Search tips
Search criteria

Results 1-13 (13)

Clipboard (0)

Select a Filter Below

Year of Publication
1.  Chitinase-3-like-1/YKL-40 as marker of circulating tumor cells 
Ex vivo expansion of circulating tumor cells (CTCs) of small cell lung cancer (SCLC) patients enabled systematic screening of secreted cytokines. Permanent CTC cultures of different patients shared secretion of chitinase-3-like-1 (CHI3L1)/YKL-40, known to be upregulated in a range of tumor entities and to be associated with increased metastasis and decreased survival. This protein lacks enzymatic activity and its mechanism of promoting tumor dissemination has not been resolved. Results from SCLC CTC cultures suggest CHI3L1 as marker and important effector of tumor cell dissemination in the peripheral blood. Furthermore, this protein may link chronic inflammation of the lung, chronic obstructive pulmonary disease (COPD) and lung cancer.
PMCID: PMC4483474  PMID: 26207216
Small cell lung cancer (SCLC); circulating tumor cells (CTCs); secretome; chitinase-3-like-1 (CHI3L1); YKL-40; chronic obstructive pulmonary disease (COPD); prognostic marker
2.  Residual Tumor Cells Are Unique Cellular Targets in Glioblastoma 
Annals of neurology  2010;68(2):264-269.
Residual tumor cells remain beyond the margins of every glioblastoma (GBM) resection. Their resistance to postsurgical therapy is considered a major driving force of mortality, but their biology remains largely uncharacterized. In this study, residual tumor cells were derived via experimental biopsy of the resection margin after standard neurosurgery for direct comparison with samples from the routinely resected tumor tissue. In vitro analysis of proliferation, invasion, stem cell qualities, GBM-typical antigens, genotypes, and in vitro drug and irradiation challenge studies revealed these cells as unique entities. Our findings suggest a need for characterization of residual tumor cells to optimize diagnosis and treatment of GBM.
PMCID: PMC4445859  PMID: 20695020
3.  The mTORC1/mTORC2 inhibitor AZD2014 enhances the radiosensitivity of glioblastoma stem-like cells 
Neuro-Oncology  2013;16(1):29-37.
The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs).
mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation.
Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments.
These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.
PMCID: PMC3870843  PMID: 24311635
AZD2014; glioblastoma; mTOR; orthotopic xenograft; Radiation; tumor stem cell
4.  The ATP-Competitive mTOR Inhibitor INK128 enhances in vitro and in vivo radiosensitivity of pancreatic carcinoma cells 
Radiotherapy remains a primary treatment modality for pancreatic carcinoma, a tumor characterized by aberrant mTOR activity. Given mTOR's regulatory role in gene translation, in this study we defined the effects of the clinically relevant, ATP-competitive mTOR inhibitor, INK128 on the radiosensitivity of pancreatic carcinoma cell lines.
Experimental Design
Clonogenic survival was used to determine the effects of INK128 on in vitro radiosensitivity on 3 pancreatic carcinoma cell lines and a normal fibroblast cell line with mTOR activity defined using immunoblots. DNA double strand breaks were evaluated according to γH2AX foci. The influence of INK128 on radiation-induced gene translation was determined by microarray analysis of polysome-bound mRNA. Leg tumor xenografts grown from pancreatic carcinoma cells were evaluated for mTOR activity, eIF4F cap complex formation and tumor growth delay.
INK128, while inhibiting mTOR activity in each of the cell lines, enhanced the in vitro radiosensitivity of the pancreatic carcinoma cells, but had no effect on normal fibroblasts. The dispersal of radiation-induced γH2AX foci was inhibited in pancreatic carcinoma cells by INK128 as were radiation-induced changes in gene translation. Treatment of mice with INK128 resulted in an inhibition of mTOR activity as well as cap-complex formation in tumor xenografts. Whereas INK128 alone had no effect of tumor growth rate, it enhanced the tumor growth delay induced by single and fractionated doses of radiation.
These results indicate that mTOR inhibition induced by INK128 enhances the radiosensitivity of pancreatic carcinoma cells and suggest that this effect involves the inhibition of DNA repair.
PMCID: PMC3947297  PMID: 24198241
Radiosensitization; mTOR; pancreatic carcinoma; INK128; xenograft
5.  P25. In vitro evaluation of the anticancer activity of ganetespib in combination with standard chemotherapeutics in small cell lung cancer (SCLC) cell lines 
Ganetespib (STA-9090) has anticancer activity due to inhibition of the chaperoning activity of HSP90 for oncogenes and other cellular proteins which support malignant growth. Especially, clinical activity as single drug was found in tumors addicted to modified kinases, such as HER2, ALK, BRAF, KIT and others. In the present work we tested the cytotoxic effects of ganetespib either alone or in combination with chemotherapeutics against a panel of small cell lung cancer cell lines.
Cytotoxicity was assessed using MTT assays, cell cycle effects by propidium iodide staining, HSP90 expression by flow cytometry and synergism of drug combinations was calculated employing the Chou-Talalay method.
The chemoresistant small cell lung cancer (SCLC) cell lines GLC16, NCI-H417 and DMS153 were highly sensitive to ganetespib (IC50 <30 nM), whereas chemosensitive GLC14 and primary SCLC26A cells exhibited chemoresistance to this drug. Ganetespib showed marked synergy in combination with cisplatin/carboplatin, etoposide and doxorubicin, whereas limited cooperative effects were observed for topotecan and the cisplatin/etoposide triple combination. Determinations of HSP90 expression using a monoclonal antibody pointed to an association of the cytotoxic effects of ganetespib with cell surface expression of this target and not with total expression of this heat shock protein.
In contrast to the first-generation HSP90 inhibitors, such as geldanamycin or 17-N-allylamino-17-demethoxygeldanamycin (17AAG), ganetespib has a far more favorable safety profile and has shown clinical anticancer activity in breast cancer, non-small cell lung cancer (NSCLC), gastrointestinal stromal tumors (GIST) and various hematological malignancies. According to our results, ganetespib is not expected to be active against primary, untreated SCLCs as single drug; however, is estimated to show cytotoxicity for pretreated SCLCs, either alone or in combinations with platinum drugs, etoposide or doxorubicin. These in vitro results are compatible with inhibition of an HSP90 client protein by ganetespib which is induced in response to prior exposure of the cells to chemotherapy in vivo.
PMCID: PMC4367771
Small cell lung cancer (SCLC); ganetespib
6.  Quantitative Influenza Follow-Up Testing (QIFT)—A Novel Biomarker for the Monitoring of Disease Activity at the Point-of-Care 
PLoS ONE  2014;9(3):e92500.
Influenza infections induce considerable disease burden in young children. Biomarkers for the monitoring of disease activity at the point-of-care (POC) are currently lacking. Recent methodologies for fluorescence-based rapid testing have been developed to provide improved sensitivities with the initial diagnosis. The present study aims to explore the utility of second-generation rapid testing during longitudinal follow-up of influenza patients (Rapid Influenza Follow-up Testing = RIFT). Signal/control fluorescent readouts (Quantitative Influenza Follow-up Testing = QIFT) are evaluated as a potential biomarker for the monitoring of disease activity at the POC.
Methods and Findings
RIFT (SOFIA) and QIFT were performed at the POC and compared to blinded RT-PCR at the National Reference Centre for Influenza. From 10/2011-4/2013, a total of 2048 paediatric cases were studied prospectively; 273 cases were PCR-confirmed for influenza. During follow-up, RIFT results turned negative either prior to PCR (68%), or simultaneously (30%). The first negative RIFT occurred after a median of 8 days with a median virus load (VL) of 5.6×10∧3 copies/ml and cycle threshold of 37, with no evidence of viral rebound. Binning analysis revealed that QIFT differentiated accurately between patients with low, medium and high viral titres. QIFT increase/decrease showed 88% agreement (sensitivity = 52%, specificity = 95%) with VL increase/decrease, respectively. QIFT-based viral clearance estimates showed similar values compared to PCR-based estimates. Variations in viral clearance rates were lower in treated compared to untreated patients. The study was limited by use of non-invasive, semi-quantitative nasopharyngeal samples. VL measurements below the limit of detection could not be quantified reliably.
During follow-up, RIFT provides a first surrogate measure for influenza disease activity. A “switch” from positive to negative values may indicate a drop in viral load below a critical threshold, where rebound is no longer expected. QIFT may provide a useful tool for the monitoring of disease burden and viral clearance at the POC.
PMCID: PMC3962407  PMID: 24658130
7.  In Vitro HIV-1 Evolution in Response to Triple Reverse Transcriptase Inhibitors & In Silico Phenotypic Analysis 
PLoS ONE  2013;8(4):e61102.
Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance.
Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments.
Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates.
Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here “in silico” from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of “wet-” and “dry-lab” experimentation may improve our understanding of HIV-1 resistance evolution in the future.
PMCID: PMC3629221  PMID: 23613794
8.  Astrocytes Enhance the Invasion Potential of Glioblastoma Stem-Like Cells 
PLoS ONE  2013;8(1):e54752.
Glioblastomas (GBMs) are characterized as highly invasive; the contribution of GBM stem-like cells (GSCs) to the invasive phenotype, however, has not been completely defined. Towards this end, we have defined the invasion potential of CD133+ GSCs and their differentiated CD133− counterparts grown under standard in vitro conditions and in co-culture with astrocytes. Using a trans-well assay, astrocytes or astrocyte conditioned media in the bottom chamber significantly increased the invasion of GSCs yet had no effect on CD133− cells. In addition, a monolayer invasion assay showed that the GSCs invaded farther into an astrocyte monolayer than their differentiated progeny. Gene expression profiles were generated from two GSC lines grown in trans-well culture with astrocytes in the bottom chamber or directly in contact with astrocyte monolayers. In each co-culture model, genes whose expression was commonly increased in both GSC lines involved cell movement and included a number of genes that have been previously associated with tumor cell invasion. Similar gene expression modifications were not detected in CD133− cells co-cultured under the same conditions with astrocytes. Finally, evaluation of the secretome of astrocytes grown in monolayer identified a number of chemokines and cytokines associated with tumor cell invasion. These data suggest that astrocytes enhance the invasion of CD133+ GSCs and provide additional support for a critical role of brain microenvironment in the regulation of GBM biology.
PMCID: PMC3551925  PMID: 23349962
9.  Antiviral Resistance and Correlates of Virologic Failure in the first Cohort of HIV-Infected Children Gaining Access to Structured Antiretroviral Therapy in Lima, Peru: A Cross-Sectional Analysis 
The impact of extended use of ART in developing countries has been enormous. A thorough understanding of all factors contributing to the success of antiretroviral therapy is required. The current study aims to investigate the value of cross-sectional drug resistance monitoring using DNA and RNA oligonucleotide ligation assays (OLA) in treatment cohorts in low-resource settings. The study was conducted in the first cohort of children gaining access to structured ART in Peru.
Between 2002–5, 46 eligible children started the standard regimen of AZT, 3TC and NFV Patients had a median age of 5.6 years (range: 0.7-14y), a median viral load of 1.7·105 RNA/ml (range: 2.1·103 – 1.2·106), and a median CD4-count of 232 cells/μL (range: 1–1591). Of these, 20 patients were classified as CDC clinical category C and 31/46 as CDC immune category 3. At the time of cross-sectional analysis in 2005, adherence questionnaires were administered. DNA OLAs and RNA OLAs were performed from frozen PBMC and plasma, RNA genotyping from dried blood spots.
During the first year of ART, 44% of children experienced virologic failure, with an additional 9% failing by the end of the second year. Virologic failure was significantly associated with the number of resistance mutations detected by DNA-OLA (p < 0.001) during cross-sectional analysis, but also with low immunologic CDC-scores at baseline (p < 0.001). Children who had been exposed to unsupervised short-term antiretrovirals before starting structured ART showed significantly higher numbers of resistance mutations by DNA-OLA (p = 0.01). Detection of M184V (3TC resistance) by RNA-OLA and DNA-OLA demonstrated a sensitivity of 0.93 and 0.86 and specificity of 0.67 and 0.7, respectively, for the identification of virologic failure. The RT mutations N88D and L90M (NFV resistance) detected by DNA-OLA correlated with virologic failure, whereas mutations at RT position 215 (AZT resistance) were not associated with virologic failure.
Advanced immunosuppression at baseline and previous exposures to unsupervised brief cycles of ART significantly impaired treatment outcomes at a time when structured ART was finally introduced in his cohort. Brief maternal exposures to with AZT +/− NVP for the prevention of mother-to-child transmission did not affect treatment outcomes in this group of children. DNA-OLA from frozen PBMC provided a highly specific tool to detect archived drug resistance. RNA consensus genotyping from dried blood spots and RNA-OLA from plasma consistently detected drug resistance mutations, but merely in association with virologic failure.
PMCID: PMC3782360  PMID: 23280237
10.  Adverse Respiratory Symptoms and Environmental Exposures Among Children and Adolescents Following Hurricane Katrina 
Public Health Reports  2011;126(6):853-860.
Children and adolescents are especially vulnerable to environmental exposures and their respiratory effects. Following Hurricane Katrina in 2005, residents experienced multiple adverse environmental exposures. We characterized the association between upper respiratory symptoms (URS) and lower respiratory symptoms (LRS) and environmental exposures among children and adolescents affected by Hurricane Katrina.
We conducted a cross-sectional study following the return of the population to New Orleans after Hurricane Katrina (October 2005 and February 2006) among a convenience sample of children and adolescents attending New Orleans health facilities. We used uni-, bi-, and multivariable analyses to describe participants, exposures, and associations with URS/LRS.
Of 1,243 participants, 47% were Caucasian, 50% were male, and 72% were younger than 11 years of age. Multiple environmental exposures were identified during and after the storm and at current residences: roof/glass/storm damage (50%), outside mold (22%), dust (18%), and flood damage (15%). Self-reported URS and LRS (76% and 36%, respectively) were higher after the hurricane than before the hurricane (22% and 9%, respectively, p<0.0001). Roof/glass/storm damage at home was associated with URS (adjusted odds ratio [AOR] = 1.59, 95% confidence interval [CI] = 1.15, 2.21) and LRS (AOR=1.35, 95% CI 1.01, 1.80), while mold growth at home was associated with LRS (AOR=1.47, 95% CI 1.02, 2.12).
Children and adolescents affected by Hurricane Katrina experienced environmental exposures associated with increased prevalence of reported URS and LRS. Additional research is needed to investigate the long-term health impacts of Hurricane Katrina.
PMCID: PMC3185321  PMID: 22043101
11.  Persistence versus Reversion of 3TC Resistance in HIV-1 Determine the Rate of Emergence of NVP Resistance 
Viruses  2012;4(8):1212-1234.
When HIV-1 is exposed to lamivudine (3TC) at inhibitory concentrations, resistant variants carrying the reverse transcriptase (RT) substitution M184V emerge rapidly. This substitution confers high-level 3TC resistance and increased RT fidelity. We established a novel in vitro system to study the effect of starting nevirapine (NVP) in 3TC-resistant/NNRTI-naïve clinical isolates, and the impact of maintaining versus dropping 3TC pressure in this setting. Because M184V mutant HIV-1 seems hypersusceptible to adefovir (ADV), we also tested the effect of ADV pressure on the same isolates. We draw four conclusions from our experiments simulating combination therapy in vitro. (1) The presence of low-dose (1 μM) 3TC prevented reversal to wild-type from an M184V mutant background. (2) Adding low-dose 3TC in the presence of NVP delayed the selection of NVP-associated mutations. (3) The presence of ADV, in addition to NVP, led to more rapid reversal to wild-type at position 184 than NVP alone. (4) ADV plus NVP selected for greater numbers of mutations than NVP alone. Inference about the “selection of mutation” is based on two statistical models, one at the viral level, more telling, and the other at the level of predominance of mutation within a population. Multidrug pressure experiments lend understanding to mechanisms of HIV resistance as they bear upon new treatment strategies.
PMCID: PMC3446758  PMID: 23012621
HIV-1; lamivudine; nevirapine; adefovir; resistance; selection; serial passage.
12.  The Brain Microenvironment Preferentially Enhances the Radioresistance of CD133+ Glioblastoma Stem-like Cells 
Neoplasia (New York, N.Y.)  2012;14(2):150-158.
Brain tumor xenografts initiated from glioblastoma (GBM) CD133+ tumor stem-like cells (TSCs) are composed of TSC and non-TSC subpopulations, simulating the phenotypic heterogeneity of GBMs in situ. Given that the discrepancies between the radiosensitivity of GBM cells in vitro and the treatment response of patients suggest a role for the microenvironment in GBM radioresistance, we compared the response of TSCs and non-TSCs irradiated under in vitro and orthotopic conditions. As a measure of radioresponse determined at the individual cell level, γH2AX and 53BP1 foci were quantified in CD133+ cells and their differentiated (CD133-) progeny. Under in vitro conditions, no difference was detected between CD133+ and CD133- cells in foci induction or dispersal after irradiation. However, irradiation of orthotopic xenografts initiated from TSCs resulted in the induction of fewer γH2AX and 53BP1 foci in CD133+ cells compared to their CD133- counterparts within the same tumor. Xenograft irradiation resulted in a tumor growth delay of approximately 7 days with a corresponding increase in the percentage of CD133+ cells at 7 days after radiation, which persisted to the onset of neurologic symptoms. These results suggest that, although the radioresponse of TSCs and non-TSCs does not differ under in vitro growth conditions, CD133+ cells are relatively radioresistant under intracerebral growth conditions. Whereas these findings are consistent with the suspected role for TSCs as a determinant of GBM radioresistance, these data also illustrate the dependence of the cellular radioresistance on the brain microenvironment.
PMCID: PMC3306260  PMID: 22431923
13.  Microenvironmental regulation of glioblastoma radioresponse 
Brain tumor xenografts initiated from human glioblastoma (GBM) stem like cells (TSCs) simulate the biological characteristics of GBMs in situ. Therefore, to determine whether the brain microenvironment affects the intrinsic radiosensitivity of GBM cells, we compared the radioresponse of GBM TSCs grown in vitro and as brain tumor xenografts.
Experimental Design
As indicators of DNA double strand breaks (DSBs), γH2AX and 53BP1 foci were defined after irradiation of two GBM TSC lines grown in vitro and as orthotopic xenografts in nude mice. Microarray analysis was performed to compare gene expression patterns under each growth condition.
Dispersal of radiation-induced γH2AX and 53BP1 foci was faster in the tumor cells grown as orthotopic xenografts compared to cells irradiated in vitro. In addition, cells irradiated in vivo were approximately 3-fold less susceptible to foci induction as compared to cells grown in vitro. Microarray analysis revealed a significant number of genes whose expression was commonly affected in the two GBM models by orthotopic growth conditions. Consistent with the decrease in sensitivity to foci induction, genes related to ROS metabolism were expressed at higher levels in the brain tumor xenografts.
γH2AX and 53BP1 foci analyses indicate that GBM cells irradiated within orthotopic xenografts have a greater capacity to repair DSBs and are less susceptible to their induction than tumor cells irradiated under in vitro growth conditions. Because DSB induction and repair are critical determinants of radiosensitivity, these results imply that the brain microenvironment contributes to GBM radioresistance.
PMCID: PMC3005074  PMID: 21037023
microenvironment; glioblastoma stem cells; radiosensitivity; γH2AX; orthotopic xenografts

Results 1-13 (13)