March 15, 2015 (Vol. 35, No. 6)

Improved Mechanistic Understanding of Novel Anti-Angiogenic Therapies

The formation of new blood vessels by angiogenesis is important for tumor growth beyond 2 mm3 in size. Over the past decade anti-angiogenic therapies that slow blood vessel growth, often by inhibiting vascular endothelial growth factor (VEGF), have shown only limited success. Most notably, the breast cancer anti-angiogenic therapy bevacizumab (Avastin) showed no improvement in survival rates, or quality of life, causing the FDA to revoke its approval for this indication in 2011.

In a preclinical study recently published in Cancer Cell, targeting angiopoietin 2 (Ang2) was tested as a potential new anticancer therapy. Inhibition of Ang2 effectively blocked metastatic progression in preclinical mouse models, and Ang2 antibody treatment combined well with low-dose metronomic chemotherapy (LDMC, see Figure 1). Mechanistically, the Ang2 blockade could be linked to quenching of the inflammatory response.

To further understand the cell signaling involved in the Ang2-mediated inhibition of metastasis, immunofluorescence staining was carried out, to show an increase in the phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT-3) following exposure of endothelial cells to rhAng-2.
STAT-3 is activated by phosphorylation, its activation being linked to tumor-promoting inflammation and antitumor immunity. Reliably quantified Western blotting was required to support the immunofluorescence data and further strengthen the conclusion.

Western blotting is an essential cell biology technique that has been used since the late 1970s to provide evidence of protein expression, modification, and assembly. However even after 30 years, determining robust quantitative data from traditional Western blotting techniques has proved challenging. For this study, GE Healthcare Life Sciences’ Amersham™ WB system was used to provide the quantitative Western blotting data required for publication.

Figure 1. (A) Schematic representation of the experimental protocol. (B) Bioluminescence imaging three weeks after the primary tumor had been removed. (C) The frequency of metastasis in the various mouse organs.

Detecting Cell Signaling with Quantitative Western Blotting

Protein phosphorylation is a fundamental mechanism in cell signaling. The addition of the phosphate group to a protein can change the protein’s activity, reactivity, and interactions with other molecules. In this study, the cell-signaling pathways resulting from rhAng2 exposure and the potential effects of this signaling on inflammatory adhesion molecule expression were investigated. STAT-3 was one of the factors studied, as it is stimulated by angiogenic factors. The tyrosine 705 residue is phosphorylated to phospho-Tyr705-STAT-3, after which the protein translocates from the cell cytoplasm to the nucleus.

Immunofluorescence staining demonstrated that, in the presence of rhAng2, STAT-3 localizes to the cell nucleus, whereas in the control samples it is located in the cytoplasm. However it was difficult to distinguish both proteins with the same molecular weight on the same Western blot using enhanced chemiluminescence (ECL) developing systems and x-ray film. By utilizing a quantitative fluorescent Western blotting system, data were generated that supported the proposed mechanism of action for anti-Ang2 therapy.

Detecting the often small differences in levels between unphosphorylated and phosphorylated proteins creates difficulties in analyzing traditional Western blots, especially when making comparisons between different cell types. When two proteins of the same molecular weight are analyzed using traditional Western blotting, a single blot may need to be stripped and re-probed in order to visualize the second protein, with a further loss of luminescence signal and change to x-ray film exposure times.

Although strict control of reaction times is required with ECL, the chemiluminescence signal is inherently unstable. Different exposure times in the darkroom may be required, and the resulting data may vary greatly due to technical differences, as opposed to observing true biological ones. When imaging x-ray films exposed to Western blots and visualized using ECL, the distance between the film and camera results in a further loss of sensitivity, which makes the imaging and analysis of phosphorylated proteins even more difficult.

The Amersham WB system used in this study is capable of multiplexed fluorescence detection. Its high-performance laser scanner allows for two different Cy™-labeled fluorescence secondary antibodies, Cy3 and Cy5, which can be used to visualize unphosphorylated and phosphorylated proteins in the same blot, with endogenous protein normalization. This can result in a significant improvement in visualization of a protein, especially as antibodies for phosphorylated proteins can be less intensely stained than other cell types.

Figure 2. (A) When rhAng2 was added to human umbilical vein endothelial cells (HUVEC), STAT-3 was phosphorylated to p-STAT-3 (red) and translocated into nuclei (blue); the cytoskeleton can be visualized in green. Controls were treated with PBS only. (B) Amersham results analyzing STAT-3 phosphorylation (Tyr 705), following stimulation with rhAng2. (C) Quantification of the ratio of p-STAT-3 to total STAT-3 protein expression, normalized to control.

When staining for only one protein of interest, total protein can be assessed using Cy5 pre-labeling, allowing for consistent measurement and normalization of protein expression while preventing issues associated with variation in endogenous protein levels. This is especially useful for comparison of protein expression between different cell lines, where endogenous protein expression might also be variable. The Cy5-based total protein measurements were useful for confirming those measurements made using the Bradford assay.

The use of fluorescence detection improved the stability of the protein signal, in addition to increasing sensitivity and providing a broader dynamic range. A clear twofold increase in P-STAT-3 after 10 mins treatment with rhAng2 was measured by the Western blotting system (Figure 2), with the transformation from raw to publishable data being uncomplicated, accurate, and conveniently fast. The blot was analyzed automatically by the Amersham™ WB system, and the data produced was immediately publishable.

The Amersham WB system has replaced the old system of Western blotting for most applications. The process is easy to teach and easy to learn, and the principles can be explained in a very short time. The traditional Western blotting workflow is streamlined, significantly reducing the steps and time required (Figure 3B), as well as providing robust quantitative analysis (Figure 3C). Western blotting has been transformed from a consuming full-day project to a standard half-day procedure; a transformation comparable to the development of qPCR for PCR—a “qWestern blot” machine.

Figure 3. (A) Amersham WB system, including Western blotting system and analyser. (B) Comparison of traditional Western blotting workflow with the Amersham WB system, showing timings required at each step. (C) Workflow for image analysis and quantification of Western blots. The time and number of manual steps required in each workflow are reduced when using the Amersham WB system, while improving the consistency of results.


Understanding cell-signaling mechanisms is essential to the development of potential new cancer treatments. A new quantitative Western blotting system was used to measure cell signaling in a study investigating anti-Ang2 exposure by measuring phosphorylation of a downstream target, STAT-3. The new system supported the consistent and quantitative measurement of often difficult-to-detect phosphorylated antibodies. It could also be used to detect proteins of the same molecular weight on the same blot, without requiring different exposure times or multiple repeats, as is the case with traditional Western blotting techniques. 

Martin Teichert ([email protected]) is a Ph.D. candidate in vascular oncology and metastasis, German Cancer Research Center.

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