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This unit details the applications of one of the more common retroviral packaging systems, based on the highly transfectable 293T cell. The packaging system employs the use of the Phoenix cell lines. Calcium phosphate-mediated transfection is described for efficient introduction of retroviral vector plasmid DNA into the cells to generate high yields of virion-containing supernatant. An alternate protocol describes a method for transfecting retroviruses that contain a vesicular stomatitis virus G (VSV G) protein. Such virions are said to be "pseudotyped" with VSV G glycoprotein. Support protocols provide a simple method for concentrating VSV-G-pseudotyped retroviruses, as well as methods for culturing, cryopreserving, thawing, and drug selecting the Phoenix packaging cell lines. Finally, several methods for transfecting adherent or suspension cells with retroviruses are described.

A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA-mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development. (Cancer Res 2005; 65(20): 9294-303)

IntroductionErythropoietin receptor (EpoR) and its cognate ligand erythropoietin (Epo) function to prevent apoptosis of erythroid progenitors, allow for erythrocyte maturation, and are essential for definitive erythropoiesis. However, expression of functional EpoR was also reported in endothelial cells (reviewed by Arcasoy 1 ). rHuEpo and other erythropoiesisstimulating agents (ESAs) were reported to stimulate nitric oxide synthase expression, induce proliferation in endothelial cell preparations, and stimulate angiogenesis in chick embryo chorioallantoic membrane, mouse uterine, and rodent tumor models through direct stimulation of endothelial EpoR.Some data also suggested that EpoR may be functionally expressed in other nonhematopoietic cells, such as cardiac myocytes, kidney, and neuronal cells, and ESAs have been reported to be cytoprotective for these cells. 1 Antiapoptotic signaling pathways downstream of EpoR were reportedly activated by ESAs to inhibit cell death associated with cytotoxic insult (eg, ischemia, reperfusion injury, and exposure to cytotoxins) both in vitro and in vivo. It has also been hypothesized that alternative ESA-binding receptor complexes, such as a heteroreceptor composed of the granulocytemacrophage colony-stimulating factor/interleukin-3 (IL-3)/IL-5 receptor ␤-common chain and EpoR, may mediate the cytoprotective activities of ESAs. 2 These reports have formed the basis for a number of clinical studies examining the "direct" action of ESAs in diseases, such as stroke and myocardial infarction.However, the data surrounding the expression of functional EpoR or alternative receptors in endothelial and other nonhematopoietic cells are conflicting and confounded for a number of reasons. First, reports describing EpoR protein expression used nonspecific antibodies, which produce false positive results. 3,4 Second, when surface EpoR was examined on nonerythroid cells using rHuEpo-binding studies, the reported receptor characteristics were very different from that known for erythroid EpoR: that is, receptor affinity was extremely low and receptor number unusually high compared with erythroid cells. [5][6][7] Although alternative ESA receptor complexes 2 could theoretically explain differences in the affinity and receptor number, other studies have found no evidence for alternative ESA receptor complexes. 8,9 In addition, there are conflicting data surrounding the presence of functional endothelial EpoR. ESAs were unable to stimulate the expression of vasoactive factors in vitro, 10 did not induce endothelial nitric oxide synthase expression or response in rats, 11 did not stimulate vasoconstriction of arterioles in humans, 12 and did not influence vascular density in rodent tumor models. 13 Other studies were confounded by cross-species inactivity of rHuEpo: rHuEpo had no effect on chicken erythroid cells 14 yet reportedly stimulated angiogenesis in a chick embryo chorioallantoic membrane assay. 15 Similarly, in some nonhematopoietic tissue protection in vivo models, ESAs were unable to ...