Siver A. Moestue

Detection of colorectal polyps in humans using an intravenously administered fluorescent peptide targeted against c-Met

Colon cancer prevention currently relies on colonoscopy using white light to detect and remove polyps, but small and flat polyps are difficult to detect and frequently missed when using this technique. Fluorescence colonoscopy combined with a fluorescent probe specific for a polyp biomarker may improve polyp detection. Here we describe GE-137, a water-soluble probe consisting of a 26–amino acid cyclic peptide that binds the human tyrosine kinase c-Met conjugated to a fluorescent cyanine dye. Intravenous administration of GE-137 leads to its accumulation specifically in c-Met–expressing tumors in mice, and it is safe and well tolerated in humans. Fluorescence colonoscopy in patients receiving intravenous GE-137 enabled visualization of all neoplastic polyps that were visible with white light (38), as well as an additional nine polyps that were not visible with white light. This first-in-human pilot study shows that molecular imaging using an intravenous fluorescent agent specific for c-Met is feasible and safe, and that it may enable the detection of polyps missed by other techniques.

Distinct choline metabolic profiles are associated with differences in gene expression for basal-like and luminal-like breast cancer xenograft models

BackgroundIncreased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood.MethodsThe concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS).The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses.The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer.ResultsIn basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (CHKA, CHKB) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (PLA2G4A) and phospholipase B1 (PLB1) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer.ConclusionsThe differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminal-like breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer.

HR MAS MR Spectroscopy in Metabolic Characterization of Cancer

One of the central hallmarks of cancer is the rapid and infinite cellular proliferation. In order to cope with increased requirement for building blocks and energy, cancer cells develop abnormal metabolic properties. Detailed assessment of cancer cell metabolism can provide biological information for use in both drug discovery and development of personalized cancer therapy. Analysis of intact tissue using high resolution magic angle spinning (HR MAS) magnetic resonance spectroscopy (MRS) gives qualitative and quantitative metabolite measures with minimal sample preparation. Multivariate statistical methods are important tools for analysis of complex MR data and have in recent years been used for analysis of HR MAS data from intact tissue. HR MAS analysis of intact tissue allows combination of metabolomic data with genomic or proteomic data, and can therefore be used both for exploring the molecular biology of cancer and for clinical improvements in cancer diagnostics, prognostics and treatment planning. In this review, the basic concepts of HR MAS are presented, and its use in characterisation of cancer metabolism is discussed with specific focus on selected pathways such as choline metabolism and glycolysis. The use of HR MAS in analysis of amino acids and lipid metabolism in cancer is also reviewed. Finally, the expected role of HR-MAS in metabolic characterisation in the near future is discussed.

MRS and MRSI guidance in molecular medicine: targeting and monitoring of choline and glucose metabolism in cancer.

MRS and MRSI are valuable tools for the detection of metabolic changes in tumors. The currently emerging era of molecular medicine, which is shaped by molecularly targeted anticancer therapies combined with molecular imaging of the effects of such therapies, requires powerful imaging technologies that are able to detect molecular information. MRS and MRSI are such technologies that are able to detect metabolites arising from glucose and choline metabolism in noninvasive in vivo settings and at higher resolution in tissue samples. The roles played by MRS and MRSI in the diagnosis of different types of cancer, as well as in the early monitoring of the tumor response to traditional chemotherapies, are reviewed. The emerging roles of MRS and MRSI in the development and detection of novel targeted anticancer therapies that target oncogenic signaling pathways or markers in choline or glucose metabolism are discussed. Copyright

Metabolic biomarkers for response to PI3K inhibition in basal-like breast cancer

IntroductionThe phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in cancer cells through numerous mutations and epigenetic changes. The recent development of inhibitors targeting different components of the PI3K pathway may represent a valuable treatment alternative. However, predicting efficacy of these drugs is challenging, and methods for therapy monitoring are needed. Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype, frequently associated with PI3K pathway activation. The objectives of this study were to quantify the PI3K pathway activity in tissue sections from xenografts representing basal-like and luminal-like breast cancer before and immediately after treatment with PI3K inhibitors, and to identify metabolic biomarkers for treatment response.MethodsTumor-bearing animals (n = 8 per treatment group) received MK-2206 (120 mg/kg/day) or BEZ235 (50 mg/kg/day) for 3 days. Activity in the PI3K/Akt/mammalian target of rapamycin pathway in xenografts and human biopsies was evaluated using a novel method for semiquantitative assessment of Aktser473 phosphorylation. Metabolic changes were assessed by ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy.ResultsUsing a novel dual near-infrared immunofluorescent imaging method, basal-like xenografts had a 4.5-fold higher baseline level of pAktser473 than luminal-like xenografts. Following treatment, basal-like xenografts demonstrated reduced levels of pAktser473 and decreased proliferation. This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors. BEZ235 also caused increased glucose and glycerophosphocholine concentrations. No response to treatment or change in metabolic profile was seen in luminal-like xenografts. Analyzing tumor sections from five patients with BLBC demonstrated that two of these patients had an elevated pAktser473 level.ConclusionThe activity of the PI3K pathway can be determined in tissue sections by quantitative imaging using an antibody towards pAktser473. Long-term treatment with MK-2206 or BEZ235 resulted in significant growth inhibition in basal-like, but not luminal-like, xenografts. This indicates that PI3K inhibitors may have selective efficacy in basal-like breast cancer with increased PI3K signaling, and identifies lactate, phosphocholine and glycerophosphocholine as potential metabolic biomarkers for early therapy monitoring. In human biopsies, variable pAktser473 levels were observed, suggesting heterogeneous PI3K signaling activity in BLBC.

Low‐molecular contrast agent dynamic contrast‐enhanced (DCE)‐MRI and diffusion‐weighted (DW)‐MRI in early assessment of bevacizumab treatment in breast cancer xenografts

To investigate the effect of bevacizumab treatment on vascular architecture and function in two xenograft models with different angiogenic properties using diffusion‐weighted magnetic resonance imaging (DW‐MRI) and dynamic contrast‐enhanced MRI (DCE‐MRI).

Metabolic reprogramming supports the invasive phenotype in malignant melanoma

Invasiveness is a hallmark of aggressive cancer like malignant melanoma, and factors involved in acquisition or maintenance of an invasive phenotype are attractive targets for therapy. We investigated melanoma phenotype modulation induced by the metastasis-promoting microenvironmental protein S100A4, focusing on the relationship between enhanced cellular motility, dedifferentiation and metabolic changes. In poorly motile, well-differentiated Melmet 5 cells, S100A4 stimulated migration, invasion and simultaneously down-regulated differentiation genes and modulated expression of metabolism genes. Metabolic studies confirmed suppressed mitochondrial respiration and activated glycolytic flux in the S100A4 stimulated cells, indicating a metabolic switch toward aerobic glycolysis, known as the Warburg effect. Reversal of the glycolytic switch by dichloracetate induced apoptosis and reduced cell growth, particularly in the S100A4 stimulated cells. This implies that cells with stimulated invasiveness get survival benefit from the glycolytic switch and, therefore, become more vulnerable to glycolysis inhibition. In conclusion, our data indicate that transition to the invasive phenotype in melanoma involves dedifferentiation and metabolic reprogramming from mitochondrial oxidation to glycolysis, which facilitates survival of the invasive cancer cells. Therapeutic strategies targeting the metabolic reprogramming may therefore be effective against the invasive phenotype.

Interplay of choline metabolites and genes in patient-derived breast cancer xenografts

IntroductionDysregulated choline metabolism is a well-known feature of breast cancer, but the underlying mechanisms are not fully understood. In this study, the metabolomic and transcriptomic characteristics of a large panel of human breast cancer xenograft models were mapped, with focus on choline metabolism.MethodsTumor specimens from 34 patient-derived xenograft models were collected and divided in two. One part was examined using high-resolution magic angle spinning (HR-MAS) MR spectroscopy while another part was analyzed using gene expression microarrays. Expression data of genes encoding proteins in the choline metabolism pathway were analyzed and correlated to the levels of choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) using Pearson’s correlation analysis. For comparison purposes, metabolic and gene expression data were collected from human breast tumors belonging to corresponding molecular subgroups.ResultsMost of the xenograft models were classified as basal-like (N = 19) or luminal B (N = 7). These two subgroups showed significantly different choline metabolic and gene expression profiles. The luminal B xenografts were characterized by a high PCho/GPC ratio while the basal-like xenografts were characterized by highly variable PCho/GPC ratio. Also, Cho, PCho and GPC levels were correlated to expression of several genes encoding proteins in the choline metabolism pathway, including choline kinase alpha (CHKA) and glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). These characteristics were similar to those found in human tumor samples.ConclusionThe higher PCho/GPC ratio found in luminal B compared with most basal-like breast cancer xenograft models and human tissue samples do not correspond to results observed from in vitro studies. It is likely that microenvironmental factors play a role in the in vivo regulation of choline metabolism. Cho, PCho and GPC were correlated to different choline pathway-encoding genes in luminal B compared with basal-like xenografts, suggesting that regulation of choline metabolism may vary between different breast cancer subgroups. The concordance between the metabolic and gene expression profiles from xenograft models with breast cancer tissue samples from patients indicates that these xenografts are representative models of human breast cancer and represent relevant models to study tumor metabolism in vivo.

Glycerophosphocholine (GPC) is a poorly understood biomarker in breast cancer

Stewart et al. (1) recently reported that EDI3 is the glycerophosphodiesterase responsible for breaking down glycerophosphocholine (GPC) to choline and G3P in mammalian cells. The authors further claim that this is of relevance in breast cancer, as this disease, according to the authors, is associated with reduced levels of GPC and a low GPC/phosphocholine (PCho) ratio. By implication, inhibition of EDI3 could increase the concentration of GPC, restore a normal GPC/PCho ratio, and reduce metastatic potential. In our opinion, this hypothesis, which is based on studies of cultured breast cancer cells, does not take recent clinical findings into account. In a cohort of 156 patients, our group has found that estrogen receptor-negative breast cancer is associated with low PCho and high GPC concentrations (2). Furthermore, we have demonstrated that a basal-like patient-derived animal model had a remarkably high GPC/PCho ratio (3), which was corroborated by analysis of tumor tissue from patients with triple-negative breast cancer. These findings clearly indicate that the most aggressive breast cancer subtypes have higher GPC concentration than subtypes with a better prognosis. Furthermore, we have studied the role of GPC as a biomarker for response to therapy. In breast cancer biopsy specimens collected from patients receiving neoadjuvant chemotherapy, we have found that a favorable response to treatment, in fact, is associated with a reduction in GPC concentration during the course of treatment (4). Finally, we have shown that reduction in GPC concentration after neoadjuvant chemotherapy is associated with long-term survival in this patient group (5). In summary, high GPC concentration and a GPC/PCho ratio >1 is associated with aggressive subtypes of breast cancer. Furthermore, a reduction in GPC levels can be predictive of response to treatment. These findings are not consistent with the hypothesis that restoration of a high GPC/PCho ratio would be beneficial in breast cancer, which forms the basis for the work of Stewart et al. (1). The abnormal metabolism of breast cancer, not surprisingly, reflects the heterogeneity of the disease. The complex role of GPC remains poorly understood, but it has become increasingly clear that high, rather than low, GPC concentrations are associated with poor prognosis in breast cancer. Thus, in our opinion, the available body of data is not taken into account. Pursuing the findings in the article (1) can, of course, lead to discovery of novel drug targets. However, research based on a hypothesis that is not consistent with clinical findings may be considered a suboptimal approach for targeting the abnormal choline metabolism in breast cancer. Inhibition of EDI3 could surely have inhibitory effects on breast cancer cells, as interference with choline phospholipid metabolism repeatedly has been associated with such effects. We therefore await further progress from Stewart et al. in this field in great anticipation, and hope that further elucidation of GPC metabolism can lead to findings of new prognostic biomarkers or drug targets.

In vivo MRI and histopathological assessment of tumor microenvironment in luminal-like and basal-like breast cancer xenografts

To explore tumor pathophysiology with special attention to the microenvironment in two molecular subtypes of human breast cancer using in vivo magnetic resonance imaging (MRI) and histopathology. The impact of tumor growth, size, and the influence of estradiol were also investigated.