There was no difference in the density of blood vessels though th

There was no difference in the density of blood vessels though there were fewer vessels per field of view observed compared to the glioblastoma sections (data not shown) and similar levels of necrosis were observed for the tumors derived from treated and control mice (Figure 2C, D). In contrast, there was a significant decrease in the selleck chemical Perifosine number of macrophages infiltrated into the tumors isolated from PKRA7-treated mice as measured by staining intensity of the mouse macrophage marker F4/80 (Figure 2E, F). These results strongly suggest that PKRA7 could inhibit pancreatic tumor growth via a different mechanism by blocking PKR1- and PKR2-expressing macrophages from infiltrating into the tumor microenvironment rather than suppressing angiogenesis, an observation consistent with the phenotypic features of human pancreatic cancer as poorly vascularized but exhibiting abundant desmoplastic fibrosis and containing large number of infiltrated myeloid cells including macrophages [10], [15].

PKRA7 Inhibits Endothelial Cell Capillary Branching and Myeloid Cell Migration The results from the in vivo xenograft studies of human glioma and pancreatic cancer cells strongly supported the notion that the anti-tumor activity of PKRA7 could be mediated via two very different mechanisms. To probe this further at the cellular level, we conducted in vitro assays to assess the impact of PKRA7 on the angiogenic activity of endothelial cells, as well as the migratory ability of myeloid cells.

To determine whether PKRA7 affects the ability of endothelial cells to form capillary tube-like network as an important indicator of angiogenesis [23], we employed immortalized human microvascular endothelial cells (IHMVECs). The cells were treated with 200 ng/ml recombinant PK2 alone or PK2 plus 1 ��g/ml PKRA7 and plated onto a thin layer of Matrigel. This concentration of PKRA7 was used in our in vitro experiments because it is close to the concentration of PKRA7 in the circulation of mice from our in vivo experiments while remaining non-toxic to cells in tissue culture (data not shown). As shown in Figure 3A, PKRA7 effectively inhibited PK2-induced capillary branching as measured by the number of connections between cells, but had no effect on VEGFA-induced capillary branching (quantification presented in Figure 3B).

Identical results were obtained using two additional endothelial cell lines: mouse embryonic endothelial cells and primary human microvascular endothelial cells (data not shown). The specificity of the anti-PK2 activity in this assay was further confirmed by the demonstration of a similar effect by the anti-PK2 polyclonal antiserum (Figure S3 and Entinostat data not shown). Taken together, these results suggest that PKRA7 can specifically inhibit the angiogenic effect of PK2 on endothelial cells.

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