Detection of phenotype-specific therapeutic vulnerabilities in breast cells using a CRISPR loss-of-function screen.

Cellular phenotype plasticity between the epithelial and mesenchymal states has been linked to metastasis and heterogeneous responses to cancer therapy, and remains a challenge for the treatment of triple-negative breast cancer (TNBC). Here, we used isogenic human breast epithelial cell lines, D492 and D492M, representing the epithelial and mesenchymal phenotypes, respectively. We employed a CRISPR-Cas9 loss-of-function screen targeting a 2240-gene 'druggable genome' to identify phenotype-specific vulnerabilities. Cells with the epithelial phenotype were more vulnerable to the loss of genes related to EGFR-RAS-MAPK signaling, while the mesenchymal-like cells had increased sensitivity to knockout of G2 -M cell cycle regulators. Furthermore, we discovered knockouts that sensitize to the mTOR inhibitor everolimus and the chemotherapeutic drug fluorouracil in a phenotype-specific manner. Specifically, loss of EGFR and fatty acid synthase (FASN) increased the effectiveness of the drugs in the epithelial and mesenchymal phenotypes, respectively. These phenotype-associated genetic vulnerabilities were confirmed using targeted inhibitors of EGFR (gefitinib), G2 -M transition (STLC), and FASN (Fasnall). In conclusion, a CRISPR-Cas9 loss-of-function screen enables the identification of phenotype-specific genetic vulnerabilities that can pinpoint actionable targets and promising therapeutic combinations.

Inhibition of O-GlcNAc transferase activates tumor-suppressor gene expression in tamoxifen-resistant 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.

R2* Relaxation Affects Pharmacokinetic Analysis of Dynamic Contrast-Enhanced MRI in Cancer and Underestimates Treatment Response at 7 T.

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.

Biomarker Discovery Using NMR-Based Metabolomics of Tissue.

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.

Glutamine to proline conversion is associated with response to glutaminase inhibition in breast cancer.

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. 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.

O-GlcNAc Transferase Inhibition Differentially Affects Breast Cancer Subtypes.

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.

NMR-Based Prostate Cancer Metabolomics.

Prostate cancer is the second most common malignancy, and the fifth leading cause of cancer-related death among men, worldwide. A major unsolved clinical challenge in prostate cancer is the ability to accurately distinguish indolent cancer types from the aggressive ones. Reprogramming of metabolism is now a widely accepted hallmark of cancer development, where cancer cells must be able to convert nutrients to biomass while maintaining energy production. Metabolomics is the large-scale study of small molecules, commonly known as metabolites, within cells, biofluids, tissues, or organisms. Nuclear magnetic resonance (NMR) spectroscopy is commonly applied in metabolomics studies of cancer. This chapter provides protocols for NMR-based metabolomics of cell cultures, biofluids (serum and urine), and intact tissue, with concurrent advice for optimal biobanking and sample preparation procedures.

NMR-based metabolomics of biofluids in cancer.

This review describes the current status of NMR-based metabolomics of biofluids with respect to cancer risk assessment, detection, disease characterization, prognosis, and treatment monitoring. While the metabolism of cancer cells is altered compared with that of non-proliferating cells, the metabolome of blood and urine reflects the entire organism. We conclude that many studies show impressive associations between biofluid metabolomics and cancer progression, but translation to clinical practice is currently hindered by lack of validation, difficulties in biological interpretation, and non-standardized analytical procedures.

APIM-peptide targeting PCNA improves the efficacy of docetaxel treatment in the TRAMP mouse model of prostate cancer.

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.

18F-Fluciclovine PET/MRI for preoperative lymph node staging in high-risk prostate cancer patients.

OBJECTIVE: To investigate the diagnostic potential of simultaneous 18F-fluciclovine PET/MRI for pelvic lymph node (LN) staging in patients with high-risk prostate cancer. METHODS: High-risk prostate cancer patients (n=28) underwent simultaneous 18F-fluciclovine PET/MRI prior to surgery. LNs were removed according to a predefined template of eight regions. PET and MR images were evaluated for presence of LN metastases according to these regions. Sensitivity/specificity for detection of LN metastases were calculated on patient and region basis. Sizes of LN metastases in regions with positive and negative imaging findings were compared with linear mixed models. Clinical parameters of PET-positive and -negative stage N1 patients were compared with the Mann-Whitney U test. RESULTS: Patient- and region-based sensitivity/specificity for detection of pelvic LN metastases was 40 %/87.5 % and 35 %/95.7 %, respectively, for MRI and 40 %/100 % and 30 %/100 %, respectively, for PET. LN metastases in true-positive regions were significantly larger than metastases in false-negative regions. PET-positive stage N1 patients had higher metastatic burden than PET-negative N1 patients. CONCLUSION: Simultaneous 18F-fluciclovine PET/MRI provides high specificity but low sensitivity for detection of LN metastases in high-risk prostate cancer patients. 18F-Fluciclovine PET/MRI scan positive for LN metastases indicates higher metastatic burden than negative scan. KEY POINTS: • 18F-Fluciclovine PET/MRI has high specificity for detection of lymph node metastasis. • 18F-Fluciclovine PET/MRI lacks sensitivity to replace ePLND. • 18F-Fluciclovine PET/MRI may be used to aid surgery and select adjuvant therapy. • 18F-Fluciclovine PET-positive patients have more extensive disease than PET-negative patients. • Size of metastatic lymph nodes is an important factor for detection.

Multiparametric characterization of response to anti-angiogenic therapy using USPIO contrast-enhanced MRI in combination with dynamic contrast-enhanced MRI.

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 (<24 h) and late changes in tumor vasculature. SSC-MRI and DCE-MRI provide complementary information and can be used to assess different aspects of vascular responses to anti-angiogenic therapies. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:1589-1600.

Combined 18F-Fluciclovine PET/MRI Shows Potential for Detection and Characterization of High-Risk Prostate Cancer.

The objective of this study was to investigate whether quantitative imaging features derived from combined 18F-fluciclovine PET/multiparametric MRI show potential for detection and characterization of primary prostate cancer. Methods: Twenty-eight patients diagnosed with high-risk prostate cancer underwent simultaneous 18F-fluciclovine PET/MRI before radical prostatectomy. Volumes of interest (VOIs) for 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 levels. Partial least-squared discriminant analysis (PLS-DA) with double leave-one-patient-out cross-validation was performed to distinguish tumors from benign tissue (BPH, prostatitis, or healthy tissue) and high-grade tumors from other tissue (low-grade tumors or benign tissue). The performance levels 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 VOIs (26 high-grade), 36 BPH VOIs, 6 prostatitis VOIs, and 37 healthy-tissue VOIs. PET/MRI performed better than MRI and PET alone for distinguishing tumors from benign tissue (AUCs of 87%, 81%, and 83%, respectively, at the voxel level and 96%, 93%, and 93%, respectively, at the VOI level) and high-grade tumors from other tissue (AUCs of 85%, 79%, and 81%, respectively, at the voxel level and 93%, 93%, and 91%, respectively, at the VOI level). T2-weighted MRI, diffusion-weighted MRI, and PET features were the 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.

Non-Invasive Prostate Cancer Characterization with Diffusion-Weighted MRI: Insight from In silico Studies of a Transgenic Mouse Model.

Diffusion-weighted magnetic resonance imaging (DWI) enables non-invasive, quantitative staging of prostate cancer via measurement of the apparent diffusion coefficient (ADC) of water within tissues. In cancer, more advanced disease is often characterized by higher cellular density (cellularity), which is generally accepted to correspond to a lower measured ADC. A quantitative relationship between tissue structure and in vivo measurements of ADC has yet to be determined for prostate cancer. In this study, we establish a theoretical framework for relating ADC measurements with tissue cellularity and the proportion of space occupied by prostate lumina, both of which are estimated through automatic image processing of whole-slide digital histology samples taken from a cohort of six healthy mice and nine transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. We demonstrate that a significant inverse relationship exists between ADC and tissue cellularity that is well characterized by our model, and that a decrease of the luminal space within the prostate is associated with a decrease in ADC and more aggressive tumor subtype. The parameters estimated from our model in this mouse cohort predict the diffusion coefficient of water within the prostate-tissue to be 2.18 × 10-3 mm2/s (95% CI: 1.90, 2.55). This value is significantly lower than the diffusion coefficient of free water at body temperature suggesting that the presence of organelles and macromolecules within tissues can drastically hinder the random motion of water molecules within prostate tissue. We validate the assumptions made by our model using novel in silico analysis of whole-slide histology to provide the simulated ADC (sADC); this is demonstrated to have a significant positive correlation with in vivo measured ADC (r2 = 0.55) in our mouse population. The estimation of the structural properties of prostate tissue is vital for predicting and staging cancer aggressiveness, but prostate tissue biopsies are painful, invasive, and are prone to complications such as sepsis. The developments made in this study provide the possibility of estimating the structural properties of prostate tissue via non-invasive virtual biopsies from MRI, minimizing the need for multiple tissue biopsies and allowing sequential measurements to be made for prostate cancer monitoring.

Metabolic Response to Everolimus in Patient-Derived Triple-Negative Breast Cancer Xenografts.

Patients with triple-negative breast cancer (TNBC) are unresponsive to endocrine and anti-HER2 pharmacotherapy, limiting their therapeutic options to chemotherapy. TNBC is frequently associated with abnormalities in the PI3K/AKT/mTOR signaling pathway; drugs targeting this pathway are currently being evaluated in these patients. However, the response is variable, partly due to heterogeneity within TNBC, conferring a need to identify biomarkers predicting response and resistance to targeted therapy. In this study, we used a metabolomics approach to assess response to the mTOR inhibitor everolimus in a panel of TNBC patient-derived xenografts (PDX) (n = 103 animals). Tumor metabolic profiles were acquired using high-resolution magic angle spinning magnetic resonance spectroscopy. Partial least-squares-discriminant analysis on relative metabolite concentrations discriminated treated xenografts from untreated controls with an accuracy of 67% (p = 0.003). Multilevel linear mixed-effects models (LMM) indicated reduced glycolytic lactate production and glutaminolysis after treatment, consistent with PI3K/AKT/mTOR pathway inhibition. Although inherent metabolic heterogeneity between different PDX models seemed to hinder prediction of treatment response, the metabolic effects following treatment were more pronounced in responding xenografts compared to nonresponders. Additionally, the metabolic information predicted p53 mutation status, which may provide complementary insight into the interplay between PI3K signaling and other drivers of disease progression.

Pharmacokinetics of Perfluorobutane after Intra-Venous Bolus Injection of Sonazoid in Healthy Chinese Volunteers.

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.

A PET/MRI study towards finding the optimal [18F]Fluciclovine PET protocol for detection and characterisation of primary prostate cancer.

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<0.05, adjusted for multiple testing). RESULTS: Twenty-eight (28) patients [median (range) age: 66 (55-72) years] were included. An early (W1: 5-10 minutes post-injection) and two late candidate windows (W2: 18-23; W3: 33-38 minutes post-injection) were selected. Late compared with early imaging was better able to distinguish between malignant and benign tissue [W3, SUVmean: tumour vs. BPH 2.5 vs. 2.0 (p<0.001), tumour vs. inflammation 2.5 vs. 1.7 (p<0.001), tumour vs. healthy tissue 2.5 vs. 2.0 (p<0.001); W1, SUVmean: tumour vs. BPH 3.1 vs. 3.1 (p=0.771), tumour vs inflammation 3.1 vs. 2.2 (p=0.021), tumour vs. healthy tissue 3.1 vs. 2.5 (p<0.001)] as well as between high-grade and low/intermediate-grade tumours (W3, SUVmean: 2.6 vs. 2.1 (p=0.040); W1, SUVmean: 3.1 vs. 2.8 (p=0.173)). These differences were relevant to the peripheral zone, but not the central gland. CONCLUSION: Late-window [18F]Fluciclovine PET imaging shows promise for distinguishing between prostate tumours and benign tissue and for assessment of tumour aggressiveness.

Impact of Freezing Delay Time on Tissue Samples for Metabolomic Studies.

INTRODUCTION: Metabolic profiling of intact tumor tissue by high-resolution magic angle spinning (HR MAS) MR spectroscopy (MRS) provides important biological information possibly useful for clinical diagnosis and development of novel treatment strategies. However, generation of high-quality data requires that sample handling from surgical resection until analysis is performed using systematically validated procedures. In this study, we investigated the effect of postsurgical freezing delay time on global metabolic profiles and stability of individual metabolites in intact tumor tissue. MATERIALS AND METHODS: Tumor tissue samples collected from two patient-derived breast cancer xenograft models (n = 3 for each model) were divided into pieces that were snap-frozen in liquid nitrogen at 0, 15, 30, 60, 90, and 120 min after surgical removal. In addition, one sample was analyzed immediately, representing the metabolic profile of fresh tissue exposed neither to liquid nitrogen nor to room temperature. We also evaluated the metabolic effect of prolonged spinning during the HR MAS experiments in biopsies from breast cancer patients (n = 14). All samples were analyzed by proton HR MAS MRS on a Bruker Avance DRX600 spectrometer, and changes in metabolic profiles were evaluated using multivariate analysis and linear mixed modeling. RESULTS: Multivariate analysis showed that the metabolic differences between the two breast cancer models were more prominent than variation caused by freezing delay time. No significant changes in levels of individual metabolites were observed in samples frozen within 30 min of resection. After this time point, levels of choline increased, whereas ascorbate, creatine, and glutathione (GS) levels decreased. Freezing had a significant effect on several metabolites but is an essential procedure for research and biobank purposes. Furthermore, four metabolites (glucose, glycine, glycerophosphocholine, and choline) were affected by prolonged HR MAS experiment time possibly caused by physical release of metabolites caused by spinning or due to structural degradation processes. CONCLUSION: The MR metabolic profiles of tumor samples are reproducible and robust to variation in postsurgical freezing delay up to 30 min.

Inhibition of O-GlcNAc transferase activity reprograms prostate cancer cell metabolism.

Metabolic networks are highly connected and complex, but a single enzyme, O-GlcNAc transferase (OGT) can sense the availability of metabolites and also modify target proteins. We show that inhibition of OGT activity inhibits the proliferation of prostate cancer cells, leads to sustained loss of c-MYC and suppresses the expression of CDK1, elevated expression of which predicts prostate cancer recurrence (p=0.00179). Metabolic profiling revealed decreased glucose consumption and lactate production after OGT inhibition. This decreased glycolytic activity specifically sensitized prostate cancer cells, but not cells representing normal prostate epithelium, to inhibitors of oxidative phosphorylation (rotenone and metformin). Intra-cellular alanine was depleted upon OGT inhibitor treatment. OGT inhibitor increased the expression and activity of alanine aminotransferase (GPT2), an enzyme that can be targeted with a clinically approved drug, cycloserine. Simultaneous inhibition of OGT and GPT2 inhibited cell viability and growth rate, and additionally activated a cell death response. These combinatorial effects were predominantly seen in prostate cancer cells, but not in a cell-line derived from normal prostate epithelium. Combinatorial treatments were confirmed with two inhibitors against both OGT and GPT2. Taken together, here we report the reprogramming of energy metabolism upon inhibition of OGT activity, and identify synergistically lethal combinations that are prostate cancer cell specific.

Diffusion-weighted MRI for early detection and characterization of prostate cancer in the transgenic adenocarcinoma of the mouse prostate model.

PURPOSE: To improve early diagnosis of prostate cancer to aid clinical decision-making. Diffusion-weighted magnetic resonance imaging (DW-MRI) is sensitive to water diffusion throughout tissues, which correlates with Gleason score, a histological measure of prostate cancer aggressiveness. In this study the ability of DW-MRI to detect prostate cancer onset and development was evaluated in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. MATERIALS AND METHODS: T2 -weighted and DW-MRI were acquired using a 7T MR scanner, 200 mm bore diameter; 10 TRAMP and 6 C57BL/6 control mice were scanned every 4 weeks from 8 weeks of age until sacrifice at 28-30 weeks. After sacrifice, the genitourinary tract was excised and sectioned for histological analysis. Histology slides registered with DW-MR images allowed for validation of DW-MR images and the apparent diffusion coefficient (ADC) as tools for cancer detection and disease stratification. An automated early assessment tool based on ADC threshold values was developed to aid cancer detection and progression monitoring. RESULTS: The ADC differentiated between control prostate ((1.86 ± 0.20) × 10(-3) mm(2) /s) and normal TRAMP prostate ((1.38 ± 0.10) × 10(-3) mm(2) /s) (P = 0.0001), between TRAMP prostate and well-differentiated cancer ((0.93 ± 0.18) × 10(-3) mm(2) /s) (P = 0.0006), and between well-differentiated cancer and poorly differentiated cancer ((0.63 ± 0.06) × 10(-3) mm(2) /s) (P = 0.02). CONCLUSION: DW-MRI is a tool for early detection of cancer, and discrimination between cancer stages in the TRAMP model. The incorporation of DW-MRI-based prostate cancer stratification and monitoring could increase the accuracy of preclinical trials using TRAMP mice.

Identification of Metastasis-Associated Metabolic Profiles of Tumors by (1)H-HR-MAS-MRS.

Tumors develop an abnormal microenvironment during growth, and similar to the metastatic phenotype, the metabolic phenotype of cancer cells is tightly linked to characteristics of the tumor microenvironment (TME). In this study, we explored relationships between metabolic profile, metastatic propensity, and hypoxia in experimental tumors in an attempt to identify metastasis-associated metabolic profiles. Two human melanoma xenograft lines (A-07, R-18) showing different TMEs were used as cancer models. Metabolic profile was assessed by proton high resolution magic angle spinning magnetic resonance spectroscopy ((1)H-HR-MAS-MRS). Tumor hypoxia was detected in immunostained histological preparations by using pimonidazole as a hypoxia marker. Twenty-four samples from 10 A-07 tumors and 28 samples from 10 R-18 tumors were analyzed. Metastasis was associated with hypoxia in both A-07 and R-18 tumors, and (1)H-HR-MAS-MRS discriminated between tissue samples with and tissue samples without hypoxic regions in both models, primarily because hypoxia was associated with high lactate resonance peaks in A-07 tumors and with low lactate resonance peaks in R-18 tumors. Similarly, metastatic and non-metastatic R-18 tumors showed significantly different metabolic profiles, but not metastatic and non-metastatic A-07 tumors, probably because some samples from the metastatic A-07 tumors were derived from tumor regions without hypoxic tissue. This study suggests that (1)H-HR-MAS-MRS may be a valuable tool for evaluating the role of hypoxia and lactate in tumor metastasis as well as for identification of metastasis-associated metabolic profiles.