Gene expression profiling identifies inflammation and angiogenesis as distinguishing features of canine hemangiosarcoma

<p>Abstract</p> <p>Background</p> <p>The etiology of hemangiosarcoma remains incompletely understood. Its common occurrence in dogs suggests predisposing factors favor its development in this species. These factors could represent a constellation of heritable characteristics that promote transformation events and/or facilitate the establishment of a microenvironment that is conducive for survival of malignant blood vessel-forming cells. The hypothesis for this study was that characteristic molecular features distinguish hemangiosarcoma from non-malignant endothelial cells, and that such features are informative for the etiology of this disease.</p> <p>Methods</p> <p>We first investigated mutations of VHL and Ras family genes that might drive hemangiosarcoma by sequencing tumor DNA and mRNA (cDNA). Protein expression was examined using immunostaining. Next, we evaluated genome-wide gene expression profiling using the Affymetrix Canine 2.0 platform as a global approach to test the hypothesis. Data were evaluated using routine bioinformatics and validation was done using quantitative real time RT-PCR.</p> <p>Results</p> <p>Each of 10 tumor and four non-tumor samples analyzed had wild type sequences for these genes. At the genome wide level, hemangiosarcoma cells clustered separately from non-malignant endothelial cells based on a robust signature that included genes involved in inflammation, angiogenesis, adhesion, invasion, metabolism, cell cycle, signaling, and patterning. This signature did not simply reflect a cancer-associated angiogenic phenotype, as it also distinguished hemangiosarcoma from non-endothelial, moderately to highly angiogenic bone marrow-derived tumors (lymphoma, leukemia, osteosarcoma).</p> <p>Conclusions</p> <p>The data show that inflammation and angiogenesis are important processes in the pathogenesis of vascular tumors, but a definitive ontogeny of the cells that give rise to these tumors remains to be established. The data do not yet distinguish whether functional or ontogenetic plasticity creates this phenotype, although they suggest that cells which give rise to hemangiosarcoma modulate their microenvironment to promote tumor growth and survival. We propose that the frequent occurrence of canine hemangiosarcoma in defined dog breeds, as well as its similarity to homologous tumors in humans, offers unique models to solve the dilemma of stem cell plasticity and whether angiogenic endothelial cells and hematopoietic cells originate from a single cell or from distinct progenitor cells.</p

Nfatc2 and Tob1 have non-overlapping function in T cell negative regulation and tumorigenesis.

Nfatc2 and Tob1 are intrinsic negative regulators of T cell activation. Nfatc2-deficient and Tob1-deficient T cells show reduced thresholds of activation; however, whether these factors have independent or overlapping roles in negative regulation of T cell responses has not been previously examined. Here, we show that Nfatc2 knockout (KO) but not Tob1 KO mice have age-associated accumulation of persistently activated T cells in vivo and expansion of the CD44+ memory cell compartment and age-associated lymphocytic infiltrates in visceral organs, without significant changes in numbers of CD4+CD25+Foxp3+ regulatory T cells (Treg). In vitro, CD4+CD25- "conventional" T cells (Tconvs) from both KO strains showed greater proliferation than wild type (WT) Tconvs. However, while Tregs from Nfatc2 KO mice retained normal suppressive function, Tregs from Tob1 KOs had enhanced suppressive activity. Nfatc2 KO Tconvs expanded somewhat more rapidly than WT Tconvs under conditions of homeostatic proliferation, but their accelerated growth capacity was negated, at least acutely, in a lymphoreplete environment. Finally, Nfatc2 KO mice developed a previously uncharacterized increase in B-cell malignancies, which was not accelerated by the absence of Tob1. The data thus support the prevailing hypothesis that Nfatc2 and Tob1 are non-redundant regulators of lymphocyte homeostasis

<i>Nfatc2</i> KO mice, but not <i>Tob1</i> KO mice show age-related accumulation of memory T cells.

<p>(A) Spleen and lymph node cells were isolated from age-matched WT, <i>Nfatc2</i> KO, and <i>Tob1</i> KO mice, and cells from each genotype and for each organ were pooled for experiments. Expression of CD44 and CD62L was measured by conventional cell surface staining in WT, <i>Nfatc2</i> KO, and <i>Tob1</i> KO T cells immediately after isolation from spleens or lymph nodes. Panels are representative two-dimensional contour plots showing CD44 and CD62L staining from young (top) and old (bottom) mice gated on CD3 T cells from representative mice as indicated. Similar data for young and old mice were obtained in 9 and 4 experiments using WT cells, 6 and 4 experiments using <i>Nfatc2</i> KO cells, and 4 and 4 experiments using <i>Tob1</i> KO cells, respectively. Means ± SD are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100629#pone-0100629-t001" target="_blank">Table 1</a>.</p

Naïve T cells from <i>Nfatc2</i> KO mice show the hyper-proliferative response phenotype.

<p>CD4 T cells were isolated from single cell suspensions of spleens and lymph nodes from WT and <i>Nfatc2</i> KO littermates by negative immunomagnetic selection. CD4⁺CD25⁻CD44^dim (naïve Tconv) cells were enriched by sorting to deplete CD4⁺CD25⁺ (Treg) and CD4⁺CD44^bright (memory) cells. Naïve Tconv cells were labeled with CFSE, mixed 1∶1 with syngeneic AgPCs and stimulated with anti-CD3. Proliferation was measured by CFSE dilution in CD4 T cells using flow cytometry after of 96 hr of culture. (A) Representative one-dimensional histograms of CFSE dilution from unstimulated (top) or from stimulated (bottom) WT and <i>Nfatc2</i> KO cells. Insets show two-dimensional contour plots of CD4 (y-axis) and CD44 (x-axis) expression. The box indicates the percent of CD4⁺CD44⁺ cells at the end of the culture period. (B) Means ± S.D. of the percent of cells in triplicate samples that underwent 0–7 divisions over 96 hr for each genotype from 2 experiments for each genotype.</p

<i>Nfatc2</i> deficiency leads to enhanced proliferation in a lymphodepleted environment of homeostatic expansion, but does not provide improved survival fitness in a competitive lymphoreplete environment.

<p>CD4⁺CD25⁻ Tconvs were prepared as described from WT and from <i>Nfatc2</i> KO mice. Sorted cells (100,000) were adoptively transferred into the tail vein of two B6.SCID (top) or two B6.CD45.1 (bottom) mice. Recipients were sacrificed 15 days later and total cell numbers recovered from spleens (SP) and lymph nodes (LN) were enumerated using a CellDyn 3500 hematology analyzer. The percent of donor cells in each recipient was then calculated based on the percent of CD4⁺CD45.2⁺ cells present in each preparation, and is represented by a symbol in the graphs. Lines indicate the mean for each recipient group.</p

<i>Nfatc2</i> and <i>Tob1</i> KO mice have increased numbers of persistently activated cells <i>in vivo</i>.

<p>Spleen and lymph node cells were isolated from age-matched WT, <i>Nfatc2</i> KO, and <i>Tob1</i> KO mice, and cells from each genotype and for each organ were pooled for experiments. Expression of CDK4 was measured by intracellular staining and expression of CD69 was measured by conventional cell surface staining of freshly isolated cells. (A). One-dimensional histograms (top) showing CDK4 expression, gated on CD4 and CD8 cells from representative <i>Nfatc2</i> KO and <i>Tob1</i> KO mice overlaid on WT controls as indicated. Dark lines in the histograms represent KOs and grey lines represent WT mice. Bar graphs (bottom) represent means ± SD of the mean fluorescence intensity (MFI) for CDK4 expression in CD4 and CD8 cells. Data summarize 15, 12, and 5 experiments using triplicate samples of WT cells, <i>Nfatc2</i> KO cells, <i>Tob1</i> KO cells, respectively, each with pooled cells from 2 or 3 mice. MFIs among different experiments showed normal distribution. Asterisks denote values that are significantly different from WT (Student t-test p<0.05). (B) One-dimensional histograms (top) showing CD69 expression, gated on CD4 and CD8 cells from representative young (left) and old (right) <i>Nfatc2</i> KO and <i>Tob1</i> KO mice overlaid on WT controls as indicated. Dark lines in the histograms represent KOs and grey lines represent WT mice. Bar graphs (bottom) represent means ± SD percent CD69⁺ cells in the CD4 and CD8 compartments. Data for young and old mice summarize 10 and 4 experiments using triplicate samples of WT cells, 6 and 8 experiments using triplicate samples of <i>Nfatc2</i> KO cells, and 4 and 3 experiments using triplicate samples of <i>Tob1</i> KO cells, respectively. The percent of positive cells among different experiments showed normal distribution. Asterisks denote values that are significantly different from WT (Student t-test p<0.05).</p