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.

https://cancerres.aacrjournals.org/content/canres/65/20/9294.full.pdf

Multiple Roles for the Receptor Tyrosine Kinase Axl in Tumor Formation

Background

Conventional flow cytometry does not allow the rapid analysis of multiple samples. This has limited its uses in drug discovery, for which the standard for throughput is 100,000 samples per day.



Methods

We describe a simple method in which commercial peristaltic tubing is connected from a commercial autosampler to a flow cytometer. The samples are delivered via a peristaltic pump from source wells in a multiwell plate. The samples are separated by air bubbles.



Results

Throughput rates approach the limit of the autosampler (up to 100 wells per minute). Using optimal tubing and flow rates, particles remain within appropriate light scatter and fluorescence gates. The carryover between wells is typically less than 5% without and 1% with a wash step. The volumes of sample delivered are in the microliter scale. The approach has been validated with instruments from three manufacturers.



Conclusions

Flow cytometry has potential throughput of 100,000 samples or more per day starting with the method described. The method is currently best suited to end-point assays. However, combined with high-speed sorting and single- cell assays, the number of assays could approach 1 billion per day. Cytometry 44:83–90, 2001. © 2001 Wiley-Liss, Inc.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/1097-0320%2820010501%2944%3A1%3C83%3A%3AAID-CYTO1085%3E3.0.CO%3B2-O

High throughput flow cytometry.

Retrovirally Delivered Random Cyclic Peptide Libraries Yield Inhibitors of Interleukin-4 Signaling in Human B Cells

The invention relates to methods and compositions utilizing Renilla green fluorescent proteins (rGFP), and Ptilosarcus green fluorescent proteins (pGFP). In particular, the invention relates to the use of Renilla GFP or Ptilosarcus GFP proteins as reporters for cell assays, particularly intracellular assays, including methods of screening libraries using rGFP or pGFP.

Methods and compositions comprising Renilla GFP

The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.

/pdf/systematic-identification-of-regulatory-proteins-critical-562n3ixb07.pdf

Systematic identification of regulatory proteins critical for T-cell activation.

Retroviral Delivery of Peptide Modulators of Cellular Functions

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Retroviral Delivery of Peptide Modulators of Cellular Functions