Monitoring and tissue collection

Mice were monitored by weighing them and measuring their primary tumor growth 3 times weekly. Tumors were measured using calipers in the longest dimension (L) and at a right angle to that axis (W). Tumor volume was calculated using the formula L × W × W × π/6. Mice were euthanized when the primary tumor reached 2500 mm³ in size or earlier if required by humane endpoints. Mice were euthanized according to protocol, were grossly examined for metastases and primary tumors, and their corresponding lung metastases were isolated and divided such that equal pieces were fixed in formalin for histologic evaluation, snap frozen for protein evaluation and preserved in RNAlater (Ambion, Life Technologies, Grand Island, NY) to assess RNA.