An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Summary

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Abstract

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

Introduction

Kaposi sarcoma (KS) is a multi-focal angioproliferative tumor affecting dermal, mucosal, and visceral sites that develops most commonly in the setting of advanced immune suppression¹. Four epidemiological forms have been described: classic, an indolent form that typically affects older people of Mediterranean and Middle Eastern heritage; iatrogenic, resulting from treatment with immunosuppressive drugs following organ transplantation; epidemic, an AIDS-defining cancer; and endemic, an HIV-independent form common in children in endemic regions in Africa. With the advent of effective combination anti-retroviral drug regimens for the treatment of ....

Protocol

NOTE: All procedures described in this protocol should be performed under BSL-2 conditions.

1. KSHV Stock Preparation

1. Prepare TNE buffer: dissolve 292.24 mg EDTA in ddH₂O, bring to 225 mL, and adjust to pH 8. Dissolve 605.7 mg Tris in ddH₂O, bring to 225 mL, and adjust to pH 8. Combine EDTA and Tris solutions, add 4.38 g NaCl, adjust final volume to 500 mL, filter sterilize, and store at 4 °C^.
2. Culture the KSHV-positive, EBV-negative primary effusion lymphoma cell line BCBL-1 in a humidified incubator at 37 °C plus 5% CO₂ in RPMI supplemented with 10% heat-inactivate....

Results

The morphology of primary EC is classically described as "cobble stone", and this morphology is not altered by expression of the papillomavirus E6 and E7 genes (Figure 1A). Expression of the E6 and E7 genes alone does not induce a transformed phenotype; thus, cells are susceptible to contact inhibition and will cease dividing upon reaching confluence in culture. The cells will however proliferate and regrow to confluence upon trypsinization and replating at a lower densit.......

Discussion

Oncogenesis is a multistep process that circumvents important safeguards within an organism³⁶. As KS lesions exist along a spectrum of chronic inflammation to true sarcomas, elucidation of certain pathophysiological processes mediated by KSHV requires that some studies be conducted in cell culture models that support transformation⁹. It should be noted that loss of contact inhibition and anchorage-dependent growth, phenotypes indicative of cellular transformation, do not re.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCBL-1 cells	NIH AIDS Reagent Program	3233
PA317 cells	ATCC	CRL-2203
Neonatal dermal microvascular endothelial cells	Lonza	CC-2505
EBM-2 Basal Medium	Lonza	CC-3156
EGM-2 BulletKit	Lonza	CC-3162
anti-KSHV LANA/ORF 73	Advanced Biotechnologies	13-210-100
TrypLETMExpress, no phenol red	ThermoFisher	12604013
RPMI
DMEM
PBS with calcium and magnesium