Selection of a highly invasive neuroblastoma cell population through long-term human cytomegalovirus infection

Abstract

The human cytomegalovirus (HCMV) is suspected to increase tumour malignancy by infection of cancer and/or stroma cells (oncomodulation). So far, oncomodulatory mechanisms have been attributed to the presence of HCMV and direct action of its gene products on cancer cells. Here, we investigated whether the prolonged presence of HCMV can result in the irreversible selection of a cancer cell population with increased malignancy. The neuroblastoma cell line UKF-NB-4 was long-term (200 passages) infected with the HCMV strain Hi91 (UKF-NB-4(Hi)) before virus eradication using ganciclovir (UKF-NB-4(HiGCV)). Global gene expression profiling of UKF-NB-4, UKF-NB-4(Hi) and UKF-NB-4(HiGCV) cells and subsequent bioinformatic signal transduction pathway analysis revealed clear differences between UKF-NB-4 and UKF-NB-4(Hi), as well as between UKF-NB-4 and UKF-NB-4(HiGCV) cells, but only minor differences between UKF-NB-4(Hi) and UKF-NB-4(HiGCV) cells. Investigation of the expression of a subset of five genes in different chronically HCMV-infected cell lines before and after virus eradication suggested that long-term HCMV infection reproducibly causes specific changes. Array comparative genomic hybridisation showed virtually the same genomic differences for the comparisons UKF-NB-4(Hi)/UKF-NB-4 and UKF-NB-4(HiGCV)/UKF-NB-4. UKF-NB-4(Hi) cells are characterised by an increased invasive potential compared with UKF-NB-4 cells. This phenotype was completely retained in UKF-NB-4(HiGCV) cells. Moreover, there was a substantial overlap in the signal transduction pathways that differed significantly between UKF-NB-4(Hi)/UKF-NB-4(HiGCV) and UKF-NB-4 cells and those differentially regulated between tumour tissues from neuroblastoma patients with favourable or poor outcome. In conclusion, we present the first experimental evidence that long-term HCMV infection can result in the selection of tumour cell populations with enhanced malignancy.

Figure 1

Establishment of UKF-NB-4^Hi and UKF-NB-4^HiGCV cells. UKF-NB-4 cells were infected once with HCMV strain Hi at MOI 10 and then subcultured without further addition of virus (UKF-NB-4^Hi). After 200 passages, UKF-NB-4^Hi cells were treated for six passages with 20 μM ganciclovir (GCV) until no HCMV IEA or late antigen (LA) expression were detectable anymore. (a) Representative pictures showing immunostaining of UKF-NB-4, UKF-NB-4^Hi or UKF-NB-4^HiGCV cells for IEA or LA. (b) Fraction of HCMV antigen-expressing cells in UKF-NB-4^Hi cells at different passages after initial infection. (c) HCMV antigen expression in GCV-treated UKF-NB-4^Hi cells at different passages in comparison with non-treated UKF-NB-4^Hi cells. Values represent mean±s.d. from three independent experiments. *P<0.05 relative to non-treated UKF-NB-4^Hi cells. (d) HCMV antigen expression in GCV-cured UKF-NB-4^Hi (UKF-NB-4^HiGCV) cells after cultivation for 10 passages in the absence of GCV in comparison with non-treated UKF-NB-4^Hi cells. n.d., not detectable.

Figure 2

Comparison of global gene expression in UKF-NB-4, UKF-NB-4^Hi and UKF-NB-4^HiGCV cells by gene microarray. (a) Hierarchical cluster analysis based on the Pearson correlation coefficient. (b) Numbers of genes significantly differentially expressed (FDR<0.05) between the investigated cell lines.

Figure 3

Array CGH of all 24 human chromosomes resulting from comparisons of UKF-NB-4^Hi cells (upper plot) and UKF-NB-4^HiGCV cells (bottom plot), both compared with UKF-NB-4 cells. Results suggest that both UKF-NB-4^Hi and UKF-NB-4^HiGCV have very similar karyotypes: whole-chromosome gain and loss (e.g., on chromosomes 6 and 18), losses and gains of chromosome segments, e.g., terminal 2p and 12q, as well as amplification and loss on individual loci.

Figure 4

Adhesion of UKF-NB-4, UKF-NB-4^Hi and UKF-NB-4^HiGCV cells to endothelial cell monolayers, transmigration and endothelial cell monolayer distruction. (a) Fractions of cells that adhered to HUVEC monolayers (in the presence or absence of an α₅β₁-blocking antibody (a, b)) were expressed as the percentage of the total number of input cells. (b) Fractions of cells that transmigrated through HUVEC monolayers (in the presence or absence of an α₅β₁-blocking antibody (a, b)) were expressed as the percentage of the total number of adherent cells. (c) Focal endothelial cell monolayer disruption (in the presence or absence of an α₅β₁-blocking antibody (a, b)) was expressed as the percentage of the cell-free area. Values represent mean±s.d. from three independent experiments. *P<0.05 relative to UKF-NB-4; ^#P<0.05 relative to the corresponding cell line in the absence of α₅β₁-blocking antibody; n.d., not detectable; AB, antibody.