Patent Application
20100291584
1. Field of Invention
Embodiments of the present invention relate to microfluidic systems, and more particularly to microfluidic systems and methods for large-scale cell culture and assay.
2. Discussion of Related Art
All references cited in this specification are incorporated herein by reference.
Astrocytic brain tumors span a wide range of neoplasms with distinct clinical, histopathological, and genetic features. Molecular genetic data that has been gathered since the prior WHO classification in 1993 suggest that individual histologically defined types of astrocytomas are even more diverse at a biological level.1 For instance, the majority of glioblastomas arise without clinical or histological evidence of a less malignant precursor lesion and these lesions have been designated primary glioblastoma. They appear in older patients (mean age, 55 yr) after a short clinical history of usually less than 3 months. These primary glioblastomas are characterized by EGFR amplification (˜40% of cases) and/or overexpression (60%), PTEN mutations (30%), p16INK4a deletion (30%-40%), MDM2 amplification (<10%) and/or overexpression (50%), and in 50%-80% of cases, loss of heterozygosity (LOH) on the entire chromosome 10. In contrast, secondary flioblastomas develop more slowly malignant progression from diffuse (WHO grade II) or anaplastic astrocytoma (WHO grade III) and appear in younger patients (mean age, 40 yr). Secondary glioblastomas contain TP53 mutations in approximately 60% of cases; more than 90% of these mutations are already present in the preceding diffuse (WHO grade II) or anaplastic astrocytoma (grade III). The pathway to secondary glioblastomas is further characterized by allelic loss of chromosomes 19q and 10q1. Histopathologically, an unambiguous distinction of these subtypes has remained elusive, but they clearly evolve through different genetic pathways1-3. It also remains to be shown whether these subtypes differ significantly with respect to prognosis, but it is likely that they will respond differently to specific novel therapies as they are developed4. As a result, ongoing clinical trials need to incorporate molecular su typing and future classification schemes will no doubt be based on such differences as well5.
Among patients with glioblastoma, the most common primary malignant brain tumor of adults, small subgroup seems to benefit from the EGFR kinase inhibitors erolotinib and gefitinib6. However, the infrequency of mutations in the EGFR kinase domain in the glioblastomas7,8 suggests that such EGFR mutations cannot account for responsiveness to EGFR kinase inhibitors9. The EGFR gene is commonly amplified in glioblastoma10, but this abnormality also does not correlate with responsiveness to EGFP kinase inhibitors9, Glioblastoma often express EGFRvIII, constitutively active genomic deletion variant of EGFR11-15. This variant of EGFR strongly and persistently activates the phosphatidylinositol 3′ kinase (PI3K) signaling pathway, which provides critical information for cell survival, proliferation, and motility16-20. Persistent PI3K signaling activated by EGFRvIII is believed to cause pathway addiction21; addicted tumor cells die if the pathway is disrupted by tyrosine kinase inhibitors. By promotering chronic dependence on PI3K signaling, EGFRvIII may sensitize glioblastoma cells to EGFR kinase inhibitors.
The PTEN (phosphatase and tensin homologue deleted in chromosome 10) tumor-suppressor protein, inhibitor of the PI3K signaling pathway, is commonly lost in glioblastoma10,17,22. This loss may promote cellular resistance to EGFR kinase-inhibitor therapy by dissociating EGFR inhibition from downstream PI3K pathway inhibition23. We hypothesized that EGFRvIII would sensitize tumors to EGFR kinase inhibitors, whereas PTEN loss would impair the response to such inhibitors23.
To test this hypothesis, we analyzed EGFRvIII and PTEN at the gene and protein levels in glioblastomas from patients before treatment with EGFR kinase inhibitors. We also searched for mutations in EGFR and in its heterodimerization partner Her2/neu, which has been reported to be mutated in glioblastoma and could also affect the response to EGFR kinase inhibitors24. We found a strong association between the coexpression of EGFRvIII and PTEN in glioblastoma cells and responsiveness to EGFR kinase inhibitors.
mTOR
Mammalian target of rapamycin (mTOR, also known as FRAP, RAFT1 and RAP1) has been identified as a key kinase acting downstream of the activation of PI3K25. Rapamycin and rapamycin derivatives that specifically block mTOR have been developed during the past 5 years as potential anticancer agents. mTOR regulates essential signal transduction pathways and is involved in coupling growth stimuli to cell-cycle progression. In response to growth-inducing signals, quiescent cells increase the translation of a subset of mRNAs, the protein products of which are required for progression through the G1 phase of the cell cycle. PI3K and AKT are the key elements of the upstream pathway that links the ligation of growth factor receptors to the phosphorylation and activation state of mTOR26,27. With regard to the role of the PI3K/AKT/mTOR pathway in the genesis and proliferation of cancer cells, elements of the PI3K/AKT/mTOR pathway have been demonstrated to be activated by the erythroblastic leukaemia viral oncogene homologue (ERB) family of surface receptors, the insulin like growth factor receptors (GFRs), and oncogenic Ras28-31.
The clinical applications of 18F-2-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography (PET) are continually expanding, especially in the field of oncology. In colorectal cancer (CRC), the diverse uses of PET include initial diagnosis, staging, restaging, and assessment of the therapeutic response32-34. PET has also been reported to offer advantages over conventional, anatomically based morphologic modalities for detecting recurrent CRC and metastatic disease, because of its capacity to provide a functional image and evidence of tumor behavior35,36. This is based on the knowledge that enhanced glucose uptake is one of the major metabolic changes characteristic of malignant tumors. Clinically, FDG is the most commonly used PET tracer. It is metabolized similarly to glucose, being transported into the cell, but once enzymatically phosphorylated, FDG-6-phosphate is metabolically trapped in tumor cells. Thus, tumors demonstrate increased commutation of FDG and can be distinguished on PET scan images by areas of increased tracer activity37. Clinically, variable FDG uptake, semiquantified as the standardized uptake value (SUV), has been seen on PET scans of tumors from the same origin, including CRC. Much research has been done on the differences in FDG uptake among tumors and the mechanism of this uptake. Emerging evidence indicates that the factors affecting FDG uptake are complicated because the specific biological characteristics of tumors determine the degree of glucose metabolism38-40. Most factors affecting FDG uptake, such as hypoxia and cell density, are thought to be associated with changes in glycolysis-related protein expression41,42. The expression of glucose transporter proteins, especially GLUT-1, which is directly involved in FDG uptake, is thought to determine the levels of FDG uptake in cancer cells43-45.
As described above, FDG is the most commonly used as a PET tracer, and transported into cells through glucose transporter (Glut). This transported FDG was phosphorylated by Hexokinase and trapped in cells. Thus FDG uptake was regulated by both Glut and hexokinase. However, interestingly, localization of Glut to cell membrane is enhanced by Akt activity. Additionally, expression of hexokinase was also regulated by Akt. This means FDG uptake is strongly related with PI3K/Akt/mTOR pathway.
In a limited number of pilot trials using RAD001 and AP23573, 18F-2-fluoro-2-deoxy-d-glucose positron emission tomography (PET) was used to monitor the glucose uptake of tumors following administration of mTOR inhibitors. In those preliminary studies, some tumors exhibited a decrease in glucose uptake that was not consistently associated with objective responses determined by radiological methods46.
As described above, the PI3K/Akt/mTOR signaling pathway is strongly associated with glucose and FDG uptake in cancer cells. That is the reason why FDG is such a great surrogate marker for evolution of therapeutic effects of inhibitors and/or drugs which target the PI3K/AkT/mTOR, signaling pathway. Additionally, as described above, brain tumors should be classified with molecular fingerprints and clinical trials should follow this classification. The PET imaging for cancer diagnosis should also cooperate with this classification. Therefore, it is not unreasonable to propose that the development of molecular therapeutics should be performed in conjunction with the development of molecular diagnostics according to the classification of molecular fingerprints of the disease. However, there is no such discovery platform currently available.
A microfluidic system according to an embodiment of the current invention has a pipette system comprising a plurality of pipettes, a microfluidic chip arranged proximate the pipette system, an imaging optical detection system arranged proximate the microfluidic chip, and an image processing system in communication with the imaging optical detection system. The microfluidic chip has a plurality of cell culture chambers defined by a body of the microfluidic chip, each cell culture chamber being in fluid connection with an input channel and an output channel defined by the microfluidic chip. The pipette system is constructed and arranged to at least one of inject fluid through the plurality of pipettes into the plurality of input channels or extract fluid through the plurality of pipettes from the plurality of output channels while the microfluidic system is in operation. A method of automated fluorescent imaging of a plurality of cell cultures includes loading a plurality of cell cultures into a plurality of cell culture chambers of a microfluidic chip, the plurality of cell culture chambers comprising a surface coating of an extracellular matrix material for immobilization of said cell cultures, applying at least one fluorescent probe to the cell cultures and incubating the cell cultures under suitable conditions to promote binding of the probe to a specific target in or on the cells, illuminating the cell cultures to cause the fluorescent probe to emit fluorescent light, and imaging light fluorescing from the cells in each of the plurality of cell culture chambers while the cell cultures remain substantially immobilized in the plurality of cell culture chambers to provide information regarding the specific target in or on the cells.
Additional features of this invention are provided in the following detailed description of various embodiments of the invention with reference to the drawings. Furthermore, the above-discussed and other attendant advantages of the present invention will become better understood by reference to the detailed description when taken in conjunction with the accompanying drawings, in which:
In recent years, micro total analysis systems (μTAS) have been of great interest to biological researchers for cellular analysis. A prominent characteristic of μTAS is the capability of constructing highly integrated/functional systems on a microchip. Therefore, many processes that were complicated in conventional cellular analysis could be integrated on stand-alone microchips. This integration resulted in short-time analysis and easy handling for operation. Moreover, these integrated microfluidic systems had advantages such as a reduction in the consumption of cells, reagents, and samples, real-time analysis, and constancy of experimental conditions47. As stated above, cellular analysis on an integrated microchip can provide numerous benefits. Thus, cellular analysis on the microchip has been rapidly spreading, for example, to applications of cell sorting48, and the introduction of genes into cells.
Poly(dimethylsiloxane) (PDMS)-based integrated microfluidics represent a large scale architecture of fluidic channels that allow for the execution and automation of sequential physical, chemical and biological processes on the same device with digital control of operations49,50. In particular, the elasticity of PDMS materials enable a parallel fabrication of the micron-scale functioning modules, such as valves, pumps and columns51, that are necessary for sequential operations. In addition, fabrication of intricate devices using this technology requires only relatively simple facilities: the fluidic and control networks are mapped using standard CAD software and transferred onto transparent photomasks. Photolithographic techniques can be used to produce a reusable mold onto which a PDMS resin is poured and cured by baking. Access to the fluidic channels can be achieved by punching holes through the bulk material, and the devices can be readily bonded to glass or silicon substrates, for example. Large arrays of active components, such as valves and pumps, can be created by stacking multiple, individually fabricated layers. When pressurized with air or inert gases, a channel on the control layer that crosses a channel on the flow layer is deflected, sealing the flow channel and stopping fluid movement. This method of valve operation also constitutes the binary switches (e.g., open or closed) of the microfluidics chip. Using this simple fabrication technology, our joint research team has demonstrated devices of remarkable diversity, including microfluidic devices with chemical reaction circuits (PCT Int. Appl. 2006, WO 2006071470 and U.S. Patent Application 2007, No. 60/876,525), an integrated microfluidic blood sampler for mice (UCLA Case No. 2005-659-1), a microfluidic platform for high sensitivity quantification of radioisotope concentrations (U.S. Provisional Patent Application, UCLA Case No. 2006-388-1) and a microfluidic platform for cell culture and assay (U.S. Provisional Patent Application No. 60/876,525 filed Dec. 22, 2006), the entire contented of which are incorporated herein by reference.
Single-cell measurements can reveal information obscured in population averages. For example, studies of variation in gene expression in individual Escherichia coli and S. cerevisiae52,53 cells have shown that only a fraction of cell-to-cell variation in the expression of reporter genes results from stochastic fluctuations in the workings of the gene expression machinery54-56, and have identified other processes and genes that account for and control the bulk of the variation54.
One means to collect single-cell data is flow cytometry, whose development began in the 1930s. Modern instruments are powerful but (i) cannot interrogate individual cells repeatedly to produce time series for each cell, (ii) cannot collect a great deal of light, owing to both the short time (typically microseconds) that the cell passes the detector and to the numerical aperture of the objective, which often collects less than 10% of the emitted light, and (iii) typically do not capture images of the cells, making it difficult to analyze cell shape, size and intracellular localization of fluorescence. Some recent work has attempted to address the last two limitations by integrating the fluorescence signal over longer times and capturing cell images with custom-built charge-coupled device (CCD) detectors57. Optical microscopy can compensate for some limitations of flow cytometry by providing abilities to revisit individual cells over time, collect emitted light for long times and capture cell images with high resolution. Automation by computer-aided cell tracking and image analysis began in the 1960s and can permit generation of some such data with high throughput57-63. However, such approaches of single cell tracking have limitations in practical applications, such as low throughput and/or poor cell viability.
According to an embodiment of the current invention, new PDMS-based microfluidic devices (see
This microfluidic platform according to some embodiments of the current invention has the potential to significantly enhance the throughput of cell analysis with microscope-based cytometry. This device can enable one to analyze the drug effects on PI3K signaling pathways in individual cancer cells and assess PET probe uptake at the same time (
An intrinsic advantage of drug screening and PET probe discovery in a microfluidic device according to some embodiments of the current invention is that we can reduce the volume of medium, antibodies, PET tracers, and so on used. Volumes as low as about 200 pL per microchamber have been found to be suitable according to some embodiments of the current invention.
In an embodiment of the current invention, signatures of the PI3K/Akt/mTOR signaling pathway are stained with immunofluorescent methods. We can then analyze drug effects on PI3K signaling pathways with a microscope-based cytometry system according to an embodiment of the current invention.
This device allows us to classify glioblastoma of patients with a molecular fingerprint analysis for individual cells. According to this classification, we can choose effective drugs for each glioblastoma patient.
At the same time, we can assess and choose an effective PET tracer for each patient with the microfluidic system according to an embodiment of the current invention. According to sonic embodiments of the current invention, we can develop the various PET tracers for each molecular event in cancer cells.
Dr. Luke Lee's group at UC Berkeley presented a high aspect ratio microfluidic device for culturing cells inside an array of microchambers with continuous perfusion of medium. The device was designed to provide a potential tool for cost-effective and automated cell culture. The single unit of the array consists of a circular microfluidic chamber 40 μm in height surrounded by multiple narrow perfusion channels 2 μm in height. The high aspect ratio (˜20) between the microchamber and the perfusion channels can offer advantages such as localization of the cells inside the microchamber as well as creating a uniform microenvironment for cell growth. Finite element methods were used to simulate flow profile and mass transfer of the device. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37° C. and were grown to confluence.
High-throughout measurement of ion-channel activity by patch damping is of considerable interest in drug discovery as a tool to characterize therapeutic molecules. Microsystems that combine high throughput with small reagent volumes have led to commercial microscale patch-clamp devices. In these devices, ion-channel recording is typically achieved by placing cells on a micrometer-sized aperture in a membrane that separates two electrodes64,65. By guiding cells onto apertures using microfluidic paths, it is possible to reduce the otherwise labor-intensive micromanipulations needed to locate cells at recording sites and to present the cell with successive stimuli66. Obtaining the high-electrical-resistance seals necessary for high quality ion-channel recording (˜109Ω) is technically challenging on both a macro- and a microscale, and microsystems have been more successful in meeting the throughput challenge.
In both conventional studies and microsystems, the analysis of single cells has typically been performed using image-based techniques and intracellular fluorescent probes (such as those that measure calcium flux67). However, the ability of integrated microfluidics to accurately manipulate, handle and analyze very small volumes has opened up new opportunities for analysis of intracellular constituents. A microfluidic device with integrated pneumatic valves capable of isolating single cells and then lysing them using a chemical lysis buffer has been shown to be capable of extracting and recovering messenger RNA from a single cell68. A similar device that also integrates electrophoretic separation can analyze amino acids from the lysed contents of a single cell69. Single cell analysis by electrophoretic separation, but with electrokinetic flowdriven cell loading, docking and lysis have also been demonstrated70.
Using an immunohistochemical analysis applied to a tissue microarray, Dr. Mische's group performed hierarchical clustering and multidimensional scaling, as well as univariate and multivariate analyses, to dissect the PI3K pathway in vivo17. The results provide the first dissection of the PI3K pathway in glioblastoma in vivo and suggest an approach to stratifying patients for targeted kinase inhibitor therapy. Additionally, DNA-microarray analysis is most useful when it can be integrated with clinical, imaging and histological data. Substantial effort is required to develop appropriate databases that contain key clinical information, including patient characteristics such as age and sex. Brain imaging is routinely undertaken and images are housed in a central database. Histological photomicrographs document cellular morphology, and clinical data are entered in real time through wireless input devices to ensure accurate and up-to-date information. Biopsy material is preserved for future analyses, linked to clinical data and used to extract RNA for large-scale expression analysis using microarrays71.
During the past 20 years, a good deal of research-directed automated microscope-based cytometry outside of clinical and pharmaceutical applications has relied on two commercial software packages, Metamorph (Molecular Devices Corporation) and ImagePro (Media Cybernetics, Inc.), to operate the microscopes, collect the data and analyze them. These packages, often used together with more general purpose analysis programs, such as Matlab (The Mathworks, Inc.) and Labview (National Instruments Corporation), probably constitute the state of the art in commercial software used for these purposes. Likewise, open-source projects can provide valuable tools for image analysis. Examples include the Open Microscopy Environment (OME), which provides file formats and metadata standards for microscope images14, Image J, a Java-based package of microscope image analysis tools15, and CellProfiler16.
The prototypical microchips for drug screening and PET probe discovery (
In some embodiments, microfluidic chips with dimensions as small as the following has been used, Chamber dimension: (100 μm (l)×100 μm (w)×20 μm (h), with a volume of 200 pL. Commonly used microfluidic chips to date have the following dimensions. Chamber dimension: (3000 μm (l)×500 μm (w)×100 μm (h), with a volume of 150 nL. In other examples, chambers as large as the following have been used according to embodiments of the current invention. Chamber dimension: (6000 μm (l)×2000 μm (w)×200 μm (h), with a volume of 2.4 μL.
U87 human glioblastoma cell line and stably growing cells from primary glioblastomas from patients treated at UCLA for brain tumors (NS 107, NS 117, NS 146) were first selected for the proof-of-concept trial. U87 cells were modified with retroviruses which have PTEN, EGFR or EGFRvIII driven by the CMV promoter to construct model cell lines for glioblastoma patients (U87-PTEN, U87-EGFR and U87-EGFRvIII).
Fibronectin (FN), which is the extracellular matrix, was coated onto the surfaces of all channels for cell adhesion. 8.0 μl of FN with a concentration of 250 μg/ml was introduced into each channel and incubated at 37° C. for 30 min. The mixture of suspended U87 cell lines obtained from regular cell culture setting was introduced into the FN-coated channels and kept in an incubator for 30 min. Then the channel was rinsed with 100 μL of cell culture medium (Dulbecco's Modified Eagle Medium+10% Bovine Calf Serum+1% Penicillin-streptomycin+1% L-glutamine). After being left overnight, cells in the channels were fixed with 4% paraformaldehyde for 15 min at room temperature. After 15 min, each channel was rinsed with PBS. To prevent non-specific binding of antibody onto the surface of each channel, blocking solution (10% normal goat serum, 0.1% Triton X-100 and 0.1% N-Dodecyl-β-D-maltoside in PBS) was loaded into each channel and incubated for 30 min. To visualize the PI3K/Akt/mTOR. signaling pathway, antibodies were used; mouse anti-hPTEN (Cascade Bioscience) at 100 μg/mL, mouse anti-hEGFR (Zymed Laboratories) at 15 μg/mL, anti-hEGFRvIII (Dako) 87 μg/mL. All antibodies were labeled with mouse IgG Labeling kits (invitrogen). Those antibodies were loaded into each channel and incubated at room temperature for 30˜60 min. After immunostaining, DAPI was loaded into each channel for nuclear staining. After completion of all staining, microchips were monitored with Nikon TE-2000 microscopy, and the images were analyzed with Metamorph® (Molecular Devices) imaging software.
In the cases of EGFR and EGFRvIII, we can also quantitatively analyze population of EGFR negative/positive cells and EGFR immunofluorescent intensity of individual cells (
To monitor glucose uptake in U87 cells, a fluorescent deoxyglucose (2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose; 2-NBDG)72,73 was used and monitored the 2-NBDG uptake by U87 and U87-PTEN cells (
We have demonstrated that this type of microfluidic device according to an embodiment of the current invention can be utilized for glioblastoma and analysis including patient samples. The microfluidic device according to some embodiments of the current invention can provide a platform to monitor individual cells. A microfluidic device according to some embodiments of the current invention can be used to provide cell analysis. A microfluidic system according to some embodiments of the current invention can also be used for high throughput drug screening. Cost reductions can be achieved for cell analysis according to some embodiments of the current invention. In addition, a microfluidic system according to some embodiments of the current invention can be used for PET probe discovery.
A microfluidic system 100 according to an embodiment of the current invention is illustrated schematically in
The microfluidic chip 104 has a plurality of cell culture chambers defined by a body of said microfluidic chip 104. Each cell culture chamber is in fluid connection with an input channel and an output channel defined by the body of the microfluidic chip. The microfluidic chip 104 can be a PDMS-based chip such as that described above in reference to
The pipette system 102 is constructed and arranged to at least one of inject fluid through the plurality of pipettes into the plurality of input channels or extract fluid through the plurality of pipettes from the plurality of output channels while the microfluidic system 100 is in operation. For example, the pipette system 102 can inject a fluid containing cells to be cultured, can inject culture media and/or drugs under investigation, and can inject other biomarkers, etc. according to some embodiments of the invention. The pipette system 102 may also extract fluid from the output channels of the microfluidic chip 104 either with the same plurality of pipettes or with another plurality of pipettes, for example for cell perfusion, etc.
The pipette system 102 can also be a robotic pipette (See
A user-friendly interface that can include a chip holder (See
The microfluidic system according to some embodiments of the invention can include manual, semi-automated and/or automated operation of the following:
The microfluidic system 100 can provide, according to some embodiments of the current invention, a system for (i) large scale cell culture for high-throughput screening, (ii) microfluidic cytometry for quantification of biomolecules with single-cell precision, (iii) signaling pathway network profiling in conjunction with cancer diagnosis and therapeutic stratification, (iv) dynamic protein quantification as an alternative to Western Blot, and other broader applications for quantitative proteomic analysis in cells.
Potential applications of the microfluidic system 100 can include, but are not limited to, the following:
Signaling network profiling according to some embodiments of the current invention can provide the following:
1) Sample preparation, including cell loading, cell culture in an incubator and media exchange for cell maintenance.
2) Immunocytochemistry, including cell fixation, permeabilization, and immunostaining.
The cell culture/assay chip shown in
Data obtained from methods according to some embodiments of the current invention may be in the form, for example, of 2-D or 3-D dot plots (X axis—intensity of one signaling node (in this case, EGFRvIII) and Y axis—intensity of the other signaling node (in this case DAPI)) for 3-D dot plots Z axis—intensity of a third signaling node (
*** Please make sure that Xe lamp is completely cooled prior to turning on the microscope, and that Xe lamp remains on at least 30 minutes during every use.
*** There are two options for viewing images:
*** a) We recommend to use “1 MHz” for “Digitizer” and “3 (4×)” for “Gain” when taking fluorescence images.
*** Before taking the fluorescence image, turn off the brightfield light with the button on the Nikon control box.
Our joint team has recently demonstrated a microfluidic cytometry platform, based on a microfluidic cell array in conjunction with a semi-automated pipette and a fluorescence microscope, for profiling of signaling events involved in the PI3K signaling pathway with single cell resolution. Our initial focus has been on the EGFR/PI3K signaling axis. Drawing on basic mechanistic studies and translating them into the clinic, we have: 1) demonstrated that coexpression of two key proteins that regulate PI3K signaling, a mutant epidermal growth factor receptor (EGFRvIII) and the PTEN tumor suppressor protein are strongly associated with clinical response of glioblastoma patients to EGFR kinase inhibitors and identified mechanisms of resistance associated with PTEN loss (Mellinghoff et al., NEJM 2005); 2) developed strategies to overcome resistance to EGFR kinase inhibitors mediated by PTEN loss (Wang et al., Cancer Res. 2006) and which are being tested in the clinic, and 3) identified additional EGFR mutations that may confer sensitivity to EGFR kinase inhibitors (Lee et al., PLoS Medicine 2006). We have also begun to recognize the challenge of acquired resistance, and to recognize the number of potential routes by which glioblastoma cells may become resistant (Mellinghoff et al., Clinical Cancer Res. 2007).
A number of genetically manipulated glioblastoma cells (i.e., U87, U87-PTEN, U87-EGFR, U87 EGFRvIII and U87-EGFRvIII/PTEN) as well as primary cells have been successfully cultured in the devices, and the expression levels of receptors (EGFR and EFGRvIII), protease (PTEN), phosphorylated kinases (p-Akt, pmTOR and p-S6) of the cells can be analyzed quantitatively. In this example, our research effort has been focused on (i) developing robust and reproducible protocols for immunostaining and image acquistion/analysis to achieve optimal dynamic range for our chip-based cytometry measurements, and (ii) creating a user-friendly interface between microfluidic cell array and a robotic pipette, allowing large-scale signaling profiling in a closely-related microenvironment.
The following describes collection of immunostaining protocols for PI3K/Akt signaling pathway with some exploration of the associated signaling network. Below, we describe some aspects of this approach to characterize molecularly heterogeneous solid glioblastoma tumor samples using this microfluidic cytometry platform.
The original microfluidic cytometry platform utilized a semi-automated pipette to handle cell loading, culture media exchange, fixation, permeabilization and antibody staining in sequence. Besides flow injection, most of the operation/process was, in fact, manually controlled. As a result, a significant amount of labor and inevitable operation error constrain further exploration of this semi-automated approach for applications require large-scale studies. In collaboration with our colleagues (Profs. Mike van Dam and Chris Behrenbruch), a user-friendly interface between microfluidic cell array and a robotic pipette (
One can implement the new immunostaining protocols for PI3K/Akt signaling pathway profiling using the robotic system according to an embodiment of the current invention. One can automate the image acquisition and processing to provide a complete solution covering sample preparation, image acquisition and data analysis for our microfluidic cytometry technology.
1b) Application of this Technology to Perform Multiparameter Measurement of Key Nodes of the PI3K Signaling Pathway Including in Molecularly Heterogeneous Samples. This Device has Obvious Implications for Glioblastoma, but is also likely to be Quite Important for Analysis of other Cancer Types. Quantitative Single Cell Detection of Upstream Signaling Proteins—EGFR, EGFRvIII and PTEN in Glioblastoma Cells:
We have made significant advances in our ability to quantify these upstream markers of PI3K signaling in glioblastoma cells, at the single cell level. Here, we now show how quantitative analysis can be performed simultaneously on multiple signaling proteins of the PI3K pathway in a highly quantitative fashion, at the level of single cell resolution. Further, we show that such data can be analyzed in a similar fashion to flow cytometry data, to facilitate direct comparisons, and to allow for analysis of signaling profiles in complex heterogeneous mixtures, including before and after molecularly targeted treatments.
Expression of the constitutively activated EGFR, EGFRvIII, and loss of the PTEN tumor suppressor have known implications for constitutive activation of PI3K signaling (Mellinghoff et al. NEJM, 2005). Thus, it is expected that these signaling proteins should be activated in the same cells in which EGFRvIII is expressed and/or PTEN is lost. Thus, we farther analyzed these downstream markers on chip in the same cells in which EGFRvIII is overexpressed and PTEN is lost. As shown below in
It has long been recognized that glioblastoma is one of the most molecularly heterogeneous of all tumors, hence the name glioblastoma multiform. This heterogeneity refers to the striking phenotypic and molecular variability of individual cells within a single tumor. Thus, one of the aims of this example is to develop tools to characterize the signaling pathways within these molecularly diverse individual cells.
To determine whether we could identify a “rare” population of cells on chip based on expression of key signaling markers, we mixed U87 (95%) and U87-PTEN-EGFR (5%) and examined the populations on chip. As shown in
Multiparametcr Measurement of Key Signaling Pathways from a Solid Clinical Tumor Sample.
Most of the advances in the field of single cell analysis have come from non-solid tumors such as leukemias and lymphomas, which can be easily dissociated into single cell suspensions for characterization. One of the chief challenges for solid cancers is to adapt these technology-based approaches to characterize signaling in solid tumor samples. We have begun to make significant progress in this area. As an important intermediate step, we have begun working with human serially passaged glioblastoma intracranial xenografts developed by C. David James (at UCSF) and him Sarkaria (at Mayo clinic) (Sarkaria et al., Mol. Cancer Ther. 2006). These models consist of serially passed human tumors that retain the key characteristics of glioblastoma, such as EGFRvIII expression, which are lost in normal culture and xenograft systems. Using this model enables us to best optimize our approaches using a renewable clinical resources which is for all intents and purposes a solid, molecularly heterogeneous clinical tumor sample, much like the ones coming straight from the operating room. Below, we demonstrate considerable progress in this area. In parallel with this, we are optimizing our preparation techniques to go from a solid tumor sample from the operating room to the chip, with no need for tissue culture (which significantly changes the molecular composition of the tumor). We have made significant progress in this area as well. We now have developed an approach to enable us to go directly from a tumor sample to a chip. The chief obstacle has been the difficulty of getting the cells to “lie down” on the solid chip. We have now developed some approaches to facilitate this, which do not change the signaling characteristics of the tumor cells.
To begin to analyze EGFRvIII and PI3K signaling in a solid clinical sample, we used GBM 39 from the James/Sarkaria model system. After obtaining a solid piece of tumor, we dissociated with collagenase and mechanical means and optimized a series of approaches for moving onto chip followed by analysis of PI3K signaling. As shown, below, we present, to the best of our knowledge, the first demonstration of single cell quantification of key nodes of the PI3K signaling pathway in a solid GBM sample. The data demonstrate a tumor with with EGFRvIII expression and significant PI3K pathway activation (see
Addressing the challenge of measuring endogenous levels of key signaling proteins—PTEN measurement. We highlight the challenge that some of our antibodies do not provide an ideal signal to noise ratio. When combined with the relatively low level of expression of certain endogenous proteins such as PTEN, this raises a challenge. We have adapted a tyramide signal amplification strategy to increase our signal to noise ratio for PTEN detection, which enables us to detect endogenous levels of PTEN while allowing us to continue to multiplex the measurement reactions. In
2. Development of Technologies to further Assess Pathways of Cancer at a Single Cell Level.
We have begun using the DEAL, DNA-encoded antibody libraries, coupled with development of new cell surface markers to develop new strategies for isolating and enriching single cell populations.
2a) DEAL technology we have validated the ability of DEAL-based approaches for sorting cells based on EGFR expression. We are currently working on incorporating new cell surface markers into this process. We have developed a panel of novel cell surface markers, validated by flow cytometry and western blotting, which we plan to incorporate into this DEAL strategy.
2b) Detection of novel cell surface proteins and secreted proteins. We have leveraged global gene expression data from clinical samples (63 GBMs) relative to 20 normal brain samples and identified potential cell surface markers that are likely to be highly overexpressed in GBM cells. We have then gone on to analyze the expression in a large cohort of GBM and grade III gliomas patients, to confirm the the mRNA level overexpression of these markers in GBM (and their significant overrexpression in GBM relative to grade III tumors). We have then gone on to examine their protein level expression in GBM cell lines, then in a series of matched glioblastoma/normal pairs from patients on whom autopsies were done. We have then begun to analyze in vitro, the potential relationship between these markers and the EGFR/PI3K signaling axis. To date, we have identified 10 potential cell surface markers including: PSCI.R1, CXCR4, and PTRPZ1 (also identified as a secreted protein—see approach 2 below).
2c) Identification and Validation of Novel Secreted Proteins:
Through global MPSS data analysis on differential gene expression of brain tissue samples from glioblastoma patients versus non-tumor patients, approximately 5000 genes were found to be differentially expressed in glioblastoma brain tissue samples. Thirty eight genes whose expression is relatively enriched in brain were selected for targeted screening of serum samples from glioblastma patients as blood diagnostic hiomarker candidates for glioblastoma. In the mass spectrometry based targeted proteomics approach that is being developed at ISB, proteins from serum samples are first digested to peptides and all peptides are labeled at the N-terminus with stable isotope labeled reagents called iTRAQ (from Applied Biosystems). Peptides from up to four samples can he differentially labeled with iTRAQ reagents for measurement of relative abundance of 100s of proteins across the four different samples. In order to overcome the difficulty of quantifying low-abundance proteins in serum by mass spectrometry due to the sample complexity, two techniques have been utilized: (1) several predominant serum proteins can be selectively removed by immunoaffinity-based depletion methods; (2) the detection of low abundance peptides by mass spectrometer is significantly enhanced by increasing the total amount of the selected peptide in the ITRAQ labeled mixture—84 peptides from 38 candidate blood protein markers were synthesized at low cost and mixed with one of the iTRAQ reagents while two peptide samples from normal and tumor patients were labeled with the other two iTRAQ reagents. The data analysis of this experiment is currently under way. As a follow up of this, we have isolated a set of 25 matched tumor normal brain paired samples and have begun screening for differentially expressed proteins by Mass Spec.
2d) Integration of these New Technologies into Molecularly Guided Clinical Trials:
We are now in the process of a clinical trial of patients treated with the VEGF inhibitor Avastin (in combination with CPT-11), we can now demonstrate that Avastin nearly quadruples time to tumor progression from a median of 56 days (95% CI=49-97) to 209 days (95% CI=122-272) and nearly doubles overall survival from a median of 184 days (95% CI=171-225) to 340 days (95% CI=251-424) for patients with recurrent malignant gliomas. The obvious clinical efficacy of avastin will most certainly change the standard of care for malignant glioma patients. In addition, the fact that we have a relatively large number of clear clinical responders (as well as non-responders), with frozen tissue (as well as paraffin tissue) obtained at the time of diagnosis, as well as frozen tissue in a subset of patients who had a clinical response, but then failed treatment, and serum obtained before treatment, during clinical response and at the time of treatment failure provides a remarkable coordinated molecular/clinical resource for identifying the molecular determinants of response to this drug and to identify effective targeted combinations. We have a relatively large number of clear clinical responders (as well as non-responders), with frozen tissue (as well as paraffin tissue) obtained at the time of diagnosis, as well as frozen tissue in a subset of patients who had a clinical response, but then failed treatment, and serum obtained before treatment, during clinical response and at the time of treatment failure. We will address the following questions: 1) does increased vascularity predispose towards clinical response to avastin (this will be done in collaboration with Dr. Luisa Iruela-Arispe, a noted expert in angiogenesis at UCLA); 2) are the downstream signaling pathways regulated by VEGF reactivated during response and by what mechanism. Are tumors which are driven by key pathways that promote VEGF expression (i.e. EGFR/PI3K signaling) more sensitive to avastin? In addition to testing lese specific hypotheses, we will apply global screening strategies. These include array CGH to detect chromosomal regions of interest associated with sensitivity and resistance (will be done at MSKCC by Dr. Mellinghoff) and integrated with global gene expression data (done both at UCLA and at MSKCC), in addition, through our NanoSystems Biology Cancer Center, we have begun collaboration with Dr. Lee Hood to use surface Plasmon resonance to facilitate the quantitative detection of thousands of proteins in a serum samples. This will enable us to identify protein signatures associated with sensitivity and acquired resistance.
3. Systems Biology Approaches Towards Identification of New GBM Targets:
3a) Targeting the Src Family Kinases: Targeting EGFR/PI3K signaling provides a proof-of-principle for the potential efficacy of molecularly targeted therapy for glioblastoma. Identifying new drug targets is a critical next step. We have developed and applied systems biology approaches to identify novel molecular targets in glioblastoma. Integrating global gene expression data from GBM samples with molecular interaction databases to identify potential targetable pathways, we have identified the Src family kinases (SFKS) Fyn, Lyn and Src as molecular targets in glioblastorna. We leveraged global transcriptome data from glioblastoma clinical samples to identify and validate the Src family kinases (SFKs) Src, Fyn and Lyn as key molecular targets We demonstrated that these kinases are overexpressed and persistently phosphorylated in up to 50% of glioblastoma patients, demonstrated using both siRNA, small molecule inhibitor and over-expression studies that the SFKs are necessary and sufficient to promote glioblastoma invasion and we have demonstrated that the small molecule inhibitor dasatinib (a compound safely given to patients with imatinib resistant CML) inhibits glioblastoma invasion, promotes tumor regression and apoptosis and greatly enhances survival in a mouse model in vivo. (See
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3b) Studying the mechanism of mTOR mediated inhibition in vivo: We conducted clinical trail of the mTOR inhibitor rapamycin in patients with relapsed, PTEN-deficient glioblastomas and identified key determinants of sensitivity and resistance (Cloughesy et al. 2007, PLoS Medicine, in press). We showed that: 1) the mTOR inhibitor rapamycin is present in potentially therapeutic levels in tumor tissue in vivo; that 2) rapamycin significantly inhibits mTOR signaling in all patients although the extent of inhibition is variable (from 10%-80% pathway inhibition) and that 3) the extent of pathway inhibition is critical. mTOR pathway inhibition of greater than 50% resulted in significantly inhibited proliferation; lower levels of mTOR inhibition did not translate into biological or clinical response, We further showed that that was not due to cell intrinsic resistance, but more likely was associated with failure of the drug to fully access its target in vivo. Finally, we studied the relationship between growth inhibition and sustained clinical response and found evidence for an Akt mediated feedback loop nearly half the patients. This Akt-mediated feedback loop was significantly associated with acquired clinical resistance. These findings have important implications. First, they indicate that in future clinical trials, it will be critical to develop approaches to document sufficient target inhibition in glioblastoma patients in order to interpret clinical activity. Second, these findings further point to the value of combined EGFR/mTOR blockade (as also suggested by item I.2. above). Third, the data on the Akt-mediated feedback loop suggests the importance of combined PI3K/mTOR inhibition to better inhibit this pathway and to suppress the upstream feedback loop. These insights will soon be put into practice in a series of clinical trials (in addition to the EGFR/mTOR Inhibitor trials). (See
3c) Measuring EGFRvIII in vivo. We developed a novel nucleic-acid based approach for reliable detection of the mutant EGFRvIII (Yoshimoto et al., Clinical Canceer Res, 2007, in press). We have previously shown that EGFRvIII (in the context of intact PTEN) sensitizes gliohlastomas to EGER inhibitors (Mellinghoff et al., NEJM 2005). EGFRvIII also presents a unique antigenic target for anti-EGFRvIII-directed vaccines. Thus, detection in clinical samples may be warranted. However, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin embedded (FFPE) clinical samples is a major challenge. We developed a real-time RT-PCR assay for EGFRvIII from routinely processed formalin fixed, paraffin-embedded glioblastoma biopsy samples that is 92% sensitive and 98% specific. This assay will be readily available to pathology laboratories everywhere to facilitate widespread testing of EGFRvIII mutation in glioblastoma patients.
B.1: Expanded microfluidcs assays. One can continue to expand our analysis on-chip of the key signaling pathways in glioblastoma according to embodiments of the current invention. One can further increase the number of signaling proteins being currently measured, assess our ability to study the effects of signal transduction pathway inhibitors on-chip, including in mixed populations of GBM cells, and integrate these chips with the cell surface sorting approaches described above. We can also begin working more with fresh tumor samples from patients to optimize approaches for quantifying the molecular heterogeneity and for optimizing on-chip culture of samples.
B2: Optimization of detection of key signaling pathways in routinely processed clinical samples (and in retrospectively frozen clinical samples). One can apply this to solid clinical tumor samples to optimize multiparameter measurement of solid clinical samples. We can also aim to develop ways of examining previously frozen samples, especially those from patients on molecularly based clinical trials, as this can allow us to make correlations between signaling states and response to targeted therapies in trials we have already performed.
B3. Assessment of whether molecular heterogeneity is global or local. The extert of molecular heterogeneity within glioblasioma has been recognized, but not rigorously studied. In fact, it is unclear whether local heterogeneity within a tumor is representative of the entire tumor, or whether spatially dispersed biopsies are required for a complete molecular characterization of a tumor. The answer to this question has obvious importance for designing rational combination therapies for patients to suppress resistance. The development of these tools can facilitate our asking this question.
B4. Pathways of a cancer at a single cell level: We can expand our DEAL approaches using antibodies to the cell surface proteins described above. We can further integrate these DEAL approaches onto the microfluidics based chips.
B5. Development of new serum markers: The UCLA, ISB and CIT groups may integrate their potential serum marker lists. UCLA and ISB can both obtain serum samples, as well as tumor samples, and we can perform larger scale analysis of potential serum proteins using the ISB-based approaches.
B6. Further identification and validation of cell surface markers: We can continue to analyze the expression of the list of potential cell surface markers, begin to determine whether sorting based on these markers (DEAL and flow based) identifies populations of GBM cells with different phenotypic and biochemical properties (and different patterns of sensitivity and resistance to targeted agents).
The current model of pathology diagnosis for cancer is based on the microscopic resemblance of cancer cells to their presumed cell of origin or its developmental precursor. Based on tissue morphological appearance, as well as the presence or absence of a few protein markers, the pathologist concludes a broad pathological diagnosis conveying tumor type and grade. Typically, the patient is treated with relatively toxic, non-specific therapies such as DNA damaging agents and radiation. While this classification and affiliated grading system has proven to be useful for predicting the overall survival for groups of patients and for communicating broad information about the disease category24-29, relatively limited insight is gained about the underlying molecular pathway lesions30. Furthemore, clinically relevant subsets that may differ significantly in their time course and responses to therapy cannot be monitored with the current classification system. Traditional pathological examination, considered to be the “gold standard” of cancer diagnostics, may not be well-suited towards molecularly targeted approaches because lineage type distinctions based on morphology do not reveal information about the underlying molecular networks.
Cancer is a disease of molecular heterogeneity31-35. The past decade has clearly demonstrated that the underlying molecular lesions in tumors of the same histological type are quite different, More importantly, patients with cancers of the same histological type may respond quite differently to therapy depending on the molecular composition of their tumor. There is emerging evidence for considerable molecular heterogeneity within individual tumors, for example a small population of tumor repopulating stem cells, which may be the key drivers of cancer recurrence. There is considerable need to develop robust quantitative tools for multi-parameter measurement of signaling networks within the individual cells of a tumor. It is now clear that any type of cancer stratifies into a set of diseases, each with its own molecular signature. Traditional pathological examination cannot distinguish these relevant tumor subsets, because they are usually microscopically identical.
The advent of targeted therapy has led to the development of a variety of target-specific drugs (e,g., kinase inhibitors36), and have demonstrated profound outcomes in several types of cancer6,37-42. The implementation of targeted therapy necessitates a new molecular diagnostic approach for identification and quantification of disease targets associated with the exact signaling pathway responsible for the malignant transformation of cancers. Conventional technologies for molecular analysis (e.g.. Western blot) that require a significant amount of tissue are constrained for dynamic and single-cell characterization. One of the critical challenges for molecular diagnosis is multi-parameter measurement of the signaling pathways within the molecularly diverse cells with single cell precision.
No two individual tumors are identical. Analyzing individual genes or proteins in isolation is unlikely to yield important breakthroughs for the diagnosis and treatment of cancer. In contrast, a systems biology approach43-46 to cancer aims to define the protein and gene “modules” and networks that are responsible for the emergent properties of cancer (i.e. their proliferative capacity, their invasive capacity, their resistance to therapeutic inhibitors). By capturing information about relationships between key elements of the system, commonalities between highly individual cancers can be understood and targeted.
The systems approach involves taking as many molecular signatures of gene and protein expression as possible as the input, as well as phenomenological information, and integrating them into a network using graphical models. As more inputs are integrated, the structure of the networks is refined enabling generation of hypotheses about how the system works (or in the case of cancer, how it has gone awry). These hypotheses can then be dynamically tested by performing a series of systematic perturbations and measuring the effects on the network (and on phenotypic properties such as growth, invasion, response to therapy, etc). This allows for modification of the hypotheses, followed by further testing and refinement. A more complete and molecular “snapshot” of the system is possible and this high-content knowledge can translate into new diagnostic and therapeutic tools thereby redefining the pathological diagnosis of cancer.
With the current suite of available technologies, a true systems level evaluation of cancer is impossible. However, our group has been at the cutting edge of an emerging set of molecular and nanotechnologies that is being integrated into a systems biology laboratory. The development and validation of the Microfluidic Image Cytometry (MIC) technology described here will facilitate a new type of pathologic examination of cancer cells and tissues; chiefly, one that incorporates a systems level approach to diagnosis.
PI3K47 is a lipid kinase that promotes diverse biological functions including cellular proliferation, survival and motility48-51. The PI3K signaling pathway regulates various cellular procosses, such as proliferation, growth, apoptosis and cytoskeletal rearrangement. The PI3K signaling pathway is frequently deregulated in a majority of human cancer types52,53, often in combination with the ER pathway54-56. The PI3K-AKT-mTOR pathway57-61 (and RAS/ERK pathway62 63) can become deregulated on the basis of oncogene activation and tumor suppressor gene losses that are commonly seen in glioblastoma64-66. A significant portion of cancer cell types contain alterations of the PTEN tumor suppressor gene7-9, a negative regulator of PI3K signaling, which results in constitutive activation of the PI3K pathway67, Upstream of PI3K, the epidermal growth factor receptor (EGFR) is commonly overexpressed39,64,68-71, frequently in association with its constitutively activated EGFRvIII variant (and other variants)71-75, often leading to deregulated PI3K and RAS/ERK signaling62. The PI3K and RAS/ERK pathways connect richly to other signaling cascades, thereby integrating signals associated with other cell surface events, stress activation pathways and extracellular matrix proteins. Clearly, the PI3K and ERK signaling pathways, and associated signaling molecules, are important therapeutic targets.
Flow cytometry76-79 can track and analyze signaling events in individual cancer cells. Flow cytometry's unique capability to quantify multiple properties of individual cells can provide information for each cell in a heterogeneous mixture. Cells are usually fixed and permeabilized to allow access by various reagents. Cells with signaling molecules of interest are detected using a fluorophore-conjugated antibody that is specific for recognition of the protein. The single-cell resolution and multi-parameter nature of flow cytometry data can produce signatures to distinguish between cancer cells and non-tumor cells. Because cells have to be dissociated for detection, flow cytometry has primarily been used to study hematological caricers80,81. Adherent tumor cells from solid tumors must be dissociated or detached prior to the flow cytometry measurement. In this way, the signaling pathways of the cells are potentially perturbed and false readouts are possible. The ultimate limitation to flow cytometry is the large number requirement of cell sample (on the order of 106) making it impossible to interface with needle biopsy and other minimally invasive biopsy sampling techniques.
Microfluidic systems are ideal platforms for handling tumor samples for many intrinsic advantages including sample economy, precise fluidic delivery, scalability and digital controllability82-87. Cell culture and cell assays can he performed in microfluidic devices72,88-97. Multiple culture chambers can be incorporated in a single chip, allowing multi-parameter analysis with high fidelity, Unlike a microplate-based cell culture system, the microfluidic chip offers a 3-D culture environment that better mimics an in-vivo microenvironment. Controlled unidirectional fluid flow improves the fidelity of biological assays. Some researchers in this field include Dr. Steven Quake at Stanford, Dr. Luke Lee at UC Berkeley, and Dr. David Beebe at the University of Wisconsin, Madison. Dr. Quake implemented a microfluidic bioreactor for long-term culture72 of mammalian cells and monitoring of extremely small populations of bacteria at the single-cell level98. A microfluidic cell culture device for mammalian cells98-102 and yeast has been developed at UCB and is now a commercial product. Dr. Beebe has achieved some interesting cellular microenvironments on a chip by exploiting diffusion-limited mixing and other phenomena unique to the microscale96,103,104. The research field is well-developed and characterized now reaching a stage where “Lab on a Chip” devices can reliably be exploited to accelerate much-needed progress in other stagnant or monumentally complex research fields, but only if driven with economic and solutions-based initiatives.
MIC technology according to some embodiments of the current invnetion can perform quantitative and multiparameter immunocytochemistry (ICC) with superb precision and data fidelity. Our objective for some applications is to detect a collection of biomarkers associated with a cancer signaling network responsible for the malignant transformation of cancer. The resulting molecular signature can aide in better cancer diagnosis and the implementation of targeted therapy. The MIC platform integrates three functional modules, including (i) an economic PDMS-based microfluidic cell array chip for supporting the culture of primary cancer cells; (ii) a semi-automated pipette or a robotic pipette for performing cell seeding/culture and ICC, and (iii) an associated data acquisition (fluorescence microscope) system plus sophisticated software for scalable high-content analysis that is well-suited for clinical cancer applications. A series of protocols has been established for measuring the expression/phosphorylation levels of six biomarkers simultaneously (EGFR, EGFRvIII, PTEN, pAKT, pS6 and pERK) in both the PI3K-Akt-mTOR and RAS-RAF-MAPK signaling pathways. Multi-parameter signaling profiles of heterogeneous cell populations from primary tumor tissue are now possible. We also demonstrated the ability of MIC technology to track dynamic changes of the signaling events in the cancer cells upon the exposure to exogenous stimuli (e.g., drugs and growth factors). We are further applying MIC technology to detect signaling events beyond a single pathway, for example, the cross-talk between the PI3K-AKT-mTOR and RAS-RAF-MAPK pathways and feedback interactions involved m-TOR, IRS1 and pAKT. Some aspects of the MIC technology is summarized as the follows:
1. Enabling in-vitro molecular diagnostics. The MIC technology can harness the advantages of microfluidics (i.e., sample economy, speed, scalability, automation and reproducibility) into a solutions driven package to facilitate a clinical pathologist's characterization of an individual's cancer at a molecular and conclusive level. We are pioneering the concept of single-cell signaling profile in a patient's cancer cells.
2. Significant sample economy. Commonly used techniques for protein quantification in biology and clinical laboratories such as Western blot and flow cytometry require at least 104 cells for a single measurement. MIC technology can quantify four signaling molecules using only 200 cells in less than 1.0 μL of media. Consumption of antibodies is dramatically reduced in MIC technology in contrast to Western blot and flow cytometry. The sample economy in MIC technology allows a perfect conjunction for analyzing precious pathology and cytology samples which have limited quantity and are unable to be analyzed by Western blot, flow cytometry, ICC and immunohistochemisny. The single-cell resolution and multi-parameter nature of the MIC data clearly distinguishes the signaling signatures of cancer cells from non-cancer cells, thereby tackling the cellular and tumor heterogeneity issues and far surpasses the performance of competing technologies.
Cost-efficient operation and easy adoption. The cost of carrying out a semi-automated MIC measurement can be fairly low. Our manufacturing strategy can lead to low cost of a MIC chip that is extremely competitive in the market for the high-value added. The intermediate automation liquid handling solution is attainable at a relatively low cost investment for the greater through-put achieved, The florescence cytometry images are acquired on most fluorescent microscopes equipped with reasonable CCD cameras and standard filter sets.
4. Add-on cell sorting capacity. The proprietary chip manufacture strategy can allow convenient incorporation of functional surfaces onto the substrates of the MIC chips. For example, a streptavidin coating or DNA array can be introduced onto the MIC chips allowing immobilization of biotinylated and DNA-tethered antibodies specific to cancer cell surface markers to achieve an on-chip cell capturing function. A sophisticated signaling profile on subsets of cancer cells is possible by first performing on-chip cell sorting followed by the MIC signaling profile on a collection of biomarkers.
Large-scale integration. MIC technology can achieve high through-put cell culture and assay. Various mammalian cell types including, but not limited to cancer cell lines and embryonic stem cells, can be cultured in chambers permitting multiple isolated experiments or in parallel or in duplicate by integrating and automating cell-handling and preparation steps. MIC technology can generate reproducible high content data and quantitative analysis, which can be utilized for applications in large-scale drug screening.
Microfluidic Imaging Cytometry (MIC) technology integrates a microfluidic cell array88,89 with a pipetting robot (for performing sample preparation and immunocytochemistry) and an automated fluorescence microscope (for image acquisition and analysis). MIC technology has been used to perform quantitative and reproducible immunocytochemistry (ICC) for multiple protein detection in a signaling network, using only a small amount of biological samples. A number of glioblastoma cell lines, as well as genetically modified and primary glioblastoma cells were used to validate the MIC technology. The expression/phosphorylation levels of six signaling proteins associated with both of the PI3K-AKT-mTOR and RAS-RAF-MAPK pathways, including receptor tyrosine kinases (EGFR and EGFRvIII), phosphotase (PTEN) and phosphorylated proteins (pAKT, pS6 and pERK) can be quantified in parallel with single-cell precision. Remarkably, the dynamic changes of those proteins upon the exposure to exogenous stimuli (e.g., growth factors and kinase inhibitors) can be kinetically monitored, providing a potent tool to better understand how glioblastoma cells respond to a combination of drug treatments. In parallel, Dr. Wu's research group has been studying the molecular mechanism of PTEN-controlled tumorigenesis. PTEN is the second most frequently deleted human tumor suppressor gene11-13,19,20. The PTEN mutation was also found to be the cause of three autosomal dominant tumor predisposition syndromes. Dr. Wu's laboratory has generated various isogenic cell lines for pathway analyses and in-vivo tumor models for understanding the molecular and genetic mechanisms underlying PTEN controlled tumorigenesis in mice22,107-109, Dr. Mischel's clinical pathology expertise bridges MIC technology to clinical applications.
An embodiement of MIC technology (
We used the cell array chips to determine the optimal surface coating, as well as the best cell feeding schedules to maintain the chip-cultured cells according to an embodiment of the current invention. A poly-L-lysine (PLL) coating on the glass/PDMS substrates was optimal for culturing parental and genetically modified U87 cell lines. For maintaining the primary glioblastoma cells dissociated from glioblastoma patient tissues, we developed a layer-by-layer coating method of Matrigel on the glass/PDMS surfaces. Further, we demonstrated that feeding cycles ranging between 12 and 36 hrs allowed for reproducible proliferation of the glioblastoma cells for at least 10 days. Viability assays (e.g.. Calcein AM or MitoTracker Red from Invitrogen) indicated cell viabilities in the devices. The growth rates of GBM were quantified by counting the cell numbers at different time points. Although inhibition of cell proliferation has been reported103 in other microfluidic cell culture settings, the growth rates of the glioblastoma cells in the cell array chip were compatible with those observed in conventional dishes110. Additionally, we have also developed a variety of direct attachment approaches to immobilize freshly dissociated/released cells on to the substrates of cell array chips. We were able to deposit a layer of tissue adhesive protein (Cell Tak™) to immobilize cells from their suspension solutions enabling the MIC measurement of freshly dissociated cells (see
In general, ICC uses various fluorophore-labeled antibodies to recognize specific protein molecules in cells. Due to high antibody reagent costs, it becomes impractical to carry out parametric assay optimization (reagent concentration, protocols . . . etc.) at the conventional macroscopic level. As a result, the ICC approach can only examine the presence/absence of target proteins in cells and the fluorescent signals in the ICC-treated samples do not reflect the absolute quantity of target proteins. The foundation of the MIC technology was built upon thoroughly optimized protocols that ensure reliable and reproducible ICC. In order to achieve measurement precision over a wide range of protein expression/phosphorylation level, we carried out systematic optimization of ICC protocols for each antibody. Five signaling proteins of interest associated with the PI3K-AKT-mTOR pathway—namely EGFR, EGFRvIII, PTEN, pAKT and pS6—were tested using the corresponding low/high-expression cell line systems110 . . . U87 vs. U87-EGFR, U87 vs. U87-EGFRvIII, U87 vs. U87-PTEN Triciribine-treated U87-EGFRvIII vs. EGFRvIII and rapamycin-treated U87 vs. U87-EGFRvIII, respectively. We were able to test a variety of ICC reagents and conditions for cellular fixation, membrane permeabilization and antibody staining, as well as the operation parameters of the semi-automated pipette for controlling the reagent/solution volumes and applied flow rates. The optimized condition utilizes 4% paraformaldehyde for cell fixation and 0.3% Triton X-100 for cell membrane permeabilization. The highly specific antibodies anti-EGFR (BD Pharmigen), anti-EGFRvIII (Dako), PTEN (Cascade), pAKT (Cell Signaling) and pS6 (Cell Signaling) was each covalently conjugated with a different fluorophore—emitting at 750 (Cy7), 647 (Cy5), 555 (TRITC), 647 (Cy5) and 488 (FITC) nm, respectively. Based on a similar approach, we were able to optimize a MIC protocol for pERK (a signaling node in the RAS-RAF-MAPK pathway, data not shown) using the respective controlled cell lines.
The MIC chip containing ICC-treated cells was mounted onto the fluorescent microscope (Nikon TE2000S) for image acquisition. Operational parameters of the microscope and CCD camera (Photomatrix. Cascade II), i.e., image exposure times and EM gains, were also optimized to attain superior signaling-to-noise ratios for the fluorescent images. The MetaMorph program (Molecular Devices) was used to quantify specific fluorescent signals in individual cells and generate cytometry histograms. In each set of measurements, a pair of histograms for both low and high-expression cell samples is generated after image analysis. The purpose of fine-tuning the MIC conditions and imaging parameters is to achieve maximum separation of the two histograms. Good separation becomes critical for quantifying multiple signaling proteins.
One of the major challenges in defining genetic lesions is measuring the loss of heterozygosity (LOH) of a particular tumor suppressor gene (e.g., PTEN) since (i) the intrinsic signals in the tissues are relatively low and (ii) the surrounding normal tissues express a normal level of tumor suppress gene, generating a significant background signal, PTEN is the second most frequently deleted tumor suppressor gene found in human cancers, and PTEN negatively regulates the PI3K-AKT-mTOR pathway. To test the feasibility of quantifying PTEN expression in primary cells (where PTEN expression levels are at 2, 1 or 0) using the MIC technology, two sets of isogenic mouse cell lines, including (i) PTEN ES lines (i.e., p8 (+/−) and CaP8 (−/−) and (ii) MEF lines (i.e,, PTENloxp/loxp (+/+) and PTENΔloxp/Δloxp (−/−). As shown in
The MIC technology is capable of parallel detection of several signaling molecules in individual cells enabling the capture of cellular heterogeneity in a tissue sample. To prove this, we carried out (
The ICC approach is often used to examine the presence/absence of target proteins in cells, but due to poor data reproducibility it is not suitable for quantification of protein expression/phospohorylation. Given the ability of MIC technology to perform quantification as described above, we attempted to apply MIC technology for determination of rapamycin pharmacodynamics. Rapamycin is a potent inhibitor targeting mTOR, a critical signaling molecule in the PI3K signaling pathway. The effect of rapamycin inhibition on mTOR can be quantified by monitoring the activation of the downstream signaling molecule pS6. The MIC technology was used to quantify rapamycin-induced down-regulation of pS6 levels in individual cells (
The successful demonstration of MIC technology using glioblastoma cell lines encouraged us to test the MIC in-vitro molecular diagnostic technology in clinical setting. We were able to bond our first validation study with an existing clinical trial focused on brain tumor targeted therapy. A group of clinically well-characterized brain cancer patients at UCLA medical school were recruited and tested. Brain tumors often contain high percentages of cancer cells, thus are an outstanding model system for validating the MIC technology. So far, we were able to carry out the quantitative/multi-parameter ICC in the MIC chips to measure molecular signatures (i.e. EGFR, EGFRvIII, PTEN, pAKT and pS6) on seven surgically removed brain tumors (i.e., glioma at different grades). To ensure a minimum level of perturbation to the cells, a mild and rapid tumor dissociation protocol developed by our joint team (15-min TryPEL Express treatment at 37° C.) was established to process freshly isolated tumor tissues. As shown in
The original MIC platform utilized a human-operated semi-automated pipette to perform sample preparation and ICC in sequence. The pipette digitally controls the flow rates and volumes, but the rest of the operation/process was, in fact, manually operated. Generally, human involvement introduces operator variability and error, therefore the semi automated MIC platform requires scale-up for applications requiring high-throughput and/or multistep studies. A user-friendly interface between the microfluidic cell array chip and a robotic pipette (
The mechanical aspect of the user-friendly interface is composed of two custom-designed components, i.e., a chip holder (
The chip holder is a key component that can ensure precise position and orientation of the microfluidic chips with respect to the coordinate system of the robotic pipetting system. Pipetting tips are precisely aligned with inlets and outlets of cell culture chambers. A combination of mechanical depressions for the chips and a clamping mechanism ensure convenient and consistent positioning of the chips with tolerances for slight differences in assembly.
The robotic pipetting system has eight individually controlled pipette tips, each capable of dispensing and withdrawing liquid samples with 5-nL precision. To integrate the MIC chip with the robotic pipetting system, we grouped the eight tips into four pairs for handling four cell culture chambers (
In addition to the mechanical aspects of the interface, there is also an important software component. We have developed a very flexible program interface that implements generic “protocols/recipes” (cell loading, media exchange, ICC, etc . . . ) composed of standardized steps configured in an XML file. Each protocol step specifies a reagent location, dispensing volume, dispensing speed, etc. The user simply selects the recipe to automatically execute. The software allows specification of which chips (and which columns per chip) should be loaded with the new media/reagent for more complex studies involving different cell types or different treatments.
A group of clinically well-characterized brain cancer patients at UCLA medical school were recruited and tested. Brain tumors often contain high percentages of cancer cells, thus are an outstanding model system for validating the MIC technology. So far, we were able to carry out the quantitative/multi-parameter ICC in the M1C chips to measure molecular signatures (i.e. EGFR, EGFRvIII, PTEN, pAKT and pS6) on twenty surgically removed brain tumors (i.e., glioma at different grades). To ensure a minimum level of perturbation to the cells, a mild and rapid tumor dissociation protocol developed by our joint team (15-min TryPEL Express treatment at 37° C.) was established to process freshly isolated tumor tissues. 3D logarithmic scatter plots were used to visualize the protein expression and phosphorylation level in each cell. Each patient has different plots distribution and clustering, which reflect intrinsic tissue heterogeneity of the tumors as well as characteristics of different cell types.
Dual Parameter Dot Plots have been used to display the relationship of the upstream proteins, PTEN and EGFR, with the downstream proteins, pAKT and pS6. The thresholds for high expression and low expression of PTEN, EGFR, pAKT and pS6 were obtained through the statistic of all the patient data. We also can obtain the percentage of cells located in each subset. A hierarchical clustering shows the four protein expression level at the same time and the similarity of each subset cells.
Data suggests that an in vitro neural stem cell (NSC) culture system composed of serum-free media supplemented with epidermal growth factor and fibroblast growth factor, when used to culture human primary tumors isolates, enriches and maintains a tumor-initiating/tumorigenic population of cells termed ‘brain tumor stem cells’ (BTSCs). These cells express neural stem cell genes (Nakano, et al.), have properties of surface attachment-free growth and form characteristic spheroid aggregates called ‘neurospheres’. Unlike their ‘non-stem’ attachment-dependent counterparts that are enriched with an in vitro culture system consisting of a serum-based media, these putative BTSCs fulfill major tenets of cancer stem cell biology in that they are self-renewing, capable of multi-potential differentiation and, when xenotransplanted, form tumors that recapitulate many aspects of the parental tumor they were derived from. Recent data suggests that the growth and ability for patient BTSC lines to be maintained over long-term passages mimic the clinical progression of the patient tumors and thus, this in vitro model serves as an independent prognostic factor (Laks, et al.).
Another component of BTSC theory, derived from the A,B and C-type neural progenitors found in normal neural development, is that these BTSCs may be more quiescent and less mitotic active than other brain tumor cellular progeny, with the most quiescent cells retaining the most ‘stem-like’ characteristics and rapidly-dividing cells having a more limited progenitor capacity. This has been posited as one of the reasons these cells are ‘chemo-resistant’ simply because, until the advent of classes of drugs known to target molecules that specifically attenuate a cell-signaling pathway, most common chemotherapeutic modality used clinically targeted cells with high mitotic activity.
The signaling pathway of interest as the proof-of-concept for the microfludic-based image cytometry (MIC) system was the phosphoinosioI 3 kinase (PI3-K) pathway since its dysregulation is implicated in both brain tumor oncogenesis and brain cancer stem cell evolution and maintenance. Simultaneous multi-nodal (pS6, PTEN, pAkt and EGFR) analysis with single cell resolution allows the study of these components in each cell in primary tumors, cultured brain tumor stem cell and non-stem cell progeny. The MIC system is picking up valuable intra and inter-group pathway differences and the identity of personalized BTSC molecular ‘signatures’ via comparison of patient brain tumor samples with their non-BTSC and BTSC-enriched progeny (at the very 1st in vitro passage) has revealed striking pathway similarities to distinguish stem (NS, ‘neurosphere’) and non-stem (SC, ‘serum cell’). Although the significance is still not clear, most notable quantitative shifts observed are lower expression of EGFR and pS6 in NS samples versus SC samples at the single cell level (
MIC-derived analysis of chemo-sensitive and chemo-resistance phenomena is yielding data which may allow further detailed characterization of BTSCs based on pathway response to chemotherapy. For instance, at least 2 novel chemotherapeutic phenomena are emerging in patient BTSC lines and this is even irrespective of the parental tumor's World Health Organization (WHO) clinical diagnosis. The first is the rapamycin-induced chemo-activation due to the release of mTOR's feedback inhibition on activated pAkt at the single cell level (
Additionally, in tests of multiple BTSC lines, a new predictive model for chemotherapeutic response reveals that signal propagation from EGFR is through Akt. The predictive power of this model initially came from the observation that when EGFR expression significantly correlates with pAkt in pretreated samples, EGFR blocking abrogates the correlation post-treatment and can quantitatively reduce activated Akt expression at the single cell level (
The invention has been described in detail with respect to various embodiments, and it will now be apparent from the foregoing to those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects, and the invention, therefore, as defined in the claims is intended to cover all such changes and modifications as fall within the true spirit of the invention.
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9. Under review: Lu, K V, Zhu S J, Dang J, Yoshimoto K, Felciano R M, Richards D R, Laurance M E, Chen Z, Caldwell J S, Shah N P, Horvath S, Nelson S F, James C D, Bergers G, Lee F, Weinmann R, Mischel P S. The Src Tyrosine Kinases Fyn and Lyn are Dasatinib-Sensitive Molecular Target in Glioblastoma Patients Submitted; revised manuscript under consideration at Cancer Cell.
This application claims priority to U.S. provisional application No. 61/006,842 filed on Feb. 1, 2008, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/32880 | 2/2/2009 | WO | 00 | 7/19/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 61006842 | Feb 2008 | US |
