Figure 5.

Non-invasive optical tumour imaging by fluorescently labelled DARPin 8h6 in PyMT tumour model. (a) Schematic representation of the imaging experiment outline with the orthotopic PyMT tumour model. (b) PyMT cells were injected into the left inguinal fat pads of FVB/N mice and allowed to grow until 100-150 mm³. C-terminally Cy5.5-labelled DARPin 8h6 or nonselective DARPin E3_5 was injected i.v. (1 nmol in 100 μL) and epi-fluorescent images were taken at designated time-points after administration. False blue-hot colouring of the radiant efficiency (radiance (photons per second per square centimetre per steradian) per incident excitation power (microwatt per square cm)) is overlaid on bright-field images. (c) Quantification of the average radiant efficiency from b in tumours and contralateral mammary glands as the means ± standard error. Regions of interest (ROI): tumour - red circle, contralateral mammary gland - black circle. (d) Tumour-to-contralateral mammary gland ratios of the average radiant efficiency in individual mice from b represented as the means ± standard error. *** p<0.001, ** p<0.01 (e, f) DARPin distribution in isolated organs 3 h and (g, h) 72 h after administration. False blue-hot images of the radiance efficiency are overlaid on bright-field images. Representative images of four mice per group are shown. The average radiant efficiency from isolated organs is quantified as the mean ± standard error. Li = liver, S = spleen, Ln = lymph nodes, T = tumour, H = heart, Lu = lungs, K = kidneys. *** p<0.001, ** p<0.01, * p<0.05 (i) Immunohistochemical analysis of cryopreserved tumours 3 h after i.v. administration of Cy5.5-labelled DARPin 8h6 (red). The tissue sections were stained with macrophage marker F4/80 (green) and for cell nuclei (DAPI, blue). Scale bar, 20 μm; inlet scale bar, 10 μm. (j) DARPin pull-down of CatB from tumour tissue 30 minutes after intratumoural administration of 5 nmol of C-terminally biotinylated DARPins. The streptavidin-bound fraction was separated using SDS-PAGE and immunoblotted against CatB. The presence of DARPins was confirmed by detection with streptavidin-HRP. Total extract from tumour (50 μg) was added to visualize different forms of CatB in the tumours. proCatB = procathepsin B, scCatB = single chain CatB, hcCatB = heavy chain CatB.