Systemic coagulation parameters in mice after treatment with vascular targeting agents

BACKGROUND: Vascular targeting of malignant tumors has become a clinically validated new treatment approach with clear patient benefit. However clinical studies have also revealed that some types of vascular targeting agents (VTAs) are prone to coagulation system side effects. It is therefore essential to predetermine coagulation parameters in preclinical studies. As of to date, this has rarely been done, predominantly due to technical issues. The goal of this study was to establish and apply a standardized process, whereby systemic coagulation activation can be routinely measured in mice. RESULTS: We have evaluated a number of sampling techniques and coagulation tests regarding their suitability for this purpose. We were able to adapt two assays measuring soluble fibrin, a marker for a prethrombotic status. Thus, soluble fibrin could be measured for the first time in mice. All assays were validated in a positive control model for systemic coagulation activation, i.e. lipopolysaccharide-induced endotoxemia. Based on our results, we selected a panel of coagulation tests, which are both feasable and informative for preclinical testing of VTAs: soluble fibrin, thrombin-antithrombin complexes, free antithrombin III, white blood cell counts and platelet counts. The effect of tumor transplants on coagulation parameters was evaluated using this panel. We then applied this set of assays in treatment studies with a VTA developed in our laboratory to investigate a potential systemic coagulation activation. CONCLUSION: We have established a standardized panel of assays that can be used to test murine blood samples for coagulation activation in preclinical studies. All tests are feasible to perform in any research laboratory without specialized equipment. In addition, this is the first report to measure soluble fibrin, an early marker of systemic coagulation activation, in mice. The panel was applied on tumor bearing mice and mice treated with a VTA. We suggest its general application for coagulation activation analyses in mice

In-Vivo Visualization of Tumor Microvessel Density and Response to Anti-Angiogenic Treatment by High Resolution MRI in Mice

Purpose: Inhibition of angiogenesis has shown clinical success in patients with cancer. Thus, imaging approaches that allow for the identification of angiogenic tumors and the detection of response to anti-angiogenic treatment are of high clinical relevance. Experimental Design: We established an in vivo magnetic resonance imaging (MRI) approach that allows us to simultaneously image tumor microvessel density and tumor vessel size in a NSCLC model in mice. Results: Using microvessel density imaging we demonstrated an increase in microvessel density within 8 days after tumor implantation, while tumor vessel size decreased indicating a switch from macro- to microvessels during tumor growth. Moreover, we could monitor in vivo inhibition of angiogenesis induced by the angiogenesis inhibitor PTK787, resulting in a decrease of microvessel density and a slight increase in tumor vessel size. Conclusions: We present an in vivo imaging approach that allows us to monitor both tumor microvessel density and tumor vessel size in the tumor. Moreover, this approach enables us to assess, early-on, treatment effects on tumor microvessel density as well as on tumor vessel size. Thus, this imaging-based strategy of validating anti-angiogenic treatment effects ha

Percent changes (relative to day 0) in T2, MDI, VSI and ADC values after 6 days and 13 days of PTK787/vehicle treatment.

<p>Percent changes (relative to day 0) in T2, MDI, VSI and ADC values after 6 days and 13 days of PTK787/vehicle treatment.</p

Comparison of the image derived values of MDI and VSI to CD31 positive endothelial cell staining.

<p>(A) a vehicle treated (upper row) tumor presenting a high microvessel density index (MDI (1/mm²)) in MRI and a low vessel index (VSI (µm)). The PTK787-treated tumor (lower row) presents low MDI values with high VSI values. (B) correlation between CD31 positive vessels per area unit and the imaged derived MDI values (n = 10).</p

Longitudinal investigation of vascular dynamics during tumor growth.

<p>Simultaneous in vivo monitoring of tumor depiction with T2-weighted imaging, microvessel density (MDI), vessel size (VSI) and apparent diffusion coefficient (ADC) during tumor growth on day 1, day 4, day 8, day 14, and day 21 (D1, D4, D8, D14, D21).</p