Murine breast tumor models

Orthotopic: female BALB/c mice were injected with 10⁴ 4T1 mammary carcinoma cells at the 4^th mammary gland at D0. Some experiments used 4T1 cells expressing eGFP following lentivirus infection or transduced with lentivirus containing CRISPR-Cas9-GFP that targeted to genes of interest. Mouse breast tumor size was measured in 3 dimensions (length × width × height) by digital caliper (Mitutoyo). To evaluate breast tumor metastasis to lungs, mouse lungs were injected with 15 % Indian ink via trachea after sacrifice and then fixed with Fekete’s solution. Lungs that were not seeded with lung metastases were black after India ink infusion but tumor metastases part displayed in white. Number of lung metastases was counted as white nodules on the lung surface.

Lung homing model of metastasis: 4T1 cells (5 × 10⁴) were injected i.v. via tail vein at D0. Tumor burdens in lungs were evaluated at D19 using the India ink method and lung metastases were counted as described above.

Autochthonous breast tumor model: Breast tumor growth of MTAG mice were monitored and measured from 10- to 20-weeks old. Because MTAG mice could develop up to 10 discrete mammary gland tumors, total tumor volume in female MTAG mice was determined as the sum of each distinct tumor volume within an individual mouse. Each breast tumor was measured in 3 dimensions (length × width × height) by digital caliper (Mitutoyo) weekly to 16-week old and then twice a week afterwards. To evaluate tumor metastasis to lungs, the left lobe of mouse lungs was fixed, embedded in paraffin, and serial sectioned. Lung sections were picked every 200 μm and lung metastases in each section were counted under microscope. Total lung metastases were the sum from all picked sections.

All tumor-bearing mice were monitored as approved by the Benaroya Research Institute Institutional Animal Care and Use Committee.