Proteomic analysis of extracellular vesicles from medullospheres reveals a role for iron in the cancer progression of medulloblastoma

  • Maja Larsen
  • Matthias Kuhlmann Kuhlmann
  • Michael Hvam
  • Kenneth Howard
Keywords: Human serum albumin (HSA), Drugs, Albumin-binding, Albumin fusions, Half-life extension, Intracellular delivery, Neonatal Fc receptor (FcRn), Molecular medicine, Targeted drug delivery

Abstract

Background: Medulloblastoma (MB) is the most common malignant childhood brain tumor with the propensity to
disseminate at an early stage, and is associated with high morbidity. New treatment strategies are needed to
improve cure rates and to reduce life-long cognitive and functional deficits associated with current therapies.
Extracellular Vesicles (EVs) are important players in cell-to-cell communication in health and diseases. A clearer
understanding of cell-to-cell communication in tumors can be achieved by studying EV secretion in
medullospheres. This can reveal subtle modifications induced by the passage from adherent to non-adherent
growth, as spheres may account for the adaptation of tumor cells to the mutated environment.
Methods: Formation of medullospheres from MB cell lines stabilized in adherent conditions was obtained through
culture conditioning based on low attachment flasks and specialized medium. EVs collected by ultracentrifugation,
in adherent conditions and as spheres, were subjected to electron microscopy, NanoSight measurements and
proteomics.
Results: Interestingly, iron carrier proteins were only found in EVs shed by CSC-enriched tumor cell population of
spheres. We used iron chelators when culturing MB cell lines as spheres. Iron chelators induced a decrease in
number/size of spheres and in stem cell populations able to initiate in vitro spheres formation.
Conclusions: This work suggests a not yet identified role of iron metabolism in MB progression and invasion and
opens the possibility to use chelators as adjuvants in anti-tumoral chemotherapy.

Downloads

Download data is not yet available.

References

Giangaspero F, Eberhart CG, Haapsalo H, Pietsch T, Wiestler OD, Ellison DW.

Medulloblastoma. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, editors.

WHO classification of tumours of the central nervous system. Lyon: IARC;

p. 132–40.

MacDonald TJ, Aguilera D, Castellino RC. The rationale for targeted

therapies in medulloblastoma. Neuro Oncol. 2014;16:9–20.

Kennedy C, Bull K, Chevignard M, Culliford D, Dörr HG, Doz F, et al. PNET4

study group of the Brain Tumour Group of The European branch of the

International Society of Paediatric Oncology (SIOP-E). Quality of survival and

growth in children and young adults in the PNET4 European controlled trial

of hyperfractionated versus conventional radiation therapy for standard-risk

medulloblastoma. Int J Radiat Oncol Biol Phys. 2014;88:292–300.

Kool M, Korshunov A, Remke M, Jones DT, Schlanstein M, Northcott PA, et al.

Molecular subgroups of medulloblastoma: an international meta-analysis of

transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3,

and Group 4 medulloblastomas. Acta Neuropathol. 2012;123:473–84.

Manoranjan B, Venugopal C, McFarlane N, Doble BW, Dunn SE,

Scheinemann K, et al. Medulloblastoma stem cells: where development and

cancer cross pathways. Pediatr Res. 2012;71:516–22.

Zanini C, Ercole E, Mandili G, Salaroli R, Poli A, Renna C, et al.

Medullospheres from DAOY, UW228 and ONS-76 cells: increased stem cell

population and proteomic modifications. PLoS ONE. 2013;8(5):e63748.

doi:10.1371/journal.pone.0063748.

Momen-Heravi F, Balaj L, Alian S, Mantel PY, Halleck AE, Trachtenberg AJ,

et al. Current methods for the isolation of extracellular vesicles. Biol Chem.

;394:1253–62.

van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R. Classification,

functions, and clinical relevance of extracellular vesicles. Pharmacol Rev.

;64:676–705.

Epple LM, Griffiths SG, Dechkovskaia AM, Dusto NL, White J, Ouellette RJ,

et al. Medulloblastoma Exosome Proteomics Yield Functional Roles for

Extracellular Vesicles. PLoS ONE. 2012;7, e42064. doi:10.1371/

journal.pone.0042064.

Chairoungdua A, Smith DL, Pochard P, Hull M, Caplan MJ. Exosome release

of β-catenin: a novel mechanism that antagonizes Wnt signaling. J Cell Biol.

;190:1079–91.

Wang T, Gilkes DM, Takano N, Xiang L, Luo W, Bishop CJ, et al. Hypoxia-inducible

factors and RAB22A mediate formation of microvesicles that stimulate breast

cancer invasion and metastasis. Proc Natl Acad Sci U S A. 2014;111:E3234–42.

Menck K, Klemm F, Gross JC, Pukrop T, Wenzel D, Binder C. Induction and

transport of Wnt 5a during macrophage-induced malignant invasion is

mediated by two types of extracellular vesicles. Oncotarget.

;4(11):2057–66.

Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: proteomic insights

and diagnostic potential. Expert Rev Proteomics. 2009;6:267–83.

Raimondo F, Morosi L, Chinello C, Magni F, Pitto M. Advances in

membranous vesicle and exosome proteomics improving biological

understanding and biomarker discovery. Proteomics. 2011;11:709–20.

Steegmann-Olmedillas JL. The role of iron in tumour cell proliferation. Clin

Transl Oncol. 2011;13:71–6.

Maggio A, Filosa A, Vitrano A, Aloj G, Kattamis A, Ceci A, et al. Iron chelation

therapy in thalassemia major: A systematic review with meta-analyses of

patients included on randomized clinical trials. Blood Cells Mol Dis.

;47:166–73.

Mandili G, Khadjavi A, Gallo V, Minero VG, Bessone L, Carta F, et al.

Characterization of the protein ubiquitination response induced by

Doxorubicin. FEBS J. 2012;279:2182–91.

Morello N, Tonoli E, Logrand F, Fiorito V, Fagoonee S, Turco E, et al.

Haemopexin affects iron distribution and ferritin expression in mouse brain.

J Cell Mol Med. 2009;13:4192–204.

Bisaro B, Montani M, Konstantinidou G, Marchini C, Pietrella L, Iezzi M, et al.

p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of

breast cancer cells. Breast Cancer Res. 2012;14:R137. doi:10.1186/bcr3342.

Natale M, Bonino D, Consoli P, Alberio T, Ravid RG, Fasano M, et al. A

meta-analysis of two-dimensional electrophoresis pattern of the Parkinson’s

disease-related protein DJ-1. Bioinformatics. 2010;26:946–52.

Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al.

Cytoscape: a software environment for integrated models of biomolecular

interaction networks. Genome Res. 2003;13:2498–504.

Maere S, Heymans K, Kuiper M. BiNGO: a cytoscape plugin to assess

overrepresentation of gene ontology categories in biological networks.

Bioinformatics. 2005;21:3448–9.

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;Chapter 3:Unit 3.22. doi: 10.1002/0471143030.cb0322s30.

Bruno S, Deregibus MC, Camussi G. The secretome of mesenchymal stromal cells:

Role of extracellular vesicles in immunomodulation. Immunol Letters. 2015.

Akers JC, Gonda D, Kim R, Carter BS, Chen CC. Biogenesis of extracellular

vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic

bodies. J Neurooncol. 2013;113:1–11. doi:10.1007/s11060-013-1084-8.

Colombo M, Raposo G, Théry C. Biogenesis and secretion of exosomes.

Curr Opin Cell Biol. 2014;29:116–25.

Azmi AS, Bao B, Sarkar FH. Exosomes in cancer development, metastasis,

and drug resistance: a comprehensive review. Cancer Metastasis Rev.

;32:623–42.

Graner MW, Alzate O, Dechkovskaia AM, Keene JD, Sampson JH, Mitchell DA,

et al. Proteomic and immunologic analyses of tumor brain exosomes. FABES J.

;23:1541–57.

Le NT, Richardson DR. The role of iron in cell cycle progression and the

proliferation of neoplastic cells. Biochim Biophys Acta. 2002;1603:31–46.

Heath JL, Weiss JM, Lavau CP, Wechsler DS. Iron Deprivation in Cancer––Potential

Therapeutic Implications. Nutrients. 2013;5:2836–59.

Ohara T, Noma K, Urano S, Watanabe S, Nishitani S, Tomono Y, et al. A

novel synergistic effect of iron depletion on antiangiogenic cancer therapy.

Int J Cancer. 2013;132:2705–13.

Lane DJ, Mills TM, Shafie NH, Merlot AM, Saleh Moussa R, Kalinowski DS,

et al. Expanding horizons in iron chelation and the treatment of cancer: role

of iron in the regulation of ER stress and the epithelial-mesenchymal

transition. Biochim Biophys Acta. 1845;2014:166–81.

Jansson PJ, Yamagishi T, Arvind A, Seebacher N, Gutierrez E, Stacy A, et al.

Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes

multidrug resistance by a novel mechanism involving the hijacking of

lysosomal P-glycoprotein (Pgp). J Biol Chem. 2015;290:9588–603.

Submit your next manuscript to BioMed Central

Published
2019-02-07
Section
Research Article