21 december 2021: Bron: UC Davis Comprehensive Cancer Center (met dank aan Clemens Löwik - Erasmus MC)

Hoe vaak worden er niet studies gepubliceerd die beloven dat er een medicijn en/of behandeling is gevonden tegen kanker. Dat kanker een behandelbare ziekte of op z'n minst een chronische ziekte kan worden. Maar tot op vandaag is die genezende behandeling nog niet gevonden helaas.

Toch wil ik deze in vitro studie hier onder jullie aandacht brengen want het lijkt een hele interessante ontdekking die een onderzoeksgroep van het UC Davis Comprehensive Cancer Center heeft gedaan.

Vrije vertaling van citagten uit hun persbericht:

Een onderzoeksteam van het UC Davis Comprehensive Cancer Center heeft een cruciaal epitoop geïdentificeerd = een eiwitgedeelte dat het grotere eiwit kan activeren op de CD95-receptor (Fas en FasL) zodat automatisch apoptose - geprogrammeerde celdood wordt ingezet en dit er daarmee voor zorgt dat beschadigde (kanker-) cellen afsterven.

CD95-receptor (Fas en FasL) worden doodsreceptoren genoemd. Deze eiwitreceptoren bevinden zich op celmembranen. Wanneer ze worden geactiveerd, geven ze een signaal af dat ervoor zorgt dat de cellen zichzelf vernietigen. Het moduleren van de CD95-receptor (Fas en FasL) kan het inzetten van een behandeling met chimere antigeenreceptor (CAR) T-celtherapie naar vormen van kanker met solide tumoren daarmee mogelijk maken, zoals bv bij eierstokkanker waarmee de onderzoekers deze ontdekking deden.

“We hebben de meest kritische epitoop gevonden voor cytotoxische Fas-signalering, evenals de antitumorfunctie van CAR T-cel-rondom de tumorcellen”, zegt Jogender Tushir-Singh, universitair hoofddocent bij de afdeling Medische Microbiologie en Immunologie en senior auteur van de studie. .
“Eerdere pogingen om deze receptor te identificeren zijn niet succesvol geweest. Maar nu we dit epitoop hebben geïdentificeerd, zou er een therapeutisch pad kunnen zijn om de CD95-receptor (Fas en FasL) in tumoren aan te pakken, ‘aldus Tushir-Singh.

Dit nieuwe vermogen om geprogrammeerde celdood te veroorzaken zou de deur kunnen openen voor verbeterde kankerbehandelingen.>>>>>>>lees verder hun persbericht

CANCER CARE October 23, 2023

Researchers identify ‘switch’ to activate cancer cell death

By Public Affairs

(SACRAMENTO)

A research team from the UC Davis Comprehensive Cancer Center has identified a crucial epitope (a protein section that can activate the larger protein) on the CD95 receptor that can cause cells to die. This new ability to trigger programmed cell death could open the door for improved cancer treatments. The findings were published Oct. 14 in the Nature journal Cell Death & Differentiation.

CD95 receptors, also known as Fas, are called death receptors. These protein receptors reside on cell membranes. When activated, they release a signal that causes the cells to self-destruct.

Modulating Fas may also extend the benefits of chimeric antigen receptor (CAR) T-cell therapy to solid tumors like ovarian cancer.

“We have found the most critical epitope for cytotoxic Fas signaling, as well as CAR T-cell bystander anti-tumor function,” said Jogender Tushir-Singh, an associate professor in the Department of Medical Microbiology and Immunology and senior author of the study.

“Previous efforts to target this receptor have been unsuccessful. But now that we’ve identified this epitope, there could be a therapeutic path forward to target Fas in tumors,” Tushir-Singh said.>>>>>>>lees verder hun persbericht

Hier nog een afbeelding uit hun persbericht / studie die het mechanisme van de CD95-receptor (Fas en FasL) weergeeft:

A graphic shows two circles in blue and gold representing tumor cells and a red circle representing CAR-T immunotherapy.

A graphic shows two circles in blue and gold representing tumor cells and a red circle representing CAR-T immunotherapy.

An antigen-negative tumor cell, shown in the golden color on the right, is killed by Fas-mediated "bystander" killing.

Het persbericht gaat nu verder in op de studie die is gepubliceerd in Nature:

Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer

Cell Death & Differentiation volume 30, pages2408–2431 (2023)Cite this article

15k Accesses
917 Altmetric
Metricsdetails

Abstract

Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.

Data availability

Our manuscript does not contain RNA-Seq or other Large Data sets. PDB structure codes used for analysis in the manuscript are available at Protein Data Bank: https://www.rcsb.org/. Detailed sequences of antibodies and other ligands used in the manuscript are provided in tables. Raw western blots are available in the Supplementary file. Requests for reagents should be directed to JT-S (jtsingh@ucdavis.edu).

References

Ashkenazi A. Directing cancer cells to self-destruct with pro-apoptotic receptor agonists. Nat Rev Drug Discov. 2008;7:1001–12.

CAS PubMed Google Scholar
Annibaldi A, Walczak H. Death receptors and their ligands in inflammatory disease and cancer. Cold Spring Harb Perspect Biol. 2020;12:a036384.
Shivange G, Mondal T, Lyerly E, Bhatnagar S, Landen CN, Reddy S, et al. A patch of positively charged residues regulates the efficacy of clinical DR5 antibodies in solid tumors. Cell Rep. 2021;37:109953.

CAS PubMed PubMed Central Google Scholar
Du G, Zhao L, Zheng Y, Belfetmi A, Cai T, Xu B, et al. Autoinhibitory structure of preligand association state implicates a new strategy to attain effective DR5 receptor activation. Cell Res. 2023;33:131–46.
Adams C, Totpal K, Lawrence D, Marsters S, Pitti R, Yee S, et al. Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5. Cell Death Differ. 2008;15:751–61.

CAS PubMed Google Scholar
Peter ME, Hadji A, Murmann AE, Brockway S, Putzbach W, Pattanayak A, et al. The role of CD95 and CD95 ligand in cancer. Cell Death Differ. 2015;22:549–59.

CAS PubMed PubMed Central Google Scholar
Yamada A, Arakaki R, Saito M, Kudo Y, Ishimaru N. Dual role of Fas/FasL-mediated signal in peripheral immune tolerance. Front Immunol. 2017;8:403.

PubMed PubMed Central Google Scholar
Shiraki K, Tsuji N, Shioda T, Isselbacher KJ, Takahashi H. Expression of Fas ligand in liver metastases of human colonic adenocarcinomas. Proc Natl Acad Sci USA. 1997;94:6420–5.

CAS PubMed PubMed Central Google Scholar
Goumas F, Rhashid K, Trauzold A, Fricke H, Kluge M, Becker T, et al. The CD95 ligand inhibitor APG 101 reduces tumor recurrence and metastasis in an adjuvant orthotopic mouse model of pancreatic cancer as well as primary tumor load in a palliative setting. Cancer Res. 2015;75:A115.
Ceppi P, Hadji A, Kohlhapp FJ, Pattanayak A, Hau A, Liu X, et al. CD95 and CD95L promote and protect cancer stem cells. Nat Commun. 2014;5:5238.
Chen L, Park SM, Tumanov AV, Hau A, Sawada K, Feig C, et al. CD95 promotes tumour growth. Nature. 2010;465:492–6.

CAS PubMed PubMed Central Google Scholar
Green DR, Droin N, Pinkoski M. Activation-induced cell death in T cells. Immunol Rev. 2003;193:70–81.

CAS PubMed Google Scholar
Melero I, Rouzaut A, Motz GT, Coukos G. T-cell and NK-cell infiltration into solid tumors: a key limiting factor for efficacious cancer immunotherapy. Cancer Discov. 2014;4:522–6.

CAS PubMed PubMed Central Google Scholar
Hegde PS, Chen DS. Top 10 challenges in cancer immunotherapy. Immunity. 2020;52:17–35.

CAS PubMed Google Scholar
Bajgain P, Torres Chavez AG, Balasubramanian K, Fleckenstein L, Lulla P, Heslop HE, et al. Secreted Fas decoys enhance the antitumor activity of engineered and bystander T cells in Fas ligand-expressing solid tumors. Cancer Immunol Res. 2022;10:1370–85.

CAS PubMed PubMed Central Google Scholar
Upadhyay R, Boiarsky JA, Pantsulaia G, Svensson-Arvelund J, Lin MJ, Wroblewska A, et al. A critical role for Fas-mediated off-target tumor killing in T-cell immunotherapy. Cancer Discov. 2021;11:599–613.

CAS PubMed Google Scholar
Anderson KG, Oda SK, Bates BM, Burnett MG, Rodgers Suarez M, Ruskin SL, et al. Engineering adoptive T cell therapy to co-opt Fas ligand-mediated death signaling in ovarian cancer enhances therapeutic efficacy. J Immunother Cancer. 2022;10:e003959.
Consonni F, Gambineri E, Favre C. ALPS, FAS, and beyond: from inborn errors of immunity to acquired immunodeficiencies. Ann Hematol. 2022;101:469–84.

CAS PubMed PubMed Central Google Scholar
Chodorge M, Zuger S, Stirnimann C, Briand C, Jermutus L, Grutter MG, et al. A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency. Cell Death Differ. 2012;19:1187–95.

CAS PubMed PubMed Central Google Scholar
Graves JD, Kordich JJ, Huang TH, Piasecki J, Bush TL, Sullivan T, et al. Apo2L/TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activity. Cancer Cell. 2014;26:177–89.

CAS PubMed Google Scholar
Hymowitz SG, Christinger HW, Fuh G, Ultsch M, O’Connell M, Kelley RF, et al. Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Mol Cell. 1999;4:563–71.

CAS PubMed Google Scholar
Tamada T, Shinmi D, Ikeda M, Yonezawa Y, Kataoka S, Kuroki R, et al. TRAIL-R2 superoligomerization induced by human monoclonal agonistic antibody KMTR2. Sci Rep. 2015;5:17936.

CAS PubMed PubMed Central Google Scholar
Fellouse FA, Li B, Compaan DM, Peden AA, Hymowitz SG, Sidhu SS. Molecular recognition by a binary code. J Mol Biol. 2005;348:1153–62.

CAS PubMed Google Scholar
Liu WF, Ramagopal U, Cheng HY, Bonanno JB, Toro R, Bhosle R, et al. Crystal structure of the complex of human FasL and its decoy receptor DcR3. Structure. 2016;24:2016–23.

CAS PubMed Google Scholar
Cha SS, Sung BJ, Kim YA, Song YL, Kim HJ, Kim S, et al. Crystal structure of TRAIL-DR5 complex identifies a critical role of the unique frame insertion in conferring recognition specificity. J Biol Chem. 2000;275:31171–7.

CAS PubMed Google Scholar
Shivange G, Urbanek K, Przanowski P, Perry JSA, Jones J, Haggart R, et al. A single-agent dual-specificity targeting of FOLR1 and DR5 as an effective strategy for ovarian cancer. Cancer Cell. 2018;34:331–45.e311.

CAS PubMed PubMed Central Google Scholar
Strauss G, Lindquist JA, Arhel N, Felder E, Karl S, Haas TL, et al. CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling. J Exp Med. 2009;206:1379–93.

CAS PubMed PubMed Central Google Scholar
Holzelova E, Vonarbourg C, Stolzenberg MC, Arkwright PD, Selz F, Prieur AM, et al. Autoimmune lymphoproliferative syndrome with somatic Fas mutations. N Engl J Med. 2004;351:1409–18.

CAS PubMed Google Scholar
Maccari ME, Schneider P, Smulski CR, Meinhardt A, Pinto F, Gonzalez-Granado LI, et al. Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations. J Allergy Clin Immunol. 2023;151:1391–401.e7.
Delgadillo DM, Cespedes-Cruz AI, Rios-Castro E, Rodriguez Maldonado MG, Lopez-Nogueda M, Marquez-Gutierrez M, et al. Differential expression of proteins in an atypical presentation of autoimmune lymphoproliferative syndrome. Int J Mol Sci. 2022;23:5366.
Del-Rey M, Ruiz-Contreras J, Bosque A, Calleja S, Gomez-Rial J, Roldan E, et al. A homozygous Fas ligand gene mutation in a patient causes a new type of autoimmune lymphoproliferative syndrome. Blood. 2006;108:1306–12.

CAS PubMed Google Scholar
Bi LL, Pan G, Atkinson TP, Zheng LX, Dale JK, Makris C, et al. Dominant inhibition of Fas ligand-mediated apoptosis due to a heterozygous mutation associated with autoimmune lymphoproliferative syndrome (ALPS) Type Ib. BMC Med Genet. 2007;8:41.
Song JH, Tse MC, Bellail A, Phuphanich S, Khuri F, Kneteman NM, et al. Lipid rafts and nonrafts mediate tumor necrosis factor related apoptosis-inducing ligand induced apoptotic and nonapoptotic signals in non small cell lung carcinoma cells. Cancer Res. 2007;67:6946–55.

CAS PubMed Google Scholar
Fadeel B, Lindberg J, Achour A, Chiodi F. A three-dimensional model of the Fas/APO-1 molecule: cross-reactivity of anti-Fas antibodies explained by structural mimicry of antigenic sites. Int Immunol. 1998;10:131–40.

CAS PubMed Google Scholar
Mondal T, Shivange GN, Tihagam RG, Lyerly E, Battista M, Talwar D, et al. Unexpected PD-L1 immune evasion mechanism in TNBC, ovarian, and other solid tumors by DR5 agonist antibodies. EMBO Mol Med. 2021;13:e12716.
Concin N, Hamilton E, Randall LM, Banerjee S, Mileshkin L, Coleman RL, et al. Uplift (Engot-Ov67/Gog-3048) a pivotal cohort of upifitamab Rilsodotin, a Napi2b-directed Adc in platinum-resistant ovarian cancer. Int J Gynecol Cancer. 2021;31:A205.

Google Scholar
Banerjee S, Drapkin R, Richardson DL, Birrer M. Targeting NaPi2b in ovarian cancer. Cancer Treat Rev. 2023;112:102489.

CAS PubMed Google Scholar
Lakhani N, Burns T, Barve M, Edenfield J, Hays J, Zarwan C, et al. Upgrade: Phase 1 combination trial of the NaPi2b-directed dolaflexin antibody drug conjugate (ADC) upifitamab rilsodotin (UpRi; XMT-1536) in patients with ovarian cancer. Gynecologic Oncol. 2022;166:S285–6.

Google Scholar
Kandalaft LE, Powell DJ Jr, Coukos G. A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer. J Transl Med. 2012;10:157.

CAS PubMed PubMed Central Google Scholar
Akimzhanov AM, Wang XM, Sun JR, Boehning D. T-cell receptor complex is essential for Fas signal transduction. Proc Natl Acad Sci USA. 2010;107:15105–10.

CAS PubMed PubMed Central Google Scholar
Bernardes N, Fialho AM. Perturbing the dynamics and organization of cell membrane components: a new paradigm for cancer-targeted therapies. Int J Mol Sci. 2018;19:3871.
Chen B, Le W, Wang Y, Li Z, Wang D, Ren L, et al. Targeting negative surface charges of cancer cells by multifunctional nanoprobes. Theranostics. 2016;6:1887–98.

CAS PubMed PubMed Central Google Scholar
Muppidi JR, Siegel RM. Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nat Immunol. 2004;5:182–9.

CAS PubMed Google Scholar
Pan L, Fu TM, Zhao W, Zhao L, Chen W, Qiu C, et al. Higher-order clustering of the transmembrane anchor of DR5 drives signaling. Cell. 2019;176:1477–89.e1414.

CAS PubMed PubMed Central Google Scholar
Endres NF, Das R, Smith AW, Arkhipov A, Kovacs E, Huang Y, et al. Conformational coupling across the plasma membrane in activation of the EGF receptor. Cell. 2013;152:543–56.

CAS PubMed PubMed Central Google Scholar
Wang L, Jin H, Jochems F, Wang S, Lieftink C, Martinez IM, et al. cFLIP suppression and DR5 activation sensitize senescent cancer cells to senolysis. Nat Cancer. 2022;3:1284–99.

CAS PubMed Google Scholar
Sato T, Irie S, Kitada S, Reed JC. Fap-1 – a protein-tyrosine-phosphatase that associates with Fas. Science. 1995;268:411–5.

CAS PubMed Google Scholar
Mongkolsapaya J, Grimes JM, Chen N, Xu XN, Stuart DI, Jones EY, et al. Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation. Nat Struct Biol. 1999;6:1048–53.

CAS PubMed Google Scholar
Banerjee S, Oza AM, Birrer MJ, Hamilton EP, Hasan J, Leary A, et al. Anti-NaPi2b antibody-drug conjugate lifastuzumab vedotin (DNIB0600A) compared with pegylated liposomal doxorubicin in patients with platinum-resistant ovarian cancer in a randomized, open-label, phase II study. Ann Oncol. 2018;29:917–23.

CAS PubMed Google Scholar
Schnell A, Bod L, Madi A, Kuchroo VK. The yin and yang of co-inhibitory receptors: toward anti-tumor immunity without autoimmunity (vol 7, pg 421, 2020). Cell Res. 2020;30:366.

PubMed PubMed Central Google Scholar
Yeku OO, Purdon TJ, Koneru M, Spriggs D, Brentjens RJ. Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment. Sci Rep. 2017;7:10541.

PubMed PubMed Central Google Scholar
Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC, Dowd P, et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature. 1998;396:699–703.

CAS PubMed Google Scholar
Paul S, Pearlman AH, Douglass J, Mog BJ, Hsiue EH, Hwang MS, et al. TCR beta chain-directed bispecific antibodies for the treatment of T cell cancers. Sci Transl Med. 2021;13:eabd3595.
Durocher Y, Butler M. Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol. 2009;20:700–7.

CAS PubMed Google Scholar
Wollebo HS, Woldemichaele B, White MK. Lentiviral transduction of neuronal cells. Methods Mol Biol. 2013;1078:141–6.

CAS PubMed PubMed Central Google Scholar
Wilson NS, Yang B, Yang A, Loeser S, Marsters S, Lawrence D, et al. An Fcgamma receptor-dependent mechanism drives antibody-mediated target-receptor signaling in cancer cells. Cancer Cell. 2011;19:101–13.

CAS PubMed Google Scholar
Takeda K, Kojima Y, Ikejima K, Harada K, Yamashina S, Okumura K, et al. Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease. Proc Natl Acad Sci USA. 2008;105:10895–900.

CAS PubMed PubMed Central Google Scholar
Kiesgen S, Messinger JC, Chintala NK, Tano Z, Adusumilli PS. Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity. Nat Protoc. 2021;16:1331–42.

CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We are thankful for UC Davis core facilities such as flow cytometry and mouse core for assistance. All studies were approved by UC Davis Biological Use Authorization (BUA). All PBMC and animal studies were approved by the Institutional Animal Care and Use Committee.

Funding

JT-S is an early career investigator of Department of Deferense Ovarian Cancer Academy (OCA). This work was also supported by NCI/NIH grant (R01CA233752) to JT-S, and by U.S. DoD Ovarian Cancer Research Program under Early Career Investigator Award (OCRP: OC180412) to JT-S.

Author information

Authors and Affiliations

Laboratory of Novel Biologics, University of California Davis, Davis, CA, USA

Tanmoy Mondal, Himanshu Gaur, Brice E. N. Wamba, Abby Grace Michalak, Camryn Stout, Matthew R. Watson, Sophia L. Aleixo & Jogender Tushir-Singh
Department of Medical Microbiology and Immunology, University of California Davis, Davis, CA, USA

Tanmoy Mondal, Himanshu Gaur, Brice E. N. Wamba, Abby Grace Michalak, Camryn Stout, Matthew R. Watson, Sophia L. Aleixo, Arjun Singh, Sanchita Bhatnagar & Jogender Tushir-Singh
Undergraduate Research Program Volunteers, University of California Davis, Davis, CA, USA

Abby Grace Michalak, Camryn Stout, Matthew R. Watson & Sophia L. Aleixo
Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, IN, USA

Salvatore Condello
Department of Chemical Engineering, University of California Davis, Davis, CA, USA

Roland Faller
Department of Obstetrics and Gynecology, UC Davis School of Medicine, Sacramento, CA, USA

Gary Scott Leiserowitz
UC Davis Comprehensive Cancer Center, UC Davis School of Medicine, Sacramento, CA, USA

Gary Scott Leiserowitz, Sanchita Bhatnagar & Jogender Tushir-Singh
Ovarian Cancer Academy Early Career Investigator at UC Davis, Davis, CA, USA

Jogender Tushir-Singh

Contributions

Conceptualization, design, data collection and assembly of data, data analysis: TM and JT-S. Experimental methodology: TM, HG, BENW, SB, and JT-S. Technical assistance: AM, CS, MRW, SLA, AS. Resources: GSL and SB. Help with editing and critical reading of the final manuscript: GSL, SB, SC and RF. Original draft: JT-S. Supervision and funding: JT-S. All Authors read and approved the final manuscript.

Corresponding author

Correspondence to Jogender Tushir-Singh.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval

Our studies did not include human participants, human data or human tissue. All studies were approved by UC Davis Biological Use Authorization (BUA). All PBMC and animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) here at UC Davis.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Material All Raw Western Files

Supplementary Figures and Legends

Supplementary Figure 1, Figure S1

Supplementary Figure 2, Figure S2

Supplementary Figure 3, Figure S3

Supplementary Figure 4, Figure S4

Supplementary Figure 5, Figure S5

Supplementary Figure 6, Figure S6

Supplementary Figure 7, Figure S7

Supplementary Figure 8, Figure S8

Supplementary Figure 9, Figure S9

Reproducibility checklist

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.