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1.
Artigo em Inglês | MEDLINE | ID: mdl-34607525

RESUMO

LASER: Capture Microdissection (LCM) uses a laser to isolate, or capture, specific cells of interest in a complex heterogeneous tissue section, under direct microscopic visualization. Recently there has been a surge of publications using LCM for tissue spatial molecular profiling relevant to a wide range of research topics. AREAS COVERED: We summarize the many advances in tissue Laser Capture Proteomics (LCP) using mass spectrometry for discovery, and protein arrays for signal pathway network mapping. This review emphasizes: a) Transition of LCM phosphoproteomics from the lab to the clinic for individualized cancer therapy, and b) the emerging frontier of LCM single cell molecular analysis combining proteomics with genomic, and transcriptomic analysis. The search strategy was based on the combination of MeSH terms with expert refinement. EXPERT OPINION: LCM is complemented by a rich set of instruments, methodology protocols, and analytical A.I. (artificial intelligence) software for basic and translational research. Resolution is advancing to the tissue single cell level. A vision for the future evolution of LCM is presented. Emerging LCM technology is combining digital and AI guided remote imaging with automation, and telepathology, to a achieve multi-omic profiling that was not previously possible.

2.
Cancer Med ; 10(20): 7233-7241, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34559451

RESUMO

INTRODUCTION: Preoperative autophagy inhibition with hydroxychloroquine (HCQ) in combination with gemcitabine in pancreatic adenocarcinoma (PDAC) has been shown to be safe and effective in inducing a serum biomarker response and increase resection rates in a previous phase I/II clinical trial. We aimed to analyze the long-term outcomes of preoperative HCQ with gemcitabine for this cohort. METHODS: A review of patients enrolled between July 2010 and February 2013 in the completed phase I/II single arm (two doses of fixed-dose gemcitabine (1500 mg/m2 ) in combination with oral hydroxychloroquine administered for 31 consecutive days until the day of surgery for high-risk pancreatic cancer) was undertaken. Progression-free survival (PFS) and overall survival analysis (OS) using Kaplan-Meier estimates were performed. RESULTS: Of 35 patients initially enrolled, 29 patients underwent surgical resection (median age at diagnosis: 62 years, 45% females). Median duration of follow-up was 7.5 years. There was a median 15% decrease in the serum CA19-9 levels following completion of neoadjuvant therapy and 83% of the cohort underwent a pancreaticoduodenectomy, 7 (24%) patients had a concomitant venous resection. On histopathology, 14 (48%) patients had at least a partial treatment response. The median PFS and OS were 11 months (95% Confidence interval [CI]: 7-28) and 31 months (95% CI: 13-47), respectively, while 9 (31%) patients survived beyond 5 years from diagnosis; a rate that compares very favorably with contemporaneous series. CONCLUSION: Compared to historical data, neoadjuvant autophagy inhibition with HCQ plus gemcitabine is associated with encouraging long-term survival for patients with PDAC.

4.
Med (N Y) ; 2(6): 736-754, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34223403

RESUMO

Background: Upregulated glucose metabolism is a common feature of tumors. Glucose can be broken down by either glycolysis or the oxidative pentose phosphate pathway (oxPPP). The relative usage within tumors of these catabolic pathways remains unclear. Similarly, the extent to which tumors make biomass precursors from glucose, versus take them up from the circulation, is incompletely defined. Methods: We explore human triple negative breast cancer (TNBC) metabolism by isotope tracing with [1,2-13C]glucose, a tracer that differentiates glycolytic versus oxPPP catabolism and reveals glucose-driven anabolism. Patients enrolled in clinical trial NCT03457779 and received IV infusion of [1,2-13C]glucose during core biopsy of their primary TNBC. Tumor samples were analyzed for metabolite labeling by liquid chromatography-mass spectrometry (LC-MS). Genomic and proteomic analyses were performed and related to observed metabolic fluxes. Findings: TNBC ferments glucose to lactate, with glycolysis dominant over the oxPPP. Most ribose phosphate is nevertheless produced by oxPPP. Glucose also feeds amino acid synthesis, including of serine, glycine, aspartate, glutamate, proline and glutamine (but not asparagine). Downstream in glycolysis, tumor pyruvate and lactate labeling exceeds that found in serum, indicating that lactate exchange via monocarboxylic transporters is less prevalent in human TNBC compared with most normal tissues or non-small cell lung cancer. Conclusions: Glucose directly feeds ribose phosphate, amino acid synthesis, lactate, and the TCA cycle locally within human breast tumors.

5.
BMC Public Health ; 21(1): 897, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980206

RESUMO

BACKGROUND: Young adulthood is a period of increasing independence for the 40% of young adults enrolled in U.S. colleges. Previous research indicates differences in how students' health behaviors develop and vary by gender, race, ethnicity, and socioeconomic status. George Mason University is a state institution that enrolls a highly diverse student population, making it an ideal setting to launch a longitudinal cohort study using multiple research methods to evaluate the effects of health behaviors on physical and psychological functioning, especially during the COVID-19 pandemic. RESULTS: Mason: Health Starts Here was developed as a longitudinal cohort study of successive waves of first year students that aims to improve understanding of the natural history and determinants of young adults' physical health, mental health, and their role in college completion. The study recruits first year students who are 18 to 24 years old and able to read and understand English. All incoming first year students are recruited through various methods to participate in a longitudinal cohort for 4 years. Data collection occurs in fall and spring semesters, with online surveys conducted in both semesters and in-person clinic visits conducted in the fall. Students receive physical examinations during clinic visits and provide biospecimens (blood and saliva). CONCLUSIONS: The study will produce new knowledge to help understand the development of health-related behaviors during young adulthood. A long-term goal of the cohort study is to support the design of effective, low-cost interventions to encourage young adults' consistent performance of healthful behaviors, improve their mental health, and improve academic performance.


Assuntos
COVID-19 , Pandemias , Adolescente , Adulto , Estudos de Coortes , Humanos , Estudos Longitudinais , Estudos Prospectivos , SARS-CoV-2 , Estudantes , Universidades , Adulto Jovem
6.
Cureus ; 13(2): e13499, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33777584

RESUMO

Thrombus characteristics are dependent on clot composition, but identification of the etiology based on histological analysis has proved inconclusive. Identification of proteomic signatures may help to differentiate between clots of different etiologies such as cardioembolic, large artery atherosclerotic, and other known etiologies, information that could enhance an individualized medicine approach to secondary stroke prevention. In this study, total protein extracts from cardioembolic (n=25) and large artery atherosclerotic (n=23) thrombus specimens were arrayed in quadruplicate on nitrocellulose slides and immunostained for 31 proteins using a Dako Autostainer (Agilent Technologies, Inc., Santa Clara, USA). We quantified 31 proteins involved in platelet and/or endothelial function, inflammation, oxidative stress, and metabolism. Pathway analysis showed more heterogeneity and protein network interactions in the cardioembolic clots but no specific correlations with clot etiology. Reverse-phase protein arrays are a powerful tool for assessing cellular interactions within the clot microenvironment and may enhance understanding of clot formation and origination. This tool could be further explored to help in identifying stroke etiology in large vessel occlusion patients with embolic stroke of an undetermined source.

7.
Clin Cancer Res ; 27(3): 807-818, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33122343

RESUMO

PURPOSE: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. EXPERIMENTAL DESIGN: Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array-determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry-based protocols. RESULTS: Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell-mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. CONCLUSIONS: TKIs differentially alter tumor cell phenotype which can impact NK cell-mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

8.
Methods Mol Biol ; 2237: 103-122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237412

RESUMO

Reverse phase protein arrays (RPPA) are used to quantify proteins and protein posttranslational modifications in cellular lysates and body fluids. RPPA technology is suitable for biomarker discovery, protein pathway profiling, functional phenotype analysis, and drug discovery mechanism of action. The principles of RPPA technology are (a) immobilizing protein-containing specimens on a coated slide in discrete spots, (b) antibody recognition of proteins, (c) amplification chemistries to detect the protein-antibody complex, and (d) quantifying spot intensity. Construction of a RPPA begins with the robotic liquid transfer of protein-containing specimens from microtiter plates onto nitrocellulose-coated slides. The robotic arrayer deposits each sample as discrete spots in an array format. Specimens, controls, and calibrators are printed on each array, thus providing a complete calibrated assay on a single slide. Each RPPA slide is subsequently probed with catalyzed signal amplification chemistries and a single primary antibody, a secondary antibody, and either fluorescent or colorimetric dyes. The focus of this chapter is to describe RPPA detection and imaging using a colorimetric (diaminobenzidine (DAB)) detection strategy.


Assuntos
Análise Serial de Proteínas/métodos , 3,3'-Diaminobenzidina/química , Animais , Anticorpos/imunologia , Linhagem Celular , Colorimetria/métodos , Humanos , Imunoensaio/métodos , Processamento de Proteína Pós-Traducional , Proteoma/imunologia , Proteoma/metabolismo
9.
Sci Rep ; 10(1): 19340, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168903

RESUMO

Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.


Assuntos
Doença de Lyme/microbiologia , Doença de Lyme/urina , Peptídeos/urina , Carrapatos , Adulto , Idoso , Algoritmos , Animais , Babesia microti/metabolismo , Biomarcadores/metabolismo , Borrelia , Eritema Migrans Crônico/microbiologia , Eritema Migrans Crônico/urina , Exantema , Feminino , Humanos , Hidrogéis/química , Infectologia , Masculino , Espectrometria de Massas , Mesocricetus , Pessoa de Meia-Idade , Peptídeos/química , Análise de Regressão , Urinálise
10.
Sci Rep ; 10(1): 13944, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811861

RESUMO

An accurate urine test for diverse populations with active tuberculosis could be transformative for preventing TB deaths. Urinary liporabinomannan (LAM) testing has been previously restricted to HIV co-infected TB patients. In this study we evaluate urinary LAM in HIV negative, pediatric and adult, pulmonary and extrapulmonary tuberculosis patients. We measured 430 microbiologically confirmed pretreatment tuberculosis patients and controls from Peru, Guinea Bissau, Venezuela, Uganda and the United States using three monoclonal antibodies, MoAb1, CS35, and A194, which recognize distinct LAM epitopes, a one-sided immunoassay, and blinded cohorts. We evaluated sources of assay variability and comorbidities (HIV and diabetes). All antibodies successfully discriminated TB positive from TB negative patients. ROAUC from the average of three antibodies' responses was 0.90; 95% CI 0.87-0.93, 90% sensitivity, 73.5% specificity (80 pg/mL). MoAb1, recognizing the 5-methylthio-D-xylofuranose(MTX)-mannose(Man) cap epitope, performed the best, was less influenced by glycosuria and identified culture positive pediatric (N = 19) and extrapulmonary (N = 24) patients with high accuracy (ROAUC 0.87, 95% CI 0.77-0.98, 0.90 sensitivity 0.80 specificity at 80 pg/mL; ROAUC = 0.96, 95% CI 0.92-0.99, 96% sensitivity, 80% specificity at 82 pg/mL, respectively). The MoAb1 antibody, recognizing the MTX-Man cap epitope, is a novel analyte for active TB detection in pediatric and extrapulmonary disease.


Assuntos
Lipopolissacarídeos/análise , Tuberculose/diagnóstico , Tuberculose/imunologia , Adulto , Coinfecção/urina , Epitopos/imunologia , Feminino , Guiné-Bissau , Infecções por HIV/urina , Humanos , Imunoensaio/métodos , Testes Imunológicos/métodos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/urina , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Peru , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , Tuberculose/classificação , Tuberculose Pulmonar/microbiologia , Uganda , Estados Unidos , Venezuela
11.
Artigo em Inglês | MEDLINE | ID: mdl-32273752

RESUMO

Background: During metastasis, tumor cells metastasize from primary tumors to distant organs via the circulatory and the lymphatic systems. There is a plethora of information about metastasis through the circulatory system, however not much information is available about the tumor cells dissemination through the lymphatic system or the lymphatic microenvironment that aids in this process in breast cancer metastasis. Purpose: The study designed to examine the tumor-derived secretome in lymph before reaching the draining lymph nodes. Methods: Using a microsurgical technique, we have collected the lymph in transit from the primary tumor en route to the regional lymph node in animals with metastatic and non-metastatic mammary carcinoma and healthy controls. The lymph samples were subjected to LC-MS/MS analysis, bioinformatics, and pathway analysis. Results: The metastatic tumor-draining lymph before its entry into the closest regional lymph node contain 26 proteins with >175-folds in abundance compared to lymph from non-metastatic tumor-bearing animals. Among these proteins were biliverdin reductase B, heat shock protein, coagulation factor XIII, lymphocytes cytosol protein 1, and aldose reductase. These proteins were not identified in the lymph from healthy animals. Pathways analysis revealed that cadherin-mediated endocytosis, acute phase response, junction signaling, gap junction, VEGF singling, and PI3K/AKT singling pathways are overrepresented in the lymph from metastatic tumor-bearing compared to the lymph from non-metastatic tumor-bearing animals. Among the significantly up-regulated proteins in the lymph from metastatic tumor-bearing animals were proteins that identified in exosomes include heat shock protein, enolase 1 alpha, S100, and biliverdin reductase B. One of the proteins significantly down-regulated in lymph from animals with metastasis is Kininogen, a known metastasis inhibitor protein. Conclusion: Proteins and exosomal proteins in lymph draining a metastatic tumor are different from those in lymph draining non-metastatic tumors, and these proteins involved in pathways that regulate tumor cells migration and invasion.

12.
Clin Cancer Res ; 26(13): 3126-3134, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32156749

RESUMO

PURPOSE: We hypothesized that autophagy inhibition would increase response to chemotherapy in the preoperative setting for patients with pancreatic adenocarcinoma. We performed a randomized controlled trial to assess the autophagy inhibitor hydroxychloroquine in combination with gemcitabine and nab-paclitaxel. PATIENTS AND METHODS: Participants with potentially resectable tumors were randomized to two cycles of nab-paclitaxel and gemcitabine (PG) alone or with hydroxychloroquine (PGH), followed by resection. The primary endpoint was histopathologic response in the resected specimen. Secondary clinical endpoints included serum CA 19-9 biomarker response and margin negative R0 resection. Exploratory endpoints included markers of autophagy, immune infiltrate, and serum cytokines. RESULTS: Thirty-four patients in the PGH arm and 30 in the PG arm were evaluable for the primary endpoint. The PGH arm demonstrated statistically improved Evans grade histopathologic responses (P = 0.00016), compared with control. In patients with elevated CA 19-9, a return to normal was associated with improved overall and recurrence-free survival (P < 0.0001). There were no differences in serious adverse events between arms and chemotherapy dose number was equivalent. The PGH arm had greater evidence of autophagy inhibition in their resected specimens (increased SQSTM1, P = 0.027, as well as increased immune cell tumor infiltration, P = 0.033). Overall survival (P = 0.59) and relapse-free survival (P = 0.55) did not differ between the two arms. CONCLUSIONS: The addition of hydroxychloroquine to preoperative gemcitabine and nab-paclitaxel chemotherapy in patients with resectable pancreatic adenocarcinoma resulted in greater pathologic tumor response, improved serum biomarker response, and evidence of autophagy inhibition and immune activity.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Cuidados Pré-Operatórios , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuminas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Feminino , Humanos , Hidroxicloroquina/administração & dosagem , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Paclitaxel/administração & dosagem , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Cuidados Pré-Operatórios/métodos , Recidiva , Análise de Sobrevida , Resultado do Tratamento
13.
Front Chem ; 8: 601477, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33614593

RESUMO

Osteoarthritis (OA) is the most common form of arthritis and the fastest growing cause of chronic disability in the world. Formation of the ternary IL-1ß /IL-1R1/IL-1RAcP protein complex and its downstream signaling has been implicated in osteoarthritis pathology. Current OA therapeutic approaches target either the cytokine IL-1ß or the primary receptor IL-1RI but do not exploit the potential of the secondary receptor IL-1RAcP. Our previous work implicated the Arg286 residue of IL-1RAcP as a key mediator of complex formation. Molecular modeling confirmed Arg286 as a high-energy mediator of the ternary IL-1ß complex architecture and interaction network. Anti-IL-1RAcP monoclonal antibodies (mAb) targeting the Arg286 residue were created and were shown to effectively reduce the influx of inflammatory cells to damaged joints in a mouse model of osteoarthritis. Inhibitory peptides based on the native sequence of IL-1RAcP were prepared and examined for efficacy at disrupting the complex formation. The most potent peptide inhibitor had an IC50 value of 304 pM in a pull-down model of complex formation, and reduced IL-1ß signaling in a cell model by 90% at 2 µM. Overall, therapies that target the Arg286 region surface of IL-1RAcP, and disrupt subsequent interactions with subunits, have the potential to serve as next generation treatments for osteoarthritis.

14.
Adv Exp Med Biol ; 1188: 1-19, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31820380

RESUMO

RPPA technology has graduated from a research tool to an essential component of clinical drug discovery research and personalized medicine. Next generations of RPPA technology will be a single clinical instrument that integrates all the steps of the workflow.


Assuntos
Medicina de Precisão , Análise Serial de Proteínas , Proteômica , Medicina de Precisão/instrumentação , Medicina de Precisão/tendências , Análise Serial de Proteínas/normas , Análise Serial de Proteínas/tendências , Pesquisa/instrumentação , Pesquisa/tendências
15.
Adv Exp Med Biol ; 1188: 61-75, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31820383

RESUMO

Reverse phase protein arrays (RPPA) are miniature dot blots constructed using robotic arrayers to deposit protein containing samples onto nitrocellulose-coated glass slides. Reverse phase protein arrays address the challenge of quantifying low-abundance proteins and posttranslationally modified proteins in cellular lysates and body fluids. RPPA technology is ideally suited to biomarker discovery, signal pathway profiling, functional phenotype analysis, and mechanism of action studies for drug discovery. Each array is fabricated with specimens, controls, and calibrators, thus providing a complete assay on each slide. Constructing a reverse phase protein array initially consists of selecting an arrayer, pin type, print head configuration, and nitrocellulose slide that is optimized for the particular specimen type and protein detection method. Herein we present the nuances of RPPA fabrication and study design using a solid pin arrayer and nitrocellulose-coated slides.


Assuntos
Análise Serial de Proteínas , Proteínas , Colódio , Descoberta de Drogas/instrumentação , Descoberta de Drogas/tendências , Impressão Tridimensional , Análise Serial de Proteínas/instrumentação , Proteínas/química
16.
Methods Enzymol ; 629: 195-217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31727241

RESUMO

HMGB1 is the most abundant non-histone nuclear protein. It regulates transcriptional access to open areas of chromatin and limits release of DNA with apoptotic death, serving to both inhibit apoptosis and promote DNA repair. When HMGB1 is translocated to the cytosol with many types of cellular stress, it is a powerful inducer of autophagy. It can also be released by activated immune cells and damaged or dying cells into the extracellular space, where it acts as a damage associated molecular pattern (DAMP) molecule, contributing to the pathogenesis and progression of cancer. Here, the most common methodologies to not only measure HMGB1 but also to effectively determine its subcellular localization, which dictates many of HMGB1's different functions, are reviewed.


Assuntos
Biomarcadores Tumorais/análise , Proteína HMGB1/análise , Neoplasias/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Autofagia/imunologia , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/imunologia , Carcinogênese/patologia , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Citosol/imunologia , Citosol/metabolismo , Progressão da Doença , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Proteína HMGB1/imunologia , Proteína HMGB1/metabolismo , Humanos , Morte Celular Imunogênica/efeitos dos fármacos , Morte Celular Imunogênica/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Microambiente Tumoral/imunologia
17.
Sci Rep ; 9(1): 17380, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31758030

RESUMO

Tumor clonal heterogeneity drives treatment resistance. But robust models are lacking that permit eavesdropping on the basic interaction network of tumor clones. We developed an in vitro, functional model of clonal cooperation using U87MG glioblastoma cells, which isolates fundamental clonal interactions. In this model pre-labeled clones are co-cultured to track changes in their individual motility, growth, and drug resistance behavior while mixed. This highly reproducible system allowed us to address a new class of fundamental questions about clonal interactions. We demonstrate that (i) a single clone can switch off the motility of the entire multiclonal U87MG cell line in 3D culture, (ii) maintenance of clonal heterogeneity is an intrinsic and influential cancer cell property, where clones coordinate growth rates to protect slow growing clones, and (iii) two drug sensitive clones can develop resistance de novo when cooperating. Furthermore, clonal communication for these specific types of interaction did not require diffusible factors, but appears to depend on cell-cell contact. This model constitutes a straightforward but highly reliable tool for isolating the complex clonal interactions that make up the fundamental "hive mind" of the tumor. It uniquely exposes clonal interactions for future pharmacological and biochemical studies.


Assuntos
Neoplasias Encefálicas/patologia , Movimento Celular , Proliferação de Células , Células Clonais/patologia , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/patologia , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Evolução Clonal/fisiologia , Células Clonais/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Heterogeneidade Genética , Genótipo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Modelos Biológicos , Transdução de Sinais/genética
18.
Expert Rev Proteomics ; 16(8): 647-664, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31353977

RESUMO

Introduction: Signal transduction cascades drive cellular proliferation, apoptosis, immune, and survival pathways. Proteins have emerged as actionable drug targets because they are often dysregulated in cancer, due to underlying genetic mutations, or dysregulated signaling pathways. Cancer drug development relies on proteomic technologies to identify potential biomarkers, mechanisms-of-action, and to identify protein binding hot spots. Areas covered: Brief summaries of proteomic technologies for drug discovery include mass spectrometry, reverse phase protein arrays, chemoproteomics, and fragment based screening. Protein-protein interface mapping is presented as a promising method for peptide therapeutic development. The topic of biosimilar therapeutics is presented as an opportunity to apply proteomic technologies to this new class of cancer drug. Expert opinion: Proteomic technologies are indispensable for drug discovery. A suite of technologies including mass spectrometry, reverse phase protein arrays, and protein-protein interaction mapping provide complimentary information for drug development. These assays have matured into well controlled, robust technologies. Recent regulatory approval of biosimilar therapeutics provides another opportunity to decipher the molecular nuances of their unique mechanisms of action. The ability to identify previously hidden protein hot spots is expanding the gamut of potential drug targets. Proteomic profiling permits lead compound evaluation beyond the one drug, one target paradigm.


Assuntos
Neoplasias/metabolismo , Proteômica/métodos , Animais , Antineoplásicos/uso terapêutico , Descoberta de Drogas , Humanos , Espectrometria de Massas , Neoplasias/tratamento farmacológico
19.
Lab Invest ; 99(5): 708-721, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659273

RESUMO

Bone tissue is critically lagging behind soft tissues and biofluids in our effort to advance precision medicine. The main challenges have been accessibility and the requirement for deleterious decalcification processes that impact the fidelity of diagnostic histomorphology and hinder downstream analyses such as fluorescence in-situ hybridization (FISH). We have developed an alternative fixation chemistry that simultaneously fixes and decalcifies bone tissue. We compared tissue morphology, immunohistochemistry (IHC), cell signal phosphoprotein analysis, and FISH in 50 patient matched primary bone cancer cases that were either formalin fixed and decalcified, or theralin fixed with and without decalcification. Use of theralin improved tissue histomorphology, whereas overall IHC was comparable to formalin fixed, decalcified samples. Theralin-fixed samples showed a significant increase in protein and DNA extractability, supporting technologies such as laser-capture microdissection and reverse phase protein microarrays. Formalin-fixed bone samples suffered from a fixation artifact where protein quantification of ß-actin directly correlated with fixation time. Theralin-fixed samples were not affected by this artifact. Moreover, theralin fixation enabled standard FISH staining in bone cancer samples, whereas no FISH staining was observed in formalin-fixed samples. We conclude that the use of theralin fixation unlocks the molecular archive within bone tissue allowing bone to enter the standard tissue analysis pipeline. This will have significant implications for bone cancer patients, in whom personalized medicine has yet to be implemented.


Assuntos
Osso e Ossos/metabolismo , Expressão Gênica , Hibridização in Situ Fluorescente/métodos , Proteoma/metabolismo , Proteômica/métodos , Animais , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Osso e Ossos/patologia , Fixadores/química , Formaldeído/química , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Reprodutibilidade dos Testes , Fixação de Tecidos/métodos
20.
BMC Cancer ; 18(1): 965, 2018 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-30305055

RESUMO

BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Lapatinib/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteína Forkhead Box O3/biossíntese , Expressão Gênica/efeitos dos fármacos , Genes erbB-2 , Humanos , Lapatinib/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico
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