Abnormal cellular metabolism is a hallmark of cancer, yet there is an absence of quantitative methods to dynamically image this powerful cellular function. Optical metabolic imaging (OMI) is a non-invasive, high-resolution, quantitative tool for monitoring cellular metabolism. OMI probes the fluorescence intensities and lifetimes of the autofluorescent metabolic co-enzymes reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). We confirm that OMI correlates with cellular glycolytic levels across a panel of human breast cell lines, using standard assays of cellular rates of glucose uptake and lactate secretion (p<0.05, r=0.89). Additionally, OMI resolves differences in the basal metabolic activity of untransformed from malignant breast cells (p<0.05), and between breast cancer sub-types (p<0.05), defined by estrogen receptor (ER) and/or HER2 expression or absence. In vivo OMI is sensitive to metabolic changes induced by inhibition of HER2 with the antibody trastuzumab (Herceptin) in HER2-overexpressing human breast cancer xenografts in mice. This response was confirmed with tumor growth curves and stains for Ki67 and cleaved caspase-3. OMI resolved trastuzumab-induced changes in cellular metabolism in vivo as early as 48 hours post-treatment (p<0.05), while FDG-PET did not resolve any changes with trastuzumab up to 12-days post-treatment (p>0.05). In addition, OMI resolved cellular sub-populations of differing response in vivo that are critical for investigating drug resistance mechanisms. Importantly, OMI endpoints remained unchanged with trastuzumab-treatment in trastuzumab-resistant xenografts (p>0.05). OMI has significant implications for rapid cellular-level assessment of metabolic response to molecular expression and drug action, which would greatly accelerate drug development studies.

Overexpression of the receptor tyrosine kinase EPH receptor A2 (EphA2) is commonly observed in aggressive breast cancer and correlates with a poor prognosis. However, while EphA2 has been reported to enhance tumorigenesis, proliferation, and MAPK activation in several model systems, other studies suggest that EphA2 activation diminishes these processes and inhibits the activity of MAPK upon ligand stimulation. In this study, we eliminated EphA2 expression in 2 transgenic mouse models of mammary carcinoma. EphA2 deficiency impaired tumor initiation and metastatic progression in mice overexpressing ErbB2 (also known as Neu) in the mammary epithelium (MMTV-Neu mice), but not in mice overexpressing the polyomavirus middle T antigen in mammary epithelium (MMTV-PyV-mT mice). Histologic and ex vivo analyses of MMTV-Neu mouse mammary epithelium indicated that EphA2 enhanced tumor proliferation and motility. Biochemical analyses revealed that EphA2 formed a complex with ErbB2 in human and murine breast carcinoma cells, resulting in enhanced activation of Ras-MAPK signaling and RhoA GTPase. Additionally, MMTV-Neu, but not MMTV-PyV-mT, tumors were sensitive to therapeutic inhibition of EphA2. These data suggest that EphA2 cooperates with ErbB2 to promote tumor progression in mice and may provide a novel therapeutic target for ErbB2-dependent tumors in humans. Moreover, EphA2 function in tumor progression appeared to depend on oncogene context, an important consideration for the application of therapies targeting EphA2. IntroductionMalignant progression of solid tumors is a complex process that involves the activation of oncogenic signaling and downregulation of tumor suppressor pathways. In addition, modulation of the tumor microenvironment, for example through neovascularization, enhances tumor cell growth and survival, promoting invasion and metastatic spread (reviewed in refs. 1-3). Oncogenic conversion, amplification, or overexpression of protooncogenes, such as those encoding cell surface receptor tyrosine kinases (RTKs) like the EGF receptor family member ErbB2, are frequently observed in human cancers and contribute to malignancy. Other pathways, such as p53 transcription factor/genome surveillance factor, negatively regulate growth, and loss of these pathway components also contributes to tumorigenesis (reviewed in refs. 3, 4). Recent evidence suggests that Eph RTKs play multiple roles in neoplastic progression, including regulation of processes intrinsic to tumor cells, and in the tumor microenvironment, such as tumor neovascularization (reviewed in refs. 5-8).

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Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40–50% of cases are positive. To uncover other potential targetable mutations, we performed whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, D816) wildtype melanoma. Surprisingly, we found a somatic BRAF L597R mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF V600 mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that 2 (4%) harbored L597 mutations and another 2 involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by MEK inhibition. A patient with BRAF L597S mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data demonstrate clinical significance to BRAF L597 mutations in melanoma.

PurposeKnowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma.MethodsThe test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5–10% mutant allele frequency) and minimal amounts of DNA (10–20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab.ResultsDirecting this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials.ConclusionWe present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.

qRT-PCR. Whole-tumor RNA was harvested with an RNeasy kit (QIAGEN), and cDNA was synthesized (High Capacity; Applied Biosystems) and amplified using the murine cDNA-specific primers (Integrated DNA Technologies) listed in Supplemental Methods, along with SYBR Green Supermix (Bio-Rad). The following primers were used: MRC1 (forward: 5′ CCCTCAGCAAGCGATGTGC 3′; reverse: 5′-GGATACTTGCCAGGT CCCCA-3′); iNOS (forward: 5′-GGAGCATCCCAAGTACGAGTGG-3′; reverse: 5′-CGGCC-CACTTCCTCCAG); IL10 (forward: 5′-GGCGCTGTCATC-GATTTCTCC; reverse: 5′-GGCCTTGTAGACACCTTGGTC); Tgfb1 (forward: 5′-CGCAACAACGCCATCTATGAG; reverse: 5′-CGG-GACAGCAATGGGGGTTC); IL4 (forward: 5′-GGTCACAGGAGAAGG-GACG; reverse: 5′-GCGAAGCACCTTGGAAGCC);, IL12b (forward: 5′-GGAGTGGGATGTGTCCTCAG; reverse: 5′-CGGGAGTCCAGTC-CACCTCT); CCL3 (forward: 5′-CCACTGCCCTTGCTGTTCTTCTCT; reverse: 5′-GGGTGTCAGCTCCATATGGCG); and Rplp0 (forward: 5′-TCCTATAAAAGGCACACGCGGGC; reverse: 5′-AGACGATGT-CACTCCAACGAGGACG). Target To generate apoptotic MCF7, cells were treated in suspension with 1 μm BKM120 plus 2 μm ABT-263 (both inhibitors from Selleck Chemicals) for 4 hours, washed 5 times with PBS to remove residual drug, and used directly for efferocytosis assays or for annexin V staining. For efferocytosis coculture assays, Raw264.7-GFP cells (10 4 /well) and PyVmT or MCF7 cells (72 hours after infection with Ad.mCherry and Ad.HS-V-TK) were seeded together in a monolayer in 24-well plates in 2% FBS and cultured for 24 hours prior to the addition PBS or gancyclovir. Cells were imaged at 8, 16, and 32 h after addition of gancyclovir. Cells were collected and counted under fluorescence after 32 hours of coculture. In some experiments, Raw264.7-GFP cells (10 4 / well) were seeded in a monolayer in 24-well plates and cultured for 24 hours prior to the addition of 10 3 live MCF7-mCherry or 10 3 dead MCF7-mCherry cells in serum-free media. Where indicated in the figures, BMS-777607 (1 μm) or a neutralizing goat anti-mouse MerTK antibody (AF591, 25 μg/ml; R&D Systems)(44) was added 2 hours prior to the addition of gancyclovir or 2 hours prior to the addition of dead MCF7 cells to macrophage monolayers. Live and dead MCF7 cells were similarly seeded without Raw264.7 cells as single cultures. Media were collected after 16 hours of coculture, passed through a 0.2-μm filter, and used neat (250 μl) to quantify murine IL-10 and IL-4 by ELISA (BioLegend) according to the manufacturer's protocol. Total remaining cells were collected after 16 hours of coculture, lysed, and RNA was collected using an RNeasy kit (QIAGEN). MethodsMice. All mice were inbred on an FVB background for more than 10 generations. WT FVB, MMTV PyVmT and MerTK -/-mice (67), originally referred to as Mer KD , were purchased from The Jackson Laboratory. Mice were genotyped by PCR of genomic DNA as previously described(30). Female virgin mice were randomized into 2 groups: (a) 1 group that remained virgin, and (b) 1 group that was bred from 42 to 44 days of age with WT male mice. Pregnancies were timed according to identification of a va...

Cancer immunotherapies that remove checkpoint restraints on adaptive immunity are gaining clinical momentum but have not achieved widespread success in breast cancers, a tumor type considered poorly immunogenic and which harbors a decreased presence of tumor-infiltrating lymphocytes. Approaches that activate innate immunity in breast cancer cells and the tumor microenvironment are of increasing interest, based on their ability to induce immunogenic tumor cell death, type I IFNs, and lymphocyte-recruiting chemokines. In agreement with reports in other cancers, we observe loss, downregulation, or mutation of the innate viral nucleotide sensor retinoic acid-inducible gene I (RIG-I/) in only 1% of clinical breast cancers, suggesting potentially widespread applicability for therapeutic RIG-I agonists that activate innate immunity. This was tested using an engineered RIG-I agonist in a breast cancer cell panel representing each of three major clinical breast cancer subtypes. Treatment with RIG-I agonist resulted in upregulation and mitochondrial localization of RIG-I and activation of proinflammatory transcription factors STAT1 and NF-κB. RIG-I agonist triggered the extrinsic apoptosis pathway and pyroptosis, a highly immunogenic form of cell death in breast cancer cells. RIG-I agonist also induced expression of lymphocyte-recruiting chemokines and type I IFN, confirming that cell death and cytokine modulation occur in a tumor cell-intrinsic manner. Importantly, RIG-I activation in breast tumors increased tumor lymphocytes and decreased tumor growth and metastasis. Overall, these findings demonstrate successful therapeutic delivery of a synthetic RIG-I agonist to induce tumor cell killing and to modulate the tumor microenvironment These findings describe the first in vivo delivery of RIG-I mimetics to tumors, demonstrating a potent immunogenic and therapeutic effect in the context of otherwise poorly immunogenic breast cancers..

EphA2 belongs to a unique family of receptor tyrosine kinases that play critical roles in development and disease. Since EphA2 is required for ephrin-A1 ligand-induced vascular remodeling and is overexpressed in a variety of vascularized human adenocarcinomas, we assessed tumor angiogenesis and metastatic progression in EphA2-deficient host animals. 4T1 metastatic mammary adenocarcinoma cells transplanted subcutaneously and orthotopically into EphA2-deficient female mice displayed decreased tumor volume, tumor cell survival, microvascular density, and lung metastasis relative to tumor-bearing littermate controls. To determine if the phenotype in EphA2-deficient mice was endothelial cell intrinsic, we also analyzed endothelial cells isolated from EphA2-deficient animals for their ability to incorporate into tumor vessels in vivo, as well as to migrate in response to tumor-derived signals in vitro. EphA2-deficient endothelial cells displayed impaired survival and failed to incorporate into tumor microvessels in vivo, and displayed impaired tumor-mediated migration in vitro relative to controls. These data suggest that host EphA2 receptor tyrosine kinase function is required in the tumor microenvironment for tumor angiogenesis and metastatic progression.