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
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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.
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.
KeywordsKSHV 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.
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).
- 3.Raeisi D, Madani SH, Zare ME (2013) Kaposi’ s sarcoma after kidney transplantation: a 21-years experience. 7:Google Scholar
- 4.Union for International Cancer Control (2014) Review of cancer medicines on the WHO List of Essential Medicines: Kaposi’s sarcomaGoogle Scholar
- 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.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
- 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.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.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
- 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
- 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
- 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
- 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.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