Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

Z. Boussadia, J. Lamberti, F. Mattei, E. Pizzi, R. Puglisi, C. Zanetti, L. Pasquini, F. Fratini, L. Fantozzi, F. Felicetti, K. Fecchi, C. Raggi, M. Sanchez, S. D'Atri, A. Carè, M. Sargiacomo, I. Parolini

Research output: Contribution to journalArticle

Abstract

Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement. © 2018 The Author(s).
Original languageEnglish
JournalJournal of Experimental and Clinical Cancer Research
Volume37
Issue number1
DOIs
Publication statusPublished - 2018

Fingerprint

Exosomes
Melanoma
Transfer (Psychology)
Neoplasms
Proteomics
Cell Movement
Cues

Keywords

  • Exosomes
  • Melanoma progression
  • Microenvironmental acidic pH
  • Tumor stage
  • confocal microscopy
  • disease exacerbation
  • exosome
  • human
  • melanoma
  • metabolism
  • metastasis
  • pathology
  • tumor cell line
  • tumor microenvironment
  • Cell Line, Tumor
  • Disease Progression
  • Humans
  • Melanoma
  • Microscopy, Confocal
  • Neoplasm Metastasis
  • Tumor Microenvironment

Cite this

@article{cb1cfb3fc26a4cadaba232833e98848a,
title = "Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules",
abstract = "Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH na{\"i}ve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement. {\circledC} 2018 The Author(s).",
keywords = "Exosomes, Melanoma progression, Microenvironmental acidic pH, Tumor stage, confocal microscopy, disease exacerbation, exosome, human, melanoma, metabolism, metastasis, pathology, tumor cell line, tumor microenvironment, Cell Line, Tumor, Disease Progression, Humans, Melanoma, Microscopy, Confocal, Neoplasm Metastasis, Tumor Microenvironment",
author = "Z. Boussadia and J. Lamberti and F. Mattei and E. Pizzi and R. Puglisi and C. Zanetti and L. Pasquini and F. Fratini and L. Fantozzi and F. Felicetti and K. Fecchi and C. Raggi and M. Sanchez and S. D'Atri and A. Car{\`e} and M. Sargiacomo and I. Parolini",
note = "Cited By :3 Export Date: 11 April 2019 CODEN: JECRD Correspondence Address: Parolini, I.; Oncology and Molecular Medicine Department, Istituto Superiore di Sanit{\`a}Italy; email: isabella.parolini@iss.it Funding details: RF-2011-02347300 Funding text 1: This work was supported by the Italian Ministry of Health (grant number RF-2011-02347300). References: Vaupel, P., Kallinowski, F., Okunieff, P., Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: A review (1989) Cancer Res, 49, pp. 6449-6465. , 1:CAS:528:DyaK3cXltlSmtQ{\%}3D{\%}3D 2684393; Wike-Hooley, J.L., Haveman, J., Reinhold, H.S., The relevance of tumour pH to the treatment of malignant disease (1984) Radiother Oncol, 2, pp. 343-366. , 1:STN:280:DyaL2M7hsVWjuw{\%}3D{\%}3D; Fukamachi, T., Ikeda, S., Wang, X., Saito, H., Tagawa, M., Kobayashi, H., Gene expressions for signal transduction under acidic conditions (2013) Genes (Basel), 4, pp. 65-85. , 1:CAS:528:DC{\%}2BC3sXktlynsrk{\%}3D; Moellering, R.E., Black, K.C., Krishnamurty, C., Baggett, B.K., Stafford, P., Rain, M., Acid treatment of melanoma cells selects for invasive phenotypes (2008) Clin Exp Metastasis, 25, pp. 411-425. , 1:CAS:528:DC{\%}2BD1cXptVWjs74{\%}3D; Rofstad, E.K., Mathiesen, B., Kindem, K., Galappathi, K., Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice (2006) Cancer Res, 66, pp. 6699-6707. , 1:CAS:528:DC{\%}2BD28XmsVamu74{\%}3D; Raposo, G., Nijman, H.W., Stoorvogel, W., Liejendekker, R., Harding, C.V., Melief, C.J., B lymphocytes secrete antigen-presenting vesicles (1996) J Exp Med, 183, pp. 1161-1172. , 1:CAS:528:DyaK28XhvV2qsLs{\%}3D; Parolini, I., Federici, C., Raggi, C., Lugini, L., Palleschi, S., De Milito, A., Microenvironmental pH is a key factor for exosome traffic in tumor cells (2009) J Biol Chem, 284, pp. 34211-34222. , 1:CAS:528:DC{\%}2BD1MXhsVymu7fN; Ekstr{\"o}m, E.J., Bergenfelz, C., Von B{\"u}low, V., Serifler, F., Carlemalm, E., J{\"o}nsson, G., Andersson, T., Leandersson, K., WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells (2014) Molecular Cancer, 13 (1), p. 88; Valenti, R., Huber, V., Filipazzi, P., Pilla, L., Sovena, G., Villa, A., Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes (2006) Cancer Res, 66, pp. 9290-9298. , 1:CAS:528:DC{\%}2BD28Xps1ahsrg{\%}3D; Peinado, H., Aleckovic, M., Lavotshkin, S., Matei, I., Costa-Silva, B., Moreno-Bueno, G., Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET (2012) Nat Med, 18, pp. 883-891. , 1:CAS:528:DC{\%}2BC38XnsFKgtLw{\%}3D; Hood, J.L., Pan, H., Lanza, G.M., Wickline, S.A., Consortium for translational research in advanced imaging and nanomedicine (C-TRAIN). Paracrine induction of endothelium by tumor exosomes (2009) Lab Investig, 89, pp. 1317-1328; Hood, J.L., San, R.S., Wickline, S.A., Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis (2011) Cancer Res, 71, pp. 3792-3801. , 1:CAS:528:DC{\%}2BC3MXmvFWluro{\%}3D; Peppicelli, S., Bianchini, F., Torre, E., Calorini, L., Contribution of acidic melanoma cells undergoing epithelial-to-mesenchymal transition to aggressiveness of non-acidic melanoma cells (2014) Clin Exp Metastasis., 31, pp. 423-433. , 1:CAS:528:DC{\%}2BC2cXls1Oitr4{\%}3D; Colombo, M., Raposo, G., Thery, C., Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles (2014) Annu Rev Cell Dev Biol, 30, pp. 255-289. , 1:CAS:528:DC{\%}2BC2cXitVeit7jJ; Kowal, J., Arras, G., Colombo, M., Jouve, M., Morath, J.P., Primdal-Bengtson, B., Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes (2016) Proc Natl Acad Sci U S A, 113, pp. E968-E977. , 1:CAS:528:DC{\%}2BC28Xit12rs70{\%}3D; Willms, E., Johansson, H.J., Mager, I., Lee, Y., Blomberg, K.E., Sadik, M., Cells release subpopulations of exosomes with distinct molecular and biological properties (2016) Sci Rep, 6, p. 22519. , 1:CAS:528:DC{\%}2BC28XjsFeks7Y{\%}3D; Lai, C.P., Kim, E.Y., Badr, C.E., Weissleder, R., Mempel, T.R., Tannous, B.A., Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters (2015) Nat Commun, 6, p. 7029. , 1:CAS:528:DC{\%}2BC2MXhtF2ksb3N; Coscia, C., Parolini, I., Sanchez, M., Biffoni, M., Boussadia, Z., Zanetti, C., Generation, quantification, and tracing of metabolically labeled fluorescent exosomes (2016) Methods Mol Biol, 1448, pp. 217-235. , 1:CAS:528:DC{\%}2BC2sXhtVahsrbO; Helmlinger, G., Yuan, F., Dellian, M., Jain, R.K., Interstitial pH and pO2 gradients in solid tumors in vivo: High-resolution measurements reveal a lack of correlation (1997) Nat Med, 3, pp. 177-182. , 1:CAS:528:DyaK2sXpsVOktw{\%}3D{\%}3D; Warburg, O., On the origin of cancer cells (1956) Science, 123, pp. 309-314. , 1:STN:280:DyaG28{\%}2FltV2ktQ{\%}3D{\%}3D; Hess, A.R., Postovit, L.M., Margaryan, N.V., Seftor, E.A., Schneider, G.B., Seftor, R.E., Focal adhesion kinase promotes the aggressive melanoma phenotype (2005) Cancer Res, 65, pp. 9851-9860. , 1:CAS:528:DC{\%}2BD2MXhtFKktL3K; Jiang, C.C., Chen, L.H., Gillespie, S., Wang, Y.F., Kiejda, K.A., Zhang, X.D., Inhibition of MEK sensitizes human melanoma cells to endoplasmic reticulum stress-induced apoptosis (2007) Cancer Res, 67, pp. 9750-9761. , 1:CAS:528:DC{\%}2BD2sXhtFOnsb{\%}2FL; Knutson, J.R., Iida, J., Fields, G.B., McCarthy, J.B., CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes (1996) Mol Biol Cell, 7, pp. 383-396. , 1:CAS:528:DyaK28XhvVSmtLc{\%}3D; Mizuno, H., Kitada, K., Nakai, K., Sarai, A., PrognoScan: A new database for meta-analysis of the prognostic value of genes (2009) BMC Med Genet, 2, p. 18; Kobayashi, T., Stang, E., Fang, K.S., De Moerloose, P., Parton, R.G., Gruenberg, J., A lipid associated with the antiphospholipid syndrome regulates endosome structure and function (1998) Nature, 392, pp. 193-197. , 1:STN:280:DyaK1c7nsFOhsw{\%}3D{\%}3D; Groot Kormelink, T., Arkesteijn, G.J., Nauwelaers, F.A., Van Den Engh, G., Nolte-'T Hoen, E.N., Wauben, M.H., Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry (2016) Cytometry A, 89, pp. 135-147; Graham, J.M., Purification of lipid rafts from cultured cells (2002) ScientificWorldJournal, 2, pp. 1662-1666; Yam, X.Y., Birago, C., Fratini, F., Di Girolamo, F., Raggi, C., Sargiacomo, M., Proteomic analysis of detergent-resistant membrane microdomains in trophozoite blood stage of the human malaria parasite plasmodium falciparum (2013) Mol Cell Proteomics, 12, pp. 3948-3961. , 1:CAS:528:DC{\%}2BC3sXhvFWrsbjE; Ishihama, Y., Oda, Y., Tabata, T., Sato, T., Nagasu, T., Rappsilber, J., Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein (2005) Mol Cell Proteomics, 4, pp. 1265-1272. , 1:CAS:528:DC{\%}2BD2MXhtVGhu7nL; The DAVID Knowledgebase, , https://david.ncifcrf.gov/, Accessed 5 Feb 2017; Albini, A., Iwamoto, Y., Kleinman, H.K., Martin, G.R., Aaronson, S.A., Kozlowski, J.M., A rapid in vitro assay for quantitating the invasive potential of tumor cells (1987) Cancer Res, 47, pp. 3239-3245. , 1:STN:280:DyaL2s3hsFKhtQ{\%}3D{\%}3D 2438036; Niu, J., Dorahy, D.J., Gu, X., Scott, R.J., Draganic, B., Ahmed, N., Integrin expression in colon cancer cells is regulated by the cytoplasmic domain of the beta6 integrin subunit (2002) Int J Cancer, 99, pp. 529-537. , 1:CAS:528:DC{\%}2BD38XjsFansL8{\%}3D; The Prognoscan Database: A New Database for Meta-analysis of the Prognostic Value of Genes, , http://www.prognoscan.org/, Accessed 26 Oct 2017; The Gene Expression Omnibus Functional Genomics Data Repository, , https://www.ncbi.nlm.nih.gov/geo, Accessed 22 Oct 2017; Pospichalova, V., Svoboda, J., Dave, Z., Kotrbova, A., Kaiser, K., Klemova, D., Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer (2015) J Extracell Vesicles, 4. , 25530; Dutta, S., Reamtong, O., Panvongsa, W., Kitdumrongthum, S., Janpipatkul, K., Sangvanich, P., Proteomics profiling of cholangiocarcinoma exosomes: A potential role of oncogenic protein transferring in cancer progression (2015) Biochim Biophys Acta, 1852, pp. 1989-1999. , 1:CAS:528:DC{\%}2BC2MXhtFehsr7M; Lotvall, J., Hill, A.F., Hochberg, F., Buzas, E.I., Di Vizio, D., Gardiner, C., Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the International Society for Extracellular Vesicles (2014) J Extracell Vesicles., 3, p. 26913; Vukovic, V., Tannock, I.F., Influence of low pH on cytotoxicity of paclitaxel, mitoxantrone and topotecan (1997) Br J Cancer, 75, pp. 1167-1172. , 1:CAS:528:DyaK2sXjtVKhsr0{\%}3D; Raghunand, N., Gillies, R.J., PH and drug resistance in tumors (2000) Drug Resist Updat, 3, pp. 39-47. , 1:CAS:528:DC{\%}2BD3cXjsV2mt7k{\%}3D; Hanahan, D., Weinberg, R.A., Hallmarks of cancer: The next generation (2011) Cell, 144, pp. 646-674. , 1:CAS:528:DC{\%}2BC3MXjsFeqtrk{\%}3D; The Exocarta Database, , http://exocarta.org/exosome_markers, Accessed 12 Feb 2017; Dror, S., Sander, L., Schwartz, H., Sheinboim, D., Barzilai, A., Dishon, Y., Melanoma miRNA trafficking controls tumour primary niche formation (2016) Nat Cell Biol, 18, pp. 1006-1017. , 1:CAS:528:DC{\%}2BC28Xhtlykt7zK; The Gene Ontology Database, , http://www.geneontology.org, Accessed 3 Mar 2017; Estrella, V., Chen, T., Lloyd, M., Wojtkowiak, J., Cornnell, H.H., Ibrahim-Hashim, A., Acidity generated by the tumor microenvironment drives local invasion (2013) Cancer Res, 73, pp. 1524-1535. , 1:CAS:528:DC{\%}2BC3sXjtlGqu78{\%}3D; Robey, I.F., Baggett, B.K., Kirkpatrick, N.D., Roe, D.J., Dosescu, J., Sloane, B.F., Bicarbonate increases tumor pH and inhibits spontaneous metastases (2009) Cancer Res, 69, pp. 2260-2268. , 1:CAS:528:DC{\%}2BD1MXjtFyqt7c{\%}3D; Fiaschi, T., Giannoni, E., Taddei, M.L., Cirri, P., Marini, A., Pintus, G., Carbonic anhydrase IX from cancer-associated fibroblasts drives epithelial-mesenchymal transition in prostate carcinoma cells (2013) Cell Cycle, 12, pp. 1791-1801. , 1:CAS:528:DC{\%}2BC3sXhvVKjsrvK; Lardner, A., The effects of extracellular pH on immune function (2001) J Leukoc Biol, 69, pp. 522-530. , 1:CAS:528:DC{\%}2BD3MXivVagurs{\%}3D 11310837; Muhsin-Sharafaldine, M.R., Saunderson, S.C., Dunn, A.C., Faed, J.M., Kleffmann, T., McLellan, A.D., Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles (2016) Oncotarget, 7, pp. 56279-56294; Logozzi, M., De Milito, A., Lugini, L., Borghi, M., Calabro, L., Spada, M., High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients (2009) PLoS One, 4, p. e5219; Alegre, E., Sanmamed, M.F., Rodriguez, C., Carranza, O., Martin-Algarra, S., Gonzalez, A., Study of circulating microRNA-125b levels in serum exosomes in advanced melanoma (2014) Arch Pathol Lab Med, 138, pp. 828-832. , 1:CAS:528:DC{\%}2BC2cXhtFKkt7zN; Lazar, I., Clement, E., Ducoux-Petit, M., Denat, L., Soldan, V., Dauvillier, S., Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines (2015) Pigment Cell Melanoma Res, 28, pp. 464-475. , 1:CAS:528:DC{\%}2BC2MXhtVGmsbjF; Huang, S.K., Darfler, M.M., Nicholl, M.B., You, J., Bemis, K.G., Tegeler, T.J., LC/MS-based quantitative proteomic analysis of paraffin-embedded archival melanomas reveals potential proteomic biomarkers associated with metastasis (2009) PLoS One, 4, p. e4430; Al-Nedawi, K., Meehan, B., Kerbel, R.S., Allison, A.C., Rak, J., Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR (2009) Proc Natl Acad Sci U S A, 106, pp. 3794-3799; Hong, B., Cho, Ji.-H., Kim, H., Choi, E.-J., Rho, S., Kim, J., Kim, Ji., Gho, Y., Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells (2009) BMC Genomics, 10 (1), p. 556; Skog, J., Wurdinger, T., Van Rijn, S., Meijer, D.H., Gainche, L., Sena-Esteves, M., Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers (2008) Nat Cell Biol, 10, pp. 1470-1476. , 1:CAS:528:DC{\%}2BD1cXhsVentbfI; Webber, J., Steadman, R., Mason, M.D., Tabi, Z., Clayton, A., Cancer exosomes trigger fibroblast to myofibroblast differentiation (2010) Cancer Res, 70, pp. 9621-9630. , 1:CAS:528:DC{\%}2BC3cXhsFamur3E",
year = "2018",
doi = "10.1186/s13046-018-0915-z",
language = "English",
volume = "37",
journal = "Journal of Experimental and Clinical Cancer Research",
issn = "0392-9078",
publisher = "BioMed Central Ltd.",
number = "1",

}

TY - JOUR

T1 - Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules

AU - Boussadia, Z.

AU - Lamberti, J.

AU - Mattei, F.

AU - Pizzi, E.

AU - Puglisi, R.

AU - Zanetti, C.

AU - Pasquini, L.

AU - Fratini, F.

AU - Fantozzi, L.

AU - Felicetti, F.

AU - Fecchi, K.

AU - Raggi, C.

AU - Sanchez, M.

AU - D'Atri, S.

AU - Carè, A.

AU - Sargiacomo, M.

AU - Parolini, I.

N1 - Cited By :3 Export Date: 11 April 2019 CODEN: JECRD Correspondence Address: Parolini, I.; Oncology and Molecular Medicine Department, Istituto Superiore di SanitàItaly; email: isabella.parolini@iss.it Funding details: RF-2011-02347300 Funding text 1: This work was supported by the Italian Ministry of Health (grant number RF-2011-02347300). References: Vaupel, P., Kallinowski, F., Okunieff, P., Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: A review (1989) Cancer Res, 49, pp. 6449-6465. , 1:CAS:528:DyaK3cXltlSmtQ%3D%3D 2684393; Wike-Hooley, J.L., Haveman, J., Reinhold, H.S., The relevance of tumour pH to the treatment of malignant disease (1984) Radiother Oncol, 2, pp. 343-366. , 1:STN:280:DyaL2M7hsVWjuw%3D%3D; Fukamachi, T., Ikeda, S., Wang, X., Saito, H., Tagawa, M., Kobayashi, H., Gene expressions for signal transduction under acidic conditions (2013) Genes (Basel), 4, pp. 65-85. , 1:CAS:528:DC%2BC3sXktlynsrk%3D; Moellering, R.E., Black, K.C., Krishnamurty, C., Baggett, B.K., Stafford, P., Rain, M., Acid treatment of melanoma cells selects for invasive phenotypes (2008) Clin Exp Metastasis, 25, pp. 411-425. , 1:CAS:528:DC%2BD1cXptVWjs74%3D; Rofstad, E.K., Mathiesen, B., Kindem, K., Galappathi, K., Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice (2006) Cancer Res, 66, pp. 6699-6707. , 1:CAS:528:DC%2BD28XmsVamu74%3D; Raposo, G., Nijman, H.W., Stoorvogel, W., Liejendekker, R., Harding, C.V., Melief, C.J., B lymphocytes secrete antigen-presenting vesicles (1996) J Exp Med, 183, pp. 1161-1172. , 1:CAS:528:DyaK28XhvV2qsLs%3D; Parolini, I., Federici, C., Raggi, C., Lugini, L., Palleschi, S., De Milito, A., Microenvironmental pH is a key factor for exosome traffic in tumor cells (2009) J Biol Chem, 284, pp. 34211-34222. , 1:CAS:528:DC%2BD1MXhsVymu7fN; Ekström, E.J., Bergenfelz, C., Von Bülow, V., Serifler, F., Carlemalm, E., Jönsson, G., Andersson, T., Leandersson, K., WNT5A induces release of exosomes containing pro-angiogenic and immunosuppressive factors from malignant melanoma cells (2014) Molecular Cancer, 13 (1), p. 88; Valenti, R., Huber, V., Filipazzi, P., Pilla, L., Sovena, G., Villa, A., Human tumor-released microvesicles promote the differentiation of myeloid cells with transforming growth factor-beta-mediated suppressive activity on T lymphocytes (2006) Cancer Res, 66, pp. 9290-9298. , 1:CAS:528:DC%2BD28Xps1ahsrg%3D; Peinado, H., Aleckovic, M., Lavotshkin, S., Matei, I., Costa-Silva, B., Moreno-Bueno, G., Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET (2012) Nat Med, 18, pp. 883-891. , 1:CAS:528:DC%2BC38XnsFKgtLw%3D; Hood, J.L., Pan, H., Lanza, G.M., Wickline, S.A., Consortium for translational research in advanced imaging and nanomedicine (C-TRAIN). Paracrine induction of endothelium by tumor exosomes (2009) Lab Investig, 89, pp. 1317-1328; Hood, J.L., San, R.S., Wickline, S.A., Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis (2011) Cancer Res, 71, pp. 3792-3801. , 1:CAS:528:DC%2BC3MXmvFWluro%3D; Peppicelli, S., Bianchini, F., Torre, E., Calorini, L., Contribution of acidic melanoma cells undergoing epithelial-to-mesenchymal transition to aggressiveness of non-acidic melanoma cells (2014) Clin Exp Metastasis., 31, pp. 423-433. , 1:CAS:528:DC%2BC2cXls1Oitr4%3D; Colombo, M., Raposo, G., Thery, C., Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles (2014) Annu Rev Cell Dev Biol, 30, pp. 255-289. , 1:CAS:528:DC%2BC2cXitVeit7jJ; Kowal, J., Arras, G., Colombo, M., Jouve, M., Morath, J.P., Primdal-Bengtson, B., Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes (2016) Proc Natl Acad Sci U S A, 113, pp. E968-E977. , 1:CAS:528:DC%2BC28Xit12rs70%3D; Willms, E., Johansson, H.J., Mager, I., Lee, Y., Blomberg, K.E., Sadik, M., Cells release subpopulations of exosomes with distinct molecular and biological properties (2016) Sci Rep, 6, p. 22519. , 1:CAS:528:DC%2BC28XjsFeks7Y%3D; Lai, C.P., Kim, E.Y., Badr, C.E., Weissleder, R., Mempel, T.R., Tannous, B.A., Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters (2015) Nat Commun, 6, p. 7029. , 1:CAS:528:DC%2BC2MXhtF2ksb3N; Coscia, C., Parolini, I., Sanchez, M., Biffoni, M., Boussadia, Z., Zanetti, C., Generation, quantification, and tracing of metabolically labeled fluorescent exosomes (2016) Methods Mol Biol, 1448, pp. 217-235. , 1:CAS:528:DC%2BC2sXhtVahsrbO; Helmlinger, G., Yuan, F., Dellian, M., Jain, R.K., Interstitial pH and pO2 gradients in solid tumors in vivo: High-resolution measurements reveal a lack of correlation (1997) Nat Med, 3, pp. 177-182. , 1:CAS:528:DyaK2sXpsVOktw%3D%3D; Warburg, O., On the origin of cancer cells (1956) Science, 123, pp. 309-314. , 1:STN:280:DyaG28%2FltV2ktQ%3D%3D; Hess, A.R., Postovit, L.M., Margaryan, N.V., Seftor, E.A., Schneider, G.B., Seftor, R.E., Focal adhesion kinase promotes the aggressive melanoma phenotype (2005) Cancer Res, 65, pp. 9851-9860. , 1:CAS:528:DC%2BD2MXhtFKktL3K; Jiang, C.C., Chen, L.H., Gillespie, S., Wang, Y.F., Kiejda, K.A., Zhang, X.D., Inhibition of MEK sensitizes human melanoma cells to endoplasmic reticulum stress-induced apoptosis (2007) Cancer Res, 67, pp. 9750-9761. , 1:CAS:528:DC%2BD2sXhtFOnsb%2FL; Knutson, J.R., Iida, J., Fields, G.B., McCarthy, J.B., CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes (1996) Mol Biol Cell, 7, pp. 383-396. , 1:CAS:528:DyaK28XhvVSmtLc%3D; Mizuno, H., Kitada, K., Nakai, K., Sarai, A., PrognoScan: A new database for meta-analysis of the prognostic value of genes (2009) BMC Med Genet, 2, p. 18; Kobayashi, T., Stang, E., Fang, K.S., De Moerloose, P., Parton, R.G., Gruenberg, J., A lipid associated with the antiphospholipid syndrome regulates endosome structure and function (1998) Nature, 392, pp. 193-197. , 1:STN:280:DyaK1c7nsFOhsw%3D%3D; Groot Kormelink, T., Arkesteijn, G.J., Nauwelaers, F.A., Van Den Engh, G., Nolte-'T Hoen, E.N., Wauben, M.H., Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry (2016) Cytometry A, 89, pp. 135-147; Graham, J.M., Purification of lipid rafts from cultured cells (2002) ScientificWorldJournal, 2, pp. 1662-1666; Yam, X.Y., Birago, C., Fratini, F., Di Girolamo, F., Raggi, C., Sargiacomo, M., Proteomic analysis of detergent-resistant membrane microdomains in trophozoite blood stage of the human malaria parasite plasmodium falciparum (2013) Mol Cell Proteomics, 12, pp. 3948-3961. , 1:CAS:528:DC%2BC3sXhvFWrsbjE; Ishihama, Y., Oda, Y., Tabata, T., Sato, T., Nagasu, T., Rappsilber, J., Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein (2005) Mol Cell Proteomics, 4, pp. 1265-1272. , 1:CAS:528:DC%2BD2MXhtVGhu7nL; The DAVID Knowledgebase, , https://david.ncifcrf.gov/, Accessed 5 Feb 2017; Albini, A., Iwamoto, Y., Kleinman, H.K., Martin, G.R., Aaronson, S.A., Kozlowski, J.M., A rapid in vitro assay for quantitating the invasive potential of tumor cells (1987) Cancer Res, 47, pp. 3239-3245. , 1:STN:280:DyaL2s3hsFKhtQ%3D%3D 2438036; Niu, J., Dorahy, D.J., Gu, X., Scott, R.J., Draganic, B., Ahmed, N., Integrin expression in colon cancer cells is regulated by the cytoplasmic domain of the beta6 integrin subunit (2002) Int J Cancer, 99, pp. 529-537. , 1:CAS:528:DC%2BD38XjsFansL8%3D; The Prognoscan Database: A New Database for Meta-analysis of the Prognostic Value of Genes, , http://www.prognoscan.org/, Accessed 26 Oct 2017; The Gene Expression Omnibus Functional Genomics Data Repository, , https://www.ncbi.nlm.nih.gov/geo, Accessed 22 Oct 2017; Pospichalova, V., Svoboda, J., Dave, Z., Kotrbova, A., Kaiser, K., Klemova, D., Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer (2015) J Extracell Vesicles, 4. , 25530; Dutta, S., Reamtong, O., Panvongsa, W., Kitdumrongthum, S., Janpipatkul, K., Sangvanich, P., Proteomics profiling of cholangiocarcinoma exosomes: A potential role of oncogenic protein transferring in cancer progression (2015) Biochim Biophys Acta, 1852, pp. 1989-1999. , 1:CAS:528:DC%2BC2MXhtFehsr7M; Lotvall, J., Hill, A.F., Hochberg, F., Buzas, E.I., Di Vizio, D., Gardiner, C., Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the International Society for Extracellular Vesicles (2014) J Extracell Vesicles., 3, p. 26913; Vukovic, V., Tannock, I.F., Influence of low pH on cytotoxicity of paclitaxel, mitoxantrone and topotecan (1997) Br J Cancer, 75, pp. 1167-1172. , 1:CAS:528:DyaK2sXjtVKhsr0%3D; Raghunand, N., Gillies, R.J., PH and drug resistance in tumors (2000) Drug Resist Updat, 3, pp. 39-47. , 1:CAS:528:DC%2BD3cXjsV2mt7k%3D; Hanahan, D., Weinberg, R.A., Hallmarks of cancer: The next generation (2011) Cell, 144, pp. 646-674. , 1:CAS:528:DC%2BC3MXjsFeqtrk%3D; The Exocarta Database, , http://exocarta.org/exosome_markers, Accessed 12 Feb 2017; Dror, S., Sander, L., Schwartz, H., Sheinboim, D., Barzilai, A., Dishon, Y., Melanoma miRNA trafficking controls tumour primary niche formation (2016) Nat Cell Biol, 18, pp. 1006-1017. , 1:CAS:528:DC%2BC28Xhtlykt7zK; The Gene Ontology Database, , http://www.geneontology.org, Accessed 3 Mar 2017; Estrella, V., Chen, T., Lloyd, M., Wojtkowiak, J., Cornnell, H.H., Ibrahim-Hashim, A., Acidity generated by the tumor microenvironment drives local invasion (2013) Cancer Res, 73, pp. 1524-1535. , 1:CAS:528:DC%2BC3sXjtlGqu78%3D; Robey, I.F., Baggett, B.K., Kirkpatrick, N.D., Roe, D.J., Dosescu, J., Sloane, B.F., Bicarbonate increases tumor pH and inhibits spontaneous metastases (2009) Cancer Res, 69, pp. 2260-2268. , 1:CAS:528:DC%2BD1MXjtFyqt7c%3D; Fiaschi, T., Giannoni, E., Taddei, M.L., Cirri, P., Marini, A., Pintus, G., Carbonic anhydrase IX from cancer-associated fibroblasts drives epithelial-mesenchymal transition in prostate carcinoma cells (2013) Cell Cycle, 12, pp. 1791-1801. , 1:CAS:528:DC%2BC3sXhvVKjsrvK; Lardner, A., The effects of extracellular pH on immune function (2001) J Leukoc Biol, 69, pp. 522-530. , 1:CAS:528:DC%2BD3MXivVagurs%3D 11310837; Muhsin-Sharafaldine, M.R., Saunderson, S.C., Dunn, A.C., Faed, J.M., Kleffmann, T., McLellan, A.D., Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles (2016) Oncotarget, 7, pp. 56279-56294; Logozzi, M., De Milito, A., Lugini, L., Borghi, M., Calabro, L., Spada, M., High levels of exosomes expressing CD63 and caveolin-1 in plasma of melanoma patients (2009) PLoS One, 4, p. e5219; Alegre, E., Sanmamed, M.F., Rodriguez, C., Carranza, O., Martin-Algarra, S., Gonzalez, A., Study of circulating microRNA-125b levels in serum exosomes in advanced melanoma (2014) Arch Pathol Lab Med, 138, pp. 828-832. , 1:CAS:528:DC%2BC2cXhtFKkt7zN; Lazar, I., Clement, E., Ducoux-Petit, M., Denat, L., Soldan, V., Dauvillier, S., Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines (2015) Pigment Cell Melanoma Res, 28, pp. 464-475. , 1:CAS:528:DC%2BC2MXhtVGmsbjF; Huang, S.K., Darfler, M.M., Nicholl, M.B., You, J., Bemis, K.G., Tegeler, T.J., LC/MS-based quantitative proteomic analysis of paraffin-embedded archival melanomas reveals potential proteomic biomarkers associated with metastasis (2009) PLoS One, 4, p. e4430; Al-Nedawi, K., Meehan, B., Kerbel, R.S., Allison, A.C., Rak, J., Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR (2009) Proc Natl Acad Sci U S A, 106, pp. 3794-3799; Hong, B., Cho, Ji.-H., Kim, H., Choi, E.-J., Rho, S., Kim, J., Kim, Ji., Gho, Y., Colorectal cancer cell-derived microvesicles are enriched in cell cycle-related mRNAs that promote proliferation of endothelial cells (2009) BMC Genomics, 10 (1), p. 556; Skog, J., Wurdinger, T., Van Rijn, S., Meijer, D.H., Gainche, L., Sena-Esteves, M., Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers (2008) Nat Cell Biol, 10, pp. 1470-1476. , 1:CAS:528:DC%2BD1cXhsVentbfI; Webber, J., Steadman, R., Mason, M.D., Tabi, Z., Clayton, A., Cancer exosomes trigger fibroblast to myofibroblast differentiation (2010) Cancer Res, 70, pp. 9621-9630. , 1:CAS:528:DC%2BC3cXhsFamur3E

PY - 2018

Y1 - 2018

N2 - Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement. © 2018 The Author(s).

AB - Background: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. Methods: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. Results: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. Conclusions: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement. © 2018 The Author(s).

KW - Exosomes

KW - Melanoma progression

KW - Microenvironmental acidic pH

KW - Tumor stage

KW - confocal microscopy

KW - disease exacerbation

KW - exosome

KW - human

KW - melanoma

KW - metabolism

KW - metastasis

KW - pathology

KW - tumor cell line

KW - tumor microenvironment

KW - Cell Line, Tumor

KW - Disease Progression

KW - Humans

KW - Melanoma

KW - Microscopy, Confocal

KW - Neoplasm Metastasis

KW - Tumor Microenvironment

U2 - 10.1186/s13046-018-0915-z

DO - 10.1186/s13046-018-0915-z

M3 - Article

VL - 37

JO - Journal of Experimental and Clinical Cancer Research

JF - Journal of Experimental and Clinical Cancer Research

SN - 0392-9078

IS - 1

ER -