Proteogenomic Markers of Chemotherapy Resistance and Response in Triple-Negative Breast Cancer.

Microscaled proteogenomics was deployed to probe the molecular basis for differential response to neoadjuvant carboplatin and docetaxel combination chemotherapy for triple-negative breast cancer (TNBC). Proteomic analyses of pretreatment patient biopsies uniquely revealed metabolic pathways, including oxidative phosphorylation, adipogenesis, and fatty acid metabolism, that were associated with resistance. Both proteomics and transcriptomics revealed that sensitivity was marked by elevation of DNA repair, E2F targets, G2-M checkpoint, interferon-gamma signaling, and immune-checkpoint components. Proteogenomic analyses of somatic copy-number aberrations identified a resistance-associated 19q13.31-33 deletion where LIG1, POLD1, and XRCC1 are located. In orthogonal datasets, LIG1 (DNA ligase I) gene deletion and/or low mRNA expression levels were associated with lack of pathologic complete response, higher chromosomal instability index (CIN), and poor prognosis in TNBC, as well as carboplatin-selective resistance in TNBC preclinical models. Hemizygous loss of LIG1 was also associated with higher CIN and poor prognosis in other cancer types, demonstrating broader clinical implications. SIGNIFICANCE: Proteogenomic analysis of triple-negative breast tumors revealed a complex landscape of chemotherapy response associations, including a 19q13.31-33 somatic deletion encoding genes serving lagging-strand DNA synthesis (LIG1, POLD1, and XRCC1), that correlate with lack of pathologic response, carboplatin-selective resistance, and, in pan-cancer studies, poor prognosis and CIN. This article is highlighted in the In This Issue feature, p. 2483.

Improved visualisation of hepatic metastases in gadoxetate disodium-enhanced MRI: Potential of contrast-optimised (phase-sensitive) inversion recovery imaging.

BACKGROUND: Detection of metastases can have a significant impact on therapy. Nevertheless, even in gadoxetate disodium-enhanced MR scans, very small hepatic metastases may be difficult to see. PURPOSE: To investigate the potential of a contrast-optimised (phase-sensitive) inversion recovery MR sequence in gadoxetate disodium-enhanced scans for detection of hepatic metastases. MATERIALS AND METHODS: With institutional review board approval and after written informed consent, 40 patients (18 male, 22 female) with suspected or known hepatic metastases were examined on a 1.5 T MR system. A T1-weighted gradient-echo volumetric-interpolated-breath-hold (VIBE) sequence was acquired as part of the standard imaging protocol 20 minutes after administration of gadoxetate disodium. Additionally, an IR sequence was acquired with an inversion time to suppress native signal from metastases. Overall image quality and delineation of lesions were assessed on VIBE as well as on magnitude-reconstructed (MAG) and phase-sensitive IR (PSIR) sequences. Lesion-to-liver contrast (LLC) was compared between VIBE and MAG images. RESULTS: Overall image quality was high in both VIBE and MAG IR sequences (VIBE 4.275; MAG 4.313), yet significantly lower in PSIR (4.038). Subjective delineation of lesions was higher on MAG and PSIR images compared to VIBE in all size groups with an overall statistically significant difference for VIBE vs. MAG vs. PSIR (p < .001) in the variance analysis. Mean LLC was 0.35±0.01 for VIBE sequences, and 0.73±0.01 for MAG. CONCLUSION: Contrast-optimised PSIR seems to improve imaging characteristics of hepatic metastases in gadoxetate disodium-enhanced scans compared to T1 gradient-echo VIBE sequences.

Feasibility of gadoxetate disodium enhanced 3D T1 MR cholangiography (MRC) with a specific inversion recovery prepulse for the assessment of the hepatobiliary system.

AIM: To compare the potential of a gadoxetate disodium enhanced navigator-triggered 3D T1 magnetic-resonance cholangiography (MRC) sequence with a specific inversion recovery prepulse to T2-weighted MRCP for assessment of the hepatobiliary system. MATERIALS AND METHODS: 30 patients (12 male, 18 female) prospectively underwent conventional navigator-triggered 3D turbo spin-echo T2-weighted MRCP and 3D T1 MRC with a specific inversion pulse to minimise signal from the liver 30 minutes after administration of gadoxetate disodium on a 1.5 T MRI system. For qualitative evaluation, biliary duct depiction was assessed segmentally following a 5-point Likert scale. Visualisation of hilar structures as well as image quality was recorded. Additionally, the extrahepatic bile ducts were assessed quantitatively by calculation of signal-to-noise ratios (SNR). RESULTS: The advantages of T1 3D MRC include reduced affection of image quality by bowel movement and robust depiction of the relative position of the extrahepatic bile ducts in relation to the portal vein and the duodenum compared to T2 MRCP. However, overall T1 3D MRC did not significantly (p > 0.05) improve the biliary duct depiction compared to T2 MRCP in all segments: Common bile duct 4.1 vs. 4.4, right hepatic duct 3.6 vs. 4.2, left hepatic duct 3.5 vs. 4.1. Image quality did not differ significantly (p > 0.05) between both sequences (3.6 vs. 3.5). SNR measurements for the hepatobiliary system did not differ significantly (p > 0.05) between navigator-triggered T1 3D MRC and T2 MRCP. CONCLUSIONS: This preliminary study demonstrates that T1 3D MRC of a specific inversion recovery prepulse has potential to complement T2 MRCP, especially for the evaluation of liver structures close to the hilum in the diagnostic work-up of the biliary system in patients receiving gadoxetate disodium.

Small BODIPY Probes for Combined Dual (19) F†MRI and Fluorescence Imaging.

The combination of the two complementary imaging modalities (19) F magnetic resonance imaging (MRI) and fluorescence imaging (FLI) possesses high potential for biological and medical applications. Herein we report the first design, synthesis, dual detection validation, and cytotoxic testing of four promising BODIPY dyes for dual (19) F MRI-fluorescence detection. Using straightforward Steglich reactions, small fluorinated alcohols were easily covalently tethered to a BODIPY dye in high yields, leaving its fluorescence properties unaffected. The synthesized compounds were analyzed with various techniques to demonstrate their potential utility in dual imaging. As expected, the chemically and magnetically equivalent trifluoromethyl groups of the agents exhibited a single NMR signal. The determined longitudinal relaxation times T1 and the transverse relaxation times T2 , both in the lower second range, enabled the imaging of four compounds in vitro. The most auspicious dual (19) F MRI-fluorescence agent was also successfully imaged in a mouse post-mortem within a 9.4 T small-animal tomograph. Toxicological assays with human cells (primary HUVEC and HepG2 cell line) also indicated the possibility for animal testing.

3D Reconstructed Cyto-, Muscarinic M2 Receptor, and Fiber Architecture of the Rat Brain Registered to the Waxholm Space Atlas.

High-resolution multiscale and multimodal 3D models of the brain are essential tools to understand its complex structural and functional organization. Neuroimaging techniques addressing different aspects of brain organization should be integrated in a reference space to enable topographically correct alignment and subsequent analysis of the various datasets and their modalities. The Waxholm Space (http://software.incf.org/software/waxholm-space) is a publicly available 3D coordinate-based standard reference space for the mapping and registration of neuroanatomical data in rodent brains. This paper provides a newly developed pipeline combining imaging and reconstruction steps with a novel registration strategy to integrate new neuroimaging modalities into the Waxholm Space atlas. As a proof of principle, we incorporated large scale high-resolution cyto-, muscarinic M2 receptor, and fiber architectonic images of rat brains into the 3D digital MRI based atlas of the Sprague Dawley rat in Waxholm Space. We describe the whole workflow, from image acquisition to reconstruction and registration of these three modalities into the Waxholm Space rat atlas. The registration of the brain sections into the atlas is performed by using both linear and non-linear transformations. The validity of the procedure is qualitatively demonstrated by visual inspection, and a quantitative evaluation is performed by measurement of the concordance between representative atlas-delineated regions and the same regions based on receptor or fiber architectonic data. This novel approach enables for the first time the generation of 3D reconstructed volumes of nerve fibers and fiber tracts, or of muscarinic M2 receptor density distributions, in an entire rat brain. Additionally, our pipeline facilitates the inclusion of further neuroimaging datasets, e.g., 3D reconstructed volumes of histochemical stainings or of the regional distributions of multiple other receptor types, into the Waxholm Space. Thereby, a multiscale and multimodal rat brain model was created in the Waxholm Space atlas of the rat brain. Since the registration of these multimodal high-resolution datasets into the same coordinate system is an indispensable requisite for multi-parameter analyses, this approach enables combined studies on receptor and cell distributions as well as fiber densities in the same anatomical structures at microscopic scales for the first time.

Cysteine S-Glutathionylation Promotes Stability and Activation of the Hippo Downstream Effector Transcriptional Co-activator with PDZ-binding Motif (TAZ).

Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP) are critical transcriptional co-activators downstream of the Hippo pathway involved in the regulation of organ size, tissue regeneration, proliferation, and apoptosis. Recent studies suggested common and distinct functions of TAZ and YAP and their diverse impact under several pathological conditions. Here we report differential regulation of TAZ and YAP in response to oxidative stress. H2O2 exposure leads to increased stability and activation of TAZ but not of YAP. H2O2 induces reversible S-glutathionylation at conserved cysteine residues within TAZ. We further demonstrate that TAZ S-glutathionylation is critical for reactive oxygen species (ROS)-mediated, TAZ-dependent TEA domain transcription factor (TEAD) trans-activation. Lysophosphatidic acid, a physiological activator of YAP and TAZ, induces ROS elevation and, subsequently, TAZ S-glutathionylation, which promotes TAZ-mediated target gene expression. TAZ expression is essential for renal homeostasis in mice, and we identify basal TAZ S-glutathionylation in murine kidney lysates, which is elevated during ischemia/reperfusion injury in vivo This induced nuclear localization of TAZ and increased expression of connective tissue growth factor. These results describe a novel mechanism by which ROS sustains total cellular levels of TAZ. This preferential regulation suggests TAZ to be a redox sensor of the Hippo pathway.

Monofluorination and Trifluoromethylation of BODIPY Dyes for Prolonged Single-Molecule Detection.

Electrophilic monofluorination with Selectfluor and nucleophilic trifluoromethylation with the Ruppert-Prakesh reagent of dimethyl-, tetramethyl- and pentamethyl-substituted boron dipyrromethenes (BODIPY) are investigated. Monofluorinated dyes are synthesized with low yields (<30%), however trifluoromethyl derivatives are obtained in moderate to high yields (≈40-90%). All compounds are characterized by steady-state and time-resolved fluorescence spectroscopy, the photostability is investigated with fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRF). Monofluorination hardly affects the spectroscopic parameters of the unsubstituted parent compounds, but distinctly enhances the photostability, whereas trifluoromethylation leads to a hypsochromic shift by up to 17 nm in both absorption and emission, slightly enhanced intersystem crossing, and higher photostability. Further development of soft fluorination and trifluoromethylation methods is therefore highly desired.

Fragmentation patterns of boron-dipyrromethene (BODIPY) dyes by electrospray ionization high-resolution tandem mass spectrometry.

RATIONALE: 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene derivatives (BODIPYs) are fluorescent organic dyes that are widely used as non-radioactive labels in biological analyses. The fragmentation behaviour of ten structurally related BODIPYs was studied using tandem mass spectrometry (MS/MS), to support the structural elucidation process during synthesis. METHODS: The BODIPYs were investigated by electrospray ionization (ESI)-MS/MS, utilizing collision-induced dissociation (CID) data from triple quadrupole MS and high-resolution, accurate mass CID data from Fourier transform ion cyclotron resonance (FTICR) experiments. RESULTS: Unusual radical molecular cations ([M](+•)) were formed directly during the ESI process. These radical species dissociated into a large range of product ions during the subsequent CID experiments. Superimposed dissociations originating from parallel [M](+•) and [M+H](+) decompositions significantly complicated the interpretation of the MS/MS spectra. CONCLUSIONS: Detailed dissociation mechanisms were proposed in this study for BODIPY dyes. The elemental formulae of CID product ions were unambiguously assigned using FTICR-MS and unique fragment ions were discovered for the rapid identification of methyl, ethyl, butyl, tert-butyl, and phenyl substituents of individual dyes in BODIPY synthesis mixtures by low-resolution MS.