Holm, David

Krisjansen, Paul E.

Ley, Carsten Dan

Lund, Eva Løbner

Rowland, Ian John

Simonsen, Helle Juhl

Søgaard, Lise Vejby

Angiogenesis is a critical process in tumour development and presents an important target for the development of a range of anti-cancer agents . To assess the in vivo efficacy of these ‘angiotherapeutics', a simple and reproducible in vivo model would be of significant value. Here we show that a gel containing pro-angiogenic agents held within a chamber may be positioned sub-cutaneously in mice and the angiogenic process followed using MR methods. This approach allows an individual animal to be followed with time in order to study the initial development of vasculature within the chambers

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In vivo monitoring of angiogenesis within Matrigel chambers using MRI 1Danish Research Centre for Magnetic Resonance, Copenhagen University Hospital, Hvidovre, Denmark 2Informatics and Mathematical Modeling, Technical University of Denmark, Lyngby, Denmark 3Institute for Molecular Pathology, Copenhagen, Denmark Methods Perspex chambers containing Matrigel, bFGF (1000 ng/ml, Sigma, Denmark) and VEGF (500 ng/ml, Sigma, Denmark) were assembled as previously described3 (figure 1). Following ra-diation sterilization, the chambers were im-planted subcutaneously on both flanks of 24 male NMRI nu/nu mice. Magnetic resonance imaging was performed using a 4.7T Varian sys-tem two weeks after implantation under hyp-norm/dormicum anesthesia. Contrast agent was administered via a cannulated tail vein (0.4mg Endorem, n=12, 0.3ml Magnevist, n=12). Following MR examination, the chambers were excised and imaged using a digital camera.  Results & Discussion Figure 2 shows significant enhancement of the Matrigel chamber following administration of GdDTPA. The region of cellular infiltration ap-parent in figure 2 (left) becomes hyperintense compared to uninfiltrated Matrigel (right). The high vascular permeability due to VEGF results in diffuse contrast enhancement. The long T2 values of the Matrigel chamber prior to implan-tation suggested the use of a T2* blood pool agent (Endorem), to provide more vascular spe-cific information. As shown in figure 3 (left), a region of cellular infiltration is seen in the up-per left region of the image. What appears to be a vessel may be seen on the dMRI differ-ence map (red ROI, middle). Some correspon-dence between the post contrast T2 map (left) and dMRI difference (middle) is seen. As shown in the time curves (right), three distinct regions could be identified. The red region appears to be a vessel where washout causes the signal to Introduction Angiogenesis is a critical process in tumour de-velopment and presents an important target for the development of a range of anti-cancer agents1,2. To assess the in vivo efficacy of these ‘an gioth erap eu tics’, a sim p le an d  rep rod u cib le in vivo model would be of significant value. Here we show that a gel containing pro-angiogenic agents held within a chamber may be positioned sub-cutaneously in mice and the angiogenic process followed using MR methods. This approach allows an individual animal to be followed with time in order to stu-dy the initial development of vasculature within the chambers. increase slightly following initial signal reduc-tion following contrast agent administration. The green and blue regions are different parts of the infiltration that might have different vascular or cell densities. The dMRI difference maps show the initial ef-fects of the contrast agent while the T2 maps show the extent of tissue infiltration and steady state effects. The difference between Figure 3 left and middle may be interpreted as an area corresponding to a vessel (red ROI) where the intravascular contrast agent clears before the post contrast T2 map is acquired. Conclusion This study has shown that it is possible to moni-tor angiogenesis using a minimally invasive mag-netic resonance method in vivo. Furthermore, a single animal may be followed longitudinally and act as its own control. The method may be developed further to study the effects of agents designed to influence the angiogenic process. References 1Gasparini G., et al., Angiogenic inhibitors: a new therapeutic strategy in oncology. Nat Clin Pract Oncol. 2005 Nov;2(11):562-77.  2Carmeliet,P, Mechanisms of angiogenesis and arteriogenesis.  Nat Med, 2003;6(4):389-95  3Ley, C.D et al., Angiogenic synergy of bFGF and VEGF is antagonized by Angiopoietin-2 in a modified in vivo matrigel assay Microvasc Res. 2004 Nov;68(3):161-8.  4Pilatus U. et al., Imaging prostate cancer invasion with multi-nuclear magnetic resonance methods: the Metabolic Boyden Chamber. Neoplasia. 2000 May-Jun;2(3):273-9.   D.A. Holm1,2, C.D. Ley3, L.V. Søgaard 1, H.J. Simonsen1, P.E. Kristjansen3, E.L. Lund3,  I.J. Rowland1 Figure 1: Schematic diagram of the assembly and imaging of the chamber Figure 2: Ex vivo optical ima-ge (left) together with in vivo pre- (middle) and post- (right) contrast (Magnevist, 0.3 ml) T1W spin echo ima-ges (TR/TE 300/3 ms) of the same Matrigel implant.  Figure 3: Left: Quantitative T2 image post contrast (multiple readout spin echo se-quence, TR=3s, TE=15-180ms in steps of 15ms, T2 values obtained by least squares fitting). Middle: dMRI difference image (gradient echo FLASH sequence, TR/TE=20/5.2 m s, Fl=  2 0 °, nt= 1 5 0 ). The im age show n is the d ifference b etw een the m ean of the 25 first and last images. Right: Time curves for the ROIs shown in the dMRI difference map (middle) are shown in the plot. Whilst a previous study by Pilatus, U. et al.4  has shown the potential of using Matrigel for study-ing cell invasion, this study has applied MRI methods to study vascular development in an in vivo model.  Contact davidh@drcmr.dk 

In vivo monitoring of angiogenesis within Matrigel chambers using MRI

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In vivo monitoring of angiogenesis within Matrigel chambers using MRI

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