151 words 35 Running Title: Immune-Based Predictive Signature for Head and Neck Cancer 36
The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients
Background Systemic cancer spread is preceded by the establishment of a permissive microenvironment in the target tissue of metastasis - the premetastatic niche. As crucial players in establishment of the pre-metastatic niche, myeloid... more
Background Systemic cancer spread is preceded by the establishment of a permissive microenvironment in the target tissue of metastasis - the premetastatic niche. As crucial players in establishment of the pre-metastatic niche, myeloid derived suppressor cells (MDSC) release S100A8/A9, an exosomal protein that contributes to metastasis, angiogenesis, and immune suppression. We report the application of antibody-based single-photon emission computed tomography (SPECT) for detection of S100A8/A9 in vivo as an imaging marker for pre-metastatic tissue priming. Methods A syngeneic model system for invasive breast cancer with (4T1.2) or without (67NR) the tendency to form lung metastasis was established in BALB/c mice. A SPECT-probe has been generated and tested for visualization of S100A9 release. Tumor-associated changes in numbers and fuction of immune cells in pre-metastatic tissue were evaluated by flow cytometry and confocal microscopy. Results S100A8/A9 imaging reflected MDSC abunda...
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Loco-regional recurrence of breast cancer poses significant clinical problems due to frequent inoperability once the chest wall is involved. Early detection of recurrence by molecular imaging agents against therapeutically targetable... more
Loco-regional recurrence of breast cancer poses significant clinical problems due to frequent inoperability once the chest wall is involved. Early detection of recurrence by molecular imaging agents against therapeutically targetable receptors, such as c-Met, would be of potential benefit. The aim of this study was to assess [(18)F]AH113804,a peptide-based molecular imaging agent with high affinity for human c-Met, for the detection of early stage loco-regional recurrence in a human basal-like breast cancer model, HCC1954. HCC1954 tumor-bearing xenograft models were established, and [(18)F]AH113804 was administered. Distribution of radioactivity was determined via positron emission tomography (PET) at 60 min post radiotracer injection. PET and CT (computerized tomography) images were acquired 10 days after tumor inoculation, to establish baseline distribution and uptake, and then on selected days after surgical tumor resection. CT images and caliper were used to determine the tumor ...
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Research Interests: Materials Science, Microfluidics, Flow Cytometry, Fluorescence, Fluorescent Dyes and Reagents, and 11 moreMedicine, Multidisciplinary, Fluorescence Resonance Energy Transfer, Humans, Phosphorylation, Fluorescent Antibody Technique, Epidermal Growth Factor, Dimerization, Microfluidic Analytical Techniques, cytometry, and fluorescent dyes
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Research Interests: Chemistry, Biosensors, Multiphoton Microscopy, A Priori Knowledge, Fluorescence Resonance Energy Transfer, and 13 moreProceedings, Protein-DNA interaction, FRET, Fluorescent Protein, BioSensors, TCSPC, Energy Transfer, Multiphoton Imaging, Fluorescent Proteins, Flim, GFP, Fluorescence Lifetime Imaging, and Molecular Interactions
Research Interests: Biochemistry, Genetics, Immunology, Biology, Cell Cycle, and 15 moreCell Adhesion, Cell Biology, Medicine, Extracellular Matrix, Cytoskeleton, Biological Sciences, Cell line, Humans, Female, Animals, Cell Signalling, Cell communication, Breast Neoplasms, binding sites, and Medical and Health Sciences
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Background Inferring molecular pathway activity is an important step towards reducing the complexity of genomic data, understanding the heterogeneity in clinical outcome, and obtaining molecular correlates of cancer imaging traits.... more
Background Inferring molecular pathway activity is an important step towards reducing the complexity of genomic data, understanding the heterogeneity in clinical outcome, and obtaining molecular correlates of cancer imaging traits. Increasingly, approaches towards pathway activity inference combine molecular profiles (e.g gene or protein expression) with independent and highly curated structural interaction data (e.g protein interaction networks) or more generally with prior knowledge pathway databases. However, it is unclear how best to use the pathway knowledge information in the context of molecular profiles of any given study. Results We present an algorithm called DART (Denoising Algorithm based on Relevance network Topology) which filters out noise before estimating pathway activity. Using simulated and real multidimensional cancer genomic data and by comparing DART to other algorithms which do not assess the relevance of the prior pathway information, we here demonstrate that...
Research Interests: Computer Science, Algorithms, Biology, Medicine, Signal Transduction, and 13 moreBiological Sciences, Humans, Computer Simulation, Mathematical Sciences, Female, BMC Bioinformatics, Mammography, Neoplasms, Gene expression profiling, Protein interaction maps, Breast Neoplasms, BMC, and lung neoplasms
Prostate cancer remains a major cause of male mortality. Genetic alteration of the PI3K/AKT/mTOR pathway is one of the key events in tumor development and progression in prostate cancer, with inactivation of the PTEN tumor suppressor... more
Prostate cancer remains a major cause of male mortality. Genetic alteration of the PI3K/AKT/mTOR pathway is one of the key events in tumor development and progression in prostate cancer, with inactivation of the PTEN tumor suppressor being very common in this cancer type. Extensive evaluation has been performed on the therapeutic potential of PI3K/AKT/mTOR inhibitors and the resistance mechanisms arising in patients with PTEN-mutant background. However, in patients with a PTEN wild-type phenotype, PI3K/AKT/mTOR inhibitors have not demonstrated efficacy, and this remains an area of clinical unmet need. In this study, we have investigated the response of PTEN wild-type prostate cancer cell lines to the dual PI3K/mTOR inhibitor DS-7423 alone or in combination with HER2 inhibitors or mGluR1 inhibitors. Upon treatment with the dual PI3K/mTOR inhibitor DS-7423, PTEN wild-type prostate cancer CWR22/22RV1 cells upregulate expression of the proteins PSMA, mGluR1, and the tyrosine kinase rece...
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Over the past decade, immunotherapy delivered novel treatments for many cancer types. However, lung cancer still leads cancer mortality, and non-small-cell lung carcinoma patients with mutant EGFR cannot benefit from checkpoint inhibitors... more
Over the past decade, immunotherapy delivered novel treatments for many cancer types. However, lung cancer still leads cancer mortality, and non-small-cell lung carcinoma patients with mutant EGFR cannot benefit from checkpoint inhibitors due to toxicity, relying only on palliative chemotherapy and the third-generation tyrosine kinase inhibitor (TKI) osimertinib. This new drug extends lifespan by 9-months vs. second-generation TKIs, but unfortunately, cancers relapse due to resistance mechanisms and the lack of antitumor immune responses. Here we explored the combination of osimertinib with anti-HER3 monoclonal antibodies and observed that the immune system contributed to eliminate tumor cells in mice and co-culture experiments using bone marrow-derived macrophages and human PBMCs. Osimertinib led to apoptosis of tumors but simultaneously, it triggered inositol-requiring-enzyme (IRE1α)-dependent HER3 upregulation, increased macrophage infiltration, and activated cGAS in cancer cells...
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N.Anilkumar and M.Parsons contributed equally to this work Coordination of protrusive and contractile cell±matrix contacts is important for cell adhesion and migration, but the mechanisms involved are not well understood. We report an... more
N.Anilkumar and M.Parsons contributed equally to this work Coordination of protrusive and contractile cell±matrix contacts is important for cell adhesion and migration, but the mechanisms involved are not well understood. We report an unexpected direct association between fascin, an actin-bundling component of ®lopodia, microspikes and lamellipodial ribs, and protein kinase Ca (PKCa), a regulator of focal adhesions. The association is detectable by protein±protein binding in vitro, by coimmunoprecipitation from cell extracts, and in live cells as ¯uorescence resonance energy transfer detected by ¯uorescence imaging lifetime microscopy. The interaction is physiologically regu-lated by the extracellular matrix context of cells, depends on activation of PKCa and is mediated by the C1B domain of PKCa. Strikingly, a fascin mutant, fascin S39D, associates constitutively with PKCa. Through use of a newly developed set of membrane-permeable peptides that separately inhibit either fascin/PKC...
Imaging spontaneous cancer cell metastasis or heterogeneous tumor responses to drug treatment in vivo is difficult to achieve. The goal was to develop a new highly sensitive and reliable preclinical longitudinal in vivo imaging model for... more
Imaging spontaneous cancer cell metastasis or heterogeneous tumor responses to drug treatment in vivo is difficult to achieve. The goal was to develop a new highly sensitive and reliable preclinical longitudinal in vivo imaging model for this purpose, thereby facilitating discovery and validation of anticancer therapies or molecular imaging agents. Methods: The strategy is based on breast cancer cells stably expressing the human sodium iodide symporter (NIS) fused to a red fluorescent protein, thereby permit-ting radionuclide and fluorescence imaging. Using whole-body nanoSPECT/CT with 99mTcO4−, we followed primary tumor growth and spontaneous metastasis in the presence or absence of etoposide treatment. NIS imaging was used to classify organs as small as in-dividual lymph nodes (LNs) to be positive or negative for metastasis, and results were confirmed by confocal fluorescence microscopy. Etoposide treatment efficacy was proven by ex vivo anticaspase
To assess the capability of fractional water content (FWC) texture analysis (TA) to generate biologically relevant information from routine PET/MRI acquisitions for colorectal cancer (CRC) patients. Thirty consecutive primary CRC patients... more
To assess the capability of fractional water content (FWC) texture analysis (TA) to generate biologically relevant information from routine PET/MRI acquisitions for colorectal cancer (CRC) patients. Thirty consecutive primary CRC patients (mean age 63.9, range 42–83 years) prospectively underwent FDG-PET/MRI. FWC tumor parametric images generated from Dixon MR sequences underwent TA using commercially available research software (TexRAD). Data analysis comprised (1) identification of functional imaging correlates for texture features (TF) with low inter-observer variability (intraclass correlation coefficient: ICC > 0.75), (2) evaluation of prognostic performance for FWC-TF, and (3) correlation of prognostic imaging signatures with gene mutation (GM) profile. Of 32 FWC-TF with ICC > 0.75, 18 correlated with total lesion glycolysis (TLG, highest: rs = −0.547, p = 0.002). Using optimized cut-off values, five MR FWC-TF identified a good prognostic group with zero mortality (lowes...
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Purpose This study assesses the potential for vascular-metabolic imaging with FluoroDeoxyGlucose (FDG)–Positron Emission Tomography/Computed Tomography (PET/CT) perfusion to provide markers of prognosis specific to the site and stage of... more
Purpose This study assesses the potential for vascular-metabolic imaging with FluoroDeoxyGlucose (FDG)–Positron Emission Tomography/Computed Tomography (PET/CT) perfusion to provide markers of prognosis specific to the site and stage of colorectal cancer. Methods This prospective observational study comprised of participants with suspected colorectal cancer categorized as either (a) non-metastatic colon cancer (M0colon), (b) non-metastatic rectal cancer (M0rectum), or (c) metastatic colorectal cancer (M+). Combined FDG-PET/CT perfusion imaging was successfully performed in 286 participants (184 males, 102 females, age: 69.60 ± 10 years) deriving vascular and metabolic imaging parameters. Vascular and metabolic imaging parameters alone and in combination were investigated with respect to overall survival. Results A vascular-metabolic signature that was significantly associated with poorer survival was identified for each patient group: M0colon – high Total Lesion Glycolysis (TLG) wit...
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Objectives: To assess the clinical outcomes of mpMRI before biopsy and evaluate the space remaining for novel biomarkers. Methods: The INNOVATE study was set up to evaluate the validity of novel fluidic biomarkers in men with suspected... more
Objectives: To assess the clinical outcomes of mpMRI before biopsy and evaluate the space remaining for novel biomarkers. Methods: The INNOVATE study was set up to evaluate the validity of novel fluidic biomarkers in men with suspected prostate cancer who undergo pre-biopsy mpMRI. We report the characteristics of this clinical cohort, the distribution of clinical serum biomarkers, PSA and PSA density (PSAD), and compare the mpMRI Likert scoring system to the Prostate Imaging–Reporting and Data System v2.1 (PI-RADS) in men undergoing biopsy. Results: 340 men underwent mpMRI to evaluate suspected prostate cancer. 193/340 (57%) men had subsequent MRI-targeted prostate biopsy. Clinically significant prostate cancer (csigPCa), i.e., overall Gleason ≥ 3 + 4 of any length OR maximum cancer core length (MCCL) ≥4 mm of any grade including any 3 + 3, was found in 96/195 (49%) of biopsied patients. Median PSA (and PSAD) was 4.7 (0.20), 8.0 (0.17), and 9.7 (0.31) ng/mL (ng/mL/mL) in mpMRI score...
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Simple Summary There is a need to demonstrate additional clinical value/utility for PET/MRI in oncology which moves beyond simple diagnosis. We believe that our currently submitted work represents an initial step in realizing the goal of... more
Simple Summary There is a need to demonstrate additional clinical value/utility for PET/MRI in oncology which moves beyond simple diagnosis. We believe that our currently submitted work represents an initial step in realizing the goal of moving PET/MRI beyond simple diagnosis. We report how texture analysis of parametric images depicting tumor fractional water content derived from routine PET/MRI Dixon acquisitions shows good inter-operator agreement, generates biologically relevant information related to total lesion glycolysis and gene mutation count, and provides prognostic information that can potentially unlock new clinical applications for patients with colorectal cancer. This research study resulted from a long-standing multi-disciplinary collaboration between the nuclear medicine physicians at our institute and experts in quantitative imaging, MRI radiology, GI histology, clinical oncology in colorectal cancer, and several gastrointestinal surgeons at various local hospitals...
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We report the development of targeted theragnostic lipid/peptide/DNA lipopolyplexes for delivery of both a tyrosine kinase inhibitor, and plasmid DNA coding for a biosensor. These are used to quantify EGFR inhibition in cancer cell lines... more
We report the development of targeted theragnostic lipid/peptide/DNA lipopolyplexes for delivery of both a tyrosine kinase inhibitor, and plasmid DNA coding for a biosensor. These are used to quantify EGFR inhibition in cancer cell lines in vivo.
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Background: Advanced Head and Neck Squamous Cell Cancer (HNSCC) is associated with a poor prognosis, and biomarkers that predict response to treatment are highly desirable. The primary aim was to predict Progression Free Survival (PFS)... more
Background: Advanced Head and Neck Squamous Cell Cancer (HNSCC) is associated with a poor prognosis, and biomarkers that predict response to treatment are highly desirable. The primary aim was to predict Progression Free Survival (PFS) with a multivariate risk prediction model. Methods: Blood samples from 56 HNSCC patients were prospectively obtained within a Phase 2 clinical trial (NCT02633800), before and after the first treatment cycle of platinum-based chemotherapy, to identify biological covariates predictive of outcome. A total of 42 baseline covariates were derived pre-treatment, which were combined with 29 covariates after one cycle of treatment. These covariates were ranked and selected by Bayesian multivariate regression to form risk scores to predict PFS, producing "baseline" and "combined" risk prediction models respectively. Results: The baseline model comprised of CD33+CD14+ monocytes, Double Negative B cells and age, in a weighted risk signature wh...
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Tumor-associated macrophages (TAMs) can exist in pro- and anti-inflammatory states. Anti-inflammatory TAMs (also referred to as M2-polarized) generally suppress antitumor immune responses and enhance the metastatic progression of cancer.... more
Tumor-associated macrophages (TAMs) can exist in pro- and anti-inflammatory states. Anti-inflammatory TAMs (also referred to as M2-polarized) generally suppress antitumor immune responses and enhance the metastatic progression of cancer. To explore the mechanisms behind this phenomenon, we isolated macrophages from mice and humans, polarized them ex vivo, and examined their functional interaction with breast cancer cells in culture and in mice. We found that anti-inflammatory TAMs promoted a metabolic state in breast cancer cells that supported various protumorigenic phenotypes. Anti-inflammatory TAMs secreted the cytokine TGF-β that, upon engagement of its receptors in breast cancer cells, suppressed the abundance of the transcription factor STAT1 and, consequently, decreased that of the metabolic enzyme succinate dehydrogenase (SDH) in the tumor cells. The decrease in SDH levels in tumor cells resulted in an accumulation of succinate, which enhanced the stability of the transcript...
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Tertiary lymphoid structures (TLSs) develop in non-lymphatic tissue in chronic inflammation and cancer. TLS can mature to lymph node (LN) like structures with germinal centers and associated vasculature. TLS neogenesis in cancer is highly... more
Tertiary lymphoid structures (TLSs) develop in non-lymphatic tissue in chronic inflammation and cancer. TLS can mature to lymph node (LN) like structures with germinal centers and associated vasculature. TLS neogenesis in cancer is highly varied and tissue dependent. The role of TLS in adaptive antitumor immunity is of great interest. However, data also show that TLS can play a role in cancer metastasis. The importance of lymphatics in cancer distant metastasis is clear yet the precise detail of how various immunosurveillance mechanisms interplay within TLS and/or draining LN is still under investigation. As part of the tumor lymphatics, TLS vasculature can provide alternative routes for the establishment of the pre-metastatic niche and cancer dissemination. The nature of the cytokine and chemokine signature at the heart of TLS induction can be key in determining the success of antitumor immunity or in promoting cancer invasiveness. Understanding the biochemical and biomechanical fa...
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226 Background: Metastatic castration resistant prostate cancer (CRPC) is dependent on persistent activation of the AR. In addition, the AR and PI3K-AKT-mTOR pathways have been shown to regulate each other through complex feedback... more
226 Background: Metastatic castration resistant prostate cancer (CRPC) is dependent on persistent activation of the AR. In addition, the AR and PI3K-AKT-mTOR pathways have been shown to regulate each other through complex feedback mechanisms. Various early phase clinical studies are currently investigating the role of PI3K-AKT-mTOR pathway inhibitors in improving outcomes in metastatic CRPC. We hypothesise that the formation of a HER3 (human epidermal growth factor receptor) heterodimer, has an important role in regulating the AR and PI3K-AKT-mTOR feedback loop and that its detection can predict response to PI3K-AKT-mTOR pathway inhibition. Methods: LNCAP cells were stably lentiviral transduced with shRNA against HER3. The effect of PI3K-AKT-mTOR inhibition by DS7423, GDC0068, GDC0941 and Everolimus on AR and its direct target gene PSA was evaluated by western blot and real-time quantitative PCR. CWR22 and 22RVI mouse xenograft tumours were stained with fluorescently labelled antibo...
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Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in... more
Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activ...
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Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M... more
Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M mutation causes over 50% of resistance; MET amplification is also common. Preclinical data suggest synergy between MET and EGFR inhibitors. We hypothesized that EGFR-MET dimerization determines response to MET inhibition, depending on EGFR mutation status, independently of MET copy number. We tested this hypothesis by generating isogenic cell lines from NCI-H1975 cells, which co-express L858R and T790M EGFR mutations, namely H1975L858R/T790M (EGFR TKI resistant); H1975L858R (sensitized) and H1975WT (wild-type). We assessed cell proliferation in vitro and tumor growth/stroma formation in derived xenograft models in response to a MET TKI (SGX523) and correlated with EGFR-MET dimerization assessed by Förster Resonance Energy Transfer (FRET). SGX523 signi...
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Cancer cells tend to metastasize first to tumor-draining lymph nodes (LN), but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here we show that in the human breast tumor... more
Cancer cells tend to metastasize first to tumor-draining lymph nodes (LN), but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here we show that in the human breast tumor microenvironment (TME) the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of LN metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3-stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13 or RANKL was sufficient to decrease LN metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME.
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Our knowledge and understanding of the tumor microenvironment (TME) have been recently expanded with the recognition of the important role of innate lymphoid cells (ILC). Three different groups of ILC have been described based on their... more
Our knowledge and understanding of the tumor microenvironment (TME) have been recently expanded with the recognition of the important role of innate lymphoid cells (ILC). Three different groups of ILC have been described based on their ability to produce cytokines that mediate the interactions between innate and adaptive immune cells in a variety of immune responses in infection, allergy, and autoimmunity. However, recent evidence from experimental models and clinical studies has demonstrated that ILC contribute to the mechanisms that generate suppressive or tolerant environments that allow tumor regression or progression. Defining the complex network of interactions and crosstalk of ILC with other immune cells and understanding the specific contributions of each type of ILC leading to tumor development will allow the manipulation of their function and will be important to develop new interventions and therapeutic strategies.
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Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals... more
Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intra-cluster distances (d). From this data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P<0.001). For comparison, the prediction accuracy was only 61% (P=0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used...
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Whilst multi-parametric magnetic resonance imaging (mp-MRI) has been a significant advance in the diagnosis of prostate cancer, scanning all patients with elevated prostate specific antigen (PSA) levels is considered too costly for... more
Whilst multi-parametric magnetic resonance imaging (mp-MRI) has been a significant advance in the diagnosis of prostate cancer, scanning all patients with elevated prostate specific antigen (PSA) levels is considered too costly for widespread National Health Service (NHS) use, as the predictive value of PSA levels for significant disease is poor. Despite the fact that novel blood and urine tests are available which may predict aggressive disease better than PSA, they are not routinely employed due to a lack of clinical validity studies. Furthermore approximately 40 % of mp-MRI studies are reported as indeterminate, which can lead to repeat examinations or unnecessary biopsy with associated patient anxiety, discomfort, risk and additional costs. We aim to clinically validate a panel of minimally invasive promising blood and urine biomarkers, to better select patients that will benefit from a multiparametric prostate MRI. We will then test whether the performance of the mp-MRI can be ...
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Imaging biomarkers (IBs) are integral to the routine management of patients with cancer. IBs used daily in oncology include clinical TNM stage, objective response and left ventricular ejection fraction. Other CT, MRI, PET and... more
Imaging biomarkers (IBs) are integral to the routine management of patients with cancer. IBs used daily in oncology include clinical TNM stage, objective response and left ventricular ejection fraction. Other CT, MRI, PET and ultrasonography biomarkers are used extensively in cancer research and drug development. New IBs need to be established either as useful tools for testing research hypotheses in clinical trials and research studies, or as clinical decision-making tools for use in healthcare, by crossing 'translational gaps' through validation and qualification. Important differences exist between IBs and biospecimen-derived biomarkers and, therefore, the development of IBs requires a tailored…
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Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures... more
Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.
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Fluorescence lifetime imaging (FLIM) is a key optical technique for imaging the photophysical environment of fluorescent probes in vivo. In addition, it also provides information in fluorescence enhancement studies that intensity imaging... more
Fluorescence lifetime imaging (FLIM) is a key optical technique for imaging the photophysical environment of fluorescent probes in vivo. In addition, it also provides information in fluorescence enhancement studies that intensity imaging alone could not provide. We review the principles and recent advances in the application of the techniques, instrumentation and molecular probe development.
HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte... more
HIV infection is associated with a disease status-dependent impairment of Ag-specific T cell responses, resulting in anergy or unchecked apoptotic cell death. beta1 integrins play an important role in the induction of T lymphocyte responses to antigenic challenge by providing a T cell costimulatory signal, and have been shown to rescue various cell types from undergoing apoptosis. We examined the integrin-triggered cell survival signal and associated pathways in CD3+ T cells derived from 69 HIV-1-infected individuals in comparison with healthy controls. We found beta1 integrin-mediated costimulation of TCR-induced T cell proliferation and protection from aberrant cell death to be absent in the majority of patients with AIDS, but intact in asymptomatic, infected individuals. The lack of integrin-mediated rescue may be partly due to an early impairment of TCR/integrin-costimulated secretion of IFN-gamma, a type 1 lymphokine that protects against TCR-induced apoptosis of T cells from H...
Research Interests: Immunology, Immunology of the Gut, Apoptosis, Signal Transduction, Humans, and 15 moreMonoclonal Antibodies, The, Integrins, T lymphocytes, Immune Tolerance, Epitopes, Focal Adhesion Kinase, Cell Adhesion Molecules, Acquired immunodeficiency syndrome, Interphase, Interferon gamma, Protein Kinase C, Drug synergism, Protein tyrosine kinases, and HIV infections
Introduction: Adrenocortical carcinoma (ACC) is a rare disease with a poor prognosis and limited therapeutic options. Aim: The aim of this study was to assess the anti-proliferative effect of combinatorial targeting of the PI3K/Akt/mTOR... more
Introduction: Adrenocortical carcinoma (ACC) is a rare disease with a poor prognosis and limited therapeutic options. Aim: The aim of this study was to assess the anti-proliferative effect of combinatorial targeting of the PI3K/Akt/mTOR pathway with different drugs and simultaneous targeting of the MEK/Raf/ERKpathway in H295R adrenocortical cancer cells. Material and Methods: The cytotoxicity of LY294002 (pan-PI3K inhibitor), everolimus (mTOR inhibitor), BEZ235 (dual PI3K/mTOR inhibitor) and U0126 (MEK1/2 inhibitor) was assessed by Alamar blue assay in the H295R cell line. After finding the maximal cytotoxic concentrations for all inhibitors, we used suboptimal concentrations for experiments where multiple agents were used simultaneously. Everolimus and BEZ235 were kindly provided by Novartis, Switzerland; LY294002 and U0126 were commercially available. Results: While everolimus, BEZ235 and LY294002 caused 20±6%, 15.6±6% and 11±1% cytotoxicity when used as single agents, co-treatmen...
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ABSTRACT
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Two distinct populations of lymphocytes have been identified: T lymphocytes, which are thymus-dependent, and B cells, first observed in the Bursa Fabricus of birds. Mammals do not have an equivalent structure, and there are varying... more
Two distinct populations of lymphocytes have been identified: T lymphocytes, which are thymus-dependent, and B cells, first observed in the Bursa Fabricus of birds. Mammals do not have an equivalent structure, and there are varying opinions as to the similarity of these cells between species. In humans, current theories are that B lymphocytes differentiate in the fetal liver and in the bone marrow of adults. Human T and B cells are most easily obtained either from peripheral blood or from biopsy of lymphoid tissues (lymph nodes, spleen, Peyer&amp;amp;#39;s patches from gut, tonsils, and adenoids).
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Standard biomarker testing of a single macroscopic disease site is unlikely to be sufficient because of tumor heterogeneity. A focus on examining global biomarker expression or activity, particularly in microscopic residual... more
Standard biomarker testing of a single macroscopic disease site is unlikely to be sufficient because of tumor heterogeneity. A focus on examining global biomarker expression or activity, particularly in microscopic residual chemotherapy-resistant disease, is needed for the appropriate selection of targeted therapies. This study was aimed at establishing a technique for the assessment of biomarkers of ovarian cancer peritoneal spread. An in-house developed fluorescent imaging device was used to detect the expression of the c-Met oncogene in ovarian cancer. A modified cyanine 5-tagged peptide, GE137, with a high in vitro affinity for the human c-Met protein, was tested in a panel of ovarian cancer cell lines. Finally, the feasibility of detecting submillimeter ovarian cancer cell peritoneal metastases in vivo was tested through the intravenous injection of GE137 into mice with tumor xenografts. Using optical imaging it was possible to detect c-Met expression in submillimeter peritonea...
Research Interests: Cancer, Flow Cytometry, Fluorescence Microscopy, Immunohistochemistry, Fluorescent Dyes and Reagents, and 14 moreMedicine, Optical Imaging, Western blotting, Humans, Mice, Female, Animals, Feasibility Studies, Cyclic peptides, Individualized Medicine, Molecular Targeted Therapy, fluorescent dyes, Peritoneal Neoplasms, and Ovarian Neoplasms
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The effects have been determined of a systematic alteration of the alkyl chain geometry of a C14 analogue of DOTMA on the detailed molecular architecture of the resulting cationic vesicles formed both in the absence and presence of 50... more
The effects have been determined of a systematic alteration of the alkyl chain geometry of a C14 analogue of DOTMA on the detailed molecular architecture of the resulting cationic vesicles formed both in the absence and presence of 50 mol% DOPE, and of the lipoplexes prepared from these vesicles using either calf thymus or plasmid DNA. The C14 DOTMA analogues studied involved cis- or trans-double bonds at positions Δ9 or Δ11, and a compound (ALK) featuring an alkyne at position C9. For all of these analogues, examination by light scattering and neutron scattering, zeta potential measurement, and negative staining electron microscopy showed that there were no significant differences in the structures or charges of the vesicles or of the resulting lipoplexes, regardless of the nature of the DNA incorporated. Differences were observed, however, between the complexes formed by the various lipids when examining the extent of complexation and release by gel electrophoresis, where the E-lipids appeared to complex the DNA more efficiently than all other lipids tested. Moreover, the lipoplexes prepared from the E-lipids were the most effective in transfection of MDA-MB-231 breast cancer cells. As indicated through confocal microscopy studies, the E-lipids also showed a higher internalisation capacity and a more diffuse cellular distribution, possibly indicating a greater degree of endosomal escape and/or nuclear import. These observations suggest that the extent of complexation is the most important factor in determining the transfection efficiency of the complexes tested. At present it is unclear why the E-lipids were more effective at complexing DNA, although it is thought that the effective area per molecule occupied by the cationic lipid and DOPE head groups, and therefore the density of positive charges on the surface of the bilayer most closely matches the negative charge density of the DNA molecule. From a consideration of the geometry of the cationic lipids it is anticipated that the head groups of the E-lipids would occupy a smaller area per molecule than the ALK or Z-lipids.
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Research Interests: Chemistry, Fluorescence, Nuclear medicine, Confocal Microscopy, DNA damage, and 15 morePositron Emission Tomography, Medicine, Humans, Female, Clinical Sciences, Neoplasms, Reproducibility of Results, Radiopharmaceuticals, Environment, Adenocarcinoma, Lymph nodes, Etoposide, neoadjuvant therapy, Breast Neoplasms, and Neoplasm metastasis
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Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The G-protein-coupled CXC chemokine receptor 4 (CXCR4) was shown to be a major determinant in organ-specific metastasis for various cancers. The C-terminus of the... more
Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The G-protein-coupled CXC chemokine receptor 4 (CXCR4) was shown to be a major determinant in organ-specific metastasis for various cancers. The C-terminus of the receptor contains phosphorylation/ubiquitination sites and residues that interact with adaptor proteins which couple to the endocytosis machinery. Post-endocytic events that lead to a decision between degradation and recycling are hitherto poorly understood. We used fluorescently tagged receptor molecules stably expressed in breast cancer cells to establish that mutations in the protein kinase C (PKC) site of CXCR4 affect not only the cellular equilibrium between ESCRT-mediated degradation and recycling pathways but also the invasive properties. Importantly, the obtained in vitro results excellently translate to a murine model of breast cancer, in which we found that mutation of the PKC motif influences the metastatic potential. Furthermore, by using fluorescence lifetime imaging (FLIM) to measure direct protein-protein interactions via Forster resonance energy transfer (FRET) we show for the first time that ligand-dependent dimerization of CXCR4 receptors is a crucial mechanistic step in the above processes. Our findings reveal a new mechanism whereby the penetrance of a mutated receptor that exhibits resistance to its desensitization and confers an enhanced cancer migratory phenotype, is reduced through the dimerization with its wildtype counterpart. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2011-1207