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. 2007 Mar;11(3):245-58.
doi: 10.1016/j.ccr.2007.01.015.

In vivo-restricted and reversible malignancy induced by human herpesvirus-8 KSHV: a cell and animal model of virally induced Kaposi's sarcoma

Agata D'Agostino Mutlu  1 , Lucas E Cavallin, Loïc Vincent, Chiara Chiozzini, Pilar Eroles, Elda M Duran, Zahra Asgari, Andrea T Hooper, Krista M D La Perle, Chelsey Hilsher, Shou-Jiang Gao, Dirk P Dittmer, Shahin Rafii, Enrique A Mesri
Affiliations

In vivo-restricted and reversible malignancy induced by human herpesvirus-8 KSHV: a cell and animal model of virally induced Kaposi's sarcoma

Agata D'Agostino Mutlu et al. Cancer Cell. 2007 Mar.

Erratum in

  • Cancer Cell. 2007 May;11(5):471

Abstract

Transfection of a Kaposi's sarcoma (KS) herpesvirus (KSHV) Bacterial Artificial Chromosome (KSHVBac36) into mouse bone marrow endothelial-lineage cells generates a cell (mECK36) that forms KS-like tumors in mice. mECK36 expressed most KSHV genes and were angiogenic, but they didn't form colonies in soft agar. In nude mice, mECK36 formed KSHV-harboring vascularized spindle cell sarcomas that were LANA+/podoplanin+, overexpressed VEGF and Angiopoietin ligands and receptors, and displayed KSHV and host transcriptomes reminiscent of KS. mECK36 that lost the KSHV episome reverted to nontumorigenicity. siRNA suppression of KSHV vGPCR, an angiogenic gene upregulated in mECK36 tumors, inhibited angiogenicity and tumorigenicity. These results show that KSHV malignancy is in vivo growth restricted and reversible, defining mECK36 as a biologically sensitive animal model of KSHV-dependent KS.

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Figures

Figure 1
Figure 1. KSHVBac36-transfection of mouse bone marrow endothelial-hematopoietic cells leads to KSHV latent and lytic gene expression
(A) Immunofluorescence staining of LANA. (B) Confocal image showing punctuated LANA staining. (C) Immunofluorescence staining of Kaposin. (D) Immunofluorescence staining of K8.1 and LANA. (E) Detailed confocal image of LANA and K8.1. (F) Heatmap representation of the Real Time RT-PCR analysis for the whole KSHV transcriptome in Bac36KSHV transfected cells. Ctrl: mock-transfected NIH3T3 cells. Red indicates highest, yellow intermediate, and black lowest relative mRNA levels. Scale bar = 10 μM
Figure 2
Figure 2. KSHVBac36-transfection of mECs induces an angiogenic phenotype
(A) ELISA-based Telomerase Repeat Amplification Protocol (TRAP) assay of mEC-V and mECK36. Bars indicate mean of duplicates +/− SEM. (B) VEGF secretion levels of mEC-V and mECK36. Bars indicate mean of duplicates +/− SEM. (C) Intradermal skin angiogenesis assay (see methods) of mEC-V and mECK36 cells. The bar graphs show the mean microvessel density (vessels/mm2) +/− SEM. Total N (both flanks) = 10. (*** indicates p<0.05). (D) KSHVBac36-induced upregulation of angiogenic gene expression determined by Real Time qRT-PCR. Bars represent mean fold increase (triplicates +/−SEM) in mRNA levels between mECK36 and mECs. (E) Appearance at a dissected inoculation site in the skin of a mouse injected with mEC cells or mECK36 cells.
Figure 3
Figure 3. mECK36 induce vascularized spindle cell sarcomas in immunocompromised mice
(A) Mice showing subcutaneous mECK36 tumor four weeks after inoculation (B) Dissection site showing green-yellow tumor (EGFP expression). (C) Subcutaneous tumor growth in nude mice injected with mECK36 cells (closed circles) or mEC-V (open squares). Data indicate mean tumor size (N=7) +/− SEM. (D) Microscopic image of an histological section of a mECK36 tumor stained with hematoxylin & eosin (H&E). (V, microvessel). (E) Image of a spindle cell sarcoma foci (T) growing in the lung (L) stained with H&E. (F) Image of an spindle cell tumor in the lungs stained with H&E. E, blood extravasation. V, microvessel. Scale bar = 50 μM
Figure 4
Figure 4. mECK36 induce KSHV-bearing spindle cell sarcomas resembling KS tumors
(A) Dissected mouse showing subcutaneous fluorescent tumor observed under long wave UV light. (B) LANA immunohistochemistry of paraffin embedded mECK36 tumors (C) Rat IgG control for LANA immuohistochemistry. (D) Top left: Confocal images showing podoplanin staining in the cytoplasm and LANA staining in the nucleus. Top right: higher magnification image showing the specked pattern of LANA immunofluorescence in podoplanin+ cells. Bottom left: colocalization of LANA with EGFP. Bottom right: an isotype-matched control. (E) Confocal images showing K8.1 staining in the membrane (left) and colocalizing with EGFP (middle). (Right) Mouse IgG control. (F) Heatmap representation of KSHV transcriptome in mECK36 tumors and mECK36 cells grown in culture quantified using Real Time qRT-PCR. C: mECK36 cells grown in culture. T1, T2, and T3: primary mECK36 tumors. Red indicates highest, yellow intermediate, and black lowest relative mRNA levels. Scale bars: 50 μM (B-C), 10 μM (D-E).
Figure 5
Figure 5. mECK36 spindle cell sarcomas express KS and angiogenic markers
(A) Frozen sections of tumors stained with indicated antibodies. (B) Paraffin-embedded tissue sections stained with indicated antibody and developed with Cy3-conjugated anti-IgG. (C) Side and forward scatter of VEGF receptors on single cell suspensions from mECK36 tumors by Flow Cytometry. (D) Cells double positive for EGFP and indicated VEGFR from tumors in (C). (E) Percentage of cells expressing VEGFR among EGFP+ (black bars) and EGFR- (white bars) population. (F) Up-regulation of angiogenic gene expression in mECK36 tumors compared to cultured mECK36. Bars represent mean fold increase (triplicates +/- SEM) in mRNA levels quantified by Real time qRT-PCR. Scale bars: 50 μM (A), 10 μM (B).
Figure 6
Figure 6. Viral and host global gene expression of mECK36 tumors resembles KS
(A) Clustering of primary KS with mEC-Bac36 tumors. Heatmap represents Real Time qRT-PCR results. Tumor 5, 3 and 7 are three mECK36 spindle cell sarcomas. BCBL Mock and TPA are BCBL-1 uninduced and induced with TPA, respectively. (B) Heatmap representation of 562 genes (q<0.05) of the human KS Signature that are also mECK36 tumor signature (equally up or down regulated). 723 genes from the human KS signature were ortholog to mouse genes present in the mouse Affymetrix Array. Among these, 691 genes were able to differentiate mECK36 spindle cell sarcomas and mouse skin (q<0.05) for at least a two fold difference. 471 genes (68%) and 91 genes (13%) were upregulated (red) and downregulated (blue), respectively, in both mECK36 tumors and human KS. Selected downregulated genes are shown in blue and upregulated genes in red. The full list of genes is available in Supplementary Table 1. (C) Distribution of mouse KS signature. Blue: genes shared between mECK36 tumors and mEC-V. Yellow: genes shared between mECK36 tumors and mECK36 in culture. Olive: genes shared by mEC-V, mECK36 in culture and tumors. Red: mECK36 tumors only. Gene intensity from the mouse KS signature was set to 100%, and the percentage of genes that contribute to the mouse KS signature was calculated using 10% difference in intensity between the different samples and tumors (GeneSpring 7 package). (D) Angiogenic gene expression in mEC-V and in mECK36 relative to that in mECK36 tumors. mRNA levels were determined by Real Time qRT-PCR. mRNA levels in tumors were set at 100% (closed bar). Overlapped are bars representing relative mRNA levels in mECK36 cells in culture (grey bars) and in mEC-V cells (open bars) (mean of triplicates +/− SEM). Relative levels were calculated as 1/ fold-increase in tumors X 100.
Figure 7
Figure 7. mECK36 tumorigenicity is reversible and strictly KSHV-dependent
(A) Many cells in colonies each originated from a single EGFP+ mECK36 cell became EGFP- when grown in the absence of hygromycin. (B) EGFP levels of cultured and explanted mECK36 grown with or without hygromycin determined by flow cytometry. mECK36 grown in hygromycin media was used as a control and set at 100% for every passage. (C) In vitro growth rate of mECK36, mEC-V, and mECK36-KSHV-Null. Doubling times were 22, 14, and 15 hours, respectively. (D) Tumor formation of mECK36-KSHV-Null cells (open circles), mECK36 cells (closed circles), and 1:1000 ratio of mECK36:mECK36-KSHV-Null (open triangles). Data indicate mean tumor size +/− SEM (N=5). Scale bar: 100 μM.
Figure 8
Figure 8. RNAi-mediated suppression of KSHV vGPCR in mECK36 blocks angiogenicity and tumorigenesis
(A) mRNA levels of KSHV genes in mECK36 tumors compared to cultured mECK36 determined by Real time qRT-PCR. Mean values (+/− SD) from 3 tumor samples were plotted. (B) RT-PCR analysis of vGPCR and GAPDH for mECK36 cells transfected with control shRNA and vGPCR shRNA vector (C) Log2 scale plot (dCT) of the actin-normalized KSHV mRNA levels of mECK36 transfected with control shRNA (horizontal) or vGPCR shRNA (vertical). The arrows indicate transcripts that are specifically downregulated and the annotation shows the identity and fold downregulation. (D) Left panel: Levels of VEGF secretion for mECK36 transfected with either control shRNA or vGPCR shRNA vector measured by ELISA. Bars indicate mean of duplicate measures +/− range. Right panel: Angiogenesis in skin for mEC-V, mECK36, mECK36 cells transfected with either control shRNA or vGPCR shRNA vector. The bar graphs show the mean microvessel density (vessels/mm2) +/− SEM. Total N (both flanks) = 10. (***) indicates significant differences between vGPCR RNAi and control RNAi groups (P<0.05) (N=10). (E) Tumor formation of mECK36 cells transfected with control shRNA (closed circles) or with vGPCR shRNA vector (open squares). Data indicate mean tumor size +/− SEM (N=10). (F) Tumor appearance after dissection and lighting using a long-wave UV lamp for mECK36 cells transfected with control shRNA or with vGPCR shRNA vector.
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