Liquid chromatography-mass spectrometry (LC-MS) metabolomics studies produce high-dimensional data that must be processed by a complex network of informatics tools to generate analysis-ready data ...sets. As the first computational step in metabolomics, data processing is increasingly becoming a challenge for researchers to develop customized computational workflows that are applicable for LC-MS metabolomics analysis. Ontology-based automated workflow composition (AWC) systems provide a feasible approach for developing computational workflows that consume high-dimensional molecular data. We used the Automated Pipeline Explorer (APE) to create an AWC for LC-MS metabolomics data processing across three use cases. Our results show that APE predicted 145 data processing workflows across all the three use cases. We identified six traditional workflows and six novel workflows. Through manual review, we found that one-third of novel workflows were executable whereby the data processing function could be completed without obtaining an error. When selecting the top six workflows from each use case, the computational viable rate of our predicted workflows reached 45%. Collectively, our study demonstrates the feasibility of developing an AWC system for LC-MS metabolomics data processing.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM, UPUK
Abstract
INTRODUCTION
Our group has developed a patented vaccine platform that combines an mRNA-nanoparticle with CXCL9 loaded polyethylene glycol (PEG) hydrogel. The HCM vaccine is given SQ and ...recruits a diverse immune cell population to deliver a large payload of mRNA. The objective of these studies was to evaluate the effects of HCM vaccination on the TME.
METHODS
C57BL/6 mice underwent intracranial implantation of KR158 or GL261 glioma cells. The HCM vaccine was formulated with total tumor mRNA, DOTAP, CXCL9 and PEG hydrogel created in collaboration with the College of Engineering. Survival studies were analyzed with the Mantel-Cox test. Flow cytometry and ELISA data was analyzed using ANOVA. Graphpad was used for statistical analysis.
RESULTS
Intracranial tumor bearing mice were treated with SQ or intracranial injection of the HCM vaccine (total tumor mRNA, DOTAP, CXCL9, PEG hydrogel). SQ HCM delivery resulted in significant survival benefit whereas intracranial delivery did not (p = 0.0012). In a separate experiment, tumors and spleens were collected for flow cytometry. A significant increase in antigen specific CD8 T cells was found. Separately, after SQ vaccination, tumors were collected for PCR analysis. Tumors treated with HCM vaccination demonstrated significant upregulation of CCL4 and CXCR6. Intracranial tumor bearing mice were treated with HCM vaccination with or without blockade of CCL4. CCL4 blockade resulted in no survival benefit of the vaccine.
CONCLUSION
The HCM vaccine results in migration of antigen specific CD8 T cells within the TME and significant increases in CCL4. HCM vaccine efficacy is dependent on CCL4.
Abstract
INTRODUCTION: Immunotherapy for GBM has resulted in limited benefits to overall survival due to the targeting of limited antigens and the hostile TME. Our group has developed a patented ...vaccine platform that combines an mRNA-nanoparticle with CXCL9 loaded polyethylene glycol (PEG) hydrogel. The HCM vaccine is given SQ and recruits a diverse immune cell population to deliver a large payload of mRNA. The objective of these studies was to evaluate the efficacy and mechanisms of action of the HCM vaccine. METHODS: C57BL/6 mice underwent intracranial implantation of KR158 glioma cells. The HCM vaccine was formulated with total tumor mRNA, DOTAP, CXCL9 and PEG hydrogel created in collaboration with the College of Engineering. Survival studies were analyzed with the Mantel-Cox test. Flow cytometry and ELISA data was analyzed using ANOVA. Graphpad was used for statistical analysis. RESULTS: KR158-glioma intracranial tumor bearing mice were treated with SQ injection of the HCM vaccine (total tumor mRNA, DOTAP, CXCL9, PEG hydrogel). KR158 tumor bearing animals are resistant to treatment with radiation, temozolomide and other immunotherapy platforms in our laboratory. Treatment animals lived significantly longer compared to control animals (35 days versus 22 days, p < 0.0001). In a separate experiment, the fat pad was collected after vaccination and flow cytometry revealed recruitment of dendritic cells (DCs), CD4 and CD8 T cells and NK cells. Large numbers of antigen specific CD8 T cells were found in the spleen and within the TME after vaccination. When NK cells were blocked (NK 1.1 blocking antibody), the survival benefit from the vaccine was completely abrogated. NK cells were found to increase antigen presentation and available IFN-gamma within the PEG hydrogel scaffold. CONCLUSION: The HCM vaccine demonstrates efficacy in resistant murine glioma models and the efficacy is dependent on recruitment of NK cells at the site of vaccination.
Abstract
The objective of this study was to develop and test a murine system for delivery of laser interstitial thermotherapy (LITT). A system was built that allows for stereotactic placement of the ...laser into a mouse brain and real-time thermometry with a custom-made thermometer holder. Tissue heating curves for Laserglow Technologies 1064nm laser experiments testing tissue heating were performed by placing the laser in tissue and temperature monitoring via thermal probe. The laser was adapted for use with a stereotactic frame utilizing a system with a Hamilton syringe and a holder for consistent and precise placement of the thermal probe. The murine experiments were performed with C57BL/6 mice and samples were analyzed with H&E staining. First, ideal laser output was established using chicken breast. Laser dial setting of 4.38 successfully achieved slow tissue heating while a setting of 4.43 and 4.50 accomplished it rapidly, reaching a thermal ablative temperature of 43C in three and a half minutes. Next, a system for laser placement into the murine brain using the stereotactic frame was built revealing that the thermometer probe would require a separate system for consistent and precise measurements of heating. A thermometer probe holder was designed and fashioned via trigonometric calculations that allowed for thermal monitoring within 3mm spacing during laser ablation. Mice were anesthetized and treated with laser ablation at laser output of 4.38 and 4.43. H&E staining of the brain tissue demonstrated clear evidence of localized ablation identifiable by visible lesions on the H&E slides. The successful development of the stereotactic system for acquisition of tissue heating data coupled with evidence of successful live brain tissue ablation in a murine model via LITT proves that our protocol is ready for research of therapeutic combinatorial effects on live tumor bearing mice.
Abstract
INTRODUCTION
We have previously demonstrated that standard dose (SD) temozolomide results in T cell exhaustion in glioblastoma. In this study, we hypothesized that cellular immunotherapies ...will prevent T cell exhaustion. We tested temozolomide treatment with adoptive T cell transfer alone, dendritic cell (DC) vaccines alone and T cell transfer in combination with DC vaccines.
METHOD
GL-261-gp100 tumor-bearing mice were treated with SD (50 mg/kg for 5 days) or metronomic dose (MD) (25 mg/kg for 10 days) temozolomide. CD3+ T cells were isolated from Pmel mice and infused intravenously. Antigen-specific DC vaccines (gp-100) were given intradermally at the concentration of 50×104. Peripheral blood T cell populations were evaluated by flow cytometry. IFN-gamma secretion was measured for evaluation of T cells function. Survival was compared between groups.
RESULTS
Survival analysis demonstrated that naïve T cell transfer alone and DC vaccine alone add no survival benefit to temozolomide treatment for tumor-bearing animals. There were no significant differences in the expression of exhaustion markers of Tim-3 and Lag-3 on T cells of animals that received a combination of activated T cells and DC vaccines in the context of temozolomide therapy compared to the animals that just received temozolomide. Combination of activated T cell transfer and DC vaccines did not increase IFN-gamma secretion from T cells compared to temozolomide treated animals. Combination of naïve T cell transfer and two DC vaccines increased IFN-gamma secretion from T cells of GL-261 gp100 tumor-bearing animals. However, naïve T cell transfer and two DC vaccines in the context of temozolomide was not potent enough to extend the survival of these tumor-bearing animals.
CONCLUSION
Combinatorial treatment with naïve antigen-specific T cells and three dendritic cell vaccine boosters in the context of temozolomide is a promising approach to preserve T cell function and avoid T cell exhaustion.
Abstract
Dendritic cell (DC) vaccine efficacy is directly related to the efficiency of DC migration to the lymph node after delivery to the patient. In this research we discovered increasing cell ...migration by utilizing sarcosine improved anti-tumor efficacy. We hypothesized that sarcosine induced cell migration was due to chemokine or cytokine signaling.
METHODS
To generate DC vaccines, DCs were harvested from bone marrow of wild type C57BL/6 mice and electroporated with OVA-mRNA. Human DCs were isolated from PBMCs. DCs were treated with sarcosine at 20mM. OT-I T cells were isolated from transgenic mice and injected intravenously into B16F10-OVA tumor bearing mice. Following T cell transfer, DC vaccines were injected intradermal. In vitro migration was analyzed via transwell migration assay. In vivo migration was evaluated by flow-cytometry and immunofluorescence microscopy. Gene expression in RNA was investigated in DCs via RT-PCR and Nanostring.
RESULTS
Sarcosine significantly increases human and murine DC migration in vitro. In vivo murine model, sarcosine-loaded DCs had significantly increased migration to both the lymph nodes and spleen after intradermal delivery. B16F10-OVA tumor bearing mice were treated with the sarcosine-loaded DC vaccines resulted in a significant survival advantage over control and naïve DC vaccines. Gene expression in RNA was investigated in DCs. CXCR2,CXCL3 and CXCL1 were found to be upregulated in sarcosine-loaded DCs. Further metabolic analysis demonstrated the upregulation of cyclooxygenase-1 and Pik3cg. In vitro DC migration in presence of CXCR2 neutralizing antibody showed sarcosine induced migration was abrogated by adding the CXCR2 neutralizing antibody in both human and murine DCs. Animals that treated with sarcosine-loaded DC showed significantly better tumor control compares to the animals receiving anti-CXCR2 antibody one hour before DC injection.
CONCLUSION
Sarcosine increases the migration of murine and human DCs via the CXC chemokine pathway. This platform can be utilized to improve existing DC vaccine strategies.
Abstract
INTRODUCTION
Intra-tumoral immunosuppression is a major cause of treatment failure for patients with glioblastoma (GBM). Our group has developed a proprietary vaccine that combines mRNA ...encapsulated in lipid nanoparticles with PEG hydrogel and a chemokine (CXCL9). This hydrogel-chemokine-mRNA (HCM) vaccine is delivered subcutaneously (SQ) and results in significant anti-tumor efficacy in syngeneic murine glioma models. The objective of this study was to evaluate the effects of the vaccine on the tumor microenvironment (TME).
METHODS
C57BL/6 mice underwent intracranial implantation of KR158-luc tumor cells and underwent SQ injection of the HCM vaccine using total tumor mRNA. Tumors were collected for analysis. Q-PCR and flow cytometry were done to evaluate the differential gene expression and CCR5 expression (receptor for CCL4) in the TME.
RESULTS
CCL4 was found to be upregulated in tumor-bearing animals treated with HCM vaccine using PCR. In a separate experiment, CCL4 blockade was given prior to HCM vaccination and this resulted in abrogation of the survival benefit of the vaccine. Next, flow cytometry was performed and microglia in the TME had the highest expression of the CCR5 receptor. CD4 T cells in the TME had significantly increased CCL4 expression after treatment with HCM vaccine.
CONCLUSION
The HCM vaccine results in significant anti-tumor efficacy in murine syngeneic murine glioma which is dependent on upregulation of CCL4 in the TME driven by CD4 T cells. The CCL4 exerts its effect through microglia expressing CCR5 in the TME. Further analysis of the effects of vaccination on microglia phenotype and function are underway.
Background
Liquid chromatography-high resolution mass spectrometry (LC-HRMS) is a popular approach for metabolomics data acquisition and requires many data processing software tools. The FAIR ...Principles – Findability, Accessibility, Interoperability, and Reusability – were proposed to promote open science and reusable data management, and to maximize the benefit obtained from contemporary and formal scholarly digital publishing. More recently, the FAIR principles were extended to include Research Software (FAIR4RS).
Aim of review
This study facilitates open science in metabolomics by providing an implementation solution for adopting FAIR4RS in the LC-HRMS metabolomics data processing software. We believe our evaluation guidelines and results can help improve the FAIRness of research software.
Key scientific concepts of review
We evaluated 124 LC-HRMS metabolomics data processing software obtained from a systematic review and selected 61 software for detailed evaluation using FAIR4RS-related criteria, which were extracted from the literature along with internal discussions. We assigned each criterion one or more FAIR4RS categories through discussion. The minimum, median, and maximum percentages of criteria fulfillment of software were 21.6%, 47.7%, and 71.8%. Statistical analysis revealed no significant improvement in FAIRness over time. We identified four criteria covering multiple FAIR4RS categories but had a low %fulfillment: (1) No software had semantic annotation of key information; (2) only 6.3% of evaluated software were registered to Zenodo and received DOIs; (3) only 14.5% of selected software had official software containerization or virtual machine; (4) only 16.7% of evaluated software had a fully documented functions in code. According to the results, we discussed improvement strategies and future directions.
PDF
