The UCSF Preclinical Therapeutics Core (PCTC) facility generates tumor-bearing animals and conduct preclinical oncology trials for UCSF Helen Diller Family Comprehensive Cancer Center investigators. Offering a variety of xenograft-based cancer models, the core provides a complete set of services that include consultation regarding experimental design, tumor cell growth in culture and generation of tumors in mice, administration of experimental agents, monitoring of tumor burden and response to therapy, and interpretation of study results.
The PCTC maintains a cryorepository of commonly used human cancer cell lines derived from multiple tumor types along with data regarding their in vivo growth characteristics. The PCTC is also a resource of expertise in small animal survival surgery, and is available to provide training in these techniques on a recharge basis. Although the core primarily functions to test experimental anti-cancer agents in vivo, it also provides animal models of human cancer for use in novel diagnostic, tumor imaging, and basic mechanistic research.
The core oversees, maintains and provides as a service a number of small animal imaging technologies housed within the barrier facility. The availability of centralized cell and animal resources, together with personnel with expertise in conducting preclinical studies, ensures appropriate experimental design and reproducibility, compliance with local and federal regulatory guidelines for tumor-bearing animals, and maximum resource utilization through coordinated animal purchasing and housing.
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The use of mice as a model of human cancer has been an important tool in the development and translation of innovative anti-cancer therapies. Our research makes use of xenograft tumor models in immune deficient (nu/nu) mice, as well as transgenic and conditional/knock-in based engineered models that target known tumor promoting pathways. Genetic and phenotypic characterization of both xenograft tissue and genetically engineered animals allows the correlation of drug-target interaction, as well as the ability to test predictions of pathway-targeted anti-tumor efficacy. To test synergistic activity, novel therapeutics -- generated both from within UCSF as well as externally -- are tested alone or in rational combinations and dosing sequences. Tumor response to therapeutics is detected by a combination of direct tumor measurement (when possible) and molecular imaging methods.
Cell and tissue culture
Tumor xenografts are most commonly generated by injecting human tumor cells grown in vitro into immuno-suppressed recipient mice. The PCTC has expertise in high volume cell culture, and maintains a large repository of commonly used tumor cell lines. The project investigator and Dr. Hann select cell lines for the study based upon known molecular features desired for the specific hypothesis being tested (e.g., Her2 over-expression, ras-mutation, etc). Core personnel are responsible for generating the xenografts via high capacity cell culture and cell implantation. In certain cases, the tumor cells are maintained and passaged in vivo. For these studies, the Core maintains tumor tissue both in vivo as well as in frozen stocks. The Core also provides grafting of primary human tumor tissue into mice derived from either cancer patient biopsy or tumor removal surgery.
Animal grafting, dosing, and data collection
A variety of methodologies are used by core personnel to generate primary tumor xenografts. In addition to subcutaneous implantation, sites of tumor cell implantation include intra-venous (tail vein), intra-cardiac (direct injection into left ventricle), and orthotopic delivery into: mammary fat pad, pancreas, spleen, prostate, bladder, and kidney capsule. In certain cases, Matrigel is injected in combination with the tumor cells. Some tumors require hormone supplementation for growth in vivo. In most cases, this is provided by an implanted, subcutaneous slow release pellet. For drug dosing, the Preclinical Therapeutics Core provides therapeutic agent delivery by a number of routes including intra-venous, intra-peritoneal, intra-tumoral, subcutaneous, and oral (by gavage). The core can also implant Alzet osmotic minipumps for continuous drug delivery (up to 28 days). Animal weight is measured twice weekly and weight loss is used as an index of toxicity. Tumor volume is typically measured twice weekly, using electronic calipers to calculate tumor volumes. In some studies in vivo imaging is used to assess tumor volume, location and proliferation of disseminated and orthotopic tumor cells, typically expressing firefly luciferase. Body fluids, such as blood, urine, feces, organs, and cells are collected as needed per experimental design; in the case of PK studies, at frequent intervals. At the close of a study, tumors are collected and processed in a myriad of ways depending on experimental design. Tumors are placed in 10% buffered formalin for transfer to the Mouse Pathology Core for histopathology and immunohistology analyses, in Trizol for RNA analyses, or snap-frozen for other types of studies (i.e., protein analysis, etc). In some studies, blood (50-100 ul) is collected by saphenous vein bleeding at weekly intervals per standard CAR procedures. All animal procedures are performed according to the UCSF-approved CAR Protocols.
Statistics
The Preclinical Therapeutics Core works closely with project investigators and UCSF statisticians in order to design studies that yield adequate statistical power while minimizing the number of animals required. The number of animals required to achieve statistical significance is based upon historical data generated in the PCTC, as well as relevant published data.
Multimodal imaging methodologies to support preclinical studies - models of metastatic disease
Metastatic disease accounts for the vast majority of morbidity and mortality in human cancer patients. The Preclinical Therapeutics Core has developed and provides as a service a number of animal models in which a combination of imaging approaches allows rapid evaluation of tumor location and overall tumor burden, including metastases. This provides a number of benefits in the context of experimental therapeutic evaluation. There is increasing evidence that tumor growth and behavior including invasion and metastasis is dependent on the local tumor environment. For example, orthotopic xenograft models have been shown to provide a more faithful recapitulation of the biology of human disease. The challenge of such models for use in preclinical therapeutic trials is the ability to non-invasively monitor tumor burden and response to therapy. We have used a combination of fluorescent and bioluminescent imaging methodologies and a multi-modal reporter construct in the tumor cells of interest to overcome this limitation. Viral transduction is used to generate reporter cell lines, and tumor burden is subsequently monitored non-invasively through the use of bioluminescent imaging based on firefly luciferase detection. This quantifiable measure of tumor burden provides a basis for study enrollment, tumor localization and response to therapy. Finally, at the conclusion of the study, post-euthanasia autopsy under direct fluorescent visualization allows sensitive detection of gross and microscopic metastatic disease. Core personnel are skilled in application of imaging technologies and data analysis of bioluminescent images. Reporter cell lines are maintained for use in studies requiring imaging technology, or investigators can request transduction of their cell line of interest.
Genetically engineered models
UCSF develops and validates many genetically engineered mouse models of human cancer. These have certain advantages over xenograft models resulting from de novo tumor development and outgrowth in situ, including the complex multi-component tissue architecture of the tumor. Often histological response to drug in xenograft models does not correlate with phase II clinical response (Johnson et al 2001), whereas mouse models may have increased predictive value in preclinical studies. The Preclinical Therapeutics Core therefore offers services that support preclinical trials in these models such as drug dosing and PK studies, animal weight and tumor measurements, whole animal imaging, tissue collection and necropsy. Although the core does not at present have the resources to maintain breeding colonies of multiple lines of engineered mice, we are in the process of assembling a comprehensive database of models that are available at UCSF in order to foster collaborations between labs actively utilizing these models, and investigators interested in undertaking preclinical trials thereon.
Pharmacokinetic and pharmacodynamic studies
The PCTC assists investigators in conducting pharmaco-kinetic (PK) and pharmaco-dynamic (PD) analysis of experimental therapeutics. In this case, the core administers the test agents, collects blood, serum or plasma as needed and provides these to the researchers for follow-up evaluation by mass spec. or other appropriate means.
Equipment
The PCTC maintains and oversees a Xenogen IVIS 100 bioluminescent imaging system. This is used for non-invasive in vivo bioluminescent imaging. By this means, whole animals harboring optical reporter cells are imaged to determine tumor location and burden. It includes a Xenogen XG-8 anesthesia system and is supported by a desktop PC. As part of its optical imaging resources, the Core also possesses a Leica MZ FLIII, fluorescent stereoscope. This includes filters for both GFP and Texas Red, and an 0.63 objective lens. It is supported by a EXFO X-Cite 120 tungsten halogen light source, cooled color Micropublisher camera, and desktop PC. By this means, fluorescent labeled cells and/or probes can be detected and documented during survival surgery and in post-euthanasia necropsy. Other major equipment available in the lab or immediately adjacent includes centrifuges, ultra centrifuges, a spectrophotometer, a scintillation counter, fluorescence light microscopes, environmental shakers, a cold room, a darkroom, power supplies, an electroporator, HPLC, FPLC, a gel electrophoresis and imaging system, a Molecular Dynamics phosphorimager, a DNA synthesizer, and a DNA sequencing machine. Confocal and deconvolution light microscopy and microarray facilities are all available through Cancer Center cores, located in the Cancer Research Building.
Computer
Core personnel have access to three computers, and shared printers stationed in the lab space; campus computer facilities database searches; libraries; Cancer Center computer support services; etc. The core also maintains a laptop computer located in the animal barrier facility and connected to the central server that links directly to balance and caliper devices used for animal weights and tumor measurements.
History of the Preclinical Therapeutics Core
A preclinical core has been in existence for many years at UCSF where it principally supported the Breast SPORE and, more recently, the U54 (RTK targeting) grant. In this context, it has acted as the primary vehicle by which UCSF Breast SPORE and U54 investigators generate pre-clinical data in vivo and mechanistically validate hypotheses generated in vitro. Under this arrangement preclinical animal tumor model systems have been used to evaluate anti-tumor efficacy, pharmacokinetics, bio-distribution and imaging potential of innovative therapeutics. As part of the UCSF Breast SPORE, the Preclinical Core played a critical role in the development and validation of a liposome-encapsulated form of doxycycline (Doxil) that has gone on to become an approved clinical agent. The Core has performed important trials in the development and translation of a number of other novel agents such as antibody-targeted nanoparticle agents, (e.g., liposomal CPT-11), anti-telomerase RNAi delivery, and rational combinations of targeted therapeutics.
Over the past three years, the core has helped design, implement and analyze 121 studies (45 of which were preclinical trials) involving over 4,400 mice. Since July 2005, the Preclinical Therapeutics Core has been a Developing Core of the UCSF Helen Diller Family Comprehensive Cancer Center, offering its services to the greater Cancer Center community. Demand for the PCTC has been growing, and is projected to increase as a consequence of both the broader user-base at UCSF, the development of an increasing number of investigational drugs, and the trend towards analysis of combinatorial drug combinations and investigation of novel technologies to enhance cancer drug delivery and efficacy in vivo. The Preclinical Therapeutics Core is now being proposed as full core as part of the recently submitted cancer center support grant.
Why do preclinical studies?
The premise of preclinical studies is that results of drug treatment studies in animals are informative for subsequent clinical drug trials. In addition to measuring anti-tumor efficacy, in vivo models provide a valuable opportunity to assess pharmacokinetics, pharmacodynamics, drug-drug interactions and preclinical toxicity, as well as initial development of in vivo biomarkers as predictors for anti-tumor efficacy. The UCSF Preclinical Therapeutics Core functions to test novel agents and approaches in all of these capacities. At UCSF there are a number of active efforts to understand the molecular basis of human cancer with the goal of generating rational agents designed to selectively target tumor cells. A growing number of these research programs are now yielding compounds with demonstrated anti-tumor activity in vitro and possible efficacy as in vivo agents. Confirmation of therapeutic efficacy therefore requires trials in living, tumor bearing animals to demonstrate selective anti-tumor activity while remaining safe to non-tumor tissues. Animal models are paramount in developing early in vivo biomarkers predictive of therapeutic response of pathway-specific drugs, a field that will see rapid expansion as the costs of undertaking full scale clinical trials escalate. The primary purpose of the Preclinical Therapeutics Core is to facilitate existing and ongoing studies of this sort, and to provide streamlined procedures for such efforts.
Model development
There is a need for improved animal models of human cancer. As our understanding of human disease becomes more comprehensive, so also does the demand grow for more accurate and sophisticated models. Experimental therapeutics that target a particular signaling pathway or tumor specific molecule must be validated in well characterized models that allow the testing of the experimental hypothesis. For this reason, the Preclinical Therapeutics Core has demonstrated success in developing and providing better animal models. Specifically, orthotopic xenograft models have been developed that more accurately recapitulate important aspects of the tumor environment. The PCTC has also pioneered metastatic models based on intravenous and arterial (intra-cardiac) tumor cell administration. Genetically engineered models, developed by investigators, are also used in therapeutic studies, in some cases using light-based readouts. Finally, sophisticated tumor imaging methodologies are employed to facilitate analysis of anti-tumor efficacy and drug development. These include use and measurement of optical readout from luminescent and fluorescent reporter tumor cell lines to assess tumor burden and response to therapy, as well as technologies such as F-18-labeled compounds followed by PET imaging to selectively label and detect tumor cells and drug biodistribution.
Additional Resources
A searchable database of core facilities at all UCSF campus locations, provided by the Clinical and Translational Science Institute at UCSF, is available here.
