An endothelial cell line infected by Kaposi’s sarcoma–associated herpes virus (KSHV) allows the investigation of Kaposi’s sarcoma and the validation of novel viral inhibitors in vitro and in vivo

  • Tatyana Dubich
  • Anna Lieske
  • Susann Santag
  • Guillaume Beauclair
  • Jessica Rückert
  • Jennifer Herrmann
  • Jan Gorges
  • Guntram Büsche
  • Uli Kazmaier
  • Hansjörg Hauser
  • Marc Stadler
  • Thomas F. Schulz
  • Dagmar WirthEmail author
Original Article


Kaposi’s sarcoma–associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), a tumor of endothelial origin predominantly affecting immunosuppressed individuals. Up to date, vaccines and targeted therapies are not available. Screening and identification of anti-viral compounds are compromised by the lack of scalable cell culture systems reflecting properties of virus-transformed cells in patients. Further, the strict specificity of the virus for humans limits the development of in vivo models. In this study, we exploited a conditionally immortalized human endothelial cell line for establishment of in vitro 2D and 3D KSHV latency models and the generation of KS-like xenograft tumors in mice. Importantly, the invasive properties and tumor formation could be completely reverted by purging KSHV from the cells, confirming that tumor formation is dependent on the continued presence of KSHV, rather than being a consequence of irreversible transformation of the infected cells. Upon testing a library of 260 natural metabolites, we selected the compounds that induced viral loss or reduced the invasiveness of infected cells in 2D and 3D endothelial cell culture systems. The efficacy of selected compounds against KSHV-induced tumor formation was verified in the xenograft model. Together, this study shows that the combined use of anti-viral and anti-tumor assays based on the same cell line is predictive for tumor reduction in vivo and therefore allows faithful selection of novel drug candidates against Kaposi’s sarcoma.

Key messages

  • Novel 2D, 3D, and xenograft mouse models mimic the consequences of KSHV infection.

  • KSHV-induced tumorigenesis can be reverted upon purging the cells from the virus.

  • A 3D invasiveness assay is predictive for tumor reduction in vivo.

  • Chondramid B, epothilone B, and pretubulysin D diminish KS-like lesions in vivo.


KSHV Drug validation 3D culture system Humanized mouse model Novel anti-viral drugs 



T.D. acknowledges the support by the HZI Grad School. Further, we thank the central animal facility (TEE) at HZI for the excellent support.

Financial support

The work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) via the Cluster of Excellence REBIRTH (From Regenerative Biology to Reconstructive Therapy) and the SFB900 (Chronic Infection).

Compliance with ethical standards

Animal experiments were performed in accordance with the ethical laws and were approved by the local authorities (permission number 33.19-42502-04-17/2480).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest. Dagmar Wirth and Hansjörg Hauser (together with Tobias May) have filed a patent concerning the technology for establishment of conditionally immortalized cell lines (PCT/EP2009/004854).

Supplementary material

109_2018_1733_MOESM1_ESM.pptx (914 kb)
ESM 1 (PPTX 914 kb)


  1. 1.
    zur Hausen H (2001) Oncogenic DNA viruses. Oncogene 20:7820–7823CrossRefGoogle Scholar
  2. 2.
    E a M, Cesarman E, Boshoff C (2010) Kaposi’s sarcoma and its associated herpesvirus. Nat Rev Cancer 10:707–719CrossRefGoogle Scholar
  3. 3.
    Raeisi D, Madani SH, Zare ME (2013) Kaposi’ s sarcoma after kidney transplantation: a 21-years experience. 7:Google Scholar
  4. 4.
    Union for International Cancer Control (2014) Review of cancer medicines on the WHO List of Essential Medicines: Kaposi’s sarcomaGoogle Scholar
  5. 5.
    Guba M, von Breitenbuch P, Steinbauer M, Koehl G, Flegel S, Hornung M, Bruns CJ, Zuelke C, Farkas S, Anthuber M, Jauch KW, Geissler EK (2002) Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 8:128–135CrossRefGoogle Scholar
  6. 6.
    Chang HH, Ganem D (2013) A unique herpesviral transcriptional program in KSHV-infected lymphatic endothelial cells leads to mTORC1 activation and rapamycin sensitivity. Cell Host Microbe 13:429–440Google Scholar
  7. 7.
    Roy D, Sin SH, Lucas A, Venkataramanan R, Wang L, Eason A, Chavakula V, Hilton IB, Tamburro KM, Damania B, Dittmer DP (2013) MTOR inhibitors block Kaposi sarcoma growth by inhibiting essential autocrine growth factors and tumor angiogenesis. Cancer Res 73:2235–2246CrossRefGoogle Scholar
  8. 8.
    Stallone G, Schena A, Infante B, di Paolo S, Loverre A, Maggio G, Ranieri E, Gesualdo L, Schena FP, Grandaliano G (2005) Sirolimus for Kaposi’s sarcoma in renal-transplant recipients. N Engl J Med 352:1317–1323CrossRefGoogle Scholar
  9. 9.
    Alkharsah KR, Singh VV, Bosco R, Santag S, Grundhoff A, Konrad A, Sturzl M, Wirth D, Dittrich-Breiholz O, Kracht M, Schulz TF (2011) Deletion of Kaposi’s sarcoma-associated herpesvirus FLICE inhibitory protein, vFLIP, from the viral genome compromises the activation of STAT1-responsive cellular genes and spindle cell formation in endothelial cells. J Virol 85:10375–10388CrossRefGoogle Scholar
  10. 10.
    Cheng F, Pekkonen P, Laurinavicius S, Sugiyama N, Henderson S, Günther T, Rantanen V, Kaivanto E, Aavikko M, Sarek G, Hautaniemi S, Biberfeld P, Aaltonen L, Grundhoff A, Boshoff C, Alitalo K, Lehti K, Ojala PM (2011) KSHV-initiated notch activation leads to membrane-type-1 matrix metalloproteinase-dependent lymphatic endothelial-to-mesenchymal transition. Cell Host Microbe 10:577–590CrossRefGoogle Scholar
  11. 11.
    Dittmer DP (2003) Transcription profile of Kaposi’s sarcoma-associated herpesvirus in primary Kaposi’s sarcoma lesions as determined by real-time PCR arrays. Cancer Res 63:2010–2015Google Scholar
  12. 12.
    Hosseinipour MC, Sweet KM, Xiong J, Namarika D, Mwafongo A, Nyirenda M, Chiwoko L, Kamwendo D, Hoffman I, Lee J, Phiri S, Vahrson W, Damania B, Dittmer DP (2014) Viral profiling identifies multiple subtypes of Kaposi’s sarcoma. MBio 5:e01633–e01614CrossRefGoogle Scholar
  13. 13.
    Coen N, Duraffour S, Snoeck R, Andrei G (2014) KSHV targeted therapy: An update on inhibitors of viral lytic replication. Viruses 6:4731–4759CrossRefGoogle Scholar
  14. 14.
    Virgin HW 4th, Latreille P, Wamsley P et al (1997) Complete sequence and genomic analysis of murine gammaherpesvirus 68. J Virol 71:5894–5904Google Scholar
  15. 15.
    Barton E, Mandal P, Speck SH (2011) Pathogenesis and host control of gammaherpesviruses: lessons from the mouse. Annu Rev Immunol 29:351–397CrossRefGoogle Scholar
  16. 16.
    Dong S, Forrest JC, Liang X (2017) Murine gammaherpesvirus 68: a small animal model for gammaherpesvirus-associated diseases. Adv Exp Med Biol 1018:225–236CrossRefGoogle Scholar
  17. 17.
    May T, Butueva M, Bantner S, Markusic D, Seppen J, MacLeod RAF, Weich H, Hauser H, Wirth D (2010) Synthetic gene regulation circuits for control of cell expansion. Tissue Eng Part A 16:441–452CrossRefGoogle Scholar
  18. 18.
    Lipps C, Badar M, Butueva M, Dubich T, Singh VV, Rau S, Weber A, Kracht M, Köster M, May T, Schulz TF, Hauser H, Wirth D (2017) Proliferation status defines functional properties of endothelial cells. Cell Mol Life Sci 74:1319–1333CrossRefGoogle Scholar
  19. 19.
    Boivin G, Gaudreau A, Routy JP (2000) Evaluation of the human herpesvirus 8 DNA load in blood and Kaposi’s sarcoma skin lesions from AIDS patients on highly active antiretroviral therapy. AIDS 14:1907–1910CrossRefGoogle Scholar
  20. 20.
    Vieira J, O’Hearn PM (2004) Use of the red fluorescent protein as a marker of Kaposi’s sarcoma-associated herpesvirus lytic gene expression. Virology 325:225–240CrossRefGoogle Scholar
  21. 21.
    Ramirez-Solis R, Rivera-Perez J, Wallace JD et al (1992) Genomic DNA microextraction: a method to screen numerous samples. Anal Biochem 201:331–335CrossRefGoogle Scholar
  22. 22.
    Shao Z, Friedlander M, Hurst CG, Cui Z, Pei DT, Evans LP, Juan AM, Tahir H, Duhamel F, Chen J, Sapieha P, Chemtob S, Joyal JS, Smith LEH (2013) Choroid sprouting assay: an ex vivo model of microvascular angiogenesis. PLoS One 8:e69552CrossRefGoogle Scholar
  23. 23.
    Ullrich A, Chai Y, Pistorius D, Elnakady YA, Herrmann JE, Weissman KJ, Kazmaier U, Müller R (2009) Pretubulysin, a potent and chemically accessible tubulysin precursor from Angiococcus disciformis. Angew Chem Int Ed Engl 48:4422–4425CrossRefGoogle Scholar
  24. 24.
    Kati S, Tsao EH, Gunther T, Weidner-Glunde M, Rothamel T, Grundhoff A, Kellam P, Schulz TF (2013) Activation of the B cell antigen receptor triggers reactivation of latent Kaposi’s sarcoma-associated herpesvirus in B cells. J Virol 87:8004–8016CrossRefGoogle Scholar
  25. 25.
    Kati S, Hage E, Mynarek M, Ganzenmueller T, Indenbirken D, Grundhoff A, Schulz TF (2015) Generation of high-titre virus stocks using BrK.219, a B-cell line infected stably with recombinant Kaposi’s sarcoma-associated herpesvirus. J Virol Methods 217:79–86CrossRefGoogle Scholar
  26. 26.
    Wang L, Damania B (2008) Kaposi’s sarcoma-associated herpesvirus confers a survival advantage to endothelial cells. Cancer Res 68:4640–4648CrossRefGoogle Scholar
  27. 27.
    Appleton MA, Attanoos RL, Jasani B (1996) Thrombomodulin as a marker of vascular and lymphatic tumours. Histopathology 29:153–157CrossRefGoogle Scholar
  28. 28.
    Kang H, Lieberman PM (2011) Mechanism of glycyrrhizic acid inhibition of Kaposi’s sarcoma-associated herpesvirus: disruption of CTCF-cohesin-mediated RNA polymerase II pausing and sister chromatid cohesion. J Virol 85:11159–11169CrossRefGoogle Scholar
  29. 29.
    El Assal R, Gurkan UA, Chen P et al (2016) 3-D microwell array system for culturing virus infected tumor cells. Sci Rep 6:39144CrossRefGoogle Scholar
  30. 30.
    Herrmann J, Fayad AA, Muller R (2017) Natural products from Myxobacteria: novel metabolites and bioactivities. Nat Prod Rep 34:135–160CrossRefGoogle Scholar
  31. 31.
    Mariggiò G, Koch S, Schulz TF (2017) Kaposi sarcoma herpesvirus pathogenesis. Philos Trans R Soc B Biol Sci 372:20160275CrossRefGoogle Scholar
  32. 32.
    Haq I-U, Dalla Pria A, Papanastasopoulos P, Stegmann K, Bradshaw D, Nelson M, Bower M (2016) The clinical application of plasma Kaposi sarcoma herpesvirus viral load as a tumour biomarker: results from 704 patients. HIV Med 17:56–61CrossRefGoogle Scholar
  33. 33.
    Mutlu AD, Cavallin LE, Vincent L et al (2007) In vivo growth-restricted and reversible malignancy induced by Human Herpesvirus-8/ KSHV: a cell and animal model of virally induced Kaposi’s sarcoma. Cancer Cell 11:245–258CrossRefGoogle Scholar
  34. 34.
    Cloutier N, van Eyll O, Janelle M-E, Lefort S, Gao SJ, Flamand L (2008) Increased tumorigenicity of cells carrying recombinant human herpesvirus 8. Arch Virol 153:93–103CrossRefGoogle Scholar
  35. 35.
    Zhang J, Zhu L, Lu X, Feldman ER, Keyes LR, Wang Y, Fan H, Feng H, Xia Z, Sun J, Jiang T, Gao SJ, Tibbetts SA, Feng P (2015) Recombinant murine gamma herpesvirus 68 carrying KSHV G protein-coupled receptor induces angiogenic lesions in mice. PLoS Pathog 11:e1005001CrossRefGoogle Scholar
  36. 36.
    An F, Folarin HM, Compitello N et al (2006) Long-term-infected telomerase-immortalized endothelial cells: a model for Kaposi’s sarcoma-associated herpesvirus latency in vitro and in vivo. J Virol 80:4833–4846CrossRefGoogle Scholar
  37. 37.
    Blacher S, Erpicum C, Lenoir B, Paupert J, Moraes G, Ormenese S, Bullinger E, Noel A (2014) Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour. PLoS One 9:e97019CrossRefGoogle Scholar
  38. 38.
    Qin Z, Dai L, Toole B, Robertson E, Parsons C (2011) Regulation of Nm23-H1 and cell invasiveness by Kaposi’s sarcoma-associated herpesvirus. J Virol 85:3596–3606CrossRefGoogle Scholar
  39. 39.
    Dai L, Qiao J, Nguyen D, Struckhoff AP, Doyle L, Bonstaff K, del Valle L, Parsons C, Toole BP, Renne R, Qin Z (2016) Role of heme oxygenase-1 in the pathogenesis and tumorigenicity of Kaposi’s sarcoma-associated herpesvirus. Oncotarget 7:10459–10471Google Scholar
  40. 40.
    Liu R, Gong M, Li X, Zhou Y, Gao W, Tulpule A, Chaudhary PM, Jung J, Gill PS (2010) Induction, regulation, and biologic function of Axl receptor tyrosine kinase in Kaposi sarcoma. Blood 116:297–305CrossRefGoogle Scholar
  41. 41.
    Jones T, Ramos da Silva S, Bedolla R, Ye F, Zhou F, Gao S (2014) Viral cyclin promotes KSHV-induced cellular transformation and tumorigenesis by overriding contact inhibition. Cell Cycle 13:845–858CrossRefGoogle Scholar
  42. 42.
    Aoki Y, Jaffe ES, Chang Y, Jones K, Teruya-Feldstein J, Moore PS, Tosato G (1999) Angiogenesis and hematopoiesis induced by Kaposi’s sarcoma-associated herpesvirus-encoded interleukin-6. Blood 93:4034–4043Google Scholar
  43. 43.
    Dong X, Cheng A, Zou Z, Yang YS, Sumpter RM Jr, Huang CL, Bhagat G, Virgin HW, Lira SA, Levine B (2016) Endolysosomal trafficking of viral G protein-coupled receptor functions in innate immunity and control of viral oncogenesis. Proc Natl Acad Sci 113:2994–2999CrossRefGoogle Scholar
  44. 44.
    Jensen KK, Manfra DJ, Grisotto MG et al (2005) The human herpes virus 8-encoded chemokine receptor is required for angioproliferation in a murine model of Kaposi’s sarcoma. J Immunol 174:3686 LP–3683694CrossRefGoogle Scholar
  45. 45.
    Grisotto MG, Garin A, Martin AP, Jensen KK, Chan P, Sealfon SC, Lira SA (2006) The human herpesvirus 8 chemokine receptor vGPCR triggers autonomous proliferation of endothelial cells. J Clin Invest 116:1264–1273CrossRefGoogle Scholar
  46. 46.
    Prakash O, Tang Z-Y, Peng X et al (2002) Tumorigenesis and aberrant signaling in transgenic mice expressing the human herpesvirus-8 K1 gene. J Natl Cancer Inst 94:926–935CrossRefGoogle Scholar
  47. 47.
    Bala K, Bosco R, Gramolelli S, Haas DA, Kati S, Pietrek M, Hävemeier A, Yakushko Y, Singh VV, Dittrich-Breiholz O, Kracht M, Schulz TF (2012) Kaposi’s sarcoma herpesvirus K15 protein contributes to virus-induced angiogenesis by recruiting PLCγ1 and activating NFAT1-dependent RCAN1 expression. PLoS Pathog 8:e1002927CrossRefGoogle Scholar
  48. 48.
    Gramolelli S, Weidner-Glunde M, Abere B, Viejo-Borbolla A, Bala K, Rückert J, Kremmer E, Schulz TF (2015) Inhibiting the recruitment of PLCγ1 to Kaposi’s sarcoma herpesvirus K15 protein reduces the invasiveness and angiogenesis of infected endothelial cells. PLoS Pathog 11:e1005105CrossRefGoogle Scholar
  49. 49.
    Braig S, Wiedmann RM, Liebl J, Singer M, Kubisch R, Schreiner L, Abhari BA, Wagner E, Kazmaier U, Fulda S, Vollmar AM (2014) Pretubulysin: a new option for the treatment of metastatic cancer. Cell Death Dis 5:e1001CrossRefGoogle Scholar
  50. 50.
    O’Reilly T, McSheehy PMJ, Wenger F et al (2005) Patupilone (epothilone B, EPO906) inhibits growth and metastasis of experimental prostate tumors in vivo. Prostate 65:231–240CrossRefGoogle Scholar
  51. 51.
    Menhofer MH, Bartel D, Liebl J, Kubisch R, Busse J, Wagner E, Müller R, Vollmar AM, Zahler S (2014) In vitro and in vivo characterization of the actin polymerizing compound chondramide as an angiogenic inhibitor. Cardiovasc Res 104:303–314CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Tatyana Dubich
    • 1
  • Anna Lieske
    • 1
  • Susann Santag
    • 2
    • 3
  • Guillaume Beauclair
    • 2
    • 3
  • Jessica Rückert
    • 2
    • 3
  • Jennifer Herrmann
    • 3
    • 4
  • Jan Gorges
    • 5
  • Guntram Büsche
    • 6
  • Uli Kazmaier
    • 5
  • Hansjörg Hauser
    • 1
  • Marc Stadler
    • 3
    • 7
  • Thomas F. Schulz
    • 2
    • 3
  • Dagmar Wirth
    • 1
    • 8
    Email author return OK on get
  1. 1.Model Systems for Infection and ImmunityHelmholtz Centre for Infection ResearchBraunschweigGermany
  2. 2.Institute of VirologyHannover Medical SchoolHannoverGermany
  3. 3.German Centre for Infection ResearchHannover-BraunschweigGermany
  4. 4.Microbial Natural ProductsHelmholtz Institute for Pharmaceutical ResearchSaarbrückenGermany
  5. 5.Institute of Organic ChemistrySaarland UniversitySaarbrückenGermany
  6. 6.Institute of PathologyHannover Medical SchoolHannoverGermany
  7. 7.Microbial DrugsHelmholtz Centre for Infection ResearchBraunschweigGermany
  8. 8.Institute of Experimental HematologyHannover Medical SchoolHannoverGermany

Personalised recommendations