Figure 3: Sustained inhibition of ErbB2 signalling triggers apoptosis in vivo.

(a) Biodistribution analysis in BT474-xenografted mice, injected with Alexa₆₈₀-conjugated and PEGylated DARPins or PBS (i.v.; 1 mg kg⁻¹). Mice were monitored on an IVIS imaging system for 10 days. Letters indicate tumour site (T) and bladder (B). (b) Tumour growth inhibition by PEGylated DARPins in SCID beige mice with pre-established BT474 xenografts. Left, treatment was started when tumours reached 200 mm³. Agents were injected three times per week (i.v.; 20 mg kg⁻¹) as indicated by arrows. All error bars are s.e.m. Right, tumour size distribution of individual treatment groups after 24 d, for later time points see Supplementary Fig. 3C (two-sided, unpaired Students t-test). (c) In situ biomarker staining in treated BT474 xenografts. PFA-fixed, paraffin-embedded tumours were analysed by detecting the indicated cellular targets. Scale bars, 50 μm. (d) Per cent ErbB2 and p-ErbB2 (Y1222) staining intensity normalized to DAPI signal from ten tumour sections per treatment. ErbB2, p-ErbB2 and DAPI staining intensities were determined with ImageJ software (two-sided, unpaired Welch’s t-test). (e) Per cent TUNEL-positive cells from ten tumour sections per treatment. BT474 tumours were stained with TUNEL and counterstained with DAPI, the total number of single cells was identified by DAPI using Cell Profiler software, and associated TUNEL signals were quantified and assigned above a threshold (70 units) as TUNEL-positive (two-sided, unpaired Welch’s t-test).