Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases

Sébastien Tabariès, Véronique Ouellet, Brian E Hsu, Matthew G Annis, April AN Rose, Liliane Meunier, Euridice Carmona, Christine E Tam, Anne-Marie Mes-Masson, Peter M Siegel

Breast cancer cells display preferences for specific metastatic sites including the bone, lung and liver. Metastasis is a complex process that relies, in part, on interactions between disseminated cancer cells and resident/infiltrating stromal cells that constitute the metastatic microenvironment. Distinct immune infiltrates can either impair the metastatic process or conversely, assist in the seeding, colonization and growth of disseminated cancer cells. Using in vivo selection approaches, we previously isolated 4T1-derived breast cancer cells that preferentially metastasize to these organs and tissues. In this study, we examined whether the propensity of breast cancer cells to metastasize to the lung, liver or bone is associated with and dependent on distinct patterns of immune cell infiltration. Immunohistocytochemistry and immunohistofluorescence approaches were used to quantify innate immune cell infiltrates within distinct metastases and depletion of Gr1(+) (Ly-6C and Ly-6G) or specifically Ly-6G(+) cells was performed to functionally interrogate the role of Ly-6G(+) infiltrates in promoting metastasis to these organs. We show that T lymphocytes (CD3(+)), myeloid-derived (Gr-1(+)) cells and neutrophils (Ly-6G(+) or NE(+)) exhibit the most pronounced recruitment in lung and liver metastases, with markedly less recruitment within bone metastatic lesions. Interestingly, these infiltrating cell populations display different patterns of localization within soft tissue metastases. T lymphocytes and granulocytic immune infiltrates are localized around the periphery of liver metastases whereas they were dispersed throughout the lung metastases. Furthermore, Gr-1(+) cell-depletion studies demonstrate that infiltrating myeloid-derived cells are essential for the formation of breast cancer liver metastases but dispensable for metastasis to the lung and bone. A specific role for the granulocytic component of the innate immune infiltrate was revealed through Ly-6G(+) cell-depletion experiments, which resulted in significantly impaired formation of liver metastases. Finally, we demonstrate that the CD11b(+)/Ly-6G(+) neutrophils that infiltrate and surround the liver metastases are polarized towards an N2 phenotype, which have previously been shown to enhance tumor growth and metastasis. Our results demonstrate that the liver-metastatic potential of breast cancer cells is heavily reliant on interactions with infiltrating Ly-6G(+) cells within the liver microenvironment.