Neurofibromatosis type 1-dependent alterations in mouse microglia function are not cell-intrinsic.

We previously discovered a sex-by-genotype defect in microglia function using a heterozygous germline knockout mouse model of Neurofibromatosis type 1 (Nf1 ± mice), in which only microglia from male Nf1 ± mice exhibited defects in purinergic signaling. Herein, we leveraged an unbiased proteomic approach to demonstrate that male, but not female, heterozygous Nf1 ± microglia exhibit differences in protein expression, which largely reflect pathways involved in cytoskeletal organization. In keeping with these predicted defects in cytoskeletal function, only male Nf1 ± microglia had reduced process arborization and surveillance capacity. To determine whether these microglial defects were cell autonomous or reflected adaptive responses to Nf1 heterozygosity in other cells in the brain, we generated conditional microglia Nf1-mutant knockout mice by intercrossing Nf1flox/flox with Cx3cr1-CreER mice (Nf1flox/wt; Cx3cr1-CreER mice, Nf1MG ± mice). Surprisingly, neither male nor female Nf1MG ± mouse microglia had impaired process arborization or surveillance capacity. In contrast, when Nf1 heterozygosity was generated in neurons, astrocytes and oligodendrocytes by intercrossing Nf1flox/flox with hGFAP-Cre mice (Nf1flox/wt; hGFAP-Cre mice, Nf1GFAP ± mice), the microglia defects found in Nf1 ± mice were recapitulated. Collectively, these data reveal that Nf1 ± sexually dimorphic microglia abnormalities are likely not cell-intrinsic properties, but rather reflect a response to Nf1 heterozygosity in other brain cells.

T lymphocytes as dynamic regulators of glioma pathobiology.

The brain tumor microenvironment contains numerous distinct types of nonneoplastic cells, which each serve a diverse set of roles relevant to the formation, maintenance, and progression of these central nervous system cancers. While varying in frequencies, monocytes (macrophages, microglia, and myeloid-derived suppressor cells), dendritic cells, natural killer cells, and T lymphocytes represent the most common nonneoplastic cellular constituents in low- and high-grade gliomas (astrocytomas). Although T cells are conventionally thought to target and eliminate neoplastic cells, T cells also exist in other states, characterized by tolerance, ignorance, anergy, and exhaustion. In addition, T cells can function as drivers of brain cancer growth, especially in low-grade gliomas. Since T cells originate in the blood and bone marrow sinuses, their capacity to function as both positive and negative regulators of glioma growth has ignited renewed interest in their deployment as immunotherapeutic agents. In this review, we discuss the roles of T cells in low- and high-grade glioma formation and progression, as well as the potential uses of modified T lymphocytes for brain cancer therapeutics.

Immune deconvolution and temporal mapping identifies stromal targets and developmental intervals for abrogating murine low-grade optic glioma formation.

BACKGROUND: Brain tumor formation and progression are dictated by cooperative interactions between neoplastic and non-neoplastic cells. This stromal dependence is nicely illustrated by tumors arising in the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome, where children develop low-grade optic pathway gliomas (OPGs). Using several authenticated Nf1-OPG murine models, we previously demonstrated that murine Nf1-OPG growth is regulated by T cell function and microglia Ccl5 production, such that their inhibition reduces tumor proliferation in vivo. While these interactions are critical for established Nf1-OPG tumor growth, their importance in tumor formation has not been explored. METHODS: A combination of bulk and single-cell RNA mouse optic nerve sequencing, immunohistochemistry, T cell assays, and pharmacologic and antibody-mediated inhibition methods were used in these experiments. RESULTS: We show that T cells and microglia are the main non-neoplastic immune cell populations in both murine and human LGGs. Moreover, we demonstrate that CD8+ T cells, the predominant LGG-infiltrating lymphocyte population, are selectively recruited through increased Ccl2 receptor (Ccr4) expression in CD8+, but not CD4+, T cells, in a NF1/RAS-dependent manner. Finally, we identify the times during gliomagenesis when microglia Ccl5 production (3-6 weeks of age) and Ccl2-mediated T cell infiltration (7-10 weeks of age) occur, such that temporally-restricted Ccl2 or Ccl5 inhibition abrogates tumor formation >3.5 months following the cessation of treatment. CONCLUSIONS: Collectively, these findings provide proof-of-concept demonstrations that targeting stromal support during early gliomagenesis durably blocks murine LGG formation.

Asthma reduces glioma formation by T cell decorin-mediated inhibition of microglia.

To elucidate the mechanisms underlying the reduced incidence of brain tumors in children with Neurofibromatosis type 1 (NF1) and asthma, we leverage Nf1 optic pathway glioma (Nf1OPG) mice, human and mouse RNAseq data, and two different experimental asthma models. Following ovalbumin or house dust mite asthma induction at 4-6 weeks of age (WOA), Nf1OPG mouse optic nerve volumes and proliferation are decreased at 12 and 24 WOA, indicating no tumor development. This inhibition is accompanied by reduced expression of the microglia-produced optic glioma mitogen, Ccl5. Human and murine T cell transcriptome analyses reveal that inhibition of microglia Ccl5 production results from increased T cell expression of decorin, which blocks Ccl4-mediated microglia Ccl5 expression through reduced microglia NFκB signaling. Decorin or NFκB inhibitor treatment of Nf1OPG mice at 4-6 WOA inhibits tumor formation at 12 WOA, thus establishing a potential mechanistic etiology for the attenuated glioma incidence observed in children with asthma.

Targeted Therapy to ß3 Integrin Reduces Chemoresistance in Breast Cancer Bone Metastases.

Breast cancer bone metastases are common and incurable. Tumoral integrin ß3 (ß3) expression is induced through interaction with the bone microenvironment. Although ß3 is known to promote bone colonization, its functional role during therapy of established bone metastases is not known. We found increased numbers of ß3+ tumor cells in murine bone metastases after docetaxel chemotherapy. ß3+ tumor cells were present in 97% of post-neoadjuvant chemotherapy triple-negative breast cancer patient samples (n = 38). High tumoral ß3 expression was associated with worse outcomes in both pre- and postchemotherapy triple-negative breast cancer groups. Genetic deletion of tumoral ß3 had minimal effect in vitro, but significantly enhanced in vivo docetaxel activity, particularly in the bone. Rescue experiments confirmed that this effect required intact ß3 signaling. Ultrastructural, transcriptomic, and functional analyses revealed an alternative metabolic response to chemotherapy in ß3-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 ß3-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 αvß3 integrins enhanced docetaxel efficacy in bone metastases. Taken together, we show that ß3 integrin induction by the bone microenvironment promotes resistance to chemotherapy through an altered metabolic response that can be defused by combination with αvß3-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.

Bone-Induced Expression of Integrin ß3 Enables Targeted Nanotherapy of Breast Cancer Metastases.

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 ß3 (ß3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, ß3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, ß3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFß signaling through SMAD2/SMAD3 was necessary for breast cancer induction of ß3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvß3 (αvß3-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 αvß3-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 αvß3 at that metastatic site. Cancer Res; 77(22); 6299-312. ©2017 AACR.