Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice.

Female mice transgenic for the rat proto-oncogene c-erb-B2, under control of the mouse mammary tumor virus (MMTV) promoter (neuN), spontaneously develop metastatic mammary carcinomas. The development of these mammary tumors is associated with increased number of GR-1(+)CD11b(+) myeloid derived suppressor cells (MDSCs) in the peripheral blood (PB), spleen and tumor. We report a complex relationship between tumor growth, MDSCs and immune regulatory molecules in non-mutated neu transgenic mice on a FVB background (FVB-neuN). The first and second tumors in FVB-neuN mice develop at a median of 265 (147-579) and 329 (161-523) days, respectively, resulting in a median survival time (MST) of 432 (201 to >500) days. During tumor growth, significantly increased number of MDSCs is observed in the PB and spleen, as well as, in infiltrating the mammary tumors. Our results demonstrate a direct correlation between tumor size and the number of MDSCs infiltrating the tumor and an inverse relationship between the frequency of CD4(+) T-cells and MDSCs in the spleen. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assessment of enzyme and cytokine transcript levels in the spleen, tumor, tumor-infiltrating non-parenchymal cells (NPCs) and mammary glands revealed a significant increase in transcript levels from grossly normal mammary glands and tumor-infiltrating NPCs during tumor progression. Tumor NPCs, as compared to spleen cells from wild-type (w/t) mice, expressed significantly higher levels of arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor (VEGF-A) and significantly lower levels of interferon (IFN)-gamma, interleukin (IL)-2 and fms-like tyrosine kinase-3 ligand (Flt3L) transcript levels. Transcript levels in the spleens of tumor-bearing (TB) mice also differed from normal mice, although to a lesser extent than transcript levels from tumor-infiltrating NPCs. Furthermore, both spleen cells and NPCs from TB mice, but not control mice, suppressed alloantigen responses by syngeneic control spleen cells. Correlative studies revealed that the number of MDSCs in the spleen was directly associated with granulocyte colony stimulating factor (G-CSF) transcript levels in the spleen; while the number of MDSCs in the tumors was directly correlated with splenic granulocyte macrophage stimulating factor (GM-CSF) transcript levels, tumor volume and tumor cell number. Together our results support a role for MDSCs in tumor initiation and progressive, T-cell depression and loss of function provide evidence which support multiple mechanisms of MDSC expansion in a site-dependent manner.

Age-related changes in cerebellar and hypothalamic function accompany non-microglial immune gene expression, altered synapse organization, and excitatory amino acid neurotransmission deficits.

We describe age-related molecular and neuronal changes that disrupt mobility or energy balance based on brain region and genetic background. Compared to young mice, aged C57BL/6 mice exhibit marked locomotor (but not energy balance) impairments. In contrast, aged BALB mice exhibit marked energy balance (but not locomotor) impairments. Age-related changes in cerebellar or hypothalamic gene expression accompany these phenotypes. Aging evokes upregulation of immune pattern recognition receptors and cell adhesion molecules. However, these changes do not localize to microglia, the major CNS immunocyte. Consistent with a neuronal role, there is a marked age-related increase in excitatory synapses over the cerebellum and hypothalamus. Functional imaging of these regions is consistent with age-related synaptic impairments. These studies suggest that aging reactivates a developmental program employed during embryogenesis where immune molecules guide synapse formation and pruning. Renewed activity in this program may disrupt excitatory neurotransmission, causing significant behavioral deficits.

A low-cost, reliable, high-throughput system for rodent behavioral phenotyping in a home cage environment.

Inexpensive, high-throughput, low maintenance systems for precise temporal and spatial measurement of mouse home cage behavior (including movement, feeding, and drinking) are required to evaluate products from large scale pharmaceutical design and genetic lesion programs. These measurements are also required to interpret results from more focused behavioral assays. We describe the design and validation of a highly-scalable, reliable mouse home cage behavioral monitoring system modeled on a previously described, one-of-a-kind system. Mouse position was determined by solving static equilibrium equations describing the force and torques acting on the system strain gauges; feeding events were detected by a photobeam across the food hopper, and drinking events were detected by a capacitive lick sensor. Validation studies show excellent agreement between mouse position and drinking events measured by the system compared with video-based observation--a gold standard in neuroscience.

Therapeutic activity of sunitinib for Her2/neu induced mammary cancer in FVB mice.

Mouse mammary tumor virus-Neu (MMTV/neu) transgenic mice on an FVB-background (FVB-neuN) have increased numbers of myeloid derived suppressor cells (MDSCs) and regulatory T-cells (T-regs) in the spleen during mammary tumor induction and progression. Using this transgenic tumor model, we assessed the therapeutic activity of sunitinib, a multi-targeted, tyrosine kinase (TK) inhibitor and its effects on immune-regulatory cells. Our preliminary results show that sunitinib at 40mg/kg/day, p.o. (per os), delayed the time to tumor induction and reduced the incidence and growth of tumors in FVB-neuN mice. In association with its therapeutic activity, sunitinib reduced the absolute number of splenic T-reg cells (CD4(+)CD25(+)CD62L(+)) and MDSCs (CD11b(+)Gr1(+)) that were increased during tumor progression with less activity in mice with gross tumors. A significant decrease in the absolute number of splenic T-regs, dendritic cells (DCs), MDSCs and hematopoietic progenitors (Lin(-)Sca1(+)CD90(dull)) was observed following sunitinib treatment. The frequency of splenic T-regs and hematopoietic progenitors, but not MDSCs was also reduced by sunitinib treatment. Additionally immune-regulatory cytokines and enzymes were down regulated by sunitinib treatment, including TGFbeta and NOS2 in the spleen cells of sunitinib treated mice as compared to untreated tumor bearing (TB) mice. We conclude that sunitinib has therapeutic activity, in association with the down regulation of MDSCs and T-regs and has a trend towards the normalization of the inflammatory cytokine levels induced by tumor progression and growth. Based on these results, we suggest that sunitinib reduction of immune suppressive cells is a critical part of its adjuvant immune therapeutic activity.

Mammary tumor heterogeneity in the expansion of myeloid-derived suppressor cells.

The tumor microenvironment is heterogeneous for the expansion and infiltration by myeloid derived suppressor cells (MDSCs) which has been hypothesized to be dependent on tumor burden. We report a relationships between tumor size, MDSCs and T-cells; using four murine mammary tumors to assess tumor growth, infiltration and gene expression. Our analysis of cellular infiltration into tumors and gene expression used collagenase dissociated tumors and density gradient isolation of non-parenchymal cells (NPCs). The frequency of splenic and peripheral blood (PB) MDSCs was tumor dependent resulting in a significantly increased number of MDSCs. The MDSC frequency inversely correlated with the frequency of CD3+ lymphocytes in the spleen, independent of the tumor studied and directly correlated with tumor burden. Tumor growth up-regulated cyclooxygenase-2 (COX-2), vascular endothelial growth factor-A (VEGF-A), granulocyte (G-) and granulocyte-monocyte-colony stimulating factor (GM-CSF), arginase-1 (ARG-1), and nitric oxide synthase-2 (NOS-2) transcription in the tumor and spleens (not VEGF-A). The frequency of splenic MDSCs directly correlated with splenic COX-2, NOS-2, and ARG-1 message levels, while COX-2 and NOS-2 transcript levels inversely correlated with splenic CD3+ cell frequency. COX-2 mRNA levels also directly correlated with the ARG-1 and NOS-2 transcript levels from tumor-infiltrating leukocytic cells, supporting prostaglandin E2 as a regulator of ARG-1 and NOS-2 transcription. In summary, MDSC numbers in the spleen and tumor microenvironment are tumor dependent, directly correlating with tumor size and inversely correlating with T-cell number. MDSCs are also directly associated with VEGF-A and G-CSF transcript levels suggesting multiple mechanisms for MDSC regulation and COX-2, NOS-2 and ARG-1 supporting multiple mechanisms of T-cell suppression.