Gregory C. Fox

Tumor-infiltrating myeloid cells contribute to the development of the immunosuppressive tumor microenvironment. Myeloid cell expression of arginase 1 (ARG1) promotes a protumor phenotype by inhibiting T cell function and depleting extracellular l-arginine, but the mechanism underlying this expression, especially in breast cancer, is poorly understood. In breast cancer clinical samples and in our mouse models, we identified tumor-derived GM-CSF as the primary regulator of myeloid cell ARG1 expression and local immune suppression through a gene-KO screen of breast tumor cell-produced factors. The induction of myeloid cell ARG1 required GM-CSF and a low pH environment. GM-CSF signaling through STAT3 and p38 MAPK and acid signaling through cAMP were required to activate myeloid cell ARG1 expression in a STAT6-independent manner. Importantly, breast tumor cell-derived GM-CSF promoted tumor progression by inhibiting host antitumor immunity, driving a significant accumulation of ARG1-expressing myeloid cells compared with lung and melanoma tumors with minimal GM-CSF expression. Blockade of tumoral GM-CSF enhanced the efficacy of tumor-specific adoptive T cell therapy and immune checkpoint blockade. Taken together, we show that breast tumor cell-derived GM-CSF contributes to the development of the immunosuppressive breast cancer microenvironment by regulating myeloid cell ARG1 expression and can be targeted to enhance breast cancer immunotherapy.

Abstract Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFβ signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site. Cancer Res; 77(22); 6299–312. ©2017 AACR.

Integrin β3 is critical for tumor invasion, neoangiogenesis, and inflammation, making it a promising cancer target. However, preclinical and clinical data of integrin β3 antagonists have demonstrated no benefit or worse outcomes. We hypothesized that integrin β3 could affect tumor immunity and evaluated tumors in mice with deletion of integrin β3 in macrophage lineage cells (β3KOM). β3KOM mice had increased melanoma and breast cancer growth with increased tumor-promoting M2 macrophages and decreased CD8(+) T cells. Integrin β3 antagonist, cilengitide, also enhanced tumor growth and increased M2 function. We uncovered a negative feedback loop in M2 myeloid cells, wherein integrin β3 signaling favored STAT1 activation, an M1-polarizing signal, and suppressed M2-polarizing STAT6 activation. Finally, disruption of CD8(+) T cells, macrophages, or macrophage integrin β3 signaling blocked the tumor-promoting effects of integrin β3 antagonism. These results suggest that effects of integrin β3 therapies on immune cells should be considered to improve outcomes. Cancer Res; 76(12); 3484-95. ©2016 AACR.

These data suggest αvβ3-NP-mediated drug delivery of a c-MYC inhibitor can reduce protumor M2-like macrophages while preserving antitumor M1-like macrophages in breast cancer.

Abstract Breast cancer bone metastases are common and incurable. Tumoral integrin β3 (β3) expression is induced through interaction with the bone microenvironment. Although β3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of β3+ tumor cells in murine bone metastases after docetaxel chemotherapy. β3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples (n = 38). High tumoral β3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral β3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact β3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in β3-expressing cells characterized by enhanced oxygen consumption, reactive oxygen species generation, and protein production. We identified mTORC1 as a candidate for therapeutic targeting of this β3-mediated, chemotherapy-induced metabolic response. mTORC1 inhibition in combination with docetaxel synergistically attenuated murine bone metastases. Furthermore, micelle nanoparticle delivery of mTORC1 inhibitor to cells expressing activated αvβ3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that β3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvβ3-targeted mTORC1 inhibitor nanotherapy. Our work demonstrates the importance of the metastatic microenvironment when designing treatments and presents new, bone-specific strategies for enhancing chemotherapeutic efficacy.