CaMKK2 in myeloid cells is a key regulator of the immune suppressive microenvironment in breast cancer

Tumor-associated myeloid cells play a pivotal role in the regulation of processes that control tumor growth and metastasis, and their accumulation in tumors is an established negative prognostic factor in breast cancer. Here, we show that inhibition of calcium/calmodulin-dependentkinase kinase 2 (CaMKK2) expression within myeloid cells inhibits tumor growth in mouse models of mammary cancer. This activity is associated with the accumulation of macrophages within the tumor microenvironment that express high levels of the major histocompatibility molecule class II molecule I-A (MHC II I-A), and granzyme positive CD8+T-cells. Treatment with specific CaMKK2 inhibitors block tumor growth, in a CD8+ T-cell dependent manner, and facilitates favorable reprogramming of the immune cell microenvironment. CaMKK2 protein expression was highest in the most aggressive subtypes of human breast cancer and, in the most malignant tumors, both tumor cells and tumor-associated macrophages express high levels of this enzyme. In aggregate, these findings implicate CaMKK2 as a macrophage-selective immune checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer. Tumor-conditioned medium E0771 mouse breast cancer cell lines were cultured in RPMI-1640 (Gibco, MA, USA) supplied with 2 mM L-glutamine, 1.5g/L sodium bicarbonate, 1.0 mM sodium pyruvate (all from Gibco, MA, USA) and 8% fetal bovine serum (FCS, Hyclone, MA, USA). Cells were maintained in a humidified 37°C CO2 incubator and passed at 70% confluence. To generate conditioned medium, medium was removed from 70% confluent cultures and replaced with fresh RPMI-1640 8% FBS. After 3 days, culture supernates were collected and aliquots stored at -80°C. The protocol used for generating BMDM has been described previously (Racioppi et al. 2012). Briefly, hind limb bones were removed from mice and crushed in a mortar with 5 ml of Hanks' balanced salt solution (Mediatech, Manassas, VA) plus 2% FBS (Gemini Bio-Products, West Sacramento, CA) supplemented with 2 mM EDTA. Bone marrow (BM) cell suspensions were passed through a 70 μm strainer (BD Falcon) and stratified on Lympholyte (Cedarlane, Burlington, NC). The low-density fraction containing BM nucleated cells was collected, and the concentration was adjusted to 2 × 10^6 cells/ml in complete medium (10% fetal bovine serum DMEM, high glucose, supplemented with glutamine, pyruvate, and HEPES, no-phenol red) containing 30% L929-conditioned medium (differentiation medium, RM). Subsequently, BM cells were cultured for 5 days in Corning® Costar® Ultra-Low attachment multi-well plates (Sigma). To establish the purity of BMDM, cells were double-stained with anti-CD11b and anti-F4/80 antibodies. Based on this analysis, more than 95% of cells co-expressed these markers, which is a characteristic of macrophages. In the same experiments, tumor-conditioned medium (20% v/v) was added to differentiation medium. BMDM were generated in the presence of RM or CM from WT and 16 Camkk2-/- mice. Four biological replicates for each genotype were generated using long bones of 16 WT and 16 Camkk2-/- mice. Microarray analysis was performed at Sequencing and Genome Technologies Shared Resource (Duke University).