEV treatment, the LPS-primed macrophages showed an increase in CD206 expression and a decrease in CD11c expression. The inhibition of COX-2 in CD206+ macrophages resulted in polarization to an M1 phenotype (Figure 3B).

Figure 3.
Figure 3.

Effects of COX-2 in MEVs on macrophage polarization in vitro. LPS-stimulated DH82 cells were co-cultured for 48 h with MEVs, extracted after transfection with siCOX-2 or siRNA, or naïve EVs (CTL). (A) mRNA Expression of M1/M2 cytokines using qRT-PCR. (B) The M1 and M2 population were evaluated by measuring CD11c+ (red) and CD206+ (green) cells, respectively, by immunofluorescence and flow cytometry. The results are shown as the mean±SD (**p<0.01, and ***p<0.001, as analyzed by one-way ANOVA).

Effects of MEV COX-2 on mRNA expression of angiogenetic factors in macrophages. To evaluate the angiogenic effects of MEVs, the levels of VEGF, an angiogenesis-stimulating factor, in macrophages were measured. There was a greater increase in the levels of VEGF mRNA in the EV-treated groups compared to the EV-untreated groups. COX-2-depleted EV-treated groups showed a significant decrease in VEGF expression (Figure 4).

Figure 4.
Figure 4.

Effects of COX-2 in MEVs on mRNA expression levels of vascular endothelial growth factor (VEGF) measured using qRT-PCR. LPS-stimulated DH82 cells were co-cultured for 48 h with MEVs. RNA was extracted after transfection with siCOX-2 or siRNA, or naïve EVs (CTL). The results are shown as the mean±SD (*p<0.05 and **p<0.01, as analyzed by one-way ANOVA).

Effects of MEV COX-2 on angiogenetic activities in endothelial cells. Considering that COX-2 originating from MEVs increased VEGF levels in LPS-primed macrophages, they were cocultured with endothelial cells to evaluate whether it affects angiogenic factors in conditions more similar to the TME. The mRNA levels of VEGF and TGF-β were significantly higher in endothelial cells cocultured with MEV-treated DH82 cells than in those cocultured with MEV-untreated DH82 cells. After COX-2 knockdown in MEVs, the levels of these cytokines decreased and were similar to those in the LPS-primed M1 DH82 groups (Figure 5).

Figure 5.
Figure 5.

Effects of COX-2 in MEVs on mRNA expression levels of angiogenic cytokines in endothelial cells after 48 h of co-culture with DH82 cells and MEVs measured using qRT-PCR. The results are shown as the mean±SD (**p<0.01, and ***p<0.001, as analyzed by one-way ANOVA).

Discussion

TME is composed of complex and diverse elements, such as the cells surrounding cancer cells, extracellular matrix, growth hormones, and signaling substances (14). In 1889, Dr. Stephen Paget presented the “seed and soil” theory and stated that the TME plays an important role as it acts as “soil” for the growth of “seeds” called cancer cells; this suggests that the TME plays an essential role in the development and progression of cancer. Therefore, as a strategy to overcome the limitation of existing anticancer therapies, which are not able to prevent cancer progression and metastasis by directly attacking cancer cells, attention is focused on developing new therapies that target the TME (15, 16). In this study, we confirmed the effects of canine MEVs on the TME and studied the role of key EV factors in the TME.

The interaction between tumor cells and the stromal environment contributes to the phenotypic polarization of TAMs (17). TAMs are known to be associated with tumor growth and metastasis in various tumors. TAMs are generally accepted to be very similar to M2 macrophages. Tumor cells interact with macrophages, and as a result, most macrophages acquire M2 phenotypes and are likely to exhibit immunosuppressive potential. Therefore, targeting TAMs and their associated molecules might be a strategy for cancer immunotherapy (18, 19).

The evaluation of markers commonly used to detect M1 (iNOS, TNF-α) and M2 (CD206, IL-10) macrophages demonstrated that canine MEVs induce M1 to M2 polarization. The levels of inflammatory cytokines increased in the culture media of LPS-stimulated M1 macrophages, and they were downregulated when cocultured with melanoma cells while the levels of anti-inflammatory cytokines were increased. Previous studies have shown that M2 macrophage polarization results in chemotherapy resistance and tumor progression (19); these results along with the results of our study suggest that MEVs can be used as a target of new anticancer drugs.

Further, the mRNA expression of several angiogenic cytokines (VEGF, TGF-β) was increased when canine endothelial cells were cocultured with MEVs. These pro-angiogenic cytokines, which promote tumor migration and proliferation and angiogenesis via vascular endothelial cells, have been targeted to suppress tumor growth and metastasis (20, 21). Previous studies have suggested that antiangiogenic therapy is one of the antitumor therapies associated with TAMs (19). Through this study, it was shown that targeting the expression of COX-2 has potential as an antiangiogenic agent, in addition, inhibition of COX-2 expression indicated that COX-2 present within canine MEVs is a key factor that regulates the TME such as TAM and endothelial cells.

Previous studies have demonstrated that TEVs play an important role in creating a TME that promotes tumor progression. They regulate immune cells to allow tumor cells to escape from immune surveillance and prepare a metastatic niche for tumor cells (22). These features were targeted in recent studies on immunotherapy (23). TEV can escape the action of the anti-tumor immune response by regulating TME-associated immune cells; this is possible because of their abilities to suppress T cell activation and drive tumor progression through the polarization towards M2-like macrophages (5). This study is the first to investigate whether MEVs could be considered as major targets for tumor growth inhibition and TME regulation, with particular focus on immunomodulation against macrophages and angiogenesis (24). Due to their immunosuppressive function, TEVs have become a novel target of chemotherapy (25).

The release of COX-2, an enzyme that converts arachidonic acid into prostaglandin endoperoxide, is induced in monocytes, endothelial cells, and tumor cells by cytokines, tumor promoters, etc. (26). As it is closely related to cell proliferation, immune regulation, angiogenesis, and metastasis, its abundance in tumor cells is strongly associated with malignancy in many cancers, such as lung, colon, breast, and ovarian cancers (27). In a recent study, COX-2 expression was found to be high in TAMs in advanced melanoma, which sheds light on its potential to be used as a marker for melanoma progression (28). To the best of our knowledge, there are currently no reports on COX-2 expression in TAMs in canine malignant melanoma. This study examined the influence of COX-2 in canine MEVs on macrophage polarization.

This study demonstrated the in vitro anti-inflammatory effects of MEVs; therefore, in vivo experiments are required. Although we generated conditions similar to the TME through transwell culture plates, other immune or stromal cells have to be assessed together, as there are numerous cell populations in the TME. Nevertheless, this is the first study to elucidate the regulators of the TME that facilitate tumor growth and the angiogenic activity of canine MEVs. Moreover, we found that COX-2 within canine MEVs plays a major role in macrophage polarization; this finding highlights an important mechanism through which MEVs affect the TME and serves as a basis for the development of TAM-based anticancer therapy through TAM reprogramming.

This study revealed that COX-2 present in MEVs is a major factor that regulates the TME, indicating that EVs and COX-2 are factors that can be targeted by new anticancer drugs.

Acknowledgements

This study was partially supported by the Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea.

Footnotes

  • ↵* These Authors contributed equally to this work.

  • Authors’ Contributions

    NHK: Data curation, formal analysis, investigation, resources, writing-original draft. JHA: Conceptualization, data curation, formal analysis, investigation, methodology, resources, writing-original draft, writing-review & editing. JHL, SMP, KBK, THK, YIO: data curation. KWS: Data curation, writing-review & editing. HYY: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, resources, software, supervision, validation, visualization, writing-review & editing.

  • Conflicts of Interest

    The Authors have no conflicts of interest to report in relation to this study.

  • Received September 20, 2022.
  • Revision received October 1, 2022.
  • Accepted October 3, 2022.

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Effects of Cyclooxygenase-2 in Canine Melanoma-derived Extracellular Vesicles on Tumor Microenvironment In Vitro
NA-HYEONG KIM, JU-HYUN AN, JEONG-HWA LEE, SU-MIN PARK, KYEONG-BO KIM, TAE-HYUN KIM, YE-IN OH, KYEONG-WON SEO, HWA-YOUNG YOUN
Anticancer Research Nov 2022, 42 (11) 5397-5405; DOI: 10.21873/anticanres.16044
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