Immune Suppressive Myeloid Cells Induced By Hypoxic Mammary Tumor Cells Persist After Primary Tumor Resection And Promote Metastatic Growth

A role for bone marrow-derived cells (BMDCs) in promoting metastatic tumor growth is emerging, with important implications for therapeutic strategies to decrease tumor metastases. While previous work has shown accumulation of CD11b+ BMDCs in the lungs of mice bearing metastatic breast tumors, questions remain about the precise identity of these cells, the factors that regulate CD11b+ cell accumulation, and the potential long-term influence of CD11b+ cells on metastatic growth. We used transplantable (4T1, 4TO7, 67NR) and spontaneous (polyomavirus middle-T; PyVmT) mammary tumor models to study the identity, induction, longevity, and function of CD11b+ BMDCs in tissues. Using flow cytometry and ex vivo immune suppression assays, we established that metastatic mammary tumors induce splenic expansion and pulmonary accumulation of functional CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) and CD11b+F4/80+ macrophages (Mϕs). MDSCs suppress T cell-mediated immune responses against tumor cells and are thought to promote tumor cell survival in vivo , while both Mϕs and MDSCs are known to have a variety of other functions that promote primary tumor growth and metastasis. Interestingly, we found that not all tumors induce MDSC accumulation. Rather, MDSC expansion in the spleen and accumulation in the lungs is dependent on poorly oxygenated (hypoxic) cells in the primary tumor. MDSCs did not accumulate in mice bearing well-oxygenated 67NR tumors or in mice with spontaneous PyVmT tumors until hypoxia had developed in the primary PyVmT tumors. Further, specifically targeting hypoxic 4T1 tumor cells in vivo with the hypoxia-activated cytotoxin tirapazamine significantly decreased MDSC levels in the spleen and lungs. Since proteins secreted by tumor cells are capable of inducing BMDC mobilization and accumulation in tissues, we used proteomics-based analyses of medium conditioned by hypoxic or normoxic tumor cells in vitro to identify and quantify several hypoxia-induced secreted proteins that are involved in promoting myeloid cell accumulation in tissues. We found that continued secretion of proteins by primary tumors is required to maintain high levels of splenic MDSC expansion, since surgical resection of metastatic primary tumors rapidly decreased MDSC expansion in the spleen and accumulation in the lungs. Interestingly, functional immune suppressive MDSCs remained elevated in the lungs for several weeks after resection of metastatic primary tumors, and these myeloid cells were associated with enhanced subsequent pulmonary metastatic tumor growth. In order to reduce the immune suppressive environment in the lungs, we used all-trans retinoic acid (ATRA) to induce the differentiation of immature MDSCs into mature Mϕs. While ATRA treatment decreased MDSC levels and increased Mϕs in the lungs, ATRA-treated mice displayed enhanced metastatic tumor growth compared to untreated mice. Consistent with these data, we found that Mϕs were 30-fold more potent suppressors of T cell-mediated immune responses than MDSCs on a per cell basis. These data highlight the influence of immune suppressive myeloid cell sub-types on tumor metastasis, and identify immune suppressive Mϕs as potential therapeutic targets. Taken together, these data suggest that hypoxia-mediated accumulation of myeloid cells in the lungs can have long-lasting implications for the growth of secondary metastatic tumors after treatment. Our data support the development of therapeutic strategies to target hypoxic tumor cells and immune suppressive myeloid cells to treat metastatic breast cancer. Citation Format: Melisa J. Hamilton, Momir Bosiljcic, Bryant T. Harbourne, Nancy E. LePard, Elizabeth C. Halvorsen, Ada Y. Kim, Judit P. Banath, Gerald Krystal, Kevin L. Bennewith. Immune suppressive myeloid cells induced by hypoxic mammary tumor cells persist after primary tumor resection and promote metastatic growth. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A9.