Cellular Immunology and Immune Metabolism Core (CIMC)
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The Cellular Immunology and Immune Metabolism Core (CIMC) of the LSU-Cancer Center was created in 2006 with the objective of providing flow cytometry support to Promising Junior Investigators (PJI) funded during Phase I of the COBRE “Mentoring Translational Researchers in Louisiana”. During Phase II, and under the scientific direction of Dr. Paulo Rodriguez, and with the oversight of Dr. Augusto Ochoa, the CIMC evolved to provide cutting edge technologies not only to COBRE-supported PJI, but also to the LSUHSC investigators and to other users in our region. The CIMC laboratory now provides comprehensive analytic flow cytometry and high-speed cell sorting services, immune function assays including antigen specific and mitogen induced cell proliferation, cytokine production, cell mediated cytotoxicity, and effector T cell frequency. During Phase II, the CIMC also acquired and developed the infrastructure and expertise to study cellular metabolism (of tumor cells and immune cells), including oxygen consumption rate and mitochondrial mass and function; and the detection of immune mediators through multiplex approaches. The state-of-the-art services provided by the CIMC and its experienced team allow the investigators to identify new biomarkers for inflammation, new mechanisms of immune dysfunction in disease, and innovative approaches to overcome immune suppression in diseases characterized by chronic inflammation, thereby increasing their chances for high impact publications and federal and foundation-related funding. These advanced technologies have positioned the CIMC as the leader laboratory in inflammation and immune research in the region, which has resulted in an increased number of users and its integration as a core laboratory at the LSUHSC School of Medicine and the LSU-Cancer Center. The main goal of Phase III is to expand the number of users by providing the most advanced technologies to study inflammation and disease as they become available, and to complete the integration of CIMC as a Cancer Center and School of Medicine Core. Thus, our proposal encompasses the potential of significantly improving the quality of translational research in the State of Louisiana.
FACILITIES AND OTHER RESOURCES
Contribution to Success: The CIMC has served the LSU-HSC scientific community studying inflammation and disease since 2006. It has been supported by funds from Phases I and II of this COBRE, the Stanley S. Scott Cancer Center, and the School of Medicine of LSU-HSC. The progressive growth and integration of the CIMC during this time has allowed us to become the premier core laboratory to conduct innovative translational research on inflammation and diseases in LSU-HSC and throughout the State of Louisiana. In addition to the support for the facilities, the COBRE, the LSU Cancer Center, and the School of Medicine have also supported the CIMC personnel, including Drs. Rodriguez and Wyczechowska. With this support, Dr. Rodriguez has created several lines of research in tumor immunology and immunotherapy, which have resulted in the funding by grants from the Ladies Leukemia League, the LSU-HSC Translational Research Initiative, the Hope on Wheels Foundation, and R21CA162133. In addition, this support has enabled the development of infrastructure necessary to provide services from standard to cutting-edge methodologies in inflammation research.
The Dean of the School of Medicine, Steve Nelson, MD, has agreed to continue providing the partial financial support for the CIMC facilities now and after the COBRE Phase III ends. Moreover, Dr. Nelson will facilitate the integration of the CIMC and other COBRE cores into the research structure of LSU-HSC (See letter of Support from Dr. Nelson).
Institutional Commitment to Success: The School of Medicine and the Cancer Center at Louisiana State University Health Sciences Center are committed to the success of CIMC as a laboratory that will facilitate the development of translational research in the field of inflammation. The LSU-Cancer Center and the School of Medicine have provided extensive support to obtain and to maintain equipment. After completion of this Phase II of the COBRE, we expect that the CIMC will be funded by the user fees (approximately 30%), LSU Cancer Center funds (40%) and School of Medicine funds (30%).
Laboratory: The CIMC is located in the LSU Health Sciences Center at 1700 Tulane Ave. R-934, New Orleans, LA. 70112. Phone (504) 210-2556. It consists of two laboratories, totaling 400 square feet on the ninth floor of the LCRC Building. Drs. Ochoa’s and Rodriguez’s laboratories and offices, as well as Dr. Wyczechowska’s office, are located on the same floor. The CIMC space contains two outfitted benches and is well equipped for immunology and metabolism experimentation. Each bench has full sets of pipettes and an electrophoresis power supply. Common lab equipment includes three refrigerator/freezers, two –20C freezers, a –80C freezer, three refrigerated microfuges, two thermocyclers, three waterbaths, a sonicator, a UV/Vis spectrophotometer, a BioRad plate reader, a pH meter, a scale and balance, a platform shaker, three centrifuges, and one conventional incubator for bacterial cultures. For cell cultures, the lab has two 2-foot laminar Biosafety flow hoods, four water-jacketed CO2 incubators, an inverted microscope, and a refrigerated centrifuge.
Computers: The CIMC laboratory has three Optiplex 620 Dell computers and two Optiplex 740 Dell computers. The laboratory is fully connected to the Internet and the LSUHSC mainframe computers. There are two scanners, a color Lexmark laser printer, and two ink color printers.
All services will be offered to investigators with approved protocols from the LSUHSC Institutional Biosafety Committee (IBC). LSUHSC has a control plan that provides policies and guidance to protect workers with occupational exposure to blood and other potentially infectious materials. All individuals associated with this proposal adhere to these policies, and all have received the Occupational Exposure to bloodborne pathogens Training and completed the Biosafety and Radioactivity Courses. Research-related incidents will be reported immediately to the IBC at LSUHSC. Such incidents include research-related accidents and illnesses, as well as inadvertent release or improper disposal of biohazardous materials.
The biohazard substances used in this study require biosafety level one or two (BL1 or 2). These include tumor cell lines, and bacteria for the expansion of recombinant DNA, among others. The proper handling, transport, and disposal of these biological and chemical materials will be implemented by following the policies, guidelines, and programs established by the IBC at LSUHSC to guarantee the biological safety of the community and the environment.
Biohazard signs are prominently placed to be easily read by all entering the laboratories. Additional biohazard signs are placed on refrigerators, biosafety hoods, and cabinets containing the reagents. Sharps materials, including needles and scalpels, will be discarded immediately into approved sharps disposal containers for biohazard disposal. All non-sharp laboratory materials utilized in experiments with biological materials (e.g., microorganisms, recombinant DNA, animal tissues, cell cultures, etc.) will be treated with 1% sodium hypochlorite prior to disposal and placed in boxes containing the international biohazard label. Pathological waste includes animal carcasses, tissues, and organs, and will be disposed by incineration, a service provided by the LSUHSC animal facility.
Chronic inflammation plays a major role in the development, progression, and metastasis of various tumors, and represents a significant limitation of every form of treatment against cancer. A similar accumulation of chronic inflammatory mediators also occurs in other pathologies including obesity, sepsis, chronic infections, and autoimmune disorders. Monitoring the immunological alterations in diseases characterized by chronic inflammation is a critical tool for the identification of pathology-linked pathways and clinical biomarkers, and for the development of new therapeutic approaches. The Cellular Immunology and Immune Metabolism Core (CIMC) from the Stanley S. Scott Cancer Center was created in 2006, with support from the COBRE “Mentoring Translational Researchers in Louisiana”, and aims to provide a customer oriented service dedicated to support the research needs of the investigators. We propose that the continuous development and implementation of novel immunological and metabolism technologies, and the expansion of the existing services, by the CIMC will be instrumental to promote high quality translational research linked to inflammation and disease, and will ensure the long-term viability of the CIMC after the COBRE phase III ends.
The long-term goal of the CIMC is to provide the latest technological approaches related to inflammation to support the needs of the scientific community. Thus, we propose to continue providing high quality assays and advice to the investigators on their research plans; to implement novel technologies, to obtain new state-of-the- art instrumentation that will allow us to keep providing superior services; and to continue the integration of the CIMC as a central core facility at LSU Health Sciences Center (LSUHSC). Our goal has been to support the development of high quality translational research by providing the best and most advanced technology to study the mechanisms of inflammation in health and disease. The results of our work during Phases I and II of this COBRE show the progressive growth and integration of the CIMC that now provides the latest technologies, ranging from standard immune assays such as multicolor flow cytometry, sorting of immune cell subsets, and detection of cell proliferation, cytokines, and cytotoxic function, to the development of new technologies such as the detection of metabolic alterations in immune cells through the XFe24 Seahorse platforms, and testing cytokines and phospho-proteins in supernatants, serum, and tissues using the multiplex systems. In addition, we continue to develop new flow cytometry applications, including the detection of mitochondrial function, reactive oxygen/ nitrogen species, uptake of glucose and fatty acids, and markers linked to immune cell metabolism. These results are expected to enable the identification of novel chronic inflammatory disease-linked pathways and biomarkers, potentially leading to new therapies. As a result of its continuous progress, the CIMC has become the premier core laboratory to conduct innovative translational research on inflammation and disease.
We plan to continue our work with the following Specific Aims:
Specific Aim 1. To provide high quality support and advanced immune technologies for the COBRE pilot projects and the overall scientific community in the State of Louisiana. We will extend the CIMC services to the scientific community at LSUHSC and other institutions associated with the Louisiana Cancer Research Consortium (LCRC), including Ochsner Health System, Tulane University, Xavier University, and Dillard University, and other biomedical research institutions in Louisiana. In addition, we propose to disseminate the services and training provided by the CIMC through web, social media, seminars, and meeting platforms to increase the advertising of our services.
Specific Aim 2. To obtain and develop novel immune and metabolic detection technologies to support innovation in inflammatory research. We will develop novel technologies for the support of the investigators, including the detection by flow cytometry of mRNA profiles and metabolic changes in specific immune populations. Moreover, we propose to expand and update our services through new state-of-the-art equipment, including the future purchase of the mass cytometry cyTOF and the Amnis imaging Flow cytometer, among others, which will allow us to continue providing our pioneered and advanced services.
Specific Aim 3. To promote the long-term viability and the institutional integration of the CIMC into the LSU Cancer Center and the School of Medicine of Louisiana State University Health Sciences Center (LSUHSC) in New Orleans. To expand the number of users, to facilitate the institutionalization of the CIMC, and to ensure the long-term viability of the CIMC after the COBRE phase III ends, we will offer an integrated research support program with innovative research services, consulting and mentoring activities, and grants submission support.
1. Develop new flow cytometry and metabolism-based applications. We will implement novel technologies in flow cytometry, including the detection of micro-RNA and mRNA in specific cell populations using the QuantiGene® FlowRNA system (eBiosciences), which uses an in vitro hybridization-based technology and allows the detection of up to three transcripts, combined with multicolor flow cytometry. This technology will integrate services with the Translational Genomics Core directed by Dr. Zabaleta. Since our flow cytometers allow detection up to 14 colors, we will be in a position to monitor the differential expression of panels of three different transcripts in multiple cell populations. This technology is in the process of standardization and will be offered to our users starting in 2015. There is increasing interest among the CIMC users to integrate flow cytometry and microscopy. Therefore, we have proposed to purchase the Amnis FlowSight platform, which will allow the quantitative analysis of cell signaling, protein translocation, internalization, morphology, cell-cell interaction, cell cycle, DNA damage, cell death, autophagy, and FISH-based assays at the single-cell level. The addition of this technology will promote interactions with the imaging core directed by Dr. Luis Del Valle.
We also plan to implement the next generation of multicolor analysis cyTOF that is predicted to eventually replace flow cytometry technologies. The cyTOF platform couples flow cytometry with mass spectrometry and uses antibodies labeled with lanthanide metals, which are typically the same antibody clone used in conventional flow cytometry. The advantages of cyTOF over conventional flow cytometry include the ability to measure more than 36 parameters at any one time, compared with a maximum of 18 parameters by fluorescence cytometry. No spectral overlap is detected from neighboring isotopes, eliminating the need for compensation matrices to correct for such overlap, as it is required in fluorescence cytometry. There is also no contaminating biological background signal when using lanthanide metals, which avoids cell autofluorescence, a common problem when using fluorescence cytometry. We also propose to establish the detection of nitric oxide and peroxynitrites in supernatants and plasma using the Scimetric YSI analyzer 2900 D, which will enable us to expand our services into nitric oxide-related research, a significant interest of LSUHSC investigators. We believe that the future implementation of these complex protocols will make our core superior to other existing flow cytometry cores, and will expedite the development of projects by our investigators, promoting efficiency, innovation, and high quality productivity in our research community. The submission of large equipment grants are planned over the coming two years.
2. Upgrade the existing Instruments within the CIMC facilities. We anticipate that the increased demand for multi-color analysis and the increased complexity of experimental designs will require upgrades of existing equipment. Therefore, we propose to:
- allocate funding to maintain the service contracts for all instruments of the CIMC.
- upgrade the LSR II with the violet UV laser, which will allow us to acquire up to 18 different parameters.
- place the FACSAria in an NIH compliant hood, which would ensure BSL-2 conditions and the safety of the operator and to allow us to analyze and sort patient samples.
- install hypoxia chambers for the XF24 and XFe 24 analyzers to allow us to determine the effects of hypoxia on the metabolism of different cell populations.
The equipment that we propose to purchase is expensive. As these technologies become more popular, we expect prices to drop. Costs for that equipment will be shared by the LSU Cancer Center and the School of Medicine. In addition, we expect that the increased number of users will allow us to apply for large equipment grants to the National Institutes of Health.
3. Advertise the innovative approaches developed by the CIMC. After the proposed technologies are standardized, they will be offered to our scientific community for service and training, as stated in Strategy 2 of Specific Aim #1. The menu of services offered by the CIMC facilities will be advertised through our webpage, the LA CaTS’ webpage, social media, the weekly “Work-in-Progress” meetings, the core’s seminar series, the IDeA/COBRE meetings, and the LCRC retreat. In addition, we will sponsor in house training to the investigators. Implementation of these sophisticated technologies in our facilities should significantly increase our market portfolio and promote highly innovative research among our users, which is expected to increase productivity and chances for successful funding.