Lung Metastasis Quantification

Hematoxylin–eosin (H&E) staining and India Ink assay were used to quantify lung metastasis. For H&E staining, mice were euthanized and were injected with 5 ml 10% neutral buffered formalin (NBF) into the trachea. The lungs were harvested, fixed with 10% NBF and paraffin embedded. For each lung sample, six 3-μm paraffin-embedded sections at 300-μm intervals were used for hematoxylin and eosin staining. Images of whole lung sections were captured using PathScan Enabler IV (Meyer), and metastases were quantified using the texture segmentation algorithm of MATLAB v2014b.

For the India Ink assay, mice were euthanized and injected with 5 ml India Ink (15% v/v diluted in PBS; Hardy Diagnostics) into the trachea. The lungs were then harvested and fixed with Fekete’s solution (for 1 L, mix 880 ml 70% ethanol, 80 ml 37% formaldehyde, and 40 ml glacial acetic acid) overnight at 4°C. The lung metastasis nodules were counted manually.