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.