CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients
Abstract
:1. Introduction
1.1. Small Extracellular Vesicles
1.2. Melanoma
1.3. Hypoxia
1.4. Carbonic Anhydrase IX
1.5. Aim of This Study
2. Results
2.1. Expression of HIF-1α and Carbonic Anhydrase IX Is Induced under Hypoxia in Melanoma Cells
2.2. Small EVs Released by Melanoma Cells under Normoxic and Hypoxic Conditions Have Comparable Particle Concentration and Median Size
2.3. Melanoma Cells Release CA-IX-Expressing sEVs during Hypoxia
2.4. CA-IX Protein Is Associated to the Membrane of sEVs Secreted by Hypoxic Melanoma Cells
2.5. CA-IX-Positive sEVs Are Found in Melanoma Liquid Biopsies and Their Capture Enables the Enrichment of BRAFV600E Mutation
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. SEV Purification
4.3. SEV Characterization: Nano Flow Cytometry
4.4. RNA Extraction
4.5. Droplet Digital PCR
4.6. Western Blot
4.7. ELISA Assay for CA-IX-Positive sEVs
4.8. Biological Samples and Patient Consent
4.9. Isolation of CA-IX-Positive sEV Population and Generic EV Population from Plasma Samples
4.10. DNA Extraction from Isolated sEVs
4.11. PCR Amplification of BRAF Gene from EV-DNA
- (a)
- BRAF WT FW: 5′-TAGGTGATTTTGGTCTAGCTACAG+T-3′;
- (b)
- BRAF WT RW: 5′-TTAATCAGTGGAAAAATAGCCTCA-3′;
- (c)
- BRAF V600E FW: 5′-TAGGTGATTTTGGTCTAGCTACAG+A-3′;
- (d)
- BRAF V600E RW: 5′-TTAATCAGTGGAAAAATAGCCTCA-3′.
- (a)
- WT: FW: 5′-TAGGTGATTTTGGTCTAGCTACAG+T-3′;
- (b)
- WT RW: 5′-TTAATCAGTGGAAAAATAGCCTCA-3′;
- (c)
- V600E FW: 5′-TAGGTGATTTTGGTCTAGCTACAG+A-3′;
- (d)
- V600E RW: 5′-TTAATCAGTGGAAAAATAGCCTCA-3′.
- (e)
- Probe: 5′-FAM-CCGAAGGGGATC+CAGACAA+CTGTTCAAACTGCCTTCGG-3BHQ1-3
4.12. Statistical Analysis
4.13. Graphical Abstract
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- van Niel, G.; D’Angelo, G.; Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 2018, 19, 213–228. [Google Scholar] [CrossRef] [PubMed]
- Kastelowitz, N.; Yin, H. Exosomes and microvesicles: Identification and targeting by particle size and lipid chemical probes. Chembiochem 2014, 15, 923–928. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Keller, S.; Sanderson, M.P.; Stoeck, A.; Altevogt, P. Exosomes: From biogenesis and secretion to biological function. Immunol. Lett. 2006, 107, 102–108. [Google Scholar] [CrossRef]
- Vlassov, A.V.; Magdaleno, S.; Setterquist, R.; Conrad, R. Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim. Biophys. Acta. 2012, 1820, 940–948. [Google Scholar] [CrossRef]
- Kalluri, R.; LeBleu, V.S. The biology, function, and biomedical applications of exosomes. Science 2020, 367, eaau6977. [Google Scholar] [CrossRef]
- McAndrews, K.M.; Kalluri, R. Mechanisms associated with biogenesis of exosomes in cancer. Mol. Cancer 2019, 18, 52. [Google Scholar] [CrossRef]
- Zhang, L.; Yu, D. Exosomes in cancer development, metastasis, and immunity. Biochim. Biophys. Acta Rev. Cancer 2019, 1871, 455–468. [Google Scholar] [CrossRef] [PubMed]
- Venturella, M.; Criscuoli, M.; Carraro, F.; Naldini, A.; Zocco, D. Interplay between Hypoxia and Extracellular Vesicles in Cancer and Inflammation. Biology 2021, 10, 606. [Google Scholar] [CrossRef]
- Whiteside, T.L. Tumor-Derived Exosomes and Their Role in Cancer Progression. Adv. Clin. Chem. 2016, 74, 103–141. [Google Scholar] [CrossRef] [Green Version]
- Tucci, M.; Mannavola, F.; Passarelli, A.; Stucci, L.S.; Cives, M.; Silvestris, F. Exosomes in melanoma: A role in tumor progression, metastasis and impaired immune system activity. Oncotarget 2018, 9, 20826–20837. [Google Scholar] [CrossRef] [Green Version]
- Li, S.; Yi, M.; Dong, B.; Tan, X.; Luo, S.; Wu, K. The role of exosomes in liquid biopsy for cancer diagnosis and prognosis prediction. Int. J. Cancer 2021, 148, 2640–2651. [Google Scholar] [CrossRef]
- Henderson, M.; Azorsa, D. The Genomic and Proteomic Content of Cancer Cell-Derived Exosomes. Front. Oncol. 2012, 2, 38. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Peinado, H.; Alečković, M.; Lavotshkin, S.; Matei, I.; Costa-Silva, B.; Moreno-Bueno, G.; Hergueta-Redondo, M.; Williams, C.; García-Santos, G.; Ghajar, C.; et al. Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat. Med. 2012, 18, 883–891. [Google Scholar] [CrossRef] [Green Version]
- van den Boorn, J.G.; Dassler, J.; Coch, C.; Schlee, M.; Hartmann, G. Exosomes as nucleic acid nanocarriers. Adv. Drug Deliv. Rev. 2013, 65, 331–335. [Google Scholar] [CrossRef] [PubMed]
- Zhu, L.; Sun, H.T.; Wang, S.; Huang, S.L.; Zheng, Y.; Wang, C.Q.; Hu, B.Y.; Qin, W.; Zou, T.T.; Fu, Y.; et al. Isolation and characterization of exosomes for cancer research. J. Hematol. Oncol. 2020, 13, 152. [Google Scholar] [CrossRef]
- Théry, C.; Amigorena, S.; Raposo, G.; Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. 2006, 30, 3.22.1–3.22.29. [Google Scholar] [CrossRef] [PubMed]
- Kok, V.C.; Yu, C.C. Cancer-Derived Exosomes: Their Role in Cancer Biology and Biomarker Development. Int. J. Nanomedicine 2020, 15, 8019–8036. [Google Scholar] [CrossRef]
- Li, G.; Mallouk, N.; Flandrin, P.; Garcin, A.; Lambert, C.; Berremila, S.A.; Habchi, H.; Mottet, N. Presence of Urinary Exosomes for Liquid Biopsy of Clear Cell Renal Cell Carcinoma: Protocol for a Pilot Feasibility Study. JMIR Res. Protoc. 2021, 10, e24423. [Google Scholar] [CrossRef]
- Saginala, K.; Barsouk, A.; Aluru, J.S.; Rawla, P.; Barsouk, A. Epidemiology of Melanoma. Med. Sci. 2021, 9, 63. [Google Scholar] [CrossRef]
- Teixido, C.; Castillo, P.; Martinez-Vila, C.; Arance, A.; Alos, L. Molecular Markers and Targets in Melanoma. Cells 2021, 10, 2320. [Google Scholar] [CrossRef]
- Davis, L.E.; Shalin, S.C.; Tackett, A.J. Current state of melanoma diagnosis and treatment. Cancer Biol. Ther. 2019, 20, 1366–1379. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Ziani, L.; Buart, S.; Chouaib, S.; Thiery, J. Hypoxia increases melanoma-associated fibroblasts immunosuppressive potential and inhibitory effect on T cell-mediated cytotoxicity. Oncoimmunology 2021, 10, 1950953. [Google Scholar] [CrossRef] [PubMed]
- Bedogni, B.; Powell, M.B. Hypoxia, melanocytes and melanoma—survival and tumor development in the permissive microenvironment of the skin. Pigment Cell Melanoma Res. 2009, 22, 166–174. [Google Scholar] [CrossRef] [PubMed]
- Lartigau, E.; Randrianarivelo, H.; Avril, M.F.; Margulis, A.; Spatz, A.; Eschwège, F.; Guichard, M. Intratumoral oxygen tension in metastatic melanoma. Melanoma Res. 1997, 7, 400–406. [Google Scholar] [CrossRef]
- Boussadia, Z.; Gambardella, A.R.; Mattei, F.; Parolini, I. Acidic and Hypoxic Microenvironment in Melanoma: Impact of Tumour Exosomes on Disease Progression. Cells 2021, 10, 3311. [Google Scholar] [CrossRef]
- D’Aguanno, S.; Mallone, F.; Marenco, M.; Del Bufalo, D.; Moramarco, A. Hypoxia-dependent drivers of melanoma progression. J. Exp. Clin. Cancer Res. 2021, 40, 159. [Google Scholar] [CrossRef]
- Dratkiewicz, E.; Simiczyjew, A.; Mazurkiewicz, J.; Ziętek, M.; Matkowski, R.; Nowak, D. Hypoxia and Extracellular Acidification as Drivers of Melanoma Progression and Drug Resistance. Cells 2021, 10, 862. [Google Scholar] [CrossRef]
- Malekan, M.; Ebrahimzadeh, M.A.; Sheida, F. The role of Hypoxia-Inducible Factor-1alpha and its signaling in melanoma. Biomed. Pharmacother. 2021, 141, 111873. [Google Scholar] [CrossRef]
- Li, L.; Ren, F.; Qi, C.; Xu, L.; Fang, Y.; Liang, M.; Feng, J.; Chen, B.; Ning, W.; Cao, J. Intermittent hypoxia promotes melanoma lung metastasis via oxidative stress and inflammation responses in a mouse model of obstructive sleep apnea. Respir. Res. 2018, 19, 28. [Google Scholar] [CrossRef] [Green Version]
- Vaupel, P.; Mayer, A. Hypoxia in cancer: Significance and impact on clinical outcome. Cancer Metastasis. Rev. 2007, 26, 225–239. [Google Scholar] [CrossRef]
- Pucciarelli, D.; Lengger, N.; Takacova, M.; Csaderova, L.; Bartosova, M.; Breiteneder, H.; Pastorekova, S.; Hafner, C. Anti-chondroitin sulfate proteoglycan 4-specific antibodies modify the effects of vemurafenib on melanoma cells differentially in normoxia and hypoxia. Int. J. Oncol. 2015, 47, 81–90. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Syrjänen, L.; Luukkaala, T.; Leppilampi, M.; Kallioinen, M.; Pastorekova, S.; Pastorek, J.; Waheed, A.; Sly, W.S.; Parkkila, S.; Karttunen, T. Expression of cancer-related carbonic anhydrases IX and XII in normal skin and skin neoplasms. APMIS 2014, 122, 880–889. [Google Scholar] [CrossRef] [PubMed]
- Andreucci, E.; Ruzzolini, J.; Peppicelli, S.; Bianchini, F.; Laurenzana, A.; Carta, F.; Supuran, C.T.; Calorini, L. The carbonic anhydrase IX inhibitor SLC-0111 sensitises cancer cells to conventional chemotherapy. J. Enzyme Inhib. Med. Chem. 2019, 34, 117–123. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Supuran, C.T. Carbonic anhydrase inhibitors as emerging agents for the treatment and imaging of hypoxic tumors. Expert Opin. Investig. Drugs 2018, 27, 963–970. [Google Scholar] [CrossRef] [PubMed]
- Federici, C.; Lugini, L.; Marino, M.L.; Carta, F.; Iessi, E.; Azzarito, T.; Supuran, C.T.; Fais, S. Lansoprazole and carbonic anhydrase IX inhibitors sinergize against human melanoma cells. J. Enzyme Inhib. Med. Chem. 2016, 31, 119–125. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chafe, S.C.; McDonald, P.C.; Saberi, S.; Nemirovsky, O.; Venkateswaran, G.; Burugu, S.; Gao, D.; Delaidelli, A.; Kyle, A.H.; Baker, J.H.E.; et al. Targeting Hypoxia-Induced Carbonic Anhydrase IX Enhances Immune-Checkpoint Blockade Locally and Systemically. Cancer Immunol. Res. 2019, 7, 1064–1078. [Google Scholar] [CrossRef] [Green Version]
- Kciuk, M.; Gielecińska, A.; Mujwar, S.; Mojzych, M.; Marciniak, B.; Drozda, R.; Kontek, R. Targeting carbonic anhydrase IX and XII isoforms with small molecule inhibitors and monoclonal antibodies. J. Enzyme Inhib. Med. Chem. 2022, 37, 1278–1298. [Google Scholar] [CrossRef]
- Dorai, T.; Sawczuk, I.S.; Pastorek, J.; Wiernik, P.H.; Dutcher, J.P. The role of carbonic anhydrase IX overexpression in kidney cancer. Eur. J. Cancer 2005, 41, 2935–2947. [Google Scholar] [CrossRef]
- Saarnio, J.; Parkkila, S.; Parkkila, A.K.; Haukipuro, K.; Pastoreková, S.; Pastorek, J.; Kairaluoma, M.I.; Karttunen, T.J. Immunohistochemical study of colorectal tumors for expression of a novel transmembrane carbonic anhydrase, MN/CA IX, with potential value as a marker of cell proliferation. Am. J. Pathol. 1998, 153, 279–285. [Google Scholar] [CrossRef] [Green Version]
- Pastorekova, S.; Gillies, R.J. The role of carbonic anhydrase IX in cancer development: Links to hypoxia, acidosis, and beyond. Cancer Metastasis Rev. 2019, 38, 65–77. [Google Scholar] [CrossRef]
- Zandberga, E.; Zayakin, P.; Ābols, A.; Pūpola, D.; Trapencieris, P.; Linē, A. Depletion of carbonic anhydrase IX abrogates hypoxia-induced overexpression of stanniocalcin-1 in triple negative breast cancer cells. Cancer Biol. Ther. 2017, 18, 596–605. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yang, J.S.; Lin, C.W.; Hsieh, Y.H.; Chien, M.H.; Chuang, C.Y.; Yang, S.F. Overexpression of carbonic anhydrase IX induces cell motility by activating matrix metalloproteinase-9 in human oral squamous cell carcinoma cells. Oncotarget 2017, 8, 83088–83099. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Marks, I.S.; Gardeen, S.S.; Kurdziel, S.J.; Nicolaou, S.T.; Woods, J.E.; Kularatne, S.A.; Low, P.S. Development of a Small Molecule Tubulysin B Conjugate for Treatment of Carbonic Anhydrase IX Receptor Expressing Cancers. Mol. Pharm. 2018, 15, 2289–2296. [Google Scholar] [CrossRef]
- Wen, J.; Yang, T.; Mallouk, N.; Zhang, Y.; Li, H.; Lambert, C.; Li, G. Urinary Exosomal CA9 mRNA as a Novel Liquid Biopsy for Molecular Diagnosis of Bladder Cancer. Int. J. Nanomedicine 2021, 16, 4805–4811. [Google Scholar] [CrossRef] [PubMed]
- Himbert, D.; Zeuschner, P.; Ayoubian, H.; Heinzelmann, J.; Stöckle, M.; Junker, K. Characterization of CD147, CA9, and CD70 as Tumor-Specific Markers on Extracellular Vesicles in Clear Cell Renal Cell Carcinoma. Diagnostics 2020, 10, 1034. [Google Scholar] [CrossRef]
- Ozensoy Guler, O.; Supuran, C.T.; Capasso, C. Carbonic anhydrase IX as a novel candidate in liquid biopsy. J. Enzym. Inhib. Med. Chem. 2020, 35, 255–260. [Google Scholar] [CrossRef] [Green Version]
- Takacova, M.; Barathova, M.; Zatovicova, M.; Golias, T.; Kajanova, I.; Jelenska, L.; Sedlakova, O.; Svastova, E.; Kopacek, J.; Pastorekova, S. Carbonic Anhydrase IX-Mouse versus Human. Int. J. Mol. Sci. 2019, 21, 246. [Google Scholar] [CrossRef] [Green Version]
- Rossi, S.; Cordella, M.; Tabolacci, C.; Nassa, G.; D’Arcangelo, D.; Senatore, C.; Pagnotto, P.; Magliozzi, R.; Salvati, A.; Weisz, A.; et al. TNF-alpha and metalloproteases as key players in melanoma cells aggressiveness. J. Exp. Clin. Cancer Res. 2018, 37, 326. [Google Scholar] [CrossRef]
- Závada, J.; Závadová, Z.; Zat’ovicová, M.; Hyrsl, L.; Kawaciuk, I. Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients. Br. J. Cancer 2003, 89, 1067–1071. [Google Scholar] [CrossRef] [Green Version]
- Eckert, A.W.; Horter, S.; Bethmann, D.; Kotrba, J.; Kaune, T.; Rot, S.; Bache, M.; Bilkenroth, U.; Reich, W.; Greither, T.; et al. Investigation of the Prognostic Role of Carbonic Anhydrase 9 (CAIX) of the Cellular mRNA/Protein Level or Soluble CAIX Protein in Patients with Oral Squamous Cell Carcinoma. Int. J. Mol. Sci. 2019, 20, 375. [Google Scholar] [CrossRef] [Green Version]
- Schütze, D.; Milde-Langosch, K.; Witzel, I.; Rody, A.; Karn, T.; Schmidt, M.; Choschzick, M.; Jänicke, F.; Müller, V. Relevance of cellular and serum carbonic anhydrase IX in primary breast cancer. J. Cancer Res. Clin. Oncol. 2013, 139, 747–754. [Google Scholar] [CrossRef] [PubMed]
- Woelber, L.; Kress, K.; Kersten, J.F.; Choschzick, M.; Kilic, E.; Herwig, U.; Lindner, C.; Schwarz, J.; Jaenicke, F.; Mahner, S.; et al. Carbonic anhydrase IX in tumor tissue and sera of patients with primary cervical cancer. BMC Cancer 2011, 11, 12. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Giuntini, G.; Monaci, S.; Cau, Y.; Mori, M.; Naldini, A.; Carraro, F. Inhibition of Melanoma Cell Migration and Invasion Targeting the Hypoxic Tumor Associated CAXII. Cancers 2020, 12, 3018. [Google Scholar] [CrossRef]
- King, H.W.; Michael, M.Z.; Gleadle, J.M. Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer 2012, 12, 421. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Umezu, T.; Tadokoro, H.; Azuma, K.; Yoshizawa, S.; Ohyashiki, K.; Ohyashiki, J.H. Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1. Blood 2014, 124, 3748–3757. [Google Scholar] [CrossRef] [Green Version]
- Liang, Y.; Lehrich, B.M.; Zheng, S.; Lu, M. Emerging methods in biomarker identification for extracellular vesicle-based liquid biopsy. J. Extracell. Vesicles 2021, 10, e12090. [Google Scholar] [CrossRef] [PubMed]
- Hur, J.Y.; Lee, J.S.; Kim, I.A.; Kim, H.J.; Kim, W.S.; Lee, K.Y. Extracellular vesicle-based EGFR genotyping in bronchoalveolar lavage fluid from treatment-naive non-small cell lung cancer patients. Transl. Lung Cancer Res. 2019, 8, 1051–1060. [Google Scholar] [CrossRef]
- Wan, Y.; Liu, B.; Lei, H.; Zhang, B.; Wang, Y.; Huang, H.; Chen, S.; Feng, Y.; Zhu, L.; Gu, Y.; et al. Nanoscale extracellular vesicle-derived DNA is superior to circulating cell-free DNA for mutation detection in early-stage non-small-cell lung cancer. Ann. Oncol. 2018, 29, 2379–2383. [Google Scholar] [CrossRef]
- Logozzi, M.; Mizzoni, D.; Capasso, C.; Del Prete, S.; Di Raimo, R.; Falchi, M.; Angelini, D.F.; Sciarra, A.; Maggi, M.; Supuran, C.T.; et al. Plasmatic exosomes from prostate cancer patients show increased carbonic anhydrase IX expression and activity and low pH. J. Enzyme Inhib. Med. Chem. 2020, 35, 280–288. [Google Scholar] [CrossRef] [Green Version]
- Logozzi, M.; Capasso, C.; Di Raimo, R.; Del Prete, S.; Mizzoni, D.; Falchi, M.; Supuran, C.T.; Fais, S. Prostate cancer cells and exosomes in acidic condition show increased carbonic anhydrase IX expression and activity. J. Enzyme Inhib. Med. Chem. 2019, 34, 272–278. [Google Scholar] [CrossRef] [Green Version]
- Horie, K.; Kawakami, K.; Fujita, Y.; Sugaya, M.; Kameyama, K.; Mizutani, K.; Deguchi, T.; Ito, M. Exosomes expressing carbonic anhydrase 9 promote angiogenesis. Biochem. Biophys. Res. Commun. 2017, 492, 356–361. [Google Scholar] [CrossRef] [PubMed]
- Allelein, S.; Medina-Perez, P.; Lopes, A.L.H.; Rau, S.; Hause, G.; Kölsch, A.; Kuhlmeier, D. Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations. Sci Rep. 2021, 11, 11585. [Google Scholar] [CrossRef] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Venturella, M.; Falsini, A.; Coppola, F.; Giuntini, G.; Carraro, F.; Zocco, D.; Chiesi, A.; Naldini, A. CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients. Int. J. Mol. Sci. 2023, 24, 6122. https://doi.org/10.3390/ijms24076122
Venturella M, Falsini A, Coppola F, Giuntini G, Carraro F, Zocco D, Chiesi A, Naldini A. CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients. International Journal of Molecular Sciences. 2023; 24(7):6122. https://doi.org/10.3390/ijms24076122
Chicago/Turabian StyleVenturella, Marta, Alessandro Falsini, Federica Coppola, Gaia Giuntini, Fabio Carraro, Davide Zocco, Antonio Chiesi, and Antonella Naldini. 2023. "CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients" International Journal of Molecular Sciences 24, no. 7: 6122. https://doi.org/10.3390/ijms24076122