Cancer Immunology, Immunotherapy

, Volume 68, Issue 1, pp 131–141 | Cite as

Fighting breast cancer stem cells through the immune-targeting of the xCT cystine–glutamate antiporter

  • Roberto Ruiu
  • Valeria Rolih
  • Elisabetta Bolli
  • Giuseppina Barutello
  • Federica Riccardo
  • Elena Quaglino
  • Irene Fiore Merighi
  • Federica Pericle
  • Gaetano Donofrio
  • Federica Cavallo
  • Laura Conti
Focussed Research Review


Tumor relapse and metastatic spreading act as major hindrances to achieve complete cure of breast cancer. Evidence suggests that cancer stem cells (CSC) would function as a reservoir for the local and distant recurrence of the disease, due to their resistance to radio- and chemotherapy and their ability to regenerate the tumor. Therefore, the identification of appropriate molecular targets expressed by CSC may be critical in the development of more effective therapies. Our studies focused on the identification of mammary CSC antigens and on the development of CSC-targeting vaccines. We compared the transcriptional profile of CSC-enriched tumorspheres from an Her2+ breast cancer cell line with that of the more differentiated parental cells. Among the molecules strongly upregulated in tumorspheres we selected the transmembrane amino-acid antiporter xCT. In this review, we summarize the results we obtained with different xCT-targeting vaccines. We show that, despite xCT being a self-antigen, vaccination was able to induce a humoral immune response that delayed primary tumor growth and strongly impaired pulmonary metastasis formation in mice challenged with tumorsphere-derived cells. Moreover, immunotargeting of xCT was able to increase CSC chemosensitivity to doxorubicin, suggesting that it may act as an adjuvant to chemotherapy. In conclusion, our approach based on the comparison of the transcriptome of tumorspheres and parental cells allowed us to identify a novel CSC-related target and to develop preclinical therapeutic approaches able to impact on CSC biology, and therefore, hampering tumor growth and dissemination.


Cancer stem cell Vaccine Tumorsphere xCT Breast cancer NIBIT 2017 



Antibody-dependent cell cytotoxicity


Aldehyde dehydrogenase


Bovine herpesvirus-4


Cancer stem cell


Extracellular domain




Reactive oxygen species




Virus-like particles



Not applicable.

Author contributions

Roberto Ruiu, Valeria Rolih, Elisabetta Bolli, and Laura Conti produced the results discussed in this review. Roberto Ruiu, Federica Cavallo and Laura Conti provided major contribution in writing and discussing the manuscript. Federica Pericle provided the VLP technology, Gaetano Donofrio the BoHV-4 technology. Elisabetta Bolli and Valeria Rolih wrote and discussed the sections involving VLP and performed the original ELISA assay reported in this review. Giuseppina Barutello and Federica Riccardo wrote and discussed the sections involving the BALB-neuT model and the translatability of the vaccine. Roberto Ruiu produced the figures. Elena Quaglino, Federica Cavallo, Federica Pericle, Irene Fiore Merighi and Laura Conti critically revised the manuscript. All authors read and approved the final version of the manuscript.


This paper was supported by a grant from the Italian Association for Cancer Research (IG 11675) to Federica Cavallo.

Compliance with ethical standards

Conflict of interest

The authors declare that no potential conflicts of interest exist.

Ethical approval

All the in vivo experiments were approved by the Italian Ministry of Health, authorization numbers 174/2015-PR, 1042/2016-PR and 500/2017-PR.

Human and animal rights

Mice used for the vaccination experiments reported in this paper were purchased from Charles River Laboratories or bred at the Molecular Biotechnology Center, University of Turin, where all mice were maintained and treated in accordance with the University Ethical Committee and European Union guidelines under Directive 2010/63.


  1. 1.
    Koren E, Fuchs Y (2016) The bad seed: Cancer stem cells in tumor development and resistance. Drug Resist Updat 28:1–12. CrossRefPubMedGoogle Scholar
  2. 2.
    Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM (2006) Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res 66:9339–9344. CrossRefPubMedGoogle Scholar
  3. 3.
    Gammaitoni L, Leuci V, Mesiano G, Giraudo L, Todorovic M, Carnevale-Schianca F, Aglietta M, Sangiolo D (2014) Immunotherapy of cancer stem cells in solid tumors: initial findings and future prospective. Expert Opin Biol Ther 14:1259–1270. CrossRefPubMedGoogle Scholar
  4. 4.
    Ning N, Pan Q, Zheng F et al (2012) Cancer stem cell vaccination confers significant antitumor immunity. Cancer Res 72:1853–1864. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Vik-Mo EO, Nyakas M, Mikkelsen BV et al. (2013) Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma. Cancer Immunol Immunother. 62: 1499–1509. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Aurisicchio L, Ciliberto G (2012) Genetic cancer vaccines: current status and perspectives. Expert Opin Biol Ther 12:1043–1058. CrossRefPubMedGoogle Scholar
  7. 7.
    Lollini PL, Cavallo F, Nanni P, Forni G (2006) Vaccines for tumour prevention. Nat Rev Cancer 6: 204–216. CrossRefPubMedGoogle Scholar
  8. 8.
    Iezzi M, Quaglino E, Amici A, Lollini PL, Forni G, Cavallo F (2012) DNA vaccination against oncoantigens: a promise. Oncoimmunology. 1: 316–325. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Conti L, Lanzardo S, Arigoni M, Antonazzo R, Radaelli E, Cantarella D, Calogero RA, Cavallo F (2013) The noninflammatory role of high mobility group box 1/Toll-like receptor 2 axis in the self-renewal of mammary cancer stem cells. FASEB J 27:4731–4744. CrossRefGoogle Scholar
  10. 10.
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988. CrossRefGoogle Scholar
  11. 11.
    Grange C, Lanzardo S, Cavallo F, Camussi G, Bussolati B (2008) Sca-1 identifies the tumor-initiating cells in mammary tumors of BALB-neuT transgenic mice. Neoplasia 10:1433–1443CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Ginestier C, Hur MH, Charafe-Jauffret E et al (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1: 555–567. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Weiswald LB, Bellet D, Dangles-Marie V (2015) Spherical cancer models in tumor biology. Neoplasia 17:1–15. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511. CrossRefGoogle Scholar
  15. 15.
    Rovero S, Boggio K, Di Carlo E, Amici A, Quaglino E, Porcedda P, Musiani P, Forni G (2001) Insertion of the DNA for the 163–171 peptide of IL1beta enables a DNA vaccine encoding p185(neu) to inhibit mammary carcinogenesis in Her-2/neu transgenic BALB/c mice. Gene Ther 8:447–452. CrossRefPubMedGoogle Scholar
  16. 16.
    Conti L, Lanzardo S, Ruiu R, Cadenazzi M, Cavallo F, Aime S, Crich SG (2016) L-Ferritin targets breast cancer stem cells and delivers therapeutic and imaging agents. Oncotarget. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lewerenz J, Hewett SJ, Huang Y et al. (2013) The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities. Antioxid Redox Signal 18: 522–555. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Galadari S, Rahman A, Pallichankandy S, Thayyullathil F (2017) Reactive oxygen species and cancer paradox: To promote or to suppress? Free Radic Biol Med 104:144–164. CrossRefGoogle Scholar
  19. 19.
    Lanzardo S, Conti L, Rooke R et al (2016) Immunotargeting of Antigen xCT Attenuates Stem-like Cell Behavior and Metastatic Progression in Breast Cancer. Cancer Res 76:62–72. CrossRefPubMedGoogle Scholar
  20. 20.
    Briggs KJ, Koivunen P, Cao S et al. (2016) Paracrine induction of HIF by glutamate in breast cancer: EglN1 senses cysteine. Cell 166: 126–139. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Bolli E, O’Rourke JP, Conti L et al (2017) A Virus-Like-Particle immunotherapy targeting Epitope-Specific anti-xCT expressed on cancer stem cell inhibits the progression of metastatic cancer in vivo. OncoImmunology. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Ma MZ, Chen G, Wang P et al (2015) Xc- inhibitor sulfasalazine sensitizes colorectal cancer to cisplatin by a GSH-dependent mechanism. Cancer Lett 368:88–96. CrossRefPubMedGoogle Scholar
  23. 23.
    Timmerman LA, Holton T, Yuneva M et al. (2013) Glutamine sensitivity analysis identifies the xCT antiporter as a common triple-negative breast tumor therapeutic target. Cancer Cell 24: 450–465. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Robe PA, Martin DH, Nguyen-Khac MT et al (2009) Early termination of ISRCTN45828668, a phase 1/2 prospective, randomized study of sulfasalazine for the treatment of progressing malignant gliomas in adults. BMC Cancer 9:372. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Ferraro B, Cisper NJ, Talbott KT, Philipson-Weiner L, Lucke CE, Khan AS, Sardesai NY, Weiner DB (2011) Co-delivery of PSA and PSMA DNA vaccines with electroporation induces potent immune responses. Hum Vaccin 7(Suppl): 120–127CrossRefPubMedGoogle Scholar
  26. 26.
    Lollini PL, De Giovanni C, Pannellini T, Cavallo F, Forni G, Nanni P (2005) Cancer immunoprevention. Future Oncol 1:57–66. CrossRefPubMedGoogle Scholar
  27. 27.
    Marin-Acevedo JA, Soyano AE, Dholaria B, Knutson KL, Lou Y (2018) Cancer immunotherapy beyond immune checkpoint inhibitors. J Hematol Oncol 11:8. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Li L, Petrovsky N (2016) Molecular mechanisms for enhanced DNA vaccine immunogenicity. Expert Rev Vacc 15: 313–329. CrossRefGoogle Scholar
  29. 29.
    Rolla S, Nicolo C, Malinarich S, Orsini M, Forni G, Cavallo F, Ria F (2006) Distinct and non-overlapping T cell receptor repertoires expanded by DNA vaccination in wild-type and HER-2 transgenic BALB/c mice. J Immunol 177:7626–7633CrossRefPubMedGoogle Scholar
  30. 30.
    Larocca C, Schlom J (2011) Viral vector-based therapeutic cancer vaccines. Cancer J 17: 359–371. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Franceschi V, Stellari FF, Mangia C, Jacca S, Lavrentiadou S, Cavirani S, Heikenwalder M, Donofrio G (2014) In vivo image analysis of BoHV-4-based vector in mice. PLoS One 9:e95779. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Donofrio G, Cavirani S, Simone T, van Santen VL (2002) Potential of bovine herpesvirus 4 as a gene delivery vector. J Virol Methods 101:49–61CrossRefPubMedGoogle Scholar
  33. 33.
    Jacca S, Rolih V, Quaglino E et al (2016) Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2(+) mammary cancer. Oncoimmunology 5:e1082705. CrossRefPubMedGoogle Scholar
  34. 34.
    Shirbaghaee Z, Bolhassani A (2016) Different applications of virus-like particles in biology and medicine: vaccination and delivery systems. Biopolymers 105: 113–132. CrossRefPubMedGoogle Scholar
  35. 35.
    Fuenmayor J, Godia F, Cervera L (2017) Production of virus-like particles for vaccines. N Biotechnol 39: 174–180. CrossRefPubMedGoogle Scholar
  36. 36.
    Ong HK, Tan WS, Ho KL (2017) Virus like particles as a platform for cancer vaccine development. PeerJ 5:e4053. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Schwarz B, Uchida M, Douglas T (2017) Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology. Adv Virus Res 97:1–60. CrossRefPubMedGoogle Scholar
  38. 38.
    Gomes AC, Mohsen M, Bachmann MF (2017) Harnessing nanoparticles for immunomodulation and vaccines. Vaccines (Basel). CrossRefPubMedCentralGoogle Scholar
  39. 39.
    Rovero S, Amici A, Di Carlo E et al (2000) DNA vaccination against rat her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. J Immunol 165:5133–5142CrossRefPubMedGoogle Scholar
  40. 40.
    Chen RS, Song YM, Zhou ZY et al (2009) Disruption of xCT inhibits cancer cell metastasis via the caveolin-1/beta-catenin pathway. Oncogene 28:599–609. CrossRefPubMedGoogle Scholar
  41. 41.
    Pulaski BA, Ostrand-Rosenberg S (2001) Mouse 4T1 breast tumor model. Curr Protoc Immunol 39: 20.2.1–20.2.16. CrossRefGoogle Scholar
  42. 42.
    Tallerico R, Conti L, Lanzardo S et al (2017) NK cells control breast cancer and related cancer stem cell hematological spread. Oncoimmunology 6:e1284718. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Conti L, Ruiu R, Barutello G, Macagno M, Bandini S, Cavallo F, Lanzardo S (2014) Microenvironment, oncoantigens, and antitumor vaccination: lessons learned from BALB-neuT mice. Biomed Res Int. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Fusella F, Secli L, Busso E et al (2017) The IKK/NF-kappaB signaling pathway requires Morgana to drive breast cancer metastasis. Nat Commun 8:1636. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Srivastava MK, Sinha P, Clements VK, Rodriguez P, Ostrand-Rosenberg S (2010) Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. Cancer Res 70:68–77. CrossRefPubMedGoogle Scholar
  46. 46.
    Dixon SJ, Patel DN, Welsch M et al (2014) Pharmacological inhibition of cystine–glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife 3:e02523. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Chen D, Fan Z, Rauh M, Buchfelder M, Eyupoglu IY, Savaskan N (2017) ATF4 promotes angiogenesis and neuronal cell death and confers ferroptosis in a xCT-dependent manner. Oncogene 36:5593–5608. CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Sato H, Tamba M, Okuno S, Sato K, Keino-Masu K, Masu M, Bannai S (2002) Distribution of cystine/glutamate exchange transporter, system x(c)-, in the mouse brain. J Neurosci 22:8028–8033CrossRefPubMedGoogle Scholar
  49. 49.
    Ottestad-Hansen S, Hu QX, Follin-Arbelet VV, Bentea E, Sato H, Massie A, Zhou Y, Danbolt NC (2018) The cystine-glutamate exchanger (xCT, Slc7a11) is expressed in significant concentrations in a subpopulation of astrocytes in the mouse brain. Glia 66: 951–970. CrossRefPubMedGoogle Scholar
  50. 50.
    Sato H, Shiiya A, Kimata M et al (2005) Redox imbalance in cystine/glutamate transporter-deficient mice. J Biol Chem 280:37423–37429. CrossRefPubMedGoogle Scholar
  51. 51.
    Massie A, Schallier A, Kim SW et al (2011) Dopaminergic neurons of system x(c)(-)-deficient mice are highly protected against 6-hydroxydopamine-induced toxicity. FASEB J 25:1359–1369. CrossRefPubMedGoogle Scholar
  52. 52.
    De Bundel D, Schallier A, Loyens E et al (2011) Loss of system x(c)- does not induce oxidative stress but decreases extracellular glutamate in hippocampus and influences spatial working memory and limbic seizure susceptibility. J Neurosci 31:5792–5803. CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Mesci P, Zaidi S, Lobsiger CS, Millecamps S, Escartin C, Seilhean D, Sato H, Mallat M, Boillee S (2015) System xC- is a mediator of microglial function and its deletion slows symptoms in amyotrophic lateral sclerosis mice. Brain 138:53–68. CrossRefPubMedGoogle Scholar
  54. 54.
    Evonuk KS, Baker BJ, Doyle RE et al. (2015) Inhibition of system Xc(-) transporter attenuates autoimmune inflammatory demyelination. J Immunol 195: 450–63. CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Shibasaki T, Iuchi Y, Okada F, Kuwata K, Yamanobe T, Bannai S, Tomita Y, Sato H, Fujii J (2009) Aggravation of ischemia-reperfusion-triggered acute renal failure in xCT-deficient mice. Arch Biochem Biophys 490:63–69. CrossRefPubMedGoogle Scholar
  56. 56.
    Kobayashi S, Kuwata K, Sugimoto T, Igarashi K, Osaki M, Okada F, Fujii J, Bannai S, Sato H (2012) Enhanced expression of cystine/glutamate transporter in the lung caused by the oxidative-stress-inducing agent paraquat. Free Radic Biol Med 53:2197–2203. CrossRefPubMedGoogle Scholar
  57. 57.
    Kang ES, Lee J, Homma T et al (2017) xCT deficiency aggravates acetaminophen-induced hepatotoxicity under inhibition of the transsulfuration pathway. Free Radic Res 51:80–90. CrossRefPubMedGoogle Scholar
  58. 58.
    Huang Y, Dai Z, Barbacioru C, Sadee W (2005) Cystine-glutamate transporter SLC7A11 in cancer chemosensitivity and chemoresistance. Cancer Res 65:7446–7454. CrossRefPubMedGoogle Scholar
  59. 59.
    Conklin KA (2004) Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr Cancer Ther 3:294–300. CrossRefGoogle Scholar
  60. 60.
    Li Y, Tan Z, Li Z, Sun Z, Duan S, Li W (2012) Impaired long-term potentiation and long-term memory deficits in xCT-deficient sut mice. Biosci Rep 32:3153–21. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Roberto Ruiu
    • 1
  • Valeria Rolih
    • 1
  • Elisabetta Bolli
    • 1
  • Giuseppina Barutello
    • 1
  • Federica Riccardo
    • 1
  • Elena Quaglino
    • 1
  • Irene Fiore Merighi
    • 1
  • Federica Pericle
    • 2
  • Gaetano Donofrio
    • 3
  • Federica Cavallo
    • 1
  • Laura Conti
    • 1
  1. 1.Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology CenterUniversity of TurinTurinItaly
  2. 2.Agilvax, IncAlbuquerqueUSA
  3. 3.Department of Medical Veterinary ScienceUniversity of ParmaParmaItaly

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