Quantification of Liver Metastases

Liver tissues were fixed, embedded in paraffin and used to prepare thin sections (5 µm) using an established protocol^{96 (link)}. Tissue sections were stained with hematoxylin and eosin and images captured with an Olympus BX51 microscope equipped with digital camera DP26 (Olympus Corporation, Tokyo, Japan). Quantification of micrometastases, defined as single-to small clusters of tumor cells, in the liver was done using H&E-stained sections and visualized in a stereo investigator system (Zeiss Imager M2 AX10, Germany). The total area of each section was scanned and measured. Digital images were used to quantify the number of tumor metastatic foci in each section and calculated as the number of foci per mm² area. Indirect immunostaining was also used to detect highly proliferative cells with a rabbit anti-mouse Ki67 antibody (Abcam, Cambridge, UK) followed by anti-rabbit-HRP (Cell signaling, Danvers, MA, USA). Peroxidase activity was determined using DAB chromogen (Dako, Carpinteria, CA, USA). Sections were then counter-stained with hematoxylin and visualized and photographed with an Olympus BX51 microscope. All slides were examined by a certified pathologist under blinded conditions.