Figure 1.
Generation of ROSA26.vFLIP;Cdh5(PAC).creERT2 mice.
(A) The strategy for inducible recombinant activation of vFLIP expression in endothelial cells in vivo is shown with a schematic representation of the ROSA26 locus before (left) and after (right) inducible recombinant activation of vFLIP expression in endothelial cells. ROSA26.vFLIP knock-in mice were bred with Cdh5(PAC).creERT2 mice to obtain transgene expression in endothelial cells upon tamoxifen injection. (B) Transgene expression was specifically detected, both by RT-PCR (left panel) and anti-FLAG immunoblotting (right panel), in lung, spleen, liver and heart derived from ROSA26.vFLIP;Cdh5(PAC).creERT2. (C) Quantitative real-time RT-PCR for vFLIP expression. Analysis was done in 2–3 month-old mice, about one month after i.p. injection of tamoxifen. (D) Flow cytometry showing percentage of cardiac endothelial cells and (E) splenic B-cells expressing EGFP. Data represent one of three experiments with similar results; at least three TG and control animals were analyzed in each experiment. Analysis was done in 2–3 month-old mice, about one month after i.p. injection of tamoxifen. TG/Endo are ROSA26.vFLIP;Cdh5(PAC).creERT2 mice; TG/B-cells represent ROSA26.vFLIP;CD19.cre mice used as control [23].
Figure 2.
Systemic endothelial abnormalities.
(A) A representative cardiac section stained with H&E, EGFP and CD34 and/or CD31, PROX1 and Ki67 shows numerous atypical spindle-like endothelial cells. Similar cells were found in the skeletal muscle (B) and in brown fat (C). Analysis was done in 2–3 month-old mice, about one month after i.p. injection of tamoxifen. Scale bar, 200 μm. TG/Endo, ROSA26.vFLIP;Cdh5(PAC).creERT2; TG/B-cells, ROSA26.vFLIP;CD19.cre mice used as control.
Figure 3.
Systemic perineurinal proliferation.
(A) Representative section of the perirenal capsule, diaphram muscle, salivary gland, and pancreas stained with H&E shows atypical proliferation of perineurinal endothelial-like cells (arrows). Analysis was done in 2–3 month-old mice, about one month after i.p. injection of tamoxifen. Scale bar, 200 μm. (B) Serum glucose levels are shown. Data represent one of three experiments with similar results (error bars, SEM); at least three TG and control animals were analyzed in each experiment.
Figure 4.
Systemic chronic inflammation.
(A) Mixed infiltrates of inflammatory cells including PMN, lymphocytes and plasma cells are shown in peritoneum and meninges (arrows). Scale bar, 200 μm. (B) Statistical analysis of event-free survival by Kaplan-Meier cumulative survival curve and the log-rank test to evaluate statistical significance. More than 100 mice in each group were followed up for up to 20 months. (C) Humoral immune response against vFLIP was tested by western blot with a pool of mouse sera (1:100 dilution) isolated from TG mice. Two independent western blots yielded the same results, and shown here is the one that was displayed also in Fig. 1B.
Figure 5.
Perturbation in serum cytokines.
Fourteen serum cytokines were analyzed in ROSA26.vFLIP;Cdh5(PAC).creERT2 mice by using a quantitative flow cytometry-based assay. Analysis was done in 2–3 month-old mice, about one month after i.p. injection of tamoxifen. Data are representative of at least three experiments with similar results (error bars, SEM); at least three TG and control animals were analyzed in each experiment. P-values derived from two-tailed unpaired Student’s t-test on the means (bars) of WT versus TG mice are shown. *P<0.05, **P<0.01 and ***P<0.005
Figure 6.
Expansion of myeloid cells with PMN-MDSC immunophenotype.
(A) Flow cytometry analysis displayed increase in CD45+CD11b+ myeloid cells in lung, spleen, liver and heart. (B) Ly6G, Ly6C, Gr1 markers and forward/side scatter parameters were used to define the following myeloid cell subsets: polymorphonuclear myeloid derived cells (PMN-MDSC), monocytic myeloid derived cells (M-MDSC), tumor-associated macrophages (TAM). Analysis was done in 2–3 month-old mice, about one month after i.p. injection of tamoxifen. Data are representative of at least three experiments with similar results (error bars, SEM); at least three TG and control animals were analyzed in each experiment. TG/Endo, ROSA26.vFLIP;Cdh5(PAC).creERT2; TG/B-cells, ROSA26.vFLIP;CD19.cre mice used as control.
Figure 7.
Model of KSHV vFLIP-mediated tumorigenesis through endothelial alterations, aberrant myeloid differentiation, and chronic proinflammatory changes in the tumor microenvironment.
Expression of vFLIP in either B-cells or endothelial cells activates several cytokines, both in vivo and in vitro, that lead to aberrant myeloid differentiation with the emergence of myeloid subsets well known to have a role in angiogenesis, tumor immune evasion and tumor progression. (Drawing of myeloid differentiation was modified from Gabrilovich et al)[43]