Researchers report on an in vitro system that could serve as a preliminary model for investigating how Kaposi’s sarcoma-associated herpesvirus (KSHV) turns healthy cells cancerous. Scientists at the University of Texas Health Science Center and colleagues have identified a rat primary cell type that KSHV can efficiently infect in vitro, and transform into characteristically spindle-shaped malignant cells within just four days, and without any manipulation.
Describing their work in the Journal of Clinical Investigation, Shou-Jiang Gao, M.D., demonstrated that the transformed cells expressed hallmark vascular endothelial, lymphatic endothelial, and mesenchymal markers associated with KSHV-infected tumor cells, and efficiently induced the formation of tumors in nude mice. Their paper is titled “Direct and efficient cellular transformation of primary rat mesenchymal precursor cells by KSHV.”
KSHV is causally linked with a number of cancers in AIDS patients, but the mechanism by which the virus triggers malignancy remains elusive. This is primarily because scientists haven’t yet developed an experimental system for the transformation of primary cells, Dr. Gao et al explain. KSHV can infect different cell types in culture, and infected vascular and lymphatic endothelial cells take on the telltale spindle shape and express markers from both lineages. However, continued growth of these infected cells relies on exogenous growth factors, and they don’t undergo malignant transformation.
Testing KHSV on a range of human, mouse, and rat primary cells, the team found that the virus could efficiently infect and transform rat metanephric mesenchymal precursor cells (MM cells). Both the uninfected MM cells and infected (KMM) cells expressed the vascular endothelial markers β-catenin, vWF, and VEGFR-1, the lymphatic endothelial markers VEGFR-3 and LYVE-1, the mesenchymal marker vimentin, and the hematopoietic precursor marker CD34. However, KMM cells exhibited upregulation of the vascular endothelial markers CD31, VCAM-1, and ICAM-1 and the lymphatic endothelial cell marker podoplanin, and downregulation of the hematopoietic precursor marker Thy1.1. Interestingly, only intermittent and low levels of infectious virions could be found in the supernatants of induced cells, suggesting an abortive viral lytic program. Similar results were observed when long-term-passage KMM cells were examined.
Notably, the pattern of cell surface marker expression didn’t change with repeated passaging of the infected KMM cells, the investigators point out. “Thus, similar to vascular and lymphatic endothelial cells, KSHV reprograms MM cells to express a mixture of vascular endothelial, lymphatic endothelial, mesenchymal, and hematopoietic precursor markers.”
In comparison with MM cells that underwent crisis after 25–28 passages, the KMM cells were immortalized, and demonstrated a smaller size, a faster growth rate, and a shorter doubling time. And while MM cell growth arrested in serum-free medium, KMM cells continued to proliferate up to day four after seeding. The KMM cells also lost their contact inhibition and formed colonies in semisolid soft agar.
Most significantly, the KMM cells also induced tumor formation in nude mice, with a 75–95% efficiency, and a latency of about 10.5–13.5 weeks. The resulting tumors demonstrated characteristic spindle-shaped cells, and contained microvessels and infiltrates of immune cells, including CD45R+ mouse B cells, which the authors note are "reminiscent" of CD8+ T cells and CD20+ B cells found in KS tumors. Most tumor cells were in the latent state, but a few had heterogeneous expression of lytic genes.
KMM tumor cells were also positive for the vascular endothelial markers β-catenin, CD31, VE-cadherin, ICAM-1, and VCAM-1, along with the lymphatic endothelial markers LYVE-1, VEGFR-3, podoplanin, ROX-1, and the hematopoietic precursor marker CD34 and mesenchymal marker vimentin. However, they were negative for Thy1.1. “Thus, KSHV appeared to downregulate Thy1.1 expression to inhibit its tumor-suppressive effect, while at the same time converting the mesenchymal precursor cells into a mixed phenotype of vascular and lymphatic endothelial cells,” the investigators state.
The authors suggest their system could be used to help understand the mechanisms underpinning KSHV-induced malignant transformation. However, they admit, their reported achievements are in rat cells, and cellular transformation of human primary cells still remains a challenge. “Whether mesenchymal precursor cells are the KSHV targets in vivo requires further investigation.”