Research Program Areas

Systems Biology

Systems biology is fundamental to the Crump Institute's goals to develop new In vitro and in vivo (imaging) molecular diagnostic technologies. By studying cells and organisms from a systemwide perspective, we learn what are the most informative events to detect and image to assess the healthy or diseased state of the patient's tissue. The Systems Biology program employs an integrated experimental and theoretical approach to study cancer and immune diseases, and to understand the interactions of microorganisms with the human host. Microbes form an intricate symbiotic system with the host. By studying the genomes and the transcriptomes of the microbes living inside humans, we aim to understand how microorganisms interact with and influence the human immune system and play a role in human health and disease. Model systems, such as normal and cancer cells in culture, and small animal models of disease, provide controlled environments where the genetic composition and surrounding conditions can be defined and analyzed in a systematic fashion. Equally important is the ability to obtain serum and tissue from patients, that directly reflect the biology of human disease. From these biological samples, large-scale systemwide measurements can be made, of gene and protein expression that produce cell communication systems and metabolic activity to carry out normal functions of our organ systems and altered ones of disease. Finally, theoretical and computational approaches are used to untangle the complex network of interactions and feedback loops within cells and tissues. Our fundamental goal is to decipher the integrated circuits within and between cells - the networks that form the computational logic, or “thinking” of the cell that determines how it will react under normal conditions, or mis-react in disease.

The Systems Biology program works collaboratively with physicians and biologists studying diseases like cancer and immune disorders to help define what molecular events need to be detected or imaged based on the biology of the affected cells and tissues, and with the technology programs of the Crump to develop new methods to make experimental and diagnostic measurements.

Principal Investigators: Thomas Graeber, Raphael Levine, and Huiying Li

Investigators: Arion Chatziioannou, Henry Huang, Caius Radu, Hsian-Rong Tseng

In vitro Molecular Diagnostics (IVMD)

Figure 1. Conceptual summary of the microfluidic image cytometry technology: (a) Dissociated glioblastoma cells obtained from a patient are introduced into a microfluidic cell array chip for multi-parameter analysis by this technology. (b) A semi-automated pipette executes cell seeding/culture and ICC. (c) The ICC-treated samples in the chips are mounted on a fluorescent microscope for image acquisition followed by analysis using an image cytometry program (i.e., Metamorph, Molecular Devices Inc.) to quantify the expression levels of signaling proteins with single-cell resolution.

Traditional pathological and molecular diagnostics of patient specimens (blood, serum, tissue), although valuable, lack specificity to define critical molecular transition points in disease to accurate identify the presence of therapeutic targets. The In vitro Molecular Diagnostics program is engaged in developing new, microfluidics-based platforms for In vitro molecular diagnostics that will offer significant advantages over conventional methods, including speed, sensitivity, the capability to simultaneously measure numerous disease-defining biological and biochemical parameters from very small biological samples. Such measurements should yield a broad scale “systems” approach to early molecular diagnosis of disease, patient stratification and treatment monitoring. Furthermore, the shift to small, portable, chip-based diagnostic platforms, will enable us to move In vitro diagnostics out of the pathology laboratory and directly into the operating room or the patient's bedside, a change which should greatly expand patients' access to cutting-edge molecular diagnostics and give physicians the tools needed to implement the concept of personalized medicine.

Crump researchers have downsized and reformatted common analytical procedures such as flow cytometry, metabolic assays, and kinase assays onto microfluidic chips. The In vitro Molecular Diagnostics team has demonstrated the ability to precisely measure expression of signaling proteins from patient samples, with single-cell resolution. Furthermore, a chip-based PET probe assay, comprised of a cell culture array and an embedded position sensitive avalanche photodiode detector(PSAPD) detector camera has also been developed and used to measure 18F- fluorodeoxyglucose (FDG) transport and phosphorylation, again with sensitivity down to single cells as a demonstration of a general approach for measuring the biochemical properties of imaging probes. Similarly, a kinase assay chip has been coupled to the PSAPD camera, enabling quantitation of phosphorylation rates for various substrates using 32P for measuring cell communication and metabolic processes.

Principal Investigators: Hsian-Rong Tseng, Arion Chatziioannou, and Thomas Graeber

Investigators: Chris Behrenbruch, Johannes Czernin, James Heath, Henry Huang, Harley Kornblum, Jorge Lazareff, Paul Mischel, Allan Pantuck, Jianyu Rao, Antoni Ribas, Michael van Dam, and Hong Wu

Chemical Synthesis Platform Technologies

Positron Emission Tomography (PET) provides a highly sensitive and accurate method for measuring the spatial and temporal distribution of radiolabeled molecular probes or tracers. These imaging probes can provide comprehensive information about processes such as metabolic activity, cell signaling, DNA replication, cell proliferation, etc, in living subjects. However, a tremendous bottleneck in the development of new molecular probes exists due to the fact that developing even a single new candidate molecular probe is a highly labor-intensive, time-consuming and expensive process.

The Chemical Synthesis Platform Technology program is focused on eliminating this roadblock, through development of flexible and user-friendly chemical synthesis platforms. Although the initial purpose is the production of novel radiolabeled compounds for PET, our approach of breaking down complex syntheses into a small set of simple unit operations, each performed in a standardized, modular fashion, can be extended to a broad variety of chemical and biological applications. In addition, we incorporate microfluidic components to take advantage of the many scientific and practical benefits of working at small scales. Recent developments include collaboration with the Preclinical Imaging Systems program to develop a novel imaging technique to monitor radiochemical reactions on-chip, and a joint project with the Molecular Imaging with ImmunoPET group to develop microfluidic chip platforms for radiolabeling biological molecules.

Principal Investigators: Michael van Dam, Clifton Shen, and Nagichettiar Satyamurthy

Investigators: Arion Chatziioannou, Hsian-Rong Tseng, Anna Wu

Molecular Diagnostics in Cancer and Immune Disorders

Cancer and immunology are intertwined at many levels. For example, cancer cells as well as immune cells (B and T lymphocytes, macrophages, and other cells that coordinately participate in immune responses), both undergo similar programs of activation, altered gene expression, enhanced proliferation and migration. Elucidating the molecular signatures associated with the transition from a normal tissue to a cancer lesion and with the activation of the immune system to fight pathogens and tumors could lead to the identification of novel biomarkers that can precisely distinguish health and disease states at molecular and cellular levels. Furthermore, while it is widely accepted that abnormal immune function is a hallmark of many diseases, including cancer, autoimmune disorders, cardiovascular diseases, diabetes, and neurological disorders, there is an increasing recognition of the immune system as an internal sensory organ capable of real-time monitoring of cells and tissues throughout the body. Deviations from normal physiology due to infection, activation of oncogenes and other types of cellular injuries can be detected by the immune system leading to programmed cellular and humoral responses. In turn, these responses result in dynamic changes in the composition of essential compartments of the immune system, including the T cell repertoire.

The Molecular Diagnostics program has developed new technologies for high-throughput analysis of the T cell repertoire in animal models and in humans. The increased sensitivity and multiplexing capability of this approach will significantly enhance our ability to monitor immune responses using novel In vitro diagnostic platforms. Furthermore, a novel PET probe discovery platform based on differential screening to select molecular probes that target proteins specific to a biological process or disease has already lead to development of a series of imaging probes for monitoring immune activation and can be used to predict responses of cancer to common chemotherapy drugs. Through a partnership with the Institute for Molecular Medicine (IMED), these tracers have been extensively evaluated in preclinical models and have been brought into the clinic for studies in patients in less than 18 months.

Principal Investigators: Caius Radu, Johannes Czernin, Nagichettiar Satyamurthy, and James Heath

Investigators: Thomas Graeber, Antoni Ribas, Ton Schumacher (Netherlands Cancer Institute, NKI), Hsian-Rong Tseng, Owen Witte, Anna Wu

Molecular Imaging Using ImmunoPET

Figure 2: Engineered antibody fragments for in vivo PET imaging of cell surface markers. The top panel shows a native, intact antibody with the variable regions that form the binding site shown in green, and the constant domains in blue. Engineered fragments depicted include single-chain variable fragments (scFv), the diabody (a dimer of scFv fragments), and minibody (fusion of scFv and CH3 domain). The lower panel shows coronal slices of co-registered microPET/microCT images with arrows indicating the tumor, all acquired at 20 h following intravenous administration of a radiolabeled engineered antibody fragment in athymic mice carrying different subcutaneous human tumor xenografts. A. Imaging of an LS174T colon cancer tumor using a carcinoembryonic antigen (CEA)-specific diabody labeled with the positron emitter 124I. B. Imaging of a B-cell lymphoma using CD20-specific minibody labeled with 124I. C. Detection of an LAPC-9 prostate cancer xenograft using a minibody that recognizes prostate stem cell antigen (PSCA), labeled with 124I.

As part of the Crump Institute's broad goal of providing platform imaging strategies for detection of any molecular biomarker of choice, we are developing engineered antibodies as a means for imaging cell surface proteins in vivo. Approximately 20% of a cell's proteins are expressed on its surface, making them accessible to antibody-based molecular imaging probes. These cell surface biomarkers include important classes of proteins such as growth factor receptors, adhesion molecules, enzymes and proteases, tissue-specific markers, differentiation and activation markers — making the cell surface highly rich in informative targets that can reveal the biological state of the cell, both in normal states and in their transitions to disease.

Antibodies of high specificity for a selected protein target can be readily isolated by methods such as phage display. The ImmunoPET group then genetically engineers these antibodies for optimal function as imaging agents - rapid, high-level targeting to specific protein and low non-specific background in surrounding tissue. The ImmunoPET team relies on collaborations with the Systems Biology and Molecular Diagnostics programs to identify informative cell-surface proteins as biomarkers in disease, particularly in cancer and immune disorders. Implementation of these strategies requires close collaboration with the Chemical Synthesis and Preclinical Imaging programs to rapidly radiolabel and evaluate these novel tracers in animal models of disease.

Principal Investigator: Anna Wu

Investigators: Chris Behrenbruch, Arion Chatziioannou, Johannes Czernin, Caius Radu, Clifton Shen, David Stout, Michael van Dam, Christine Wu

Preclinical Imaging Systems

Preclinical imaging at the Crump Institute spans domains from In vitro diagnostics and imaging cell cultures, through in vivo imaging of mouse models of disease, using microPET, microCT, and optical imaging modalities. Overall, the focus of the Preclinical Imaging Systems program is to develop innovative technologies that enable the visualization and measurement of biological processes to facilitate research on the molecular mechanisms of cells in health and disease. To further these goals, highly sensitive silicon-based solid state imaging sensors (Position Sensitive Avalanche Photodiode Detectors, or PSAPDs), are being developed that can detect radiolabeled probes at amounts as low as a few picocuries. These devices are integrated with microfluidic chips in collaborations with the In vitro Molecular Diagnostics and Chemical Synthesis Platform program areas to produce novel imaging devices for In vitro assays in arrays of cell cultures in chips. For in vivo imaging, multimodality instrumentation is being designed and optimized for combining PET with anatomical imaging (such as CT) and optical imaging of mouse models of disease. These state-of-the-art technologies are made available to Crump investigators in coordination with the Preclinical Imaging Technology Center.

Principal Investigators: Arion F. Chatziioannou and David B. Stout

Investigators: Chris Behrenbruch, Thomas Graeber, Henry Huang, Hsian-Rong Tseng, Christine Wu

Tracer and Pharmacokinetic Modeling

Figure 2: Illustration of a new strategy of controlling the incubation medium infusion schedule to allow the measurement of the transport and reaction kinetics (e.g., phosphorylation) of PET probes in multiple wells simultaneously in an integrated microfluidics chip with a camera consisting of an array Si avalanche photodiodes (PSAPD) embedded in the chip for detecting beta particles. Panel A shows the microfluidic chip design that contains 16 cell culture wells (1-16). Panel B shows that the radioactivity recorded from each of the wells with the PSAPD camera (in this example, only eight wells contained cells) at one point in time, although data were collected as a function of time. Panel C shows the kinetic measurement for one of the wells when a strategy of using multiple cycles of tracer incubation (TI) and removal (TR) was employed to overcome the high background associated with the relatively large medium volume in the wells to improve the S/N for estimating the model parameters. The Xs at the top and bottom of each cycle are the average radioactivity measured within each TI or TR cycle period. The width of the bars is the time duration of each cycle. The seesaw solid lines are regression fit by the standard FDG compartmental model. The close match of the model fitting and the kinetic measurement indicated the adequacy of the model for extracting information from the measured kinetics (k1 - forward and k2 - reverse transport; and k3 - phosphorylation and k4 - dephosphorylation).

Figure 3: An overview of the small animal microPET imaging with a microfluidic blood sampling device. The cartoon demonstrates the concept of our design and the connections of the microfluidic chip to its operational environment. The injection of an imaging probe and the sampling of multiple blood samples are automated with a click of a button on the PC control system.

Figure 4: A tumor study in a mouse using the sequential images with microPET of the F-18 labeled probes FLT for imaging DNA replication and cell proliferation that was IV injected first and then 60 minutes later FDG was IV injected to image glycolysis. Along with continuous imaging, blood samples were obtained by a microfluidic blood sampler: (A) images - tumor shown with arrows; (B) blood curves; (C) tumor tissue curves; and (D) kinetic data for FLT and FDG in the tumor after separation of the individual kinetic curves from the composite curves in B and C. These data were then used to calculate the rate constants for transport (k1 and k2) and phosphorylation (k3) and dephosphorylation (k4).

The ultimate goal of the Crump Institute for Molecular Imaging is to develop technologies and methods for measurement of biological functions in living subjects. These efforts enable the quantitation of precise molecular events in cells with spatial information regarding their location within specific tissues and organ systems and temporal information (changes over time) to determine the rates of biological processes and biochemical reactions. In order to facilitate interpretation of results, image reconstruction, archiving, and user-friendly display are essential.

Our goals, however, extend beyond providing visualization of experimental data to converting images into assays of biological processes through the quantitative measurement capability of PET. The experimental setting in this program area ranges from the development of assays under the controlled In vitro conditions of kinetic measurement with radiolabeled probes in arrays of cell cultures on an integrated microfluidic chip to a living subject. The Tracer and Pharmacokinetic Modeling program centralizes the analysis and interpretation of kinetic In vitro measurements on a chip and in vivo measurements with microPET, by developing kinetic models for new tracers, and simplifying and automating procedures to render them accessible and transparent to investigators with varied, non-mathematical backgrounds. Where needed, new technologies and methods are developed and introduced, such as a microfluidic blood sampler and cell separation system to provide programmed, timed, plasma sampling requiring minimal amounts of blood from animal studies to determine the delivery of the radiolabeled probe to tissues throughout the body as the plasma input function for the kinetic models. As a result, accurate measurements can be made of the biochemistry, metabolism, and signaling in cells and tissues, within a living subject. Importantly, these measurements can be made over time, such as during the course of development of disease, or before and after a targeted treatment is administered, in order to more precisely measure the biology of disease and to assess the effectiveness of various therapeutic interventions.

Principal Investigator: S.C. (Henry) Huang

Investigators: Chris Behrenbruch, Arion Chatziioannou, Johannes Czernin, Caius Radu, David Stout, David Truong, Hsian-Rong Tseng, KP Wong, Christine Wu