A MALDI Mass Spectrometry Imaging Sample Preparation Method for Venous Thrombosis with Initial Lipid Characterization of Lab-Made and Murine Clots.

Venous thromboembolism (VTE) and its complications affect over 900,000 people in the U.S. annually, with a third of cases resulting in fatality. Despite such a high incidence rate, venous thrombosis research has not led to significant changes in clinical treatments, with standard anti-coagulant therapy (heparin followed by a vitamin K antagonist) being used since the 1950s. Mechanical thrombectomy is an alternative strategy for treating venous thrombosis; however, clinical guidelines for patient selection have not been well-established or accepted. The effectiveness of both treatments is impacted by the heterogeneity of the thrombus, including the mechanical properties of its cellular components and its molecular makeup. A full understanding of the complex interplay between disease initiation and progression, biochemical molecular changes, tissue function, and mechanical properties calls for a multiplex and multiscale approach. In this work, we establish a protocol for using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to characterize spatial heterogeneity of biomolecules in lab-made blood clots and ex vivo murine thrombi. In this work, we compared (1) tissue preservation and cryosectioning methods, (2) various matrixes, 9-aminoacridine hydrochloride monohydrate (9AA), 2,5-dihydroxybenzoic acid (DHB), and alpha-cyano-4-hydroxycinnamic acid matrix (CHCA), (3) plasma-rich versus red-blood-cell rich lab-made blood clots, and (4) lab-made blood clots versus ex vivo murine thrombi. This project is the first step in our work to combine mass spectrometry imaging with biomechanical testing of blood clots to improve our understanding of VTE.

Targeting de novo lipogenesis and the Lands cycle induces ferroptosis in KRAS-mutant lung cancer.

Mutant KRAS (KM), the most common oncogene in lung cancer (LC), regulates fatty acid (FA) metabolism. However, the role of FA in LC tumorigenesis is still not sufficiently characterized. Here, we show that KMLC has a specific lipid profile, with high triacylglycerides and phosphatidylcholines (PC). We demonstrate that FASN, the rate-limiting enzyme in FA synthesis, while being dispensable in EGFR-mutant or wild-type KRAS LC, is required for the viability of KMLC cells. Integrating lipidomic, transcriptomic and functional analyses, we demonstrate that FASN provides saturated and monounsaturated FA to the Lands cycle, the process remodeling oxidized phospholipids, such as PC. Accordingly, blocking either FASN or the Lands cycle in KMLC, promotes ferroptosis, a reactive oxygen species (ROS)- and iron-dependent cell death, characterized by the intracellular accumulation of oxidation-prone PC. Our work indicates that KM dictates a dependency on newly synthesized FA to escape ferroptosis, establishing a targetable vulnerability in KMLC.

Embryonic expression of cyclooxygenase-2 causes malformations in axial skeleton.

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. In this report, the direct effect of COX-2 expression on embryonic development is examined in a novel COX-2 transgenic mouse model that ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired precartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is probably responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos, suggesting that COX-2 may induce apoptosis via the up-regulation of p53. Our results demonstrate that the aberrant COX-2 signaling during embryonic development is teratogenic and suggest a possible association of COX-2 with fetal malformations of unknown etiology.

Combining Chemistry and Engineering for Hepatocellular Carcinoma: Nano-Scale and Smaller Therapies.

Primary liver cancer, or hepatocellular carcinoma (HCC), is a major worldwide cause of death from carcinoma. Most patients are not candidates for surgery and medical therapies, including new immunotherapies, have not shown major improvements since the modest benefit seen with the introduction of sorafenib over a decade ago. Locoregional therapies for intermediate stage disease are not curative but provide some benefit. However, upon close scrutiny, there is still residual disease in most cases. We review the current status for treatment of intermediate stage disease, summarize the literature on correlative histopathology, and discuss emerging methods at micro-, nano-, and pico-scales to improve therapy. These include transarterial hyperthermia methods and thermoembolization, along with microfluidics model systems and new applications of mass spectrometry imaging for label-free analysis of pharmacokinetics and pharmacodynamics.

Cross-sectional areas of deep/core veins are smaller at lower core body temperatures.

The cardiovascular system plays a crucial role in thermoregulation. Deep core veins, due to their large size and role in returning blood to the heart, are an important part of this system. The response of veins to increasing core temperature has not been adequately studied in vivo. Our objective was to noninvasively quantify in C57BL/6 mice the response of artery-vein pairs to increases in body temperature. Adult male mice were anesthetized and underwent magnetic resonance imaging. Data were acquired from three colocalized vessel pairs (the neck [carotid/jugular], torso [aorta/inferior vena cava (IVC)], periphery [femoral artery/vein]) at core temperatures of 35, 36, 37, and 38°C. Cross-sectional area increased with increasing temperature for all vessels, excluding the carotid. Average area of the jugular, aorta, femoral artery, and vein linearly increased with temperature (0.10, 0.017, 0.017, and 0.027 mm2 /°C, respectively; P < 0.05). On average, the IVC has the largest venous response for area (18.2%/°C, vs. jugular 9.0 and femoral 10.9%/°C). Increases in core temperature from 35 to 38 °C resulted in an increase in contact length between the aorta/IVC of 29.3% (P = 0.007) and between the femoral artery/vein of 28.0% (P = 0.03). Previously unidentified increases in the IVC area due to increasing core temperature are biologically important because they may affect conductive and convective heat transfer. Vascular response to temperature varied based on location and vessel type. Leveraging noninvasive methodology to quantify vascular responses to temperature could be combined with bioheat modeling to improve understanding of thermoregulation.

The putative tumor suppressor Tsc-22 is downregulated early in chemically induced hepatocarcinogenesis and may be a suppressor of Gadd45b.

Tsc-22 is a novel tumor suppressor gene that represents a new class of transcription factors that has transcriptional repressor activity. We found Tsc-22 downregulation in livers from B6C3F1 mice following treatment for 2 weeks with carcinogenic doses of the antianxiety drug oxazepam (2500 ppm) or the peroxisome proliferator Wyeth-14,643 (500 ppm) but not with two other carcinogens such as o-nitrotoluene or methyleugenol or three noncarcinogens including p-nitrotoluene, eugenol, or acetaminophen. The expression of Tsc-22 was also repressed in B6C3F1 mouse liver tumors that were induced by several chemicals from 2-year carcinogenicity studies as well as in spontaneous liver tumors. To identify potential Tsc-22 target genes in mouse liver, we transfected small interference RNA (SiRNA) designed to inhibit Tsc-22 into murine liver BNL-CL.2 cells. We selected two potential transcriptional targets of Tsc-22, growth arrest and DNA damage-inducible gene 45 beta (Gadd45b) and leucine zipper, putative tumor suppressor 2 (Lzts2) to test based on our previous complementary DNA microarray studies, showing that expression of these cancer-associated genes was increased when Tsc-22 was repressed. SiRNA treatment of BNL-CL.2 cells with Tsc-22 oligonucleotides but not nonspecific oligonucleotides decreased RNA and protein expression of Tsc-22 by 80-90%, while expression of Gadd45b gene, but not Lzts2, was increased over time after an initial decrease. Treatment of these cells with oxazepam for 48 h also resulted in decreased Tsc-22 and increased Gadd45b expression. These data provide evidence that Tsc-22 is a suppressor of Gadd45b expression, which may contribute to an early antiapoptotic response.

Pol iota is a candidate for the mouse pulmonary adenoma resistance 2 locus, a major modifier of chemically induced lung neoplasia.

In this study, we performed systematic candidate gene analyses of the Pulmonary adenoma resistance 2 locus. Differential gene expression in lung tissues and nucleotide polymorphisms in coding regions between A/J and BALB/cJ mice were examined using reverse transcription-PCR and direct sequencing. Although not all genes in the interval were analyzed at this moment due to the recent database updating, we have found that the Pol iota gene, encoding the DNA polymerase iota, contains 25 nucleotide polymorphisms in its coding region between A/J and BALB/cJ mice, resulting in a total of ten amino acid changes. Primer extension assays with purified BALB/cJ and A/J proteins in vitro demonstrate that both forms of Pol iota are active but that they may differ in substrate discrimination, which may affect the formation of Kras2 mutations in mouse lung tumors. Altered expression of POL iota protein and an amino acid-changing nucleotide polymorphism were observed in human lung cancer cells, suggesting a possible role in the development of lung cancer. Thus, our data support the Pol iota gene as a modifier of lung tumorigenesis by altering DNA polymerase activity.

Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats.

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 cancer-related genes that were categorized into binding activity, growth and proliferation, cell cycle progression, apoptosis, and invasion and metastasis. The microarray data were validated by positive correlation with quantitative real-time RT-PCR on 16 selected genes including igf1, tgfb3 and nov. Important carcinogenic pathways involved in RPM formation included insulin-like growth factor 1 (IGF-1), p38 MAPkinase, Wnt/beta-catenin and integrin signaling pathways. This study demonstrated that mesotheliomas in rats exposed to o-NT- and BCA were similar to mesotheliomas in humans, at least at the cellular and molecular level.

Drug-induced expression of nonsteroidal anti-inflammatory drug-activated gene/macrophage inhibitory cytokine-1/prostate-derived factor, a putative tumor suppressor, inhibits tumor growth.

A common in vitro response for many chemopreventive and antitumor agents, including some cyclooxygenase inhibitors, is the increased expression of nonsteroidal anti-inflammatory drug-activated gene (NAG)-1/macrophage inhibitory cytokine (MIC)-1/prostate-derived factor (PDF). The experimental anticancer drug 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F203) was a potent inducer of NAG-1 expression, and in MCF-7 cells, it inhibited cell growth and induced apoptosis. NAG-1 small interfering RNA blocked NAG-1 expression and 5F203-induced apoptosis in MCF-7 cells, indicating that NAG-1 may mediate the apoptosis and anticancer activity. One mechanism by which 5F203 increases NAG-1 expression is by increasing the stability of NAG-1 mRNA, dependent of de novo protein synthesis. Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was increased by 5F203, and inhibition of ERK1/2 phosphorylation abolished the induction of NAG-1 protein expression and increased the stability of NAG-1 mRNA. Thus, 5F203 regulates NAG-1 expression by a unique mechanism compared with other drugs. A mouse orthotopic mammary tumor model was used to determine whether 5F203 increased NAG-1 expression in vivo and suppressed tumor growth. Treatment of the mice with Phortress, the prodrug of 5F203, increased the in vivo expression of NAG-1 as measured by real-time reverse transcription-polymerase chain reaction from RNA obtained by needle biopsy, and the expression correlated with a reduction of tumor volume. These results confirm that NAG-1 suppresses tumor growth, and its in vivo expression can be controlled by treating mice with anticancer drugs, such as Phortress. Drugs that target NAG-1 could lead to a unique strategy for the development of chemotherapeutic and chemopreventive agents.

Unique patterns of gene expression changes in liver after treatment of mice for 2 weeks with different known carcinogens and non-carcinogens.

Previously we demonstrated that the mouse liver tumor response to the non-genotoxic carcinogens oxazepam and Wyeth-14,643 involved more differences than similarities in changes in early gene expression. In this study we used quantitative real-time PCR and oligonucleotide microarray analysis to identify genes that were up- or down-regulated in mouse liver early after treatment with different known carcinogens, including oxazepam (125 and 2500 p.p.m.), o-nitrotoluene (1250 and 5000 p.p.m.) and methyleugenol (75 mg/kg/day), or the non-carcinogens p-nitrotoluene (5000 p.p.m.), eugenol (75 mg/kg/day) and acetaminophen (6000 p.p.m.). Starting at 6 weeks of age, mice were treated with the different compounds for 2 weeks in the diet, at which time the livers were collected. First, expression of 12 genes found previously to be altered in liver after 2 weeks treatment with oxazepam and/or Wyeth-14,643 was examined in livers from the various chemical treatment groups. These gene expression changes were confirmed for the livers from the oxazepam-treated mice in the present study, but were not good early markers for all the carcinogens in this study. In addition, expression of 20 842 genes was assessed by oligonucleotide microarray [n = 4 livers/group, 2 hybridizations/liver (with fluor reversals)] and the results were analyzed using the Rosetta Resolver System and GeneSpring software. The analyses revealed that several cancer-related genes, including Fhit, Wwox, Tsc-22 and Gadd45b, were induced or repressed in unique patterns for specific carcinogens and not altered by the non-carcinogens. The data indicate that even if the tumor response, including molecular alterations, is similar, such as for oxazepam and methyleugenol, early gene expression changes appear to be carcinogen specific and seem to involve apoptosis and cell cycle-related genes.

Map kinase activation correlates with K-ras mutation and loss of heterozygosity on chromosome 6 in alveolar bronchiolar carcinomas from B6C3F1 mice exposed to vanadium pentoxide for 2 years.

Previous work showed a correlation between K-ras mutation and loss of heterozygosity (LOH) on chromosome 6 in the region of K-ras in lung carcinomas from B6C3F1 mice. We hypothesized that mitogen-activated protein kinase (MAPK) would be activated only in those lung neoplasms with both K-ras mutation and LOH. As MAPK activity can be correlated directly with signal detection using antibodies to phosphorylated MAPK, we were able to analyze lung carcinomas from B6C3F1 mice for the presence or absence of MAPK activity by western analysis. Vanadium pentoxide-induced mouse lung carcinomas, which had been shown to have a high frequency of K-ras mutations and LOH on chromosome 6 and for which frozen tumor tissue was available, were used for this study. Total MAPK expression levels were similar between normal lung and lung carcinomas. Phospho-MAPK was elevated in five of six lung carcinoma samples examined in which K-ras mutations and chromosome 6 LOH were identified and in four of five carcinomas with K-ras mutations that lacked LOH. Phospho-MAPK was undetectable or weakly expressed in seven carcinomas examined without K-ras mutations and in normal lung. By immunohistochemistry three K-ras positive/LOH negative samples exhibited multifocal areas of nuclear and cytoplasmic staining for phospho-MAPK. Large amounts of non-staining fibroblasts, lymphocytes and macrophages were also observed in these tumors. Two of these lung carcinomas were microdissected and chromosome 6 LOH was detected in regions of phospho-MAPK positive cells. These results suggest that MAPK is activated during vanadium pentoxide-induced B6C3F1 mouse lung tumorigenesis following K-ras mutation and loss of the wild-type K-ras allele.

Expression of potential beta-catenin targets, cyclin D1, c-Jun, c-Myc, E-cadherin, and EGFR in chemically induced hepatocellular neoplasms from B6C3F1 mice.

In this study we used liver neoplasms induced by several chemical carcinogens to investigate potential nuclear targets associated with beta-catenin/Wnt signaling and potential membrane-associated beta-catenin binding partners. Strong expression of cyclin D1, in a pattern similar to that observed previously for beta-catenin, was observed by Western analysis for all five hepatoblastomas examined regardless of treatment. Increased expression of cyclin D1 was also detected in 12 of 35 (34%) hepatocellular neoplasms. Ten of 15 tumors (67%) that had mutations in the Catnb gene had upregulation of cyclin D1, while only 2 of 20 tumors (10%) without Catnb mutations had increased cyclin D1 expression. Immunohistochemical analysis confirmed strong expression of cyclin D1 in most nuclei of hepatoblastomas and scattered nuclear staining in hepatocellular tumors that had Catnb mutations. Increased c-Jun expression was observed in 19 of 30 (63%) hepatocellular tumors and all hepatoblastomas, although upregulation was not completely correlated with Catnb mutation. C-Myc expression was not increased in the tumors. Reduced expression of E-cadherin, which interacts with beta-catenin at the membrane, was observed in some tumors, but this did not correlate with Catnb mutation. Expression of the epidermal growth factor receptor, which may have a role in beta-catenin tyrosine phosphorylation, was lower in some tumors than in normal tissue depending on chemical treatment. The results provide evidence that increased expression of cyclin D1 and c-Jun may provide an advantage during tumor progression and in the transition from hepatocellular neoplasms to hepatoblastomas. Moreover, it is likely increased cyclin D1 expression results at least in part from Catnb mutation, beta-catenin accumulation, and increased Wnt signaling.

Predominant K-ras codon 12 G --> A transition in chemically induced lung neoplasms in B6C3F1 mice.

Based on long-term toxicity and carcinogenicity studies in B6C3F1 mice conducted by the National Toxicology Program, 2,2-Bis(bromomethyl)-1,3-propanediol (BMP) and tetranitromethane (TNM) have been identified as carcinogens. Following 2 yr of exposure to 312, 625, or 1,250 ppm BMP in feed, or exposure to 0.5 or 2 ppm TNM by inhalation, increased incidences of lung neoplasms were observed in B6C3F1 mice at all exposure concentrations compared to unexposed mice. The present study characterizes genetic alterations in the K-ras protooncogene in BMP- and TNM-induced lung neoplasms, respectively, and compares the findings to spontaneous lung neoplasms from corresponding control mice. The frequencies of the K-ras mutations were 57% (29/51) in BMP-induced lung neoplasms compared to 15% (3/20) in lung neoplasms from dosed feed control mice, and 54% (14/26) in TNM-induced lung neoplasms compared to 60% (3/5) in lung neoplasms from inhalation control mice. G --> A transitions at the second base of the K-ras codon 12 (GGT --> GAT) were the most frequent pattern of K-ras mutations identified in BMP-induced (20/29) and TNM-induced lung neoplasms (13/14), which differed from the mutational patterns identified in the lung neoplasms from unexposed control mice. These results indicate that mutations in the K-ras gene are involved in B6C3F1 lung carcinogenesis following BMP- and TNM-exposure, and the high frequency and specificity of the ras mutation profile in lung neoplasms (G --> A transition) may be due to in vivo genotoxicity by the parent compounds or their metabolites.

Changes in global gene and protein expression during early mouse liver carcinogenesis induced by non-genotoxic model carcinogens oxazepam and Wyeth-14,643.

We hypothesized that the mouse liver tumor response to non-genotoxic carcinogens would involve some common early gene and protein expression changes that could ultimately be used to predict chemical hepatocarcinogenesis. In order to identify a panel of genes to test, we analyzed global differences in gene and protein expression in livers from B6C3F1 mice following dietary treatment with two rodent carcinogens, the benzodiazepine anti-anxiety drug oxazepam (2500 p.p.m.) and the hypolipidemic agent Wyeth (Wy)-14,643 (500 p.p.m.) compared with livers from untreated mice. Male mice were exposed for 2 weeks and 1, 3 or 6 months to oxazepam or Wy-14,643 in an age-matched study design. By histopathological evaluation, no liver preneoplastic foci or tumors were detected at 6 months in treated or control groups. By cDNA microarray analysis [NIEHS Mouse Chip (8700 genes); n = 3 individual livers/group, four hybridizations/sample], expression of 36 genes or 220 genes were changed relative to control livers following 6 months of oxazepam or Wy-14,643 treatment, respectively. To obtain a more comprehensive picture of gene/protein expression changes, we also conducted a proteomics study by 2D-gel electrophoresis followed by matrix assisted laser desorption/ionization-mass spectrometry on cytoplasmic, nuclear, and microsomal subcellular fractions of the same liver samples utilized for the cDNA microarray analysis. Real-time PCR, western blot analysis and immunohistochemistry were utilized for validation and to expand the results to other time points. Cyp2b20, growth arrest- and damage-inducible gene beta (Gadd45beta), tumor necrosis factor alpha-induced protein 2 and insulin-like growth factor binding protein 1 (Igfbp5) genes and proteins were upregulated by oxazepam, and Cyp2b20, Cyclin D1, proliferating cell nuclear antigen, Igfbp5, Gadd45beta and cell death-inducing DNA fragmentation factor alpha subunit-like effector A exhibited higher expression after Wy-14,643 treatment. Most of these genes/proteins were also deregulated at 2 weeks. There appeared to be more distinct than common changes in the expression of carcinogenesis-related genes/proteins between the two compounds, suggesting that the major carcinogenic pathways are different for these compounds and may be distinct for different chemical classes.