RESUMO
BACKGROUND & AIMS: Gastric carcinogenesis develops within a sequential carcinogenic cascade from precancerous metaplasia to dysplasia and adenocarcinoma, and oncogenic gene activation can drive the process. Metabolic reprogramming is considered a key mechanism for cancer cell growth and proliferation. However, how metabolic changes contribute to the progression of metaplasia to dysplasia remains unclear. We have examined metabolic dynamics during gastric carcinogenesis using a novel mouse model that induces Kras activation in zymogen-secreting chief cells. METHODS: We generated a Gif-rtTA;TetO-Cre;KrasG12D (GCK) mouse model that continuously induces active Kras expression in chief cells after doxycycline treatment. Histologic examination and imaging mass spectrometry were performed in the GCK mouse stomachs at 2 to 14 weeks after doxycycline treatment. Mouse and human gastric organoids were used for metabolic enzyme inhibitor treatment. The GCK mice were treated with a stearoyl- coenzyme A desaturase (SCD) inhibitor to inhibit the fatty acid desaturation. Tissue microarrays were used to assess the SCD expression in human gastrointestinal cancers. RESULTS: The GCK mice developed metaplasia and high-grade dysplasia within 4 months. Metabolic reprogramming from glycolysis to fatty acid metabolism occurred during metaplasia progression to dysplasia. Altered fatty acid desaturation through SCD produces a novel eicosenoic acid, which fuels dysplastic cell hyperproliferation and survival. The SCD inhibitor killed both mouse and human dysplastic organoids and selectively targeted dysplastic cells in vivo. SCD was up-regulated during carcinogenesis in human gastrointestinal cancers. CONCLUSIONS: Active Kras expression only in gastric chief cells drives the full spectrum of gastric carcinogenesis. Also, oncogenic metabolic rewiring is an essential adaptation for high-energy demand in dysplastic cells.
Assuntos
Metabolismo Energético , Ácidos Graxos , Metaplasia , Organoides , Proteínas Proto-Oncogênicas p21(ras) , Neoplasias Gástricas , Animais , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/genética , Humanos , Ácidos Graxos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Organoides/metabolismo , Camundongos , Modelos Animais de Doenças , Carcinogênese/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Celulas Principais Gástricas/metabolismo , Celulas Principais Gástricas/patologia , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Transformação Celular Neoplásica/genética , Camundongos Transgênicos , Glicólise , Adenocarcinoma/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/genética , Progressão da Doença , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/genéticaRESUMO
Mice subjected to cold or caloric deprivation can reduce body temperature and metabolic rate and enter a state of torpor. Here we show that administration of pyruvate, an energy-rich metabolic intermediate, can induce torpor in mice with diet-induced or genetic obesity. This is associated with marked hypothermia, decreased activity, and decreased metabolic rate. The drop in body temperature correlates with the degree of obesity and is blunted by housing mice at thermoneutrality. Induction of torpor by pyruvate in obese mice relies on adenosine signaling and is accompanied by changes in brain levels of hexose bisphosphate and GABA as detected by mass spectroscopy-based imaging. Pyruvate does not induce torpor in lean mice but results in the activation of brown adipose tissue (BAT) with an increase in the level of uncoupling protein-1 (UCP1). Denervation of BAT in lean mice blocks this increase in UCP1 and allows the pyruvate-induced torpor phenotype. Thus, pyruvate administration induces torpor in obese mice by pathways involving adenosine and GABA signaling and a failure of normal activation of BAT.
Assuntos
Tecido Adiposo Marrom/metabolismo , Obesidade/fisiopatologia , Ácido Pirúvico , Torpor/fisiologia , Proteína Desacopladora 1/metabolismo , Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Resistência à Insulina , Masculino , Camundongos Endogâmicos C57BL , Camundongos ObesosRESUMO
BACKGROUND & AIMS: A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the presence of a dense desmoplastic reaction (stroma) that impedes drug delivery to the tumor. Attempts to deplete the tumor stroma have resulted in formation of more aggressive tumors. We have identified signal transducer and activator of transcription (STAT) 3 as a biomarker of resistance to cytotoxic and molecularly targeted therapy in PDAC. The purpose of this study is to investigate the effects of targeting STAT3 on the PDAC stroma and on therapeutic resistance. METHODS: Activated STAT3 protein expression was determined in human pancreatic tissues and tumor cell lines. In vivo effects of AZD1480, a JAK/STAT3 inhibitor, gemcitabine or the combination were determined in Ptf1a(cre/+);LSL-Kras(G12D/+);Tgfbr2(flox/flox) (PKT) mice and in orthotopic tumor xenografts. Drug delivery was analyzed by matrix-assisted laser desorption/ionization imaging mass spectrometry. Collagen second harmonic generation imaging quantified tumor collagen alignment and density. RESULTS: STAT3 activation correlates with decreased survival and advanced tumor stage in patients with PDAC. STAT3 inhibition combined with gemcitabine significantly inhibits tumor growth in both an orthotopic and the PKT mouse model of PDAC. This combined therapy attenuates in vivo expression of SPARC, increases microvessel density, and enhances drug delivery to the tumor without depletion of stromal collagen or hyaluronan. Instead, the PDAC tumors demonstrate vascular normalization, remodeling of the tumor stroma, and down-regulation of cytidine deaminase. CONCLUSIONS: Targeted inhibition of STAT3 combined with gemcitabine enhances in vivo drug delivery and therapeutic response in PDAC. These effects occur through tumor stromal remodeling and down-regulation of cytidine deaminase without depletion of tumor stromal content.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Microambiente Tumoral , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Colágeno/metabolismo , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Humanos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Terapia de Alvo Molecular , Estadiamento de Neoplasias , Osteonectina/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Pirazóis/metabolismo , Pirimidinas/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Estromais/metabolismo , Células Estromais/patologia , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) elucidates molecular distributions in thin tissue sections. Absolute pixel-to-pixel quantitation has remained a challenge, primarily lacking validation of the appropriate analytical methods. In the present work, isotopically labeled internal standards are applied to tissue sections to maximize quantitative reproducibility and yield accurate quantitative results. We have developed a tissue model for rifampicin (RIF), an antibiotic used to treat tuberculosis, and have tested different methods of applying an isotopically labeled internal standard for MALDI IMS analysis. The application of the standard and subsequently the matrix onto tissue sections resulted in quantitation that was not statistically significantly different from results obtained using HPLC-MS/MS of tissue extracts. Quantitative IMS experiments were performed on liver tissue from an animal dosed in vivo. Each microspot in the quantitative images measures the local concentration of RIF in the thin tissue section. Lower concentrations were detected from the blood vessels and around the portal tracts. The quantitative values obtained from these measurements were comparable (>90% similarity) to HPLC-MS/MS results obtained from extracts of the same tissue.
Assuntos
Fígado/química , Rifampina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Cromatografia Líquida de Alta Pressão , RatosRESUMO
Helicobacter pylori colonization of the human stomach is a strong risk factor for gastric cancer. To investigate H. pylori-induced gastric molecular alterations, we used a Mongolian gerbil model of gastric carcinogenesis. Histologic evaluation revealed varying levels of atrophic gastritis (a premalignant condition characterized by parietal and chief cell loss) in H. pylori-infected animals, and transcriptional profiling revealed a loss of markers for these cell types. We then assessed the spatial distribution and relative abundance of proteins in the gastric tissues using imaging mass spectrometry and liquid chromatography with tandem mass spectrometry. We detected striking differences in the protein content of corpus and antrum tissues. Four hundred ninety-two proteins were preferentially localized to the corpus in uninfected animals. The abundance of 91 of these proteins was reduced in H. pylori-infected corpus tissues exhibiting atrophic gastritis compared with infected corpus tissues exhibiting non-atrophic gastritis or uninfected corpus tissues; these included numerous proteins with metabolic functions. Fifty proteins localized to the corpus in uninfected animals were diffusely delocalized throughout the stomach in infected tissues with atrophic gastritis; these included numerous proteins with roles in protein processing. The corresponding alterations were not detected in animals infected with a H. pylori ∆cagT mutant (lacking Cag type IV secretion system activity). These results indicate that H. pylori can cause loss of proteins normally localized to the gastric corpus as well as diffuse delocalization of corpus-specific proteins, resulting in marked changes in the normal gastric molecular partitioning into distinct corpus and antrum regions.IMPORTANCEA normal stomach is organized into distinct regions known as the corpus and antrum, which have different functions, cell types, and gland architectures. Previous studies have primarily used histologic methods to differentiate these regions and detect H. pylori-induced alterations leading to stomach cancer. In this study, we investigated H. pylori-induced gastric molecular alterations in a Mongolian gerbil model of carcinogenesis. We report the detection of numerous proteins that are preferentially localized to the gastric corpus but not the antrum in a normal stomach. We show that stomachs with H. pylori-induced atrophic gastritis (a precancerous condition characterized by the loss of specialized cell types) exhibit marked changes in the abundance and localization of proteins normally localized to the gastric corpus. These results provide new insights into H. pylori-induced gastric molecular alterations that are associated with the development of stomach cancer.
Assuntos
Gastrite Atrófica , Gastrite , Infecções por Helicobacter , Helicobacter pylori , Lesões Pré-Cancerosas , Neoplasias Gástricas , Animais , Humanos , Gastrite Atrófica/induzido quimicamente , Neoplasias Gástricas/patologia , Gerbillinae , Mucosa Gástrica/patologia , Gastrite/patologia , Atrofia/patologia , Infecções por Helicobacter/complicações , Lesões Pré-Cancerosas/patologia , Carcinogênese/patologiaRESUMO
Oxidation of membrane phospholipids is associated with inflammation, neurodegenerative disease, and cancer. Oxyradical damage to phospholipids results in the production of reactive aldehydes that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme that catalyzes the conversion of the peptide bond of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael adducts at the active site residues His-157 and Cys-113. Time and concentration dependencies indicate that Cys-113 is the primary site of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and Western blotting with anti-Pin1 antibody. Furthermore, orbitrap MS data support the adduction of Cys-113 in the Pin1 active site upon HNE treatment of MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.
Assuntos
Aldeídos/metabolismo , Neoplasias da Mama/metabolismo , Peptidilprolil Isomerase/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células , Química Click , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peroxidação de Lipídeos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , RNA Interferente Pequeno/genéticaRESUMO
The mouse cecum has emerged as a model system for studying microbe-host interactions, immunoregulatory functions of the microbiome, and metabolic contributions of gut bacteria. Too often, the cecum is falsely considered as a uniform organ with an evenly distributed epithelium. We developed the cecum axis (CecAx) preservation method to show gradients in epithelial tissue architecture and cell types along the cecal ampulla-apex and mesentery-antimesentery axes. We used imaging mass spectrometry of metabolites and lipids to suggest functional differences along these axes. Using a model of Clostridioides difficile infection, we show how edema and inflammation are unequally concentrated along the mesenteric border. Finally, we show the similarly increased edema at the mesenteric border in two models of Salmonella enterica serovar Typhimurium infection as well as enrichment of goblet cells along the antimesenteric border. Our approach facilitates mouse cecum modeling with detailed attention to inherent structural and functional differences within this dynamic organ.
Assuntos
Microbioma Gastrointestinal , Animais , Camundongos , Ceco , Epitélio , Células Caliciformes , Interações entre Hospedeiro e MicrorganismosRESUMO
Adequate oxygen delivery to the heart during stress is essential for sustaining cardiac function. Acute increases in myocardial oxygen demand evoke coronary vasodilation and enhance perfusion via functional upregulation of smooth muscle voltage-gated K+ (Kv) channels. Because this response is controlled by Kv1 accessory subunits (i.e., Kvß), which are NAD(P)(H)-dependent aldo-keto reductases, we tested the hypothesis that oxygen demand modifies arterial [NAD(H)]i, and that resultant cytosolic pyridine nucleotide redox state influences Kv1 activity. High-resolution imaging mass spectrometry and live-cell imaging reveal cardiac workload-dependent increases in NADH:NAD+ in intramyocardial arterial myocytes. Intracellular NAD(P)(H) redox ratios reflecting elevated oxygen demand potentiate native coronary Kv1 activity in a Kvß2-dependent manner. Ablation of Kvß2 catalysis suppresses redox-dependent increases in Kv1 activity, vasodilation, and the relationship between cardiac workload and myocardial blood flow. Collectively, this work suggests that the pyridine nucleotide sensitivity and enzymatic activity of Kvß2 controls coronary vasoreactivity and myocardial blood flow during metabolic stress.
Assuntos
NAD , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Músculo Liso , Nucleotídeos , Oxirredução , Oxigênio , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , PiridinasRESUMO
Global efforts aimed at preventing human immunodeficiency virus type one (HIV-1) infection in vulnerable populations appear to be stalling, limiting our ability to control the epidemic. Long-acting, controlled drug administration from subdermal implants holds significant potential by reducing the compliance burden associated with frequent dosing. We, and others, are exploring the development of complementary subdermal implant technologies delivering the potent prodrug, tenofovir alafenamide (TAF). The current report addresses knowledge gaps in the preclinical pharmacology of long-acting, subdermal TAF delivery using several mouse models. Systemic drug disposition during TAF implant dosing was explained by a multi-compartment pharmacokinetic (PK) model. Imaging mass spectrometry was employed to characterize the spatial distribution of TAF and its principal five metabolites in local tissues surrounding the implant. Humanized mouse studies determined the effective TAF dose for preventing vaginal and rectal HIV-1 acquisition. Our results represent an important step in the development of a safe and effective TAF implant for HIV-1 prevention.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Adenina , Alanina/uso terapêutico , Animais , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controle , Camundongos , Tenofovir/análogos & derivados , Tenofovir/uso terapêuticoRESUMO
The metabolic dependencies of cancer cells have substantial potential to be exploited to improve the diagnosis and treatment of cancer. Creatine riboside (CR) is identified as a urinary metabolite associated with risk and prognosis in lung and liver cancer. However, the source of high CR levels in patients with cancer as well as their implications for the treatment of these aggressive cancers remain unclear. By integrating multiomics data on lung and liver cancer, we have shown that CR is a cancer cell-derived metabolite. Global metabolomics and gene expression analysis of human tumors and matched liquid biopsies, together with functional studies, revealed that dysregulation of the mitochondrial urea cycle and a nucleotide imbalance were associated with high CR levels and indicators of a poor prognosis. This metabolic phenotype was associated with reduced immune infiltration and supported rapid cancer cell proliferation that drove aggressive tumor growth. CRhi cancer cells were auxotrophic for arginine, revealing a metabolic vulnerability that may be exploited therapeutically. This highlights the potential of CR not only as a poor-prognosis biomarker but also as a companion biomarker to inform the administration of arginine-targeted therapies in precision medicine strategies to improve survival for patients with cancer.
Assuntos
Neoplasias Hepáticas , Ribonucleosídeos , Arginina/metabolismo , Creatina/análogos & derivados , Creatina/urina , Humanos , Ribonucleosídeos/urinaRESUMO
Analysis and quantification of analytes in biological systems is a critical component of metabolomic investigations of cell function. The most widely used methods employ chromatographic separation followed by mass spectrometric analysis, which requires significant time for sample preparation and sequential chromatography. We introduce a novel high-throughput, separation-free methodology based on MALDI mass spectrometry that allows for the parallel analysis of targeted metabolomes. Proof-of-concept is demonstrated by analysis of prostaglandins and glyceryl prostaglandins. Derivatization to incorporate a charged moiety into ketone-containing prostaglandins dramatically increases the signal-to-noise ratio relative to underivatized samples. This resulted in an increased dynamic range (15-2000 fmol on plate) and improved linearity (r(2) = 0.99). The method was adapted for high-throughput screening methods for enzymology and drug discovery. Application to cellular metabolomics was also demonstrated.
Assuntos
Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Ensaios de Triagem em Larga Escala , Cetonas/química , Metaboloma , Camundongos , Prostaglandinas/análiseRESUMO
Helicobacter pylori colonization of the stomach is a strong risk factor for the development of stomach cancer and peptic ulcer disease. In this study, we tested the hypothesis that H. pylori infection triggers alterations in gastric lipid composition. Mongolian gerbils were experimentally infected with H. pylori for 3 months. Conventional histologic staining revealed mucosal inflammation in stomachs from the H. pylori-infected animals but not in stomachs from uninfected control animals. Atrophic gastritis (a premalignant condition characterized by loss of corpus-specific parietal and chief cells), gastric mucosal hyperplasia, dysplasia, and/or gastric cancer were detected in stomachs from several infected animals. We then used imaging mass spectrometry to analyze the relative abundance and spatial distribution of gastric lipids. We detected ions corresponding to 36 distinct lipids that were differentially abundant when comparing gastric tissues from H. pylori-infected animals with tissues from uninfected animals. Liquid chromatography-tandem mass spectrometry analysis of lipid extracts from homogenized gastric tissues provided additional supportive evidence for the identification of several differentially abundant lipids. Sixteen of the differentially abundant lipids were localized mainly to the gastric corpus in stomachs from uninfected animals and were markedly reduced in abundance in stomachs from H. pylori-infected animals with severe disease (atrophic gastritis and dysplasia or gastric cancer). These findings indicate that H. pylori infection can lead to alterations in gastric lipid composition and constitute a new approach for identifying biomarkers of gastric atrophy and premalignant changes. IMPORTANCE H. pylori colonization of the stomach triggers a cascade of gastric alterations that can potentially culminate in stomach cancer. The molecular alterations that occur in gastric tissue prior to development of stomach cancer are not well understood. We demonstrate here that H. pylori-induced premalignant changes in the stomach are accompanied by extensive alterations in gastric lipid composition. These alterations are predicted to have important functional consequences relevant to H. pylori-host interactions and the pathogenesis of gastric cancer.
Assuntos
Gastrite Atrófica/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori , Neoplasias Gástricas/etiologia , Animais , Modelos Animais de Doenças , Gastrite Atrófica/patologia , Gerbillinae , Metabolismo dos Lipídeos , Masculino , Estômago/patologiaRESUMO
To date, proteomic analyses on gastrointestinal cancer tissue samples have been performed using surgical specimens only, which are obtained after a diagnosis is made. To determine if a proteomic signature obtained from endoscopic biopsy samples could be found to assist with diagnosis, frozen endoscopic biopsy samples collected from 63 gastric cancer patients and 43 healthy volunteers were analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. A statistical classification model was developed to distinguish tumor from normal tissues using half the samples and validated with the other half. A protein profile was discovered consisting of 73 signals that could classify 32 cancer and 22 normal samples in the validation set with high predictive values (positive and negative predictive values for cancer, 96.8% and 91.3%; sensitivity, 93.8%; specificity, 95.5%). Signals overexpressed in tumors were identified as alpha-defensin-1, alpha-defensin-2, calgranulin A, and calgranulin B. A protein profile was also found to distinguish pathologic stage Ia (pT1N0M0) samples (n = 10) from more advanced stage (Ib or higher) tumors (n = 48). Thus, protein profiles obtained from endoscopic biopsy samples may be useful in assisting with the diagnosis of gastric cancer and, possibly, in identifying early stage disease.
Assuntos
Biomarcadores Tumorais/análise , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/metabolismo , Biópsia , Estudos de Casos e Controles , Defensinas/metabolismo , Gastroscopia/métodos , Humanos , Complexo Antígeno L1 Leucocitário/metabolismo , Modelos Estatísticos , Sensibilidade e Especificidade , Neoplasias Gástricas/patologiaRESUMO
Clear cell renal cell carcinoma (ccRCC) and chromophobe renal cell carcinoma (chRCC) are relatively common tumors that can have significant risk for mortality. Treatment and prognostication in renal cell carcinoma (RCC) are dependent upon correct histologic typing. ccRCC and chRCC are generally straightforward to diagnose based on histomorphology alone. However, high-grade ccRCC and chRCC can sometimes resemble each other morphologically, particularly in small biopsies. Multiple immunostains and/or colloidal iron stain are sometimes required to differentiate the two. Imaging mass spectrometry (IMS) allows simultaneous spatial mapping of thousands of biomarkers, using formalin-fixed paraffin-embedded tissue sections. In this study, we evaluate the ability of IMS to differentiate between World Health Organization/International Society for Urological Pathology grade 3 ccRCC and chRCC. IMS spectra from a training set of 14 ccRCC and 13 chRCC were evaluated via support vector machine algorithm with a linear kernel for machine learning, building a classification model. The classification model was applied to a separate validation set of 6 ccRCC and 6 chRCC, with 19 to 20, 150-µm diameter tumor foci in each case sampled by IMS. Most evaluated tumor foci were classified correctly as ccRCC versus chRCC (99% accuracy, kappa=0.98), demonstrating that IMS is an accurate tool in differentiating high-grade ccRCC and chRCC.
Assuntos
Carcinoma de Células Renais/diagnóstico por imagem , Neoplasias Renais/diagnóstico por imagem , Espectrometria de Massas , Imagem Molecular , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Distinguishing low-grade phyllodes tumor from fibroadenoma is practically challenging due to their overlapping histologic features. However, the final interpretation is essential to surgeons, who base their management on the final pathology report. Patients who receive a diagnosis of fibroadenoma might not undergo any additional intervention while lumpectomy with wide margins is the standard of care for phyllodes tumor, which can have significant cosmetic consequences. We studied the clinical, immunophenotypic, and proteomics profiles of 31 histologically confirmed low-grade phyllodes tumor and 30 fibroadenomas. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) and immunohistochemistry for Ki-67, p53, ß-catenin, and E-cadherin were performed on all cases. After the mass spectra for all 31 cases of low-grade phyllodes tumor and 30 cases of fibroadenoma were collected, an average peak value for all cases was generated. There was no significant difference in the overall mass spectra pattern in any of the peaks identified. There was also overlap in the percentage of cells staining positive for Ki-67, p53, ß-catenin, and E-cadherin. The two groups of patients showed no statistically significant difference in age, tumor size, or disease-free survival. Neither group developed malignant transformation, distant metastases, or disease-related mortality. We have demonstrated low-grade phyllodes tumor and fibroadenoma to show significant overlapping clinical and proteomics features.
RESUMO
Lack of durable response to cytotoxic chemotherapy is a major contributor to the dismal outcomes seen in pancreatic ductal adenocarcinoma (PDAC). Extensive tumor desmoplasia and poor vascular supply are two predominant characteristics which hinder the delivery of chemotherapeutic drugs into PDAC tumors and mediate resistance to therapy. Previously, we have shown that STAT3 is a key biomarker of therapeutic resistance to gemcitabine treatment in PDAC, which can be overcome by combined inhibition of the Src and EGFR pathways. Although it is well-established that concurrent EGFR and Src inhibition exert these antineoplastic properties through direct inhibition of mitogenic pathways in tumor cells, the influence of this combined therapy on stromal constituents in PDAC tumors remains unknown. In this study, we demonstrate in both orthotopic tumor xenograft and Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mouse models that concurrent EGFR and Src inhibition abrogates STAT3 activation, increases microvessel density, and prevents tissue fibrosis in vivo. Furthermore, the stromal changes induced by parallel EGFR and Src pathway inhibition resulted in improved overall survival in PKT mice when combined with gemcitabine. As a phase I clinical trial utilizing concurrent EGFR and Src inhibition with gemcitabine has recently concluded, these data provide timely translational insight into the novel mechanism of action of this regimen and expand our understanding into the phenomenon of stromal-mediated therapeutic resistance. IMPLICATIONS: These findings demonstrate that Src/EGFR inhibition targets STAT3, remodels the tumor stroma, and results in enhanced delivery of gemcitabine to improve overall survival in a mouse model of PDAC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/metabolismo , Quinases da Família src/antagonistas & inibidores , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Dasatinibe/administração & dosagem , Dasatinibe/farmacologia , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/administração & dosagem , Cloridrato de Erlotinib/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/metabolismo , GencitabinaRESUMO
Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.
Assuntos
Barreira Hematoencefálica , Iduronato Sulfatase , Animais , Encéfalo , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Lisossomos , CamundongosRESUMO
The direct analysis of tissues using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) enables both endogenous and exogenous compounds present in tissues to be detected with molecular specificity while maintaining their spatial orientation. This unique combination, coupled with excellent sensitivity and rapid analysis time, presents many potential advantages to a wide range of applications in diverse biological fields. Recent advances have shown how the technique can be applied to cancer research, neuroscience and pharmaceutical development. Examples include the use of unique protein profiles to classify human tumor tissues and predict patient outcomes, the discovery of protein changes in mouse cerebellum as a function of development, and the two-dimensional visualization of the distribution of a drug and first-pass metabolites in rat whole-body sections.
Assuntos
Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Extratos de Tecidos/análise , Animais , Biomarcadores/análise , Cerebelo/química , Cerebelo/metabolismo , Cerebelo/patologia , Humanos , Camundongos , Neoplasias/química , Neoplasias/metabolismo , Neoplasias/patologia , Neurociências/métodos , Preparações Farmacêuticas/análise , Preparações Farmacêuticas/química , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Ratos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/tendências , Tecnologia Farmacêutica/métodos , Distribuição Tecidual , Extratos de Tecidos/química , Extratos de Tecidos/metabolismoRESUMO
Exposure to noise or ototoxic agents can result in degeneration of cells in the sensory epithelium and auditory nerve, as well as non-sensory cells of the cochlear lateral wall. However, the molecular mechanisms underlying this pathology remain unclear. The purpose of this study was to localize and identify proteins in the cochlea that are responsive to noise or ototoxic exposure using a complementary proteo-transcriptomic approach. MALDI imaging of cochlear sections revealed numerous protein signals with distinct cochlear localization patterns in both cochlear injury models, of which six were chosen for further investigation. A query of proteomic databases identified 709 candidates corresponding to m/z values for the six proteins. An evaluation of mRNA expression data from our previous studies of these injured models indicated that 208 of the candidates were affected in both injury models. Downstream validation analyses yielded proteins with confirmatory distributions and responses to injury. The combined analysis of MALDI imaging with gene expression data provides a new strategy to identify molecular regulators responsive to cochlear injury. This study demonstrates the applicability of MALDI imaging for investigating protein localization and abundance in frozen sections from animals modeling cochlear pathology.