Single-cell tracking as a tool for studying EMT-phenotypes.

This article demonstrates that label-free single-cell video tracking is a useful approach for in vitro studies of Epithelial-Mesenchymal Transition (EMT). EMT is a highly heterogeneous process, involved in wound healing, embryogenesis and cancer. The process promotes metastasis, and increased understanding can aid development of novel therapeutic strategies. The role of EMT-associated biomarkers depends on biological context, making it challenging to compare and interpret data from different studies. We demonstrate single-cell video tracking for comprehensive phenotype analysis. In this study we performed single-cell video tracking on 72-h long recordings. We quantified several behaviours at a single-cell level during induced EMT in MDA-MB-468 cells. This revealed notable variations in migration speed, with different dose-response patterns and varying distributions of speed. By registering cell morphologies during the recording, we determined preferred paths of morphological transitions. We also found a clear association between migration speed and cell morphology. We found elevated rates of cell death, diminished proliferation, and an increase in mitotic failures followed by re-fusion of sister-cells. The method allows tracking of phenotypes in cell lineages, which can be particularly useful in epigenetic studies. Sister-cells were found to have significant similarities in their speeds and morphologies, illustrating the heritability of these traits.

Visualization of Small Intact Proteins in Breast Cancer FFPE Tissue.

Molecular visualization of metabolites, lipids, and proteins by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is becoming an in-demand analytical approach to aid the histopathological analysis of breast cancer. Particularly, proteins seem to play a role in cancer progression, and specific proteins are currently used in the clinic for staging. Formalin-fixed paraffin-embedded (FFPE) tissues are ideal for correlating the molecular markers with clinical outcomes due to their long-term storage. So far, to obtain proteomic information by MSI from this kind of tissue, antigen retrieval and tryptic digestion steps are required. In this chapter, we present a protocol to spatially detect small proteins in tumor and necrotic regions of patient-derived breast cancer xenograft FFPE tissues without employing any on-tissue digestion. This protocol can be used for other kinds of FFPE tissue following specific optimization of the sample preparation phases.

Metabolic profiling of colorectal cancer organoids: A comparison between high-resolution magic angle spinning magnetic resonance spectroscopy and solution nuclear magnetic resonance spectroscopy of polar extracts.

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.

Classification and biomarker identification of prostate tissue from TRAMP mice with hyperpolarized 13C-SIRA.

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.

Argininosuccinate lyase is a metabolic vulnerability in breast development and cancer.

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.

Reproducible Lipid Alterations in Patient-Derived Breast Cancer Xenograft FFPE Tissue Identified with MALDI MSI for Pre-Clinical and Clinical Application.

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.

EMT-Derived Alterations in Glutamine Metabolism Sensitize Mesenchymal Breast Cells to mTOR Inhibition.

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.

Detection of Recurrent Prostate Cancer With 18F-Fluciclovine PET/MRI.

OBJECTIVE: Simultaneous PET/MRI combines soft-tissue contrast of MRI with high molecular sensitivity of PET in one session. The aim of this prospective study was to evaluate detection rates of recurrent prostate cancer by 18F-fluciclovine PET/MRI. METHODS: Patients with biochemical recurrence (BCR) or persistently detectable prostate specific antigen (PSA), were examined with simultaneous 18F-fluciclovine PET/MRI. Multiparametric MRI (mpMRI) and PET/MRI were scored on a 3-point scale (1-negative, 2-equivocal, 3-recurrence/metastasis) and detection rates (number of patients with suspicious findings divided by total number of patients) were reported. Detection rates were further stratified based on PSA level, PSA doubling time (PSAdt), primary treatment and inclusion criteria (PSA persistence, European Association of Urology (EAU) Low-Risk BCR and EAU High-Risk BCR). A detailed investigation of lesions with discrepancy between mpMRI and PET/MRI scores was performed to evaluate the incremental value of PET/MRI to mpMRI. The impact of the added PET acquisition on further follow-up and treatment was evaluated retrospectively. RESULTS: Among patients eligible for analysis (n=84), 54 lesions were detected in 38 patients by either mpMRI or PET/MRI. Detection rates were 41.7% for mpMRI and 39.3% for PET/MRI (score 2 and 3 considered positive). There were no significant differences in detection rates for mpMRI versus PET/MRI. Disease detection rates were higher in patients with PSA≥1ng/mL than in patients with lower PSA levels but did not differ between patients with PSAdt above versus below 6 months. Detection rates in patients with primary radiation therapy were higher than in patients with primary surgery. Patients categorized as EAU Low-Risk BCR had a detection rate of 0% both for mpMRI and PET/MRI. For 15 lesions (27.8% of all lesions) there was a discrepancy between mpMRI score and PET/MRI score. Of these, 10 lesions scored as 2-equivocal by mpMRI were changed to a more definite score (n=4 score 1 and n=6 score 3) based on the added PET acquisition. Furthermore, for 4 of 10 patients with discrepancy between mpMRI and PET/MRI scores, the added PET acquisition had affected the treatment choice. CONCLUSION: Combined 18F-fluciclovine PET/MRI can detect lesions suspicious for recurrent prostate cancer in patients with a range of PSA levels. Combined PET/MRI may be useful to select patients for appropriate treatment, but is of limited use at low PSA values or in patients classified as EAU Low-Risk BCR, and the clinical value of 18F-fluciclovine PET/MRI in this study was too low to justify routine clinical use.

Cytosolic Phospholipase A2 Alpha Regulates TLR Signaling and Migration in Metastatic 4T1 Cells.

Metastatic disease is the leading cause of death in breast cancer patients. Disrupting the cancer cell's ability to migrate may be a strategy for hindering metastasis. Cytosolic phospholipase A2 α (cPLA2α), along with downstream proinflammatory and promigratory metabolites, has been implicated in several aspects of tumorigenesis, as well as metastasis, in various types of cancer. In this study, we aim to characterize the response to reduced cPLA2α activity in metastatic versus non-metastatic cells. We employ an isogenic murine cell line pair displaying metastatic (4T1) and non-metastatic (67NR) phenotype to investigate the role of cPLA2α on migration. Furthermore, we elucidate the effect of reduced cPLA2α activity on global gene expression in the metastatic cell line. Enzyme inhibition is achieved by using a competitive pharmacological inhibitor, cPLA2α inhibitor X (CIX). Our data show that 4T1 expresses significantly higher cPLA2α levels as compared to 67NR, and the two cell lines show different sensitivity to the CIX treatment with regards to metabolism and proliferation. Inhibition of cPLA2α at nontoxic concentrations attenuates migration of highly metastatic 4T1 cells, but not non-metastatic 67NR cells. Gene expression analysis indicates that processes such as interferon type I (IFN-I) signaling and cell cycle regulation are key processes regulated by cPLA2a in metastatic 4T1 cells, supporting the findings from the biological assays. This study demonstrates that two isogenic cancer cell lines with different metastatic potential respond differently to reduced cPLA2α activity. In conclusion, we argue that cPLA2α is a potential therapeutic target in cancer and that enzyme inhibition may inhibit metastasis through an anti-migratory mechanism, possibly involving Toll-like receptor signaling and type I interferons.

Anti-angiogenic therapy affects the relationship between tumor vascular structure and function: A correlation study between micro-computed tomography angiography and dynamic contrast enhanced MRI.

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.

Estrogen Receptor α Promotes Breast Cancer by Reprogramming Choline Metabolism.

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.

Anti-vascular effects of the cytosolic phospholipase A2 inhibitor AVX235 in a patient-derived basal-like breast cancer model.

BACKGROUND: Group IVA cytosolic phospholipase A2 (cPLA2α) plays an important role in tumorigenesis and angiogenesis. It is overexpressed in basal-like breast cancer (BLBC), which is aggressive and usually triple-negative, making it unresponsive to current targeted therapies. Here, we evaluated the anti-angiogenic effects of a specific cPLA2α inhibitor, AVX235, in a patient-derived triple-negative BLBC model. METHODS: Mice bearing orthotopic xenografts received i.p. injections of AVX235 or DMSO vehicle daily for 1 week and then every other day for up to 19 days. Six treated and six control mice were terminated after 2 days of treatment, and the tumors excised for high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) and prostaglandin E2 (PGE2) enzyme immunoassay (EIA) analysis. A 1-week imaging study was performed on a separate cohort of mice. Longitudinal dynamic contrast enhanced (DCE)-MRI was performed before, after 4 days, and after 1 week of treatment. The mice were then perfused with a radiopaque vascular casting agent, and the tumors excised for micro-CT angiography. Subsequently, tumors were sectioned and stained with lectin and for Ki67 or α-smooth muscle actin to quantify endothelial cell proliferation and vessel maturity, respectively. Partial least squares discriminant analysis was performed on the multivariate HR MAS MRS data, and non-parametric univariate analyses using Mann-Whitney U tests (α = 0.05) were performed on all other data. RESULTS: Glycerophosphocholine and PGE2 levels, measured by HR MAS MRS and EIA, respectively, were lower in treated tumors after 2 days of treatment. These molecular changes are expected downstream effects of cPLA2α inhibition and were followed by significant tumor growth inhibition after 8 days of treatment. DCE-MRI revealed that AVX235 treatment caused a decrease in tumor perfusion. Concordantly, micro-CT angiography showed that vessel volume fraction, density, and caliber were reduced in treated tumors. Moreover, histology showed decreased endothelial cell proliferation and fewer immature vessels in treated tumors. CONCLUSIONS: These results demonstrate that cPLA2α inhibition with AVX235 resulted in decreased vascularization and perfusion and subsequent inhibition of tumor growth. Thus, cPLA2α inhibition may be a potential new therapeutic option for triple-negative basal-like breast cancer.

Metabolic effects of signal transduction inhibition in cancer assessed by magnetic resonance spectroscopy.

Despite huge efforts in development of drugs targeting oncogenic signalling, the number of such drugs entering clinical practice to date remains limited. Rational use of biomarkers for drug candidate selection and early monitoring of response to therapy may accelerate this process. Magnetic resonance spectroscopy (MRS) can be used to assess metabolic effects of drug treatment both in vivo and in vitro, and technological advances are continuously increasing the utility of this non-invasive method. In this review, we summarise the use of MRS for monitoring the effect of targeted anticancer drugs, and discuss the potential role of MRS in the context of personalised cancer treatment.