Method Article

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

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DOI:

10.3791/54828

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August 25th, 2017

 ,  ,  ,  , 
1Division of Pediatric Infectious Diseases, University of Minnesota Medical School, 2Vaccine and Gene Therapy Institute, Oregon Health and Science University

In This Article

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 HIV, epidemic KS is much less commonly diagnosed in developing countries. However, the clinically aggressive endemic and epidemic forms remain among the most commonly diagnosed cancers in many African countries2,3,4. Therefore, identification of effective pathogenesis-targeted drugs for treatment of KS is a research priority.

Histologically, KS lesions are characterized by extensive but abnormal neovascularization whereby spindle cells of EC origin form discontinuous vascular networks5. These abnormal vessels ("vascular slits") allow extravasation of erythrocytes, which give lesions their characteristic color. Additionally, lesions contain numerous leukocytes that characterize chronic inflammation (i.e., lymphocytes, macrophages, and plasma cells). Regression of KS lesions following immune reconstitution has been described, suggesting that KS has features of both a hyper-proliferative lesion and a true tumor6,7,8,9.

KS herpesvirus (KSHV), the causative agent of KS, was identified in 199410. Since then many in vitro cell culture models have been developed to enable pathogenesis studies, including cells explanted from tumor biopsy material and primary or telomerase-expressing EC infected with KSHV in vitro11,12,13,14,15,16,17,18. None of the currently available models fully recapitulates the KS tumor microenvironment, but all have contributed valuable knowledge to our understanding of the pathobiology of KSHV infection. Unlike the other known tumorigenic human herpesvirus Epstein-Barr virus (EBV), KSHV does not readily transform cells in culture following de novo infection19,20,21,22. However, this limitation has been overcome by transducing primary human EC of either mixed microvascular or lymphatic origin with the E6 and E7 genes from human papillomavirus type 16 prior to infection with KSHV23,24. Expression of these exogenous oncogenes dramatically increases the transforming potential of KSHV in vitro in part by providing further inhibition of the retinoblastoma protein and p5323,24. This EC transduction method has allowed multiple laboratories to identify key alterations in host cell gene expression that are induced by KSHV infection and that appear to facilitate KS cell survival and proliferation25,26,27,28,29,30,31,32. The protocols described herein are straightforward and highly reproducible, and will result in the generation of age- and passage-matched KSHV-infected EC and mock-infected controls that can be cultured for far longer than primary cells and will allow for the investigation of oncogenic mechanisms employed by KSHV. Although the protocol includes a method for production of wild type KSHV from the primary effusion lymphoma cell line BCBL-1, E6/E7-immortalized EC are also highly susceptible to infection with recombinant BACmid derived KSHV-BAC1630. Protocols for preparation of BAC16 are described elsewhere33,34.

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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-inactivated fetal bovine serum (FBS) and antimicrobials to approximately 1 to 1.2 x 106 cells per mL.
  3. To induce KSHV production, split cultures 1:2 with fresh medium and add Phorbol 12-Myristate 13-Acetate (PMA) to a final concentration of 20 ng/mL and incubate cultures for 5 days. As an alternative, treat cells with sodium butyrate (NaB; 0.1 mM) for 3 days to induce lytic viral replication.
  4. To harvest viral particles, first centrifuge the culture supernatant at 300 x g for 5 min at room temperature to pellet the cells and transfer supernatants to fresh tubes.
  5. To further clarify the supernatant so as to avoid transfer of cellular debris, centrifuge at 2,500 x g for 10 min at 4 °C.
    NOTE: As an alternative to high speed centrifugation for clarification of culture supernatants, filtration through a sterile 0.45 µm filter can be performed. We have observed a significant drop in titer using filtration; therefore, we routinely perform the second centrifugation step instead.
  6. Next, overlay 5 mL of 25% sucrose solution in TNE buffer with approximately 30 mL of the clarified culture supernatant in 6 ultracentrifuge tubes.
  7. Centrifuge balanced tubes at 75,000 x g for 2 h at 4 °C under vacuum.
  8. Decant culture supernatant and blot the rim of each tube to remove as much supernatant and sucrose solution as possible, then resuspend the virus pellet, which may not be visible, in 150 µL of TNE buffer.
  9. Pool all resuspended virus, mix well, and freeze 25 µL aliquots at -80 °C.

2. Transduction of EC with E6 and E7 Papillomavirus Genes

NOTE: We routinely use standard tissue culture flasks for growing primary EC. However, if unsatisfactory growth of EC are obtained then the use of commercial culture flasks should be considered.

  1. Culture primary human dermal microvascular or lymphatic EC in a humidified incubator at 37°C plus 5% CO2 in EC growth medium (EGM; contains EC basal medium [EBM] supplemented with EGM-2 BulletKit [containing FBS, hydrocortisone, human fibroblast growth factor-B, vascular EC growth factor, insulin-like growth factor-1, ascorbic acid, human epithelial growth factor, antibiotics, and heparin]) in a T75 cell culture flask to approximately 50% confluence.
  2. For transduction, culture PA317 LXSN 16E6E7 cells24,35 in a humidified incubator at 37°C plus 5% CO2 in DMEM supplemented with 10% FBS and antimicrobials in a T150 cell culture flask until they are approximately 90% confluent. Then incubate overnight in 16 mL of medium.
  3. Clarify the PA317 medium by centrifuging at 300 x g for 5 min at room temperature.
  4. Remove the EGM from the EC culture and overlay cells with 12 mL of the clarified PA317 medium and incubate for 4 h.
    NOTE: Centrifuging 16 mL of conditioned medium at 300 x g for 5 min results in a compact pellet that is not disturbed by the careful subsequent removal of 12 mL of clarified supernatant. However, if transfer of PA317 LXSN 16E6E7 cells to primary EC cultures is a concern (the packaging cell line will quickly outgrow EC), then filtration of the conditioned supernatant through a 0.45 µm filter before transfer can be performed.
  5. Replace 6 mL of the PA317 medium with fresh EGM and incubate overnight.
  6. Refeed EC with 12 mL fresh EGM and incubate a further 48 h.
  7. To sub-culture the EC, remove medium and wash with 12 mL of PBS without cations, and add 3 mL of commercial enzymatic dissociation solution (e.g., TrypLE) by and incubate at 37 °C for 3 min.
  8. Transfer cell suspension to a 15 mL conical tube and centrifuge at 300 x g for 5 min at room temperature. Resuspend the resulting cell pellet in fresh EGM and divide evenly into 3 x T75 flasks with a final volume of 12 mL EGM per flask.
  9. To select for EC transduced with E6 and E7, add G418 to a final concentration of 200 µg/mL for two passages, after which the transduced EC can be plated for infection or frozen in liquid nitrogen.

3. Infection of Transduced EC with KSHV

  1. On the day prior to infection harvest EC by enzymatic digestion using the commercial enzymatic dissociation solution as described above. Resuspend cell pellet in 1 mL of EGM. Use 5 µL of cells to prepare a 1/10 dilution in Trypan blue. Count live cells using a hemocytometer and seed 2.5 x 105 live cells in 2 mL of EGM per well in 6 well plates.
  2. On the day of infection, remove EGM and wash cells with 3 mL of PBS with calcium and magnesium per well, and then add 2 mL EBM.
    NOTE: It is essential to use EBM rather than EGM during infection, as the heparin in EGM will inhibit binding of viral particles to and subsequent infection of target EC.
  3. To infect cells with KSHV, add 5 to 20 µL virus stock to each well and swirl plate to mix. For mock-infected cells add an equal volume of TNE buffer to each well.
  4. Centrifuge plates at 400 x g for 30 min at room temperature and then incubate the plates at 37 °C for 90 min.
  5. If the goal of the study is to investigate early events requiring a synchronous viral infection, e.g., early events in de novo infection, remove the viral inoculum at this point. Rinse and refeed the cells with 2 mL fresh EGM. Otherwise, add 2 mL EGM and incubate cultures overnight.
  6. Refeed cells with 2 mL EGM the day after infection and then every other day.
  7. When cells are approximately 90% confluent, harvest by enzymatic digestion using commercial enzymatic dissociation solution as described above, then pool the cells from three wells into a T75, noting both the total passage number and the passage post-infection.
  8. Expand mock- and KSHV-infected EC with 1:3 splits for at least two more passages at which time cells can be used in experiments or frozen in liquid nitrogen.
    NOTE: As with splitting of EC transduced with E6 and E7, maintenance cultures of mock- and KSHV-infected cells should be split before reaching confluence (~85 to 90%).

4. Confirming Infection with KSHV by Immunofluorescence

NOTE: Detection of the KSHV latency-associated nuclear antigen (LANA-1/ORF73) provides a reliable quantitative measure of infection. Anti-LANA antibodies are commercially available.

  1. The day prior to staining, plate two wells each with mock- or KSHV-infected EC on a 24-well plate at 1 x 105 cells per well in 1 mL EGM and incubate overnight.
  2. Wash cells twice with 500 µL of PBS plus calcium and magniseum. Wash for 30 s on a rocking platform.
  3. In an externally-ventillated fume hood, fix cells with 500 µL of 4% paraformaldehyde for 15 min at room temperature.
  4. Wash cells three times with 500 µL of wash buffer (0.1% Triton X-100 plus 0.02% goat serum in PBS plus cations).
  5. Block cells with 500 µL of 2% goat serum in wash buffer for 30 min at room temperature.
  6. Wash cells three times with 500 µL of wash buffer.
  7. Label one well of mock- or KSHV-infected EC with 200 µL of primary antibody diluted 1:100 in wash buffer for 30 min at room temperature on a rocker. Incubate the remaining two wells in 200 µL of wash buffer only.
  8. Wash cells three times with 500 µL of wash buffer.
  9. Label all four wells with 200 µL of secondary antibody diluted 1:100 in wash buffer for 30 min at room temperature on a rocker.
  10. Wash cells three times with 500 µL of wash buffer.
  11. To each well apply 20 µL of mounting medium containing DAPI and a coverslip and evaluate staining using an inverted fluorescent microscope.
  12. Determine the percent infection of KSHV-infected cells by counting the number of LANA-positive nuclei in at least 200 cells. Infection with recombinant KSHV-Bac16 can also be monitored via observation of cultures for expression of GFP.

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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 lowe...

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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...

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Acknowledgements

This work was supported by K12 HD068322 (SCM); R01 CA179921 and P51 OD011092 (AVM); and award 14PRE20320014 from the America Heart Association (SB).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
BCBL-1 cellsNIH AIDS Reagent Program3233
PA317 cellsATCCCRL-2203
Neonatal dermal microvascular endothelial cellsLonzaCC-2505
EBM-2 Basal MediumLonzaCC-3156
EGM-2 BulletKitLonzaCC-3162
anti-KSHV LANA/ORF 73Advanced Biotechnologies13-210-100
TrypLETMExpress, no phenol redThermoFisher12604013
RPMI
DMEM
PBS with calcium and magnesium

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KSHV InfectionEndothelial CellsViral OncogenesisCell TransformationE6 E7 ExpressionViral Particle PurificationSoft Agar AssayAnoikis ResistanceSpindle Cell Morphology

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