 |  | Effects of antibody-mediated EGF-receptor inhibition on ERK1/2 isoform phosphorylation in organoid cultures (Indivumed, AACR 2012) | [概要表示] |
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Targeted anti-cancer therapy using small molecules or therapeutic antibodies is important to improve the treatment options of individual cancer patients whose tumor show specific expression patterns of respective target proteins. In order to enhance the development of new targeted drugs, novel and highly predictive in vitro drug testing models are needed which closely reflect the characteristics of each individual tumor.
Towards this end, Indivumed has developed a preclinical drug testing platform based on freshly cultivated tumor tissue slices which enables a detailed investigation of functional effects of classical chemotherapeutic drugs, small molecules and therapeutic antibodies in a natural tumor microenvironment. In addition, this multifunctional in vitro model permits the evaluation of target expression and analysis of signaling pathway activities.
The aim of the present study was to analyze and verify the functionality of an anti-EGFR antibody in colorectal cancer tissue slices using our recently developed drug testing platform. As readout of treatment effects changes in the expression and phosphorylation status of selected signaling proteins from two EGFR-related downstream pathways, the MAPK and Akt pathways, were evaluated by Meso Scale Discovery (MSD) assays and immunohistochemistry. To further analyze the complex regulation of phosphorylation pattern in more detail, we integrated the new NanoPro 1000 technology in our pathway analysis, enabling the identification of distinct isoform phosphorylations. This approach should help to extend the knowledge about individual drug responses among patients to further advance
personalized medicine. |
| | Development of a Robust Nanoimmunoassay and Immunohistochemical Assay for ASNS (MD Anderson, AACR 2012) | [概要表示] |
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The enzyme-drug L-asparaginase (L-ASP) has been used for four decades to treat acute lymphoblastic leukemia. However, its unique mechanism of action is still poorly understood, and its clinical efficacy has proven unpredictable. Those problems have prompted a continuing search for biomarkers that predict L-ASP response. We previously found that the expression of asparagine synthetase (ASNS) is strongly negatively correlated with L-ASP anticancer activity in ovarian cancer cell lines, suggesting that L-ASP might be effective against a low-ASNS subset of ovarian cancers if salient characteristics of the cell lines reflect clinical ovarian tumors. However, quantitatively robust, single-antibody assays for ASNS expression have been absent from the literature. We therefore used a capillary-based isoelectric focusing (IEF) platform (the NanoPro 1000) to screen twelve ASNS antibodies for their specificity and sensitivity. Only two antibodies exhibited completely on-target activity (as shown by signal ablation by ASNS siRNA) and sufficient sensitivity. The on-target activity corresponded to a single band on Western blot and a single peak on the NanoPro 1000, suggesting the existence of just one ASNS protein isoform. Optimized, final NanoPro assay conditions yielded less than 8% CV, a 160-fold dynamic range, and Z′-factor of 0.82, indicating a robust assay that is amenable to high-throughput screening. We next used the best ASNS antibody to develop an immunohistochemistry (IHC) assay for ASNS. As with the NanoPro assay, optimized IHC conditions yielded a large dynamic range of staining intensity, and staining was completely ablated by ASNS siRNA. To test the hypothesis that subsets of various cancer types express very low levels of ASNS, we have initiated ASNS IHC of more than 20 tissue arrays representing a wide variety of cancer types. Using a 3-point scoring system (0 = negative, 1 = low, 2 = high), among the tumor samples assayed, 90/136 (66%) of bladder cancer, 63/133 (47%) of bone cancer, 32/149 (22%) of breast cancer, 29/115 (25%) of brain cancer, 51/168 (30%) of colon cancer, 2/85 (2%) of endocrine system cancer, 23/99 (23%) of liver cancer, 7/64 (11%) of head and neck cancer, 7/136 (5%) of lung cancer, 13/53 (25%) of lymphoma, 1/25 (4%) of bone marrow lymphoma, 2/35 (6%) of lymphoma from spleen, 9/109 (8%) of melanoma, 81/396 (21%) of ovarian cancer, 3/29 (10%) of uterine cancer, 27/73 (37%) of pancreatic cancer, 5/119 (4%) of prostate cancer, 10/125 (8%) of renal cancer, 25/138 (18%) of testicular cancer, and 8/39 (21%) of thyroid cancer were ASNS-negative (score = 0), suggesting that a subset of each cancer type may be sensitive to the drug L-asparaginase. Efforts are underway to apply the NanoPro assay to the NCI-60 cell line panel and to continue performing ASNS IHC to survey tissue arrays for ASNS expression. |
| | The use of nanoimmunoassay (NIA) technology to predict response to
insulin‐like growth factor‐1 receptor (IGF1R) inhibition in head and neck squamous cell carcinoma (HNSCC) (University of Virginia, AACR 2012) | [概要表示] |
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Background: Signaling from the IGF1R plays a role in resistance to anti‐cancer therapy in HNSCC. Thus, targeted inhibition of the IGF1R holds substantial therapeutic potential. While several inhibitors of the IGF1R are in clinical trials, there is no biomarker that predicts tumor responsiveness to anti-IGF1R therapy. Such a predictive biomarker is likely to be a component of the most prominent downstream signaling cascades from the IGF1R, which include the MEK/ERK or PI3K/AKT pathways that principally regulate proliferation and survival, respectively.
Hypothesis: Short‐term changes in the activation status of downstream signaling proteins will be predictive of long‐term tumor response to inhibitors of the IGF1R, and these changes will be detectable in minimal tissue samples using NIA technology. |
| | Use of Nano-ImmunoAssay to Generate Rapid, Quantitative Nanoscale Proteomic Profiling of the Hypoxia Pathway in Renal Cell Carcinoma Clinical Specimens (Stanford University, ASCO 2012) | [概要表示] |
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Novel inhibitors of the hypoxia pathway [VEGF, PDGF] achieve response rates of 30-57% in renal cell carcinoma (RCC); yet threshold levels of targets and downstream signaling proteins have not been identified as biomarkers to guide treatment.
Methods: To profile hypoxia proteins in RCC clinical specimens, we have developed the use of automated nanoscale immunoassays for charge-based protein separation (NIA, NanoPro 1000) and charge-based
protein separation (Simple Western, Sally). To decrease the amount of tissue and invasive procedures required to obtain cells for analysis, we optimized assays to profile specimens acquired by fine needle aspiration (FNA).
Results: We used Simple Western to quantify proteins of the MAPK (ERK1, ERK2, pERK1, pERK2, MEK2), PI3K (S6, GSK3b, AKT2, pan-AKT) and STAT pathways (p-STAT5) and loading controls (tubulin, HSP-70) in more than 200 FNA's from solid tumors including RCC. Profiles can be completed overnight after receiving the specimen. Unique to NIA, we also analyzed percent phosphorylation and resolved differences in even a single phosphorylation in FNA specimens, allowing us to group tumors based upon different patterns of phosphorylation and percent phosphorylation.
Conclusions: Rapid and quantitative nanoproteomic profiling in very small amounts of clinical specimen is enabling translational studies for novel diagnostic and predictive biomarkers. |
| | Application of a novel nano-immunoassay platform to assess changes in cIAP1 in response to the SMAC-mimetic, LCL161. Novartis Poster AACR 2011. | [概要表示] |
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Application of a novel nano-immunoassay platform to assess changes in cIAP1 in response to the SMAC-mimetic, LCL161 |
| | Assay Development on the NanoPro Platform: 4E-BP1 and 4E-BP2 (SBS 2010) | [概要表示] |
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We present the development of novel nanoimmunoassays for the translational repressor proteins 4E-BP1 and 4E-BP2 using NanoPro technology. Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase pathway regulate 4E-BP1 activity, making 4E-BP1 a focal point for these two important signaling pathways. 4E-BP2 regulation is poorly understood, partially due to the lack of specific anti-phospho 4E-BP2 antibodies. Our assay, developed on the Cell Biosciences NanoPro platform, enables detailed differential investigation of 4E-BP1 and 4E-BP2 phosphorylation and signal transduction. |
| | A rapid screening method for monitoring signaling changes in the monocyte cell line U937 (AACR 2009) | [概要表示] |
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Here we describe a precise screening assay that quantifies changes in phosphorylation of proteins in samples from as few as 100 cells, which is simple, rapid, and relatively low in cost. A nano-immunoassay system (Cell Biosciences) was used to measure changes in expression and activation of relevant signaling proteins, including MEK, ERK and STATs in U937 monocyte cells before and after cytokine treatment. A single pan-specific antibody was used to distinguish between the phosphorylated and non-phosphorylated protein isoforms, as the nano-immunoassay (NIA) method separates different phosphorylated forms of a protein based on their isoelectric point. In parallel, phospho-protein FACS analysis, which is the current state of the art for measuring multiple signaling pathways, was performed to compare changes in expression and phosphorylation of the signaling proteins. Phospho-protein FACS analysis is expensive and requires considerable technical expertise, which limits its application for large numbers of samples. This novel nano-immunoassay screening method is currently being employed at the Stanford Human Immune Monitoring Core (HIMC) and is being used for high-throughput screening of compounds that influence monocyte activation, monocyte/macrophage differentiation and analysis of various disease states in small primary tissue samples. Practical examples will be given. |
| | Application of a nanoimmunoassay platform to assess changes in EGFR-dependent signaling pathways in lung cancer cell line: surgical resections and laser-capture microdissection from patient-derived tumor cells exposed to EGFR tyrosine kinase inhibitors (AACR 2009) | [概要表示] |
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To fully enable the vision of 'bench-to-bedside' requires the development of not only novel therapies, but novel techniques for evaluating their efficacy in cell lines, animal models and primary tumor material. This report describes our development of a technique employing a nanoimmunoassay platform for the analysis of signaling protein activation in primary non-small lunch cancer (NSCLC) solid tumors. |
| | Adoption Success: Expansion Of Rapid Screening for Monitoring Signaling Changes (AACR 2009) | [概要表示] |
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Cell Biosciences' novel nano-immunoassay screening method is being adopted in an ever-increasing range of institutions, thanks to an innovative collaborative effort collaborative effort between Stanford's Human Immune Monitoring Core (HIMC), Comprehensive Cancer Center, and Cell Biosciences.
The precise screening assay quantifies changes in phosphorylated and non-phosphorylated protein isoforms in tiny samples. Notably, the assays are simple, rapid, and relatively low in cost.
The centralized location of the instrument, and unique collaborative environment have enabled rapid development and adoption of Firefly assays - first within Stanford, and now extending to other institutions, both academic and commercial. |
| | Independent Measurement of MEK Phosphoforms by Capillary Immunoassay (AACR 2008) | [概要表示] |
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Activation of the MAPK pathway involves a complicated web of MEK phosphorylations. The two MEK isoforms are regulated by at least 3 other enzymes — PAK, RAF and ERK. Up until now it has been impossible to quantitate and determine the stoichiometry of the various multiply phosphorylated MEK forms. We have developed a new capillary immunoassay which resolves the different MEK variants, and allows measurement of the relative abundance of each form with a single antibody. This measurement of how the multiply phosphorylated forms change gives insight into how the dynamics of pathway feedback and activity change in response to drug treatment.
We have shown that signaling upstream of MEK kinase is inhibited by negative feedback in tumor cells in which the pathway is driven by HER kinases. In these cells, MEK1 is phosphorylated at ERK-- and PAK-dependent sites (T292, S298), whereas phosphorylation on RAF-dependent sites is undetectable. A selective MEK inhibitor inhibits ERK phosphorylation, relieves the negative feedback and activates MEK phosphorylation in these cells. Under these conditions, phosphorylation of both kinases on the RAF dependent sites (S217, S222) is markedly induced. Thus, inhibition of MEK/MAPK signaling in these cells abrogates upstream feedback of the pathway and results in a complex change in phosphorylation of MEK due to multiple kinases.
The capillary immunoassay allows determination of complex changes in phosphorylation of MEK kinase by PAK, RAF and ERK kinases in response to MEK inhibition. This technique will be useful in mapping pathway network response to targeted drugs in vitro and in vivo. |
| | Measurement of Oncoproteins in Primary Hematopoietic Malignancies Pre- and Post-Therapy Using a Nano-Immunoassay System (AACR 2008) | [概要表示] |
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Oncoprotein quantification in clinical specimens is important for the diagnosis of specific hematopoietic malignancies as well as the development and monitoring of effective therapies that target oncoproteins. Current protein detection methods require large samples, precluding routine serial tumor sampling to assess changes in oncoprotein levels. Here we demonstrate the use of a nano-immunoassay system (Firefly™ system) to distinguish between patient specimens of Burkitt's vs. Follicular lymphoma by characterizing patterns of MYC and BCL2 expression. Changes in the expression and activation of a variety of onco/signaling proteins including ERK, MEK, STAT and JNK in malignant hematopoietic (CML) cells and patient samples before and after treatment with therapeutic agents that impact oncogenic signaling pathways are also shown. The expression levels of the different proteins were measured with high sensitivity in samples as small as 400 cells. A key benefit of this technology is that it separates protein isoforms based on its isoelectric point. Using this assay, we were able to distinguish and quantify the phosphorylated and non-phosphorylated forms of each protein with a single antibody. Thus, we have developed a novel technique which can precisely evaluate the activity levels of signaling proteins in oncogenic pathways from very small samples. |
| | Measuring Tyrosine Kinase Inhibitor Effects on Cell Signaling Pathways (Beatson 2008) | [概要表示] |
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A recent wave of anti-cancer compounds that target tyrosine kinases (TKIs) has been moving through the drug development pipeline. Assessment and screening of lead compounds in simple model systems is relatively straight forward. Until recently, however, determining the impact of these compounds in complex biology of patient-derived cells and tissues has been diffcult. Proposed genetic or protein biomarkers can act as surrogates to a response, but measuring the signaling pathway in both the target cells and surrounding normal tissue will provide a more direct metric. This has proven diffcult due to the limited nature of primary material and complexity of tissue structure. Here we describe a novel nano-immunoassay platform (Firefly™) that has two significant advantages over traditional immunoassays: (1) extremely sensitive protein detection, and (2) physical isoform separation, which allows for quantitation of protein isoforms as well as post-translational modifications such as phosphorylation.
Applications of this technology that will be described include:
1. Effect of TKIs on signaling in punch biopsies of non-small cell lung cancer cells
2. Signaling pathway response from chronic myleogenous leukemia patients to therapies targeted to the bcr/abl translocation |
| | Protein Expression and Cell Signaling Quantified in Rare Cells (ISSCR 2008) | [概要表示] |
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The discovery of tumor stem cells in acute myeloid leukemia a decade ago initiated a field of research has accelerated growth in the past few years. Researchers are now describing tumor stem cells in a variety of hematopoietic and solid tumors. The impetus for much of this research is the desire to identify targets for drug intervention in these critical tumor populations. The molecular pathways that functionally define these cells are important therapeutic targets. Tumor stem cells are rare and provide insufficient material to use standard assay methods. Although DNA microarray and/or qPCR are used to study tumor stem cells, their rare nature limits quantitative protein analysis. This creates a gap in our knowledge since many proteins, such as beta-catenin or MAPK signaling proteins, are not regulated at the transcriptional level, but through post-translational modifications (phosphorylation, ubiquitination, etc.). Here we describe a technique utilizing a nanoimmunoassay platform (Firefly?) to measure tumor stem cell proteins. Transitional tumor stem cells (TCC+) were sorted from a patient tumor and lysed for analysis. A lysate of 400 cells was subjected to isoelectric focusing and immobilization. Immunodetection was performed and quantitation of signal was measured using HRP-labeled secondary chemiluminescence reagents. Here we report beta-catenin protein concentrations of 192 ng/mg of total protein in the tumor stem cells, which was undetectable in 'non-stem' tumor cells. Comparisons of protein levels and the degree of phosphorylation are made between these samples, other tumor cell lines and hematopoetic stem cells. |
