Quantitative proteomic analysis of murine white adipose tissue for peritoneal cancer metastasis.

Cancer metastasis risk increases in older individuals, but the mechanisms for this risk increase are unclear. Many peritoneal cancers, including ovarian cancer, preferentially metastasize to peritoneal fat depots. However, there is a dearth of studies exploring aged peritoneal adipose tissue in the context of cancer. Because adipose tissue produces signals which influence several diseases including cancer, proteomics of adipose tissue in aged and young mice may provide insight into metastatic mechanisms. We analyzed mesenteric, omental, and uterine adipose tissue groups from the peritoneal cavities of young and aged C57BL/6J mouse cohorts with a low-fraction SDS-PAGE gelLC-MS/MS method. We identified 2308 protein groups and quantified 2167 groups, among which several protein groups showed twofold or greater abundance differences between the aged and young cohorts. Cancer-related gene products previously identified as significant in another age-related study were found altered in this study. Several gene products known to suppress proliferation and cellular invasion were found downregulated in the aged cohort, including R-Ras, Arid1a, and heat shock protein ß1. In addition, multiple protein groups were identified within single cohorts, including the proteins Cd11a, Stat3, and Ptk2b. These data suggest that adipose tissue is a strong candidate for analysis to identify possible contributors to cancer metastasis in older subjects. The results of this study, the first of its kind using uterine adipose tissue, contribute to the understanding of the role of adipose tissue in age-related alteration of oncogenic pathways, which may help elucidate the mechanisms of increased metastatic tumor burden in the aged. Graphical abstract We analyzed mesenteric, omental, and uterine adipose tissue groups from the peritoneal cavities of young and aged C57BL/6J mouse cohorts with a low-fraction SDS-PAGE gelLC-MS/MS method. These fat depots are preferential sites for many peritoneal cancers. The results of this study, the first of its kind using uterine adipose tissue, contribute to the understanding of the role of adipose tissue in age-related alteration of oncogenic pathways, which may help elucidate the mechanisms of increased metastatic tumor burden in the aged.

Multicellular Tumor Spheroids Combined with Mass Spectrometric Histone Analysis To Evaluate Epigenetic Drugs.

Multicellular tumor spheroids (MCTS) are valuable in vitro tumor models frequently used to evaluate the penetration and efficacy of therapeutics. In this study, we evaluated potential differences in epigenetic markers, i.e., histone post-translational modifications (PTMs), in the layers of the HCT116 colon carcinoma MCTS. Cells were grown in agarose-coated 96 well plates, forming reproducible 1-mm-diameter MCTS. The MCTS were fractionated into three radially concentric portions, generating samples containing cells from the core, the mid and the external layers. Using mass spectrometry (MS)-based proteomics and EpiProfile, we quantified hundreds of histone peptides in different modified forms; by combining the results of all experiments, we quantified the abundance of 258 differently modified peptides, finding significant differences in their relative abundance across layers. Among these differences, we detected higher amounts of the repressive mark H3K27me3 in the external layers, compared to the core. We then evaluated the epigenetic response of MCTS following UNC1999 treatment, a drug targeting the enzymes that catalyze H3K27me3, namely, the polycomb repressive complex 2 (PRC2) subunits enhancer of zeste 1 (EZH1) and enhancer of zeste 2 (EZH2). UNC1999 treatment resulted in significant differences in MCTS diameter under drug treatment of varying duration. Using matrix-assisted laser desorption/ionization (MALDI) imaging, we determined that the drug penetrates the entire MCTS. Proteomic analysis revealed a decrease in abundance of H3K27me3, compared to the untreated sample, as expected. Interestingly, we observed a comparable growth curve for MCTS under constant drug treatment over 13 days with those treated for only 4 days at the beginning of their growth. We thus demonstrate that MS-based proteomics can define significant differences in histone PTM patterns in submillimetric layers of three-dimensional (3D) cultures. Moreover, we show that our model is suitable for monitoring drug localization and regulation of histone PTMs after drug treatment.

Insights into the adaptive response of Arabidopsis thaliana to prolonged thermal stress by ribosomal profiling and RNA-Seq.

BACKGROUND: Environmental stress puts organisms at risk and requires specific stress-tailored responses to maximize survival. Long-term exposure to stress necessitates a global reprogramming of the cellular activities at different levels of gene expression. RESULTS: Here, we use ribosome profiling and RNA sequencing to globally profile the adaptive response of Arabidopsis thaliana to prolonged heat stress. To adapt to long heat exposure, the expression of many genes is modulated in a coordinated manner at a transcriptional and translational level. However, a significant group of genes opposes this trend and shows mainly translational regulation. Different secondary structure elements are likely candidates to play a role in regulating translation of those genes. CONCLUSIONS: Our data also uncover on how the subunit stoichiometry of multimeric protein complexes in plastids is maintained upon heat exposure.

Bottom-up proteomic analysis of single HCT 116 colon carcinoma multicellular spheroids.

RATIONALE: Proteomic analysis of single multicellular spheroids has not been previously reported. As three-dimensional cell cultures are an increasingly popular model system for biological research, there is interest in obtaining proteomic profiles of these samples. We investigated the proteome of single HCT 116 multicellular spheroids using protocols optimized for small sample sizes. METHODS: Six biological replicates were analyzed via microscopy for size. Total protein content was assessed via the bicinchoninic acid assay (BCA assay). Five separate biological replicate spheroids were analyzed via mass spectrometry in technical duplicate. An ultra-performance liquid chromatography (UPLC) system coupled with an LTQ Orbitrap Velos was used for peptide separation, analysis, and identification. RESULTS: The average diameter of six replicate HCT 116 spheroids was 940 ± 30 µm and the average total protein amount was determined to be 39 ± 4 µg. At least 1300 protein groups were identified in each single LC/MS/MS run with 10% of the material from each single spheroid loaded. Database search results showed variation between spheroid protein group identifications. Pearson correlations show that the disparity in identifications is due to random variations in spectra and protocol. CONCLUSIONS: We detected more than 1350 protein groups in each replicate HCT 116 spheroid. While some variation was detected between replicates, differences in the number of protein groups identified were determined to be the result of random variations in mass spectra acquisition.

Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples.

Proteins regulate many cellular functions and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. However, with mass-limited samples like tumor biopsies, it can be challenging to obtain sufficient amounts of proteins to generate high-quality mass spectrometric data. Techniques developed for macroscale quantities recover sufficient amounts of protein from milligram quantities of starting material, but sample losses become crippling with these techniques when only microgram amounts of material are available. To combat this challenge, proteomicists have developed micro-scale techniques that are compatible with decreased sample size (100 µg or lower) and still enable excellent proteome coverage. Extraction, contaminant removal, protein quantitation, and sample handling techniques for the microgram protein range are reviewed here, with an emphasis on liquid chromatography and bottom-up mass spectrometry-compatible techniques. Also, a range of biological specimens, including mammalian tissues and model cell culture systems, are discussed.

Thermal diffusivity imaging with the thermal lens microscope.

A coaxial thermal lens microscope was used to generate images based on both the absorbance and thermal diffusivity of histological samples. A pump beam was modulated at frequencies ranging from 50 kHz to 5 MHz using an acousto-optic modulator. The pump and a CW probe beam were combined with a dichroic mirror, directed into an inverted microscope, and focused onto the specimen. The change in the transmitted probe beam's center intensity was detected with a photodiode. The photodiode's signal and a reference signal from the modulator were sent to a high-speed lock-in amplifier. The in-phase and quadrature signals were recorded as a sample was translated through the focused beams and used to generate images based on the amplitude and phase of the lock-in amplifier's signal. The amplitude is related to the absorbance and the phase is related to the thermal diffusivity of the sample. Thin sections of stained liver and bone tissues were imaged; the contrast and signal-to-noise ratio of the phase image was highest at frequencies from 0.1-1 MHz and dropped at higher frequencies. The spatial resolution was 2.5 µm for both amplitude and phase images, limited by the pump beam spot size.

Chemical Analysis of Morphological Changes in Lysophosphatidic Acid-Treated Ovarian Cancer Cells.

Ovarian cancer (OvCa) cells are reported to undergo biochemical changes at the cell surface in response to treatment with lysophosphatidic acid (LPA). Here we use scanning electron microscopy (SEM) and multiplex coherent anti-Stokes Raman scattering (CARS) imaging via supercontinuum excitation to probe morphological changes that result from LPA treatment. SEM images show distinct shedding of microvilli-like features upon treatment with LPA. Analysis of multiplex CARS images can distinguish between molecular components, such as lipids and proteins. Our results indicate that OvCa429 and SKOV3ip epithelial ovarian cancer cells undergo similar morphological and chemical responses to treatment with LPA. The microvilli-like structures on the surface of multicellular aggregates (MCAs) are removed by treatment with LPA. The CARS analysis shows a distinct decrease in protein and increase in lipid composition on the surface of LPA-treated cells. Importantly, the CARS signals from cellular sheddings from MCAs with LPA treatment are consistent with cleavage of proteins originally present. Mass spectrometry on the cellular sheddings show that a large number of proteins, both membrane and intracellular, are present. An increased number of peptides are detected for the mesenchymal cell line relative to the epithelial cell indicating a differential response to LPA treatment with cancer progression.

Bacteria differently regulate mRNA abundance to specifically respond to various stresses.

Environmental stress is detrimental to cell viability and requires an adequate reprogramming of cellular activities to maximize cell survival. We present a global analysis of the response of Escherichia coli to acute heat and osmotic stress. We combine deep sequencing of total mRNA and ribosome-protected fragments to provide a genome-wide map of the stress response at transcriptional and translational levels. For each type of stress, we observe a unique subset of genes that shape the stress-specific response. Upon temperature upshift, mRNAs with reduced folding stability up- and downstream of the start codon, and thus with more accessible initiation regions, are translationally favoured. Conversely, osmotic upshift causes a global reduction of highly translated transcripts with high copy numbers, allowing reallocation of translation resources to not degraded and newly synthesized mRNAs.