The overall goal of this procedure is to prepare age-and passage-matched mock in KSHV infected endothelial cells to enable the study of KSHV-associated changes in host cell gene expression including alterations driving malignant transformation in the context of appropriate controls. This method can help define the key pathophysiological changes in host cell biology that lead to KSHV-associated cancers, thus identifying novel therapeutic targets for disease prevention or treatment. The main advantage of this system is that malignant transformation of endothelial cells following infection with KSHV occurs rapidly and reproducibly allowing the study of early events in the oncogenic process with age-and passage-matched control cells.
Though this model of viral oncogenesis can provide insight into Kaposi Sarcoma development, it can also be applied to other cancer types. Because Kaposi Sarcoma is a vascular tumor, the implications of this cell culture model extend toward understanding other disease processes driven by pathological angiogenesis. To begin, prepare and filter sterilized TNE buffer as described in the text protocol.
Then store the solution at four degrees Celsius. Culture the KSHV positive EBV negative primary effusion lymphoma cell line BCBL-1 in a T150 flask filled with 60 milliliters of RPMI medium. Maintain the cells at 37 degrees Celsius 5%carbon dioxide to obtain a suspension of approximately 1 to 1.2 times 10 to the sixth cells per milliliter.
Once the proper density is reached, split the cells one to two with fresh medium and add PMA to a final concentration of 20 nanograms per milliliter to induce KSHV production. Then incubate the cultures for five days. After five days of incubation, harvest viral particles by spinning the cell suspensions at 300 times g for five minutes at room temperature.
Then transfer the supernatants to a new tube. Centrifuge the supernatants once more at 2, 500 times g at four degrees Celsius for 10 minutes to remove cellular debris. Now that it is clarified, overlay five milliliters of 25%sucrose solution in TNE with approximately 30 milliliters of the supernatant in six ultracentrifuge tubes.
Centrifuge the balanced tubes at 75, 000 times g for two hours at four degrees Celsius under vacuum. Once the centrifugation is completed, decant the supernatant and blot the rim of each tube to remove as much of the liquid as possible. Then resuspend the pelleted virus in 150 microliters of TNE buffer and pool all the samples.
Mix the pooled suspension thoroughly and divide it into 25 microliter aliquots. Store the stock virus at minus 80 degrees Celsius. Culture primary human dermal microvascular or lymphatic endothelial cells in EGM medium in a T75 flask.
Maintain the cell culture at 37 degrees Celsius 5%carbon dioxide until approximately 50%confluence is reached. Prior to endothelial cells transduction, culture PA317 LXSN 16E6E7 cells in a T150 cell culture flask filled with 20 milliliters of DMEM at 37 degrees Celsius 5%carbon dioxide. Once approximately 90%confluence is achieved, replace the medium from PA317 cell culture with 16 milliliters of fresh medium and incubate the cells overnight.
Following an overnight incubation, collect the medium from the PA317 cell culture and clarify it by centrifugation at 300 times g for five minutes at room temperature. Replace the EGM medium from 50%confluent endothelial cell culture with 12 milliliters of the clarified PA317 medium. Incubate the cells for four hours.
Then replace six milliliters of the PA317 medium with six milliliters of fresh EGM medium and incubate the cells overnight. Afterward, remove the medium from endothelial cell culture and feed the cells with 12 milliliters of fresh EGM. Continue to incubate the cells for an additional 48 hours.
To subculture endothelial cells, remove the medium and wash the cells with 12 milliliters of cation-free PBS. Then add three milliliters of a commercial enzymatic dissociation solution and incubate the cells at 37 degrees Celsius for three minutes. Transfer the cell suspension to a 15 milliliter conical tube and centrifuge the cells at 300 times g for five minutes at room temperature.
Once pelleted, resuspend the cells in fresh EGM medium and divide the suspension evenly into three T75 flasks. Adjust the volume of the cultures to 12 milliliters of EGM per flask. Then add 200 micrograms per milliliter of G418 to select transduced endothelial cells.
Harvest the transduced endothelial cells by enzymatic digestion as previously shown. Resuspend endothelial cell pellet in one milliliter of EGM medium. Then mix five microliters of the cell suspension with 45 microliters of Trypan Blue Solution and count viable cells using a hemocytometer.
Seed 2.5 times 10 to the fifth viable cells in two milliliters of EGM medium per well of a six-well plate. On the following day, remove the EGM medium and wash the cells in each well with three milliliters of PBS with calcium and magnesium. Then add two milliliters of EBM medium to each well.
To infect cells with KSHV, add five to 20 microliters of the virus stock to each well and swirl the plate to mix the suspensions. Add an equal volume of TNE buffer to each well containing mock-infected cells. After centrifuging the plate at 400 times g for 30 minutes at room temperature, incubate it at 37 degrees Celsius for 90 minutes.
Then add two milliliters of EGM medium to each well and incubate the cultures overnight. Following the incubation, remove the EGM medium from each well and replace it with two milliliters of fresh EGM. When cells reach an approximately 90%confluence, harvest them by enzymatic digestion as previously shown.
Pool the cells from three wells into a T75 flask labeled with the number of total passage in the passage post infection. Prior to testing or freezing, expand mock and KSHV-infected endothelial cell cultures by splitting them one to three for at least two additional passages. Presented here is a microscopic image of confluent mock-infected endothelial cells revealing classic cobblestone appearance.
Cellular division in these cells is inhibited upon contact when the culture reaches confluence. KSHV-infected endothelial cells develop elongated spindle-like morphology reminiscent of Kaposi Sarcoma spindle cells. Unlike the mock-infected cells, KSHV-infected endothelial cells continue to proliferate even when they reach confluence leading to the development of multilayered cell foci.
When losing contact with the substrate, mock-infected endothelial cells undergo an apoptotic cell death, anoikis, plus they do not form colonies in soft agar. On the contrary, KSHV-infected cells are resistant to anoikis and growing colonies when cultured in soft agar. The development of the spindle-shaped morphology in KSHV-infected endothelial cells is accompanied by the expression of viral latency protein ORF73 and to a lesser extent a viral processivity factor ORF59.
When infected with KSHV BAC16 tagged with a green fluorescent protein, endothelial cells developed spindle-shaped morphology and demonstrate GFP expression confirming the casual relationship between KSHV infection in the observed phenotype. After its development, this cell culture model paved the way for researchers to assess alterations of host cell mRNA and protein expression induced by KSHV infection. Consequently, key pathogenic mechanisms underlying KSHV-mediated oncogenesis representing promising therapeutic targets have been identified.
Don't forget, KSHV is a BSL-2 agent and should be handled with appropriate biosafety containment procedures and practices.
