Optical projection tomography for three-dimensional analysis of cancer models following anti-vascular therapy Angela d’Esposito 1 , S. Peter Johnson 1 , Barbara Pedley 2 , Adrien Desjardins 3 , Mark F. Lythgoe 1* , Simon Walker-Samuel 1* 1 UCL, CABI, 72 Huntley Street, London, WC1E 6DD, UK, 2 UCL Cancer Institute, 72 Huntley Street, London, WC1E 6DD, UK, 3 UCL Department of Medical Physics and Biomedical Engineering, 1 angela.d’[email protected], * Joint senior authors Keywords: colorectal cancer, vascular disrupting agent, optical projection tomography Introduction: Colorectal cancer is the second most common cancer in both men and women in the UK 1 . In the past decade, low molecular weight vascular disrupting agents (VDA) have been shown to cause rapid and selective blockage of the established tumor vasculature, leading to cancer cell death 2 . This study proves the ability of Optical Projection Tomography (OPT) to detect the therapeutic effect of OXi4503 on the vessel architecture of subcutaneous colorectal tumours, in three-dimensions. Methods: Animal Model: Two human colorectal carcinoma cell lines (SW1222 and LS147T) were injected in 12 MF1 nu/nu mice (1x10 6 cells). After 10 days, 6 mice were injected with OXi4503 combretastatin (250μl, 4 mg/ml) and 6 with sterile saline (250μl). Ex-vivo Preparation: After 48 hours animals were perfuse-fixed following intravenous injection of 100μg of lectin-AlexaFluor 647, to fluorescently label blood vessels. Optical clearing of the tumours was achieved with BABB (1:2 benzyl alcohol: benzyl benzoate). OPT: Transmission (white light) and emission (NIR: exciter 655/40 nm, emitter 716/40 nm), isotropic resolution 5 μm, 0.9 degree angular increments, exposure times 400÷600 ms. Image Analysis: OPT images were reconstructed with NRecon software. Lectin signal (blood vessels) was segmented from background autofluorescence using simple thresholding, and skeletonised with a thinning algorithm (Amira 5.4 and ImageJ). Results: Individual tumour blood vessels were easily visualised on OPT images. Skeletonisation of vessels revealed no difference in vessel size distribution or between tumour types, but blood volume in LS174T tumours was signficantly lower than in SW1222 tumours (p<0.05, Wilcoxon rank sum). OXI4503 at a dose of 40mg/kg caused blockage of central tumour blood vessels at 48 hrs after drug administration (Fig. 1). Also, a significant reduction of vessel volume, and an increase in average vessel size within tumours was observed (p<0.05, Wilcoxon rak sum). Figure 1: 3D renderings of the blood vessels, segmented from OPT data, and colour-coded for vessel size. Example control and treated SW1222 tumours are shown in (a) and (b), respectively; example control and treated LS174T tumours are shown in (c) and (d). Conclusions: In this study, 3-dimensional OPT images of tumour microvasculature were produced by labelling with a fluorescent marker and performing optical clearing. The detailed analysis of the vasculature afforded by this technique allowed differences in vessel size and blood volume to be measured between control and treated tumours, This 3-dimensional histological approach enables a more complete and quantitative analysis than could be performed using conventional 2-dimensional approaches. References: 1. Forner, Lancet, 2012, 2. Tozer, Nat Rev Cancer, 2005 a b c d
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Optical projection tomography for three-dimensional analysis of cancer models following anti-vascular therapy
Angela d’Esposito1, S. Peter Johnson1, Barbara Pedley2, Adrien Desjardins3, Mark F. Lythgoe1*, Simon Walker-Samuel1*
1UCL, CABI, 72 Huntley Street, London, WC1E 6DD, UK, 2UCL Cancer Institute, 72 Huntley Street, London, WC1E 6DD, UK, 3UCL Department of Medical Physics and
Biomedical Engineering, 1angela.d’[email protected], * Joint senior authors Keywords: colorectal cancer, vascular disrupting agent, optical projection tomography Introduction: Colorectal cancer is the second most common cancer in both men and women in the UK1. In the past decade, low molecular weight vascular disrupting agents (VDA) have been shown to cause rapid and selective blockage of the established tumor vasculature, leading to cancer cell death2. This study proves the ability of Optical Projection Tomography (OPT) to detect the therapeutic effect of OXi4503 on the vessel architecture of subcutaneous colorectal tumours, in three-dimensions. Methods: Animal Model: Two human colorectal carcinoma cell lines (SW1222 and LS147T) were injected in 12 MF1 nu/nu mice (1x106 cells). After 10 days, 6 mice were injected with OXi4503 combretastatin (250µl, 4 mg/ml) and 6 with sterile saline (250µl). Ex-vivo Preparation: After 48 hours animals were perfuse-fixed following intravenous injection of 100µg of lectin-AlexaFluor 647, to fluorescently label blood vessels. Optical clearing of the tumours was achieved with BABB (1:2 benzyl alcohol: benzyl benzoate). OPT: Transmission (white light) and emission (NIR: exciter 655/40 nm, emitter 716/40 nm), isotropic resolution 5 µm, 0.9 degree angular increments, exposure times 400÷600 ms. Image Analysis: OPT images were reconstructed with NRecon software. Lectin signal (blood vessels) was segmented from background autofluorescence using simple thresholding, and skeletonised with a thinning algorithm (Amira 5.4 and ImageJ). Results: Individual tumour blood vessels were easily visualised on OPT images. Skeletonisation of vessels revealed no difference in vessel size distribution or between tumour types, but blood volume in LS174T tumours was signficantly lower than in SW1222 tumours (p<0.05, Wilcoxon rank sum). OXI4503 at a dose of 40mg/kg caused blockage of central tumour blood vessels at 48 hrs after drug administration (Fig. 1). Also, a significant reduction of vessel volume, and an increase in average vessel size within tumours was observed (p<0.05, Wilcoxon rak sum).
Figure 1: 3D renderings of the blood vessels, segmented from OPT data, and colour-coded
for vessel size. Example control and treated SW1222 tumours are shown in (a) and (b), respectively; example control and treated LS174T tumours are shown in (c) and (d).
Conclusions: In this study, 3-dimensional OPT images of tumour microvasculature were produced by labelling with a fluorescent marker and performing optical clearing. The detailed analysis of the vasculature afforded by this technique allowed differences in vessel size and blood volume to be measured between control and treated tumours, This 3-dimensional histological approach enables a more complete and quantitative analysis than could be performed using conventional 2-dimensional approaches. References: 1. Forner, Lancet, 2012, 2. Tozer, Nat Rev Cancer, 2005
a b c d
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