The Klemke laboratory leverages state-of-the-art facilities and expertise within the areas of proteomics, informatics, immunology, oncology, intravital microscopy, advanced tissue processing, and cell biology, to work with clients to screen for lead compounds, test cell-based therapeutics, determine their efficacy, and unravel their MOA.  To inquire about research contract services, contact Dr. Richard Klemke at rklemke@ucsd.edu.

Dr. Klemke’s laboratory at UCSD and the Moores Cancer Center has conducted numerous “research related services” for many different real and virtual biotech companies over the years. The provision of such services fits with the research and service missions of the University. University-based services are more cost-effective compared to industrial CROs and include the utilization of specialized expertise of the faculty and/or specialized instrumentation. Quite often, this will include testing of certain biologics and pharmaceutical agents and other proprietary materials for the outside company (sponsor). For the purposes of this type of agreement, services are defined as “specifically-designed” projects and are confined to those projects that provides services involving classification, diagnostics, or testing of a sponsor’s data, samples, mechanisms, procedures, or products. These services are performed using University resources, including personnel, equipment, or facilities. Confidentiality:If the service(s) contemplated under this agreement require the provision of Sponsor’s proprietary information, the terms of confidentiality shall be set forth in a Confidentiality Agreement executed by both parties. Patent Rights: In performing a Service Agreement, the University is simply involved in the provision of services involving sponsor-provided information and not in the creation of new knowledge or new technologies. Consequently, the University will make no claims on patent rights in regard to sponsor-owned materials or information provided for the Project.

Laboratory services provided by the Klemke Lab and associates:

• Preclinical animal testing using chickens, mice, and/or zebrafish models of human diseases combined with high-resolution tumor and vessel imaging technologies.  A particular strength is in transgenic mouse and zebrafish models of oncology, angiogenesis, and inflammation. Our extensive animal systems and our ability to monitor key cancer progression parameters are ideal for evaluating the MOA and the efficacy of cell-based therapeutics, prodrugs and lead compounds in the oncology, immune oncology, angiogenesis, and inflammation arenas.

1. Subcutaneous PDX and xenograft models (usually human tumor samples or cell lines transplanted into immuno-compromised rodents).
2. Commercial or client-produced cell lines of interest.
3. Orthotopic immune competent tumor models in mice, which are syngeneic cells implanted directly into organs of interest including pancreatic and breast.
4. Brain tumor progression model in rodents, chickens, and zebrafish.
5. Metastatic models, which are used for testing cells and drugs that block or promote unwanted metastases.
6. Spontaneous xenograft and syngeneic metastatic models in rodents.
7. Syngeneic subcutaneous and orthotopic models, which are rodent tumors transplanted into hosts of the same genetic background to study immunotherapy and biological pathways in a relevant immune competent environment.
8. Melanoma B16 model of tumor formation, immunity, and brain metastasis in syngeneic rodents.

Common endpoints in oncology and immune oncology studies include: 

Tumor size reduction and remission 
Tumor growth kinetics and progression and determination of local and distant metastases 
Immune effector cell profiling of primary tumors and metastatic sites 
Kaplan-Meier survival plots and disease outcome determinations
Changes in serum, solid tissue biomarker profiles, and immune cell profiles followed by detailed bioinformatics to uncover drug targets, impacted pathways, clinical correlates, and disease relevance, patient outcome, etc

• Angiogenesis assays and intravital high-resolution vascular imaging:

1. 3D mouse aortic ring and vascular sprouting
2. Mouse developmental eye model and vessel maturation
3. Mouse subcutaneous angioreactor model with pre-specified angiogenic factors and conditions
4. Chicken CAM model
5. Zebrafish intersegmental vessel assay, which is highly amenable to drug screening and determining MOA of low abundant and costly lead compounds

Common endpoints in angiogenesis studies include:
Vessel density, vessel length, size and diameter
Number of branch points
Pericyte numbers and vessel coverage
Permeability and vessel leak including evans blue and fluorescent lectin methods
Basement membrane integrity and composition
Intravital real-time quantitative analyses of endothelial cell sprouting, migration, and proliferation (zebrafish model)

• In vitro cell-based assays, immune assays, and biochemical tests:

1. Cytotoxicity analysis
2. IC50 dose-reponse determination with patient-derived cancer cells, established normal and cancer cell lines, NIH cancer cell panels, human tumor-derived fibroblasts, etc
3. Cell growth and cell cycle analyses
4. Apoptosis determination and death pathway identification
5. Cancer and endothelial cell migration and immune cell homing to tumors
6. Virus propagation, titer determinations, plaque assays, virus-induced tumor lysis
7. Soft-agar growth and colony formation of tumor cells and immune cells
8. Cancer stem cell (CSC) functions and marker identification, 3D sphere formation and reseeding, FACS profiling of cell surface markers and transcriptional activity of GFP-tagged stem cell reporters (TOP-GFP, Oct-4, Nanog, etc). Immune cell profiling by FACS and PCR
9. Cytokine multiplex and profiling analyses
10. Standard and custom ELISA and splenocyte immunoassays
11. qPCR, mRNA profiling and deep sequencing

• Quantitative proteomics, informatics, and signal transduction: We offer full proteomics services for the isolation, identification quantification, and analysis of protein signaling pathways and networks of interest.  We use the latest high specification equipment such as; the LTQ-Orbitrap XL, LTQ-LTD equipped with ETD, MALDI-Tof

1. SILAC
2. TMTs™/ iTRAQ™
3. HPLC protein separation
4. Sample preparation
5. Liquid chromatography
6. Protein identification from single or multiple gel bands/spots
7. Large-scale protein profiling of complex mixtures including cell lysates and tissues using LC/MS/MS
8. ETD analysis of PTMs combined with detailed PTM mapping/profiling
9. PY Antibody and/or IMAC Phosphoprotein enrichment and phosphoproteomics
10. Phosphosite identification and kinase activation determinations
11. Informatics capabilities developed in house and on the Web included:
    1. FatiGo+ to search against signaling pathway databases BioCarta (www.biocarta.com/genes/index.asp) and KEGG database (173 total signaling pathways identified to date in human, www.genome.jp/kegg/), or functional domain databases (www.ebi.ac.uk/interpro/index.html). FatiGo+ is used to search the Gene Ontology (GO) database to reveal functional information based on protein subcellular localization (e.g. cytoskeleton, membrane, metabolic, nucleus) and described function(s) (metabolic enzyme, GTPase, Signal transduction, RNA/DNA processing, etc).
    2. Ingenuity Pathway Analysis (IPA) and Knowledge database programs (www.ingenuity.com).  This powerful program can be used to find biological and functional relationships among the data sets in regard to single protein signatures as well as large protein data sets.  IPA is also an excellent database to interrogate the literature and to generate signal transduction pathways, interactomes, and to uncover potential drug actions and targets.  
    3. Phosphosite Identification and Kinase Assignment.  Specific phosphorylation sites and the putative kinases that phosphorylated them are assigned and functionally analyzed using the following open source phospho-informatics programs. PhosphoSitePlus (http://www.phosphosite.org/homeAction.do) and NetPhorest (http://netphorest.info/), and PhosphoScore (http://dir.nhlbi.nih.gov/papers/lkem/phosphoscore/), are used to identify and analyze biological function and regulatory significance of specific phosphorylation sites and their specific binding domains.  These tools are used for mining and interpreting large phosphoprotein data sets in the context of biological regulation, drug action, tissue distribution, subcellular localization, protein binding domains, sequences, motifs, and specific diseases including cancer.
    4. BlastPro and PhosphoBlast developed in our laboratory are used to identify and compare conserved protein, gene, and phosphorylation sites reported in the literature across diverse experimental platforms, species, laboratories and cellular systems.
    5. The NetworKIN http://networkin.info/version_2_0/search.php) program not only takes into consideration established kinase recognition motifs to assign kinase activity, but also considers higher order contextual information including cellular localization and known kinase-protein interactions in relation to its known protein members and substrates present in the dataset. This algorithm is particularly useful in understanding how specific therapeutic agents impact kinase activation and signal transduction networks at the systems level.  

• Pathology Services.  The Klemke lab has full capability to process, section, stain and provide detailed interpretation of tissues collected as part of in vivo pharmacology and disease specific studies.  Importantly, we leverage UCSD’s Moores cancer biorepository and Tissue Technology Center (http://pathology.ucsd.edu/human_and_animal_tissue_technology_center.html) of resected and archived patient samples (declassified), which include detailed pathology reports, TNM classification, and de-identified patient outcome information.  We also have the ability to evaluate the expression of candidate biomarkers using staged human tissue samples, patient-derived PDX cell lines, and various GEM models maintained in house. Our combined comprehensive approach reveals clinically relevant biomarkers that truly reflect the impact of therapeutics on disease development and progression.  

1. Routine and advanced tissue technology processing for all human and mouse tissue, analytical microscopy, and advanced tissue culture methods with hypoxic conditions and microscopic culture videography.  The Biorepository processing lab and advanced tissue technology facility houses a Ventana Medical Systems Discovery Ultra Biomarker Automated Slide Preparation System, a Li-Cor Odyssey Infrared Imaging System (with In-Cell Western and Fluorescent-labeled ELISA modules) for Western antibody validation, dual-mode UV-Vis micro-volume spectrophotometer (Nanodrop 2000c), 2 automatic tissue processors; 2 embedding stations; and both motorized and manual microtomes.
2. Specialized in situ hybridization using multiple detection systems: immunohistochemistry, immunofluorescence high-resolution multi-color staining and imaging of tissue sections, fixed cells, 3D cultures of cells, and cleared tissue samples.  ICH, ISH, FISH and Qdot immunofluorescence techniques operate in house. The capability to perform IF/FISH or IHC/ISH techniques on the same tissue section permits the greatest flexibility in the localization of biomarker expression.  The system provides extensive options to perform multiple assays (up to 30 slides per run) for more accurate semi-quantitative Immunohistochemistry / immunofluorescence and an efficient capability for methods development to validate and to develop new biomarkers.