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An R-script for the weighted correlation network analysis. This file is a .R file which can be opened in RStudio.

Western blot showing levels of IDH2 in D492 and D492M. A) IDH2 and β-actin intensity levels in D492 and D492M. B) IDH2 band intensity normalized to the β-actin loading control in D492 and D492M (n=4). Student's two-tailed t-test was used to estimate significance of results. The difference between D492 and D492M in normalized IDH2 band intensity was not significant (p = 0.08).

UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with A) 1-13C-glutamine and B) 5-13C-glutamine for 6 hours. Results are shown as mean + standard deviation (n = 3).

Summarized significance (-log(p-value)) of drug module members Spearman correlation to levels of A) Reduced glutathione (GSH) and B) Oxidized glutathione (GSSG). The p-values in the title represents significance values from Kruskal-Wallis tests for differences in significance value distributions between modules.

UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with 1,2-13C-glucose for 6 hours. Results are shown as mean + standard deviation (n = 3).

Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention. Here we used 13C carbon analogs of glucose and glutamine to examine differences in their utilization within central carbon and lipid metabolism following EMT in breast epithelial cell lines. We found that there are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of the TCA-cycle, characterized by increased mitochondrial IDH2-mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Using weighted correlation network analysis, we identified cancer drugs whose efficacy against the NCI-60 Human Tumor Cell Line panel is significantly associated with GSH abundance and confirmed these in vitro. We report that EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to mTOR inhibitors.Implications:EMT in breast cells causes an increased demand for glutamine for fatty acid biosynthesis, altering its contribution to glutathione biosynthesis, which sensitizes the cells to mTOR inhibitors.

Dependency of D492 and D492M on glucose and glutamine. Cell number of D492 and D492M in full H14 medium, H14 without glucose and H14 without glutamine. Values are presented as mean + standard error (n{greater than or equal to}6). Asterisks represent significance of pairwise comparison between full medium and the depleted medium.

Estrogen receptor α (ERα) is a key regulator of breast growth and breast cancer development. Here, we report how ERα impacts these processes by reprogramming metabolism in malignant breast cells. We employed an integrated approach, combining genome-wide mapping of chromatin-bound ERα with estrogen-induced transcript and metabolic profiling, to demonstrate that ERα reprograms metabolism upon estrogen stimulation, including changes in aerobic glycolysis, nucleotide and amino acid synthesis, and choline (Cho) metabolism. Cho phosphotransferase CHPT1, identified as a direct ERα-regulated gene, was required for estrogen-induced effects on Cho metabolism, including increased phosphatidylcholine synthesis. CHPT1 silencing inhibited anchorage-independent growth and cell proliferation, also suppressing early-stage metastasis of tamoxifen-resistant breast cancer cells in a zebrafish xenograft model. Our results showed that ERα promotes metabolic alterations in breast cancer cells mediated by its target CHPT1, which this study implicates as a candidate therapeutic target. Cancer Res; 76(19); 5634–46. ©2016 AACR.

Microarray and qPCR data showing no induction of PEMT mRNA levels by estrogen in MCF7 and T47D cells

Changes of Cho-containing metabolites upon E2 stimulation

Changed levels of intracellular metabolites in MCF7 and T47 D cells in response to estrogen

Metabolic profiles of breast cancer cells in response to estrogen treatment

Supplementary Materials and Methods and Supplementary Figure and Table Legends

Association between CHPT1 expression in breast cancer samples and clinicopathological data.

The core set of direct ERalpha regulated genes in breast cancer cells

In this study, we probed the importance of O-GlcNAc transferase (OGT) activity for the survival of tamoxifen-sensitive (TamS) and tamoxifen-resistant (TamR) breast cancer cells. Tamoxifen is an antagonist of estrogen receptor (ERα), a transcription factor expressed in over 50% of breast cancers. ERα-positive breast cancers are successfully treated with tamoxifen; however, a significant number of patients develop tamoxifen-resistant disease. We show that in vitro development of tamoxifen-resistance is associated with increased sensitivity to the OGT small molecule inhibitor OSMI-1. Global transcriptome profiling revealed that TamS cells adapt to OSMI-1 treatment by increasing the expression of histone genes. This is known to mediate chromatin compaction. In contrast, TamR cells respond to OGT inhibition by activating the unfolded protein response and by significantly increasing ERRFI1 expression. ERRFI1 is an endogenous inhibitor of ERBB-signaling, which is a known driver of tamoxifen-resistance. We show that ERRFI1 is selectively downregulated in ERα-positive breast cancers and breast cancers driven by ERBB2. This likely occurs via promoter methylation. Finally, we show that increased ERRFI1 expression is associated with extended survival in patients with ERα-positive tumors (p = 9.2e−8). In summary, we show that tamoxifen-resistance is associated with sensitivity to OSMI-1, and propose that this is explained in part through an epigenetic activation of the tumor-suppressor ERRFI1 in response to OSMI-1 treatment. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Patient-derived cancer cells cultured in vitro are a cornerstone of cancer metabolism research. More recently, the introduction of organoids has provided the research community with a more versatile model system. Physiological structure and organization of the cell source tissue are maintained in organoids, representing a closer link to in vivo tumor models. High-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) is a commonly applied analytical approach for metabolic profiling of intact tissue, but its use has not been reported for organoids. The aim of the current work was to compare the performance of HR MAS MRS and extraction-based nuclear magnetic resonance (NMR) in metabolic profiling of wild-type and tumor progression organoids (TPOs) from human colon cancer, and further to investigate how the sequentially increased genetic alterations of the TPOs affect the metabolic profile. Sixteen metabolites were reliably identified and quantified both in spectra based on NMR of extracts and HR MAS MRS of intact organoids. The metabolite concentrations from the two approaches were highly correlated (r = 0.94), and both approaches were able to capture the systematic changes in metabolic features introduced by the genetic alterations characteristic of colorectal cancer progression (e.g., increased levels of lactate and decreased levels of myo-inositol and phosphocholine with an increasing number of mutations). The current work highlights that HR MAS MRS is a well-suited method for metabolic profiling of intact organoids, with the additional benefit that the nondestructive nature of HR MAS enables subsequent recovery of the organoids for further analyses based on nucleic acids or proteins.

Epithelial-to-mesenchymal transition (EMT) is fundamental to both normal tissue development and cancer progression. We hypothesized that EMT plasticity defines a range of metabolic phenotypes and that individual breast epithelial metabolic phenotypes are likely to fall within this phenotypic landscape. To determine EMT metabolic phenotypes, the metabolism of EMT was described within genome-scale metabolic models (GSMMs) using either transcriptomic or proteomic data from the breast epithelial EMT cell culture model D492. The ability of the different data types to describe breast epithelial metabolism was assessed using constraint-based modeling which was subsequently verified using 13C isotope tracer analysis. The application of proteomic data to GSMMs provided relatively higher accuracy in flux predictions compared to the transcriptomic data. Furthermore, the proteomic GSMMs predicted altered cholesterol metabolism and increased dependency on argininosuccinate lyase (ASL) following EMT which were confirmed in vitro using drug assays and siRNA knockdown experiments. The successful verification of the proteomic GSMMs afforded iBreast2886, a breast GSMM that encompasses the metabolic plasticity of EMT as defined by the D492 EMT cell culture model. Analysis of breast tumor proteomic data using iBreast2886 identified vulnerabilities within arginine metabolism that allowed prognostic discrimination of breast cancer patients on a subtype-specific level. Taken together, we demonstrate that the metabolic reconstruction iBreast2886 formalizes the metabolism of breast epithelial cell development and can be utilized as a tool for the functional interpretation of high throughput clinical data.

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects.

Hyperpolarized 13C isotope resolved spectroscopy boosts NMR signal intensity, which improves signal detection and allows metabolic fluxes to be analyzed. Such hyperpolarized flux data may offer new approaches to tissue classification and biomarker identification that could be translated in vivo. Here we used hyperpolarized stable isotope resolved analysis (SIRA) to measure metabolite specific 13C isotopic enrichments in the central carbon metabolism of mouse prostate. Prostate and tumor tissue samples were acquired from transgenic adenocarcinomas of the mouse prostate (TRAMP) mice. Before euthanasia, mice were injected with [U–13C]glucose intraperitoneally (i.p.). Polar metabolite extracts were prepared, and hyperpolarized 1D-13C NMR spectra were obtained from normal prostate (n = 19) and cancer tissue (n = 19) samples. Binary classification and feature analysis was performed to make a separation model and to investigate differences between samples originating from normal and cancerous prostate tissue, respectively. Hyperpolarized experiments were carried out according to a standardized protocol, which showed a high repeatability (CV = 15%) and an average linewidth in the 1D-13C NMR spectra of 2 ± 0.5 Hz. The resolution of the hyperpolarized 1D-13C spectra was high with little signal overlap in the carbonyl region and metabolite identification was easily accomplished. A discrimination with 95% success rate could be made between samples originating from TRAMP mice prostate and tumor tissue based on isotopomers from uniquely identified metabolites. Hyperpolarized 13C-SIRA allowed detailed metabolic information to be obtained from tissue specimens. The positional information of 13C isotopic enrichments lead to easily interpreted features responsible for high predictive classification of tissue types. This analytical approach has matured, and the robust experimental protocols currently available allow systematic tracking of metabolite flux ex vivo.

Cancer cells often depend on microenvironment signals from molecules such as cytokines for proliferation and metabolic adaptations. PRL‐3, a cytokine‐induced oncogenic phosphatase, is highly expressed in multiple myeloma cells and associated with poor outcome in this cancer. We studied whether PRL‐3 influences metabolism. Cells transduced to express PRL‐3 had higher aerobic glycolytic rate, oxidative phosphorylation, and ATP production than the control cells. PRL‐3 promoted glucose uptake and lactate excretion, enhanced the levels of proteins regulating glycolysis and enzymes in the serine/glycine synthesis pathway, a side branch of glycolysis. Moreover, mRNAs for these proteins correlated with PRL‐3 expression in primary patient myeloma cells. Glycine decarboxylase (GLDC) was the most significantly induced metabolism gene. Forced GLDC downregulation partly counteracted PRL‐3‐induced aerobic glycolysis, indicating GLDC involvement in a PRL‐3‐driven Warburg effect. AMPK, HIF‐1α, and c‐Myc, important metabolic regulators in cancer cells, were not mediators of PRL‐3’s metabolic effects. A phosphatase‐dead PRL‐3 mutant, C104S, promoted many of the metabolic changes induced by wild‐type PRL‐3, arguing that important metabolic effects of PRL‐3 are independent of its phosphatase activity. Through this study, PRL‐3 emerges as one of the key mediators of metabolic adaptations in multiple myeloma. © 2021 The Authors. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

The association between lipid metabolism and long-term outcomes is relevant for tumor diagnosis and therapy. Archival material such as formalin-fixed and paraffin embedded (FFPE) tissues is a highly valuable resource for this aim as it is linked to long-term clinical follow-up. Therefore, there is a need to develop robust methodologies able to detect lipids in FFPE material and correlate them with clinical outcomes. In this work, lipidic alterations were investigated in patient-derived xenograft of breast cancer by using a matrix-assisted laser desorption ionization mass spectrometry (MALDI MSI) based workflow that included antigen retrieval as a sample preparation step. We evaluated technical reproducibility, spatial metabolic differentiation within tissue compartments, and treatment response induced by a glutaminase inhibitor (CB-839). This protocol shows a good inter-day robustness (CV = 26 ± 12%). Several lipids could reliably distinguish necrotic and tumor regions across the technical replicates. Moreover, this protocol identified distinct alterations in the tissue lipidome of xenograft treated with glutaminase inhibitors. In conclusion, lipidic alterations in FFPE tissue of breast cancer xenograft observed in this study are a step-forward to a robust and reproducible MALDI-MSI based workflow for pre-clinical and clinical applications.

Background: Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention.Methods: We utilize 13C carbon analogs of glucose and glutamine to examine difference in internal flux patterns of metabolites in central carbon and lipid metabolism following EMT in breast epithelial cell lines. Furthermore, an isotopomer spectral analysis, 13C-metabolic flux analysis and weighted correlation network analysis are utilized for investigation of the alterations in metabolic functionality following EMT in breast.Results: There are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of TCA-cycle flux characterized by increased mitochondrial IDH2 -mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Our network approach identified specific subtype of cancer drugs that are significantly associated with GSH abundance and we confirmed this in vitro.Conclusions: EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to PI3K/Akt/mTOR inhibitors.

Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention. Here we used 13C carbon analogs of glucose and glutamine to examine differences in their utilization within central carbon and lipid metabolism following EMT in breast epithelial cell lines. We found that there are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of the TCA-cycle, characterized by increased mitochondrial IDH2-mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Using weighted correlation network analysis, we identified cancer drugs whose efficacy against the NCI-60 Human Tumor Cell Line panel is significantly associated with GSH abundance and confirmed these in vitro. We report that EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to mTOR inhibitors. Implications: EMT in breast cells causes an increased demand for glutamine for fatty acid biosynthesis, altering its contribution to glutathione biosynthesis, which sensitizes the cells to mTOR inhibitors.

BACKGROUND Steady state susceptibility contrast (SSC)-MRI provides information on vascular morphology but is a rarely used method. PURPOSE To investigate the utility of the ultrasmall superparamagnetic iron oxide particles (USPIOs) GEH121333 for measuring tumor response to bevacizumab and compare this with gadolinium-based DCE-MRI. STUDY TYPE Prospective preclinical animal model study. ANIMAL MODEL Mice bearing subcutaneous TOV-21G human ovarian cancer xenografts treated with bevacizumab (n = 9) or saline (n = 9). FIELD STRENGTH/SEQUENCE Imaging was performed on a 7T Bruker Biospec. For SSC-MRI with GEH121333 we acquired R1 -maps (RARE-sequence with variable TR), R2 -maps (multi-spin echo), and R2*-maps (multi-gradient echo). Additionally, R1 and R2 maps were measured on the days after USPIO injection. For DCE-MRI with gadodiamide we acquired 200 T1 -weighted images (RARE-sequence). ASSESSMENT ΔR1 , ΔR2 , and ΔR2* maps were computed from SSC-MRI. DCE-MRI was analysed using the extended Tofts model. STATISTICAL TESTS Results from pre- and 3 days posttreatment SSC-MRI were compared using paired-sample t-tests. Treatment and control groups were compared using independent sample t-tests. Performance of SSC- and DCE-MRI was compared using multivariate partial least squares discriminant analysis. RESULTS Already one day after treatment and USPIO injection, R1 and R2 values were lower in treated (R1 = 0.49 ± 0.03s-1 , R2 = 23.07 ± 1.49s-1 ) compared with control tumors (R1 = 0.52 ± 0.02s-1 , R2 = 24.98 ± 1.01s-1 ), indicating lower USPIO accumulation. Posttreatment SSC-MRI displayed significantly decreased tumor blood volume (change in ΔR2 = -0.43 ± 0.26s-1 , P = 0.001) and vessel density (change in Q = -0.032 ± 0.020s-1/3 , P = 0.002). DCE-MRI showed among others lower Ktrans in treated tumors (control = 0.064 ± 0.011min-1 , tx = 0.046 ± 0.008min-1 , P = 0.002). Multivariate analysis suggests that SSC-MRI was slightly inferior to DCE-MRI in distinguishing treated from control tumors (accuracy = 75%, P = 0.058 versus 80%, P = 0.028), but a combination of both was best (accuracy = 85%; P = 0.003). DATA CONCLUSION SSC-MRI with GEH121333 is sensitive to early (

The editors are publishing this note to alert readers to concerns about [this article][1] ([1][2]). The editors were made aware of duplicated Western blot bands in Fig. 4A. Specifically, two sets of Western blot bands are duplicates: (i) T47D CCTα in the top right panel and T47D cadherin in the

Post-translational modification of intracellular proteins with a single N-acetylglucosamine sugar (O-GlcNAcylation) regulates signaling, proliferation, metabolism and protein stability. In breast cancer, expression of the enzyme that catalyzes O-GlcNAcylation – O-GlcNAc-transferase (OGT), and the extent of protein O-GlcNAcylation, are upregulated in tumor tissue, and correlate with cancer progression. Here we compare the significance of O-GlcNAcylation in a panel of breast cancer cells of different phenotypes. We find a greater dependency on OGT among triple-negative breast cancer (TNBC) cell lines, which respond to OGT inhibition by undergoing cell cycle arrest and apoptosis. Searching for the cause of this response, we evaluate the changes in the proteome that occur after OGT inhibition or knock-down, employing a reverse-phase protein array (RPPA). We identify transcriptional repressor - hairy and enhancer of split-1 (HES1) - as a mediator of the OGT inhibition response in the TNBC cells. Inhibition of OGT as well as the loss of HES1 results in potent cytotoxicity and apoptosis. The study raises a possibility of using OGT inhibition to potentiate DNA damage in the TNBC cells. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Introduction Glutaminase inhibitors target cancer cells by blocking the conversion of glutamine to glutamate, thereby potentially interfering with anaplerosis and synthesis of amino acids and glutathione. The drug CB-839 has shown promising effects in preclinical experiments and is currently undergoing clinical trials in several human malignancies, including triple-negative breast cancer (TNBC). However, response to glutaminase inhibitors is variable and there is a need for identification of predictive response biomarkers. The aim of this study was to determine how glutamine is utilized in two patient-derived xenograft (PDX) models of breast cancer representing luminal-like/ER+ (MAS98.06) and basal-like/triple-negative (MAS98.12) breast cancer and to explore the metabolic effects of CB-839 treatment. Experimental MAS98.06 and MAS98.12 PDX mice received CB-839 (200 mg/kg) or drug vehicle two times daily p.o. for up to 28 days (n = 5 per group), and the effect on tumor growth was evaluated. Expression of 60 genes and seven glutaminolysis key enzymes were determined using gene expression microarray analysis and immunohistochemistry (IHC), respectively, in untreated tumors. Uptake and conversion of glutamine were determined in the PDX models using HR MAS MRS after i.v. infusion of [5-13C] glutamine when the models had received CB-839 (200 mg/kg) or vehicle for 2 days (n = 5 per group). Results Tumor growth measurements showed that CB-839 significantly inhibited tumor growth in MAS98.06 tumors, but not in MAS98.12 tumors. Gene expression and IHC analysis indicated a higher proline synthesis from glutamine in untreated MAS98.06 tumors. This was confirmed by HR MAS MRS of untreated tumors demonstrating that MAS98.06 used glutamine to produce proline, glutamate, and alanine, and MAS98.12 to produce glutamate and lactate. In both models, treatment with CB-839 resulted in accumulation of glutamine. In addition, CB-839 caused depletion of alanine, proline, and glutamate ([1-13C] glutamate) in the MAS98.06 model. Conclusion Our findings indicate that TNBCs may not be universally sensitive to glutaminase inhibitors. The major difference in the metabolic fate of glutamine between responding MAS98.06 xenografts and non-responding MAS98.12 xenografts is the utilization of glutamine for production of proline. We therefore suggest that addiction to proline synthesis from glutamine is associated with response to CB-839 in breast cancer. Graphical abstract The effect of glutaminase inhibition in two breast cancer patient-derived xenograft (PDX) models. 13C HR MAS MRS analysis of tumor tissue from CB-839-treated and untreated models receiving 13C-labeled glutamine ([5-13C] Gln) shows that the glutaminase inhibitor CB-839 is causing an accumulation of glutamine (arrow up) in two PDX models representing luminal-like breast cancer (MAS98.06) and basal-like breast cancer (MAS98.12). In MAS98.06 tumors, CB-839 is in addition causing depletion of proline ([5-13C] Pro), alanine ([1-13C] Ala), and glutamate ([1-13C] Glu), which could explain why CB-839 causes tumor growth inhibition in MAS98.06 tumors, but not in MAS98.12 tumors.

Background The majority of patients with advanced cancer develop cachexia, a weight loss syndrome that severely reduces quality of life and limits survival. Our understanding of the underlying mechanisms that cause the condition is limited, and there are currently no treatment options that can completely reverse cachexia. Several tumour‐derived factors and inflammatory mediators have been suggested to contribute to weight loss in cachectic patients. However, inconsistencies between studies are recurrent. Activin A and interleukin 6 (IL‐6) are among the best studied factors that seem to be important, and several studies support their individual role in cachexia development. Methods We investigated the interplay between activin A and IL‐6 in the cachexia‐inducing TOV21G cell line, both in culture and in tumours in mice. We previously found that the human TOV21G cells secrete IL‐6 that induces autophagy in reporter cells and cachexia in mice. Using this established cachexia cell model, we targeted autocrine activin A by genetic, chemical, and biological approaches. The secretion of IL‐6 from the cancer cells was determined in both culture and tumour‐bearing mice by a species‐specific ELISA. Autophagy reporter cells were used to monitor the culture medium for autophagy‐inducing activities, and muscle mass changes were evaluated in tumour‐bearing mice. Results We show that activin A acts in an autocrine manner to promote the synthesis and secretion of IL‐6 from cancer cells. By inhibiting activin A signalling, the production of IL‐6 from the cancer cells is reduced by 40–50% (up to 42% reduction on protein level, P = 0.0048, and 48% reduction on mRNA level, P = 0.0308). Significantly reduced IL‐6 secretion (P < 0.05) from the cancer cells is consistently observed when using biological, chemical, and genetic approaches to interfere with the autocrine activin A loop. Inhibiting activin signalling also reduces the ability of the cancer cells to accelerate autophagy in non‐cancerous cells (up to 43% reduced autophagy flux, P = 0.0006). Coherent to the in vitro data, the use of an anti‐activin receptor 2 antibody in cachectic tumour‐bearing mice reduces serum levels of cancer cell‐derived IL‐6 by 62% (from 417 to 159 pg/mL, P = 0.03), and, importantly, it reverses cachexia and counteracts loss of all measured muscle groups (P < 0.0005). Conclusions Our data support a functional link between activin A and IL‐6 signalling pathways and indicate that interference with activin A‐induced IL‐6 secretion from the tumour has therapeutic potential for cancer‐induced cachexia. © 2019 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders Journal of Cachexia, Sarcopenia and Muscle (2019) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jcsm.12489 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

NMR-based metabolomics has shown promise in the diagnosis of diseases as it enables identification and quantification of metabolic biomarkers. Using high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy, metabolic profiles from intact tissue specimens can be obtained with high spectral resolution. In addition, HR-MAS NMR requires minimal sample preparation and the sample is kept intact for subsequent analyses. In this chapter, we describe a typical protocol for NMR-based metabolomics of tissue samples. We cover all major steps ranging from tissue sample collection to determination of biomarkers, including experimental precautions taken to ensure reproducible and reliable reporting of data in the area of clinical application.

Effective transverse relaxivity of gadolinium-based contrast agents is often neglected in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Here, we assess time and tissue dependence of R2* enhancement and its impact on pharmacokinetic parameter quantification and treatment monitoring. Multiecho DCE-MRI was performed at 7 T on mice bearing subcutaneous TOV-21G human ovarian cancer xenografts (n = 8) and on the transgenic adenocarcinoma of the mouse prostate (TRAMP) model (n = 7). Subsequently, the TOV-21G tumor-bearing mice were treated with bevacizumab and rescanned 2 days later. Pharmacokinetic analysis (extended Tofts model) was performed using either the first echo signal only (standard single-echo DCE-MRI) or the estimated signal at TE = 0 derived from exponential fitting of R2* relaxation (R2*-corrected). Neglecting R2* enhancement causes underestimation of Gd-DOTA concentration (peak enhancement underestimated by 9.4%–16% in TOV-21G tumors and 13%–20% in TRAMP prostates). Median Ktrans and ve were underestimated in every mouse (TOV-21G Ktrans: 11%–19%, TOV-21G ve: 5.3%–8.9%, TRAMP Ktrans: 8.6%–19%, TRAMP ve: 12%–21%). Bevacizumab treatment reduced Ktrans in all TOV-21G tumors after 48 hours. Treatment effect was significantly greater in all tumors after R2* correction (median change of −0.050 min−1 in R2*-corrected Ktrans vs. −0.037 min−1 in uncorrected Ktrans). R2* enhancement in DCE-MRI is both time- and tissue-dependent and may not be negligible at 7 T in tissue with high Ktrans. This has consequences for the use of Ktrans and other DCE-MRI parameters as biomarkers, because treatment effect size can be underestimated when R2* enhancement is neglected.

Purpose To compare the effects of two anti-angiogenic drugs, bevacizumab and a cytosolic phospholipase A2-α inhibitor (AVX235), on the relationship between vascular structure and dynamic contrast enhanced (DCE)-MRI measurements in a patient-derived breast cancer xenograft model. Methods Mice bearing MAS98.12 tumors were randomized into three groups: bevacizumab-treated (n = 9), AVX235-treated (n = 9), and control (n = 8). DCE-MRI was performed pre- and post-treatment. Median initial area under the concentration-time curve (IAUC60 ) and volume transfer constant (Ktrans ) were computed for each tumor. Tumors were excised for ex vivo micro-CT (computed tomography) angiography, from which the vascular surface area (VSA) and fractional blood volume (FBV) were computed. Spearman correlation coefficients (ρ) were computed to evaluate the associations between the DCE-MRI and micro-CT parameters. Results With the groups pooled, IAUC60 and Ktrans correlated significantly with VSA (ρ = 0.475 and 0.527; P = 0.019 and 0.008). There were no significant correlations within the control group. There were various significant correlations within the treatment groups, but the correlations in the bevacizumab group were of opposite sign, for example, Ktrans versus FBV: AVX235, ρ = 0.800 (P = 0.014); bevacizumab, ρ = -0.786 (P = 0.023). Conclusion DCE-MRI measurements can highly depend on vascular structure. The relationship between vascular structure and function changed markedly after anti-angiogenic treatment. Magn Reson Med 78:1513-1522, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Purpose: [18F]Fluciclovine PET imaging shows promise for the assessment of prostate cancer. The purpose of this PET/MRI study is to optimise the PET imaging protocol for detection and characterisation of primary prostate cancer, by quantitative evaluation of the dynamic uptake of [18F]Fluciclovine in cancerous and benign tissue. Methods: Patients diagnosed with high-risk primary prostate cancer underwent an integrated [18F]Fluciclovine PET/MRI exam before robot-assisted radical prostatectomy with extended pelvic lymph node dissection. Volumes-of-interest (VOIs) of selected organs (prostate, bladder, blood pool) and sub-glandular prostate structures (tumour, benign prostatic hyperplasia (BPH), inflammation, healthy tissue) were delineated on T2-weighted MR images, using whole-mount histology samples as a reference. Three candidate windows for optimal PET imaging were identified based on the dynamic curves of the mean and maximum standardised uptake value (SUVmean and SUVmax, respectively). The statistical significance of differences in SUV between VOIs were analysed using Wilcoxon rank sum tests (p

Estrogen receptor α (ERα) is a key regulator of breast growth and breast cancer development. Here, we report how ERα impacts these processes by reprogramming metabolism in malignant breast cells. We employed an integrated approach, combining genome-wide mapping of chromatin-bound ERα with estrogen-induced transcript and metabolic profiling, to demonstrate that ERα reprograms metabolism upon estrogen stimulation, including changes in aerobic glycolysis, nucleotide and amino acid synthesis, and choline (Cho) metabolism. Cho phosphotransferase CHPT1, identified as a direct ERα-regulated gene, was required for estrogen-induced effects on Cho metabolism, including increased phosphatidylcholine synthesis. CHPT1 silencing inhibited anchorage-independent growth and cell proliferation, also suppressing early-stage metastasis of tamoxifen-resistant breast cancer cells in a zebrafish xenograft model. Our results showed that ERα promotes metabolic alterations in breast cancer cells mediated by its target CHPT1, which this study implicates as a candidate therapeutic target. Cancer Res; 76(19); 5634–46. ©2016 AACR.

Cytosolic phospholipase A2α (cPLA2α) has been shown to be elevated in breast cancer and is a potential biomarker in the differentiation of molecular sub-types. Using a cPLA2α activatable fluorophore, DDAO arachidonate, we explore its ability to function as a contrast agent in fluorescence-guided surgery. In cell lines ranging in cPLA2α expression and representing varying breast cancer sub-types, we show DDAO arachidonate activates with a high correlation to cPLA2α expression level. Using a control probe, DDAO palmitate, in addition to cPLA2α inhibition and genetic knockdown, we show that this activation is a result of cPLA2α activity. In mouse models, using an ex vivo tumor painting technique, we show that DDAO arachidonate activates to a high degree in basal-like versus luminal-like breast tumors and healthy mammary tissue. Finally, we show that using an in vivo model, orthotopic basal-like tumors give significantly high probe activation compared to healthy mammary fat pads and surrounding tissue. Together we conclude that cPLA2α activatable fluorophores such as DDAO arachidonate may serve as a useful contrast agent for the visualization of tumor margins in the fluorescence-guided surgery of basal-like breast cancer. © 2019 The Author(s). Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License

Altered metabolism is now considered an emerging hallmark of cancer. The rapid proliferation rate of cancer cells is associated with a specific metabolic demand; the need to convert nutrients into biomass while maintaining energy production. Abnormal metabolism in cancer is increasingly recognized as a potential target for treatment in itself, but also as a source for novel biomarkers that can be used in diagnosis. Magnetic resonance spectroscopy (MRS) is one of several techniques that can be used for studies of cancer metabolism. Although MRS has limited analytical sensitivity, it is possible to assay a relatively large number of metabolites simultaneously, and the method is by nature quantitative. In cancer research, high-resolution magic-angle spinning (HR-MAS) MRS has become a popular tool as it allows analysis of intact tissue specimens with a spectral resolution similar to what can be achieved in cell extracts. HR-MAS MRS is highly suited for studies of cancer metabolomics in large patient cohorts as the need for sample preparation is limited and the tissue specimens are available for follow-up studies after the MRS analysis. In this chapter, we describe the current experience with use of HR-MAS MRS in studies of cancer metabolomics.

// Caroline K. Sogaard 1, 7 , Siver A. Moestue 2, 3, 4 , Morten B. Rye 1, 7 , Jana Kim 2, 5 , Anala Nepal 1 , Nina-Beate Liabakk 1 , Siri Bachke 1 , Tone F. Bathen 2, 5 , Marit Otterlei 1, 6, 7 and Deborah K. Hill 2, 5 1 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway 2 Department of Circulation and Medical Imaging, Norwegian University of Science and Technology (NTNU), Trondheim, Norway 3 Department of Laboratory Medicine, Women’s and Children’s Health, Norwegian University of Science and Technology (NTNU), Trondheim, Norway 4 Department of Pharmacy, Faculty of Health Sciences, Nord University, Namsos, Norway 5 Department of Radiology, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway 6 APIM Therapeutics A/S, Trondheim, Norway 7 Clinic of Surgery, St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway Correspondence to: Marit Otterlei, email: marit.otterlei@ntnu.no Deborah K. Hill, email: deborah.hill@ntnu.no Keywords: magnetic resonance imaging; gene expression; apoptosis; MAPK; PI3K/AKT Received: June 07, 2017 Accepted: November 06, 2017 Published: January 27, 2018 ABSTRACT Docetaxel is the chemotherapeutic choice for metastatic hormone-refractory prostate cancer, however, it only marginally improves the survival rate. The purpose of the present study was to examine if a peptide targeting the cellular scaffold protein PCNA could improve docetaxel’s efficacy. We found that docetaxel given in combination with a cell penetrating peptide containing the AlkB homolog 2 PCNA interacting motif (APIM-peptide), reduced the prostate volume and limited prostate cancer regrowth in vivo in the immunocompetent transgenic adenocarcinoma model of prostate cancer (TRAMP). In accordance with this, we found that the APIM-peptide enhanced the efficacy of docetaxel in vitro . Gene expression analysis on prostate cancer cell lines indicated that the combination of docetaxel and APIM-peptide alters expression of genes involved in cellular signaling, apoptosis, and prostate cancer development. These changes were not detected in single agent treated cells. Our results suggest that targeting PCNA and thereby affecting multiple cellular pathways simultaneously has the potential to improve docetaxel therapy of advanced prostate cancer.

Metabolomics is the branch of “omics” technologies that involves high‐throughput identification and quantification of small‐molecule metabolites in the metabolome. NMR‐based spectroscopy of biofluids represents a potential method for non‐invasive characterization of cancer. While the metabolism of cancer cells is altered compared with normal non‐proliferating cells, the metabolome of several biofluids (e.g. blood and urine) reflects the metabolism of the entire organism. This review provides an update on the current status of NMR metabolomics analysis of biofluids with respect to: (i) cancer risk assessment; (ii) cancer detection; (iii) disease characterization and prognosis; and (iv) treatment monitoring. We conclude that many studies show impressive associations between biofluid metabolomics and cancer progression, and suggest that NMR metabolomics can be used to provide information with prognostic or predictive value. However, translation of these findings to clinical practice is currently hindered by a lack of validation, difficulties in biological interpretation, and non‐standardized analytical procedures. Locked until 19.4.2019 due to copyright restrictions. This is the peer reviewed version of the following article: [NMR‐based metabolomics of biofluids in cancer], which has been published in final form at [https://doi.org/10.1002/nbm.3927]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

Published version available in Cancer Letters. 2015;366(1):71-83. 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.

The majority of cancer patients with advanced disease experience weight loss, including loss of lean body mass. Severe weight loss is characteristic for cancer cachexia, a condition that significantly impairs functional status and survival. The underlying causes of cachexia are incompletely understood, and currently no therapeutic approach can completely reverse the condition. Autophagy coordinates lysosomal destruction of cytosolic constituents and is systemically induced by starvation. We hypothesized that starvation-mimicking signaling compounds secreted from tumor cells may cause a systemic acceleration of autophagy during cachexia. We found that IL-6 secreted by tumor cells accelerates autophagy in myotubes when complexed with soluble IL-6 receptor (trans-signaling). In lung cancer patients, were cachexia is prevalent, there was a significant correlation between elevated IL-6 expression in the tumor and poor prognosis of the patients. We found evidence for an autophagyinducing bioactivity in serum from cancer patients and that this is clearly associated with weight loss. Importantly, the autophagy-inducing bioactivity was reduced by interference with IL-6 trans-signaling. Together, our findings suggest that IL-6 trans-signaling may be targeted in cancer cachexia. © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/)

Magnetic resonance (MR) can be exploited in a variety of ways to obtain a wide range of anatomical and physiological information in a safe and noninvasive manner. This makes MR imaging (MRI) and spectroscopy (MRS) valuable tools in cancer research and clinical oncology, among other fields. This chapter provides a basic introduction to MR physics and describes how different in vivo MR techniques are used to noninvasively characterize tumors and the tumor microenvironment. Two of the most commonly utilized techniques are contrast-enhanced MRI and diffusion-weighted MRI. Contrast-enhanced MRI methods are used to evaluate tumor vascularization and vascular function by measuring the kinetics and distribution of intravenously administered contrast agents. Diffusion-weighted MRI is sensitive to the diffusion of water molecules in the tissue, from which inferences about tumor cellularity and tissue microstructure can be made. Blood-oxygen-level-dependent MRI can distinguish between oxygenated and deoxygenated blood as a proxy to tumor oxygenation. In addition, efforts have been made to develop targeted contrast agents to directly image hypoxia. MRS can be used to measure the levels of various metabolites such as lactate and choline that are involved in metabolic reprogramming in cancer. Both endogenous and exogenous pH-sensitive indicators enable spectroscopic measurement of tumor pH. While this chapter does not provide an exhaustive overview of the MR methods used for cancer characterization, it discusses both clinical and experimental techniques that highlight the versatility of MR as a tool for exploring some key aspects of the tumor microenvironment.

Sonazoid is an ultrasound contrast agent based on microbubbles (MB) containing perfluorobutane (PFB) gas. Sonazoid is approved in Japan, Korea and Norway for contrast-enhanced ultrasonography of focal liver lesions and focal breast lesions (Japan only). The objective of this study was to determine the pharmacokinetics (PKs) and safety of Sonazoid in Chinese healthy volunteers (HVs) and to evaluate the potential for ethnic differences in PKs between Chinese and Caucasian HVs. Sonazoid was administered as an intra-venous bolus injection at the clinical dose of 0.12 μL or 0.60 μL MB/kg body weight to two groups of eight Chinese HVs. Expired air and blood samples were collected and analyzed using a validated gas chromatographic tandem mass spectrometry method, and the main PK parameters were calculated. The highest PFB concentrations in blood were observed shortly after intra-venous administration of Sonazoid, and elimination of PFB was rapid. In the 0.12 μL MB/kg body weight cohort, PFB concentrations above the limit of quantification were observed for only 10 to 15 min post-injection. In the 0.60 μL MB/kg body weight cohort, PFB concentrations above the limit of quantification were observed for 60 min post-injection, and the shape of the elimination curve suggested a biphasic elimination profile. The maximum observed concentration (Cmax) values of PFB in blood were 2.3 ± 1.1 and 19.1 ± 9.2 ng/g for the 0.12 and 0.60 μL MB/kg body weight dose groups (mean ± standard deviation). Area under the curve values were 10.1 ± 2.7 and 90.1 ± 38.3 ng × min/g for the 0.12 and 0.60 μL MB/kg body weight dose groups. Cmax values of PFB in exhaled air were 0.35 ± 0.2 and 2.4 ± 0.7 ng/mL for the 0.12 and 0.60 μL MB/kg body weight dose groups. Assessment of laboratory parameters, vital signs, oxygen saturation and electrocardiograms revealed no changes indicative of a concern. The PK profile and safety data generated in the Chinese HVs were comparable to previous data for Caucasian HVs. © 2017 The Authors. Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology. Printed in the USA. This is an open access article under the CC BY-NC-ND license

The objective of this study is to investigate if quantitative imaging features derived from combined 18F-Fluciclovine Positron Emission Tomograpy (PET) / multiparametric Magnetic Resonance Imaging (MRI) show potential for detection and characterization of primary prostate cancer. Methods: Twenty-eight (28) patients diagnosed with high-risk prostate cancer underwent simultaneous 18F-Fluciclovine PET/MRI before radical prostatectomy. Volumes-of-interest (VOIs) of prostate tumors, benign prostatic hyperplasia (BPH) nodules, prostatitis, and healthy tissue were delineated on T2-weighted images using histology as a reference. Tumor VOIs were marked as high-grade (≥ Gleason Grade group 3) or not. MRI and PET features were extracted on the voxel and VOI-level. Partial least-squared discriminant analysis (PLS-DA) with double leave-one-patient-out cross validation was performed to classify tumor from benign tissue (BPH, prostatitis, healthy tissue) and high-grade tumor from other tissue (low-grade tumor, benign tissue). The performances of PET, MRI, and combined PET/MRI features were compared using the area under the receiver operating characteristic curve (AUC). Results: Voxel and VOI features were extracted from 40 tumor (26 high-grade), 36 BPH, 6 prostatitis, and 37 healthy tissue VOIs. PET/MRI performed better than MRI and PET for classification of tumor vs benign tissue (voxel: AUC 87%, 81%, and 83%; VOI: AUC 96%, 93%, and 93%, respectively) and high-grade tumor vs other tissue (voxel: AUC 85%, 79%, and 81%; VOI: AUC 93%, 93%, and 91%, respectively). T2-weighted MRI, diffusion-weighted MRI and PET features were most important for classification. Conclusion: Combined 18F-Fluciclovine PET/multiparametric MRI shows potential for improving detection and characterization of high-risk prostate cancer, in comparison to MRI and PET alone. © 2017. This is the authors’ accepted and refereed manuscript to the article. The final publication is available at http://jnm.snmjournals.org/content/early/2017/10/05/jnumed.117.198598