47 research outputs found
Nanostructured polystyrene well plates allow unbiased high-throughput characterization of circulating tumor cells
No full textRapid, reliable and unbiased circulating tumor cell (CTC) isolation and molecular characterization methods are urgently required for implementation in routine clinical diagnostic and prognostic procedures. We report on the development of a novel unbiased CTC detection approach that combines high-throughput automated microscopy with a simple yet efficient approach for achieving a high level of tumor cell binding in standard tissue culture polystyrene (PS) well plates. A single 5 min high-power oxygen plasma treatment was used to create homogeneous nanoscale roughness on standard PS tissue culture plates and, in turn, drastically enhance the binding of a range of tumor cells. After physical adsorption of an adlayer of poly-l-lysine, binding yields above 97% were obtained at 2 h for all tumor cell lines used in the study. Morphological analysis of the cells confirmed strong adherence to the nanorough PS substrates. Clinically relevant concentrations of a highly metastatic breast cancer cell line, used as model for CTCs, could be reliably detected among blood cells on the nanorough polystyrene plates using an automated microscopy system. The approach was then successfully used to detect CTCs in the blood of a stage IIIc colorectal cancer patient. By combining the high binding abilities of nanorough PS well plates with the high-throughput nature of high-content analysis systems, this methodology has great potential toward enabling unbiased routine clinical analysis of CTCs. It could be applied, once clinically validated, in any clinical center equipped with an automated microscopy facility at a fraction of the cost of current CTC isolation technologies.Yuan Wan, Marnie Winter, Bahman Delalat, Jennifer E. Hardingham, Phulwinder K. Grover, Joseph Wrin, Nicolas H. Voelcker, Timothy J. Price, and Benjamin Thierr
Development and Evaluation of Mouse Monoclonal Antibodies Against Human C1q
No full textThe complement protein C1q plays an important role in breast cancer susceptibility, development, and progression. Mice genetically deficient in C1qA exhibit an eighty-five percent decrease in mammary tumour incidence when administered the chemical carcinogen DMBA. Similarly, in the aggressive MMTV-PyMT model of mouse mammary cancer, C1qA null mutant mice exhibit two thirds of the tumour burden seen in wild type mice and a third of the late-stage carcinoma at eighteen weeks of age. There may also be a role for C1q in human breast cancer as women genetically deficient in C1q have a decreased incidence of breast cancer. C1q plays a key role in macrophage-mediated efferocytic uptake of dying mammary epithelial cells, thereby preventing apoptotic cells from becoming necrotic and releasing pro-inflammatory cytoplasmic components. Persistence of necrotic cells alters the immune response during cancer initiation, as evidenced by an increase in cytotoxic T cells mobilised to the mammary gland of C1q null mutant mice in response to carcinogen DMBA. The over-arching goal of this research project was to generate and characterise an inhibitor of C1q-mediated efferocytosis. A monoclonal antibody was chosen as the C1q inhibitor, to further explore the role of C1q in breast cancer and as a potential first step in development of a new breast cancer therapeutic. Human C1q was inoculated into C1qA null mutant mice to generate an antibody-mediated immune response to C1q. Three fusions of immune splenocytes from these mice yielded a total of 2,776 cultures of which 2,017 contained viable hybridomas. Antibody binding by enzyme-linked immunosorbent assay (ELISA) included both linear epitopes (contiguous amino acids) and conformational epitopes (binding to a three-dimensional structure), making this an ideal screening strategy. Screening of these cultures by ELISA identified eight hybridomas that bound C1q. Of these, four were successfully expanded and cultures established from single cells. Thus, four candidate monoclonal antibodies were generated: BHI1-1G4, BHI1-4D3, BHI3-3F6, and BHI3-8B9. Characterisation of these antibodies was performed to determine the specificity of their binding conditions. Binding of the monoclonal antibodies in assays that involve denaturation of proteins and presentation of linear epitopes was not observed. These assays included western blotting, immunohistochemistry on normal breast sections, and immunocytochemistry on a fixed macrophage cell line. An assay with potential to display conformational epitopes, immunocytochemistry on unfixed macrophages, also did not demonstrate monoclonal binding. The antibodies were also tested for binding to C1q by immunoprecipitation. This assay can detect conformational epitopes, and all four monoclonal antibodies were demonstrated to bind soluble C1q by this method. Combined, these studies suggested that all four candidate monocloncal antibodies bind only to intact native C1q and do not bind to denatured antigen. Identification of a monoclonal antibody that inhibits C1q-mediated efferocytosis required development of a bioassay that quantifies the functional activity of candidate antibodies. An in vitro efferocytosis assay was developed involving fluorescent green-labelled macrophages co-cultured with fluorescent red-labelled MDA-MB-231 breast cancer cells induced to undergo apoptosis by cross-linking the TRAIL receptor 2. Co-localisation of red and green fluorescence as an indicator of efferocytosis was investigated in bone marrow-derived macrophages from C1qA replete and null mice. Reduced efferocytosis was observed in macrophages where C1q was absent and was quantified using ImageJ software involving digital masking of images and Boolean calculation of overlap. Hybridoma supernatants from candidate antibodies BHI1-1G4 and BHI1-4D3 were tested in the assay using the mouse macrophage cell line RAW 264.7 labelled green and dying breast cancer cells labelled red. Commercially available anti-C1q antibody 9A7 and hybridoma supernatant from non-C1q binding cell line BHI3-2C12 were also assessed. The antibodies exhibited variable capacity to affect C1q-mediated phagocytosis however whether a specific candidate monoclonal antibody could effectively inhibit C1q was inconclusive due to a high degree of variability in the timing of apoptotic cell uptake. This research led to the generation of four candidate monoclonal antibodies with potential for further pre-clinical research. Future work should concentrate on purification of the antibodies in order to improve investigation of their inhibitory capacity in C1q-mediated efferocytosis bioassays. Studies in mouse mammary cancer models would also provide valuable data on the potential of these candidate antibodies for downstream clinical applications in breast cancer patients.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202
Prognostic impact and the relevance of PTEN copy number alterations in patients with advanced colorectal cancer (CRC) receiving bevacizumab
No full textArticle first published online: 25 MAR 2013Loss of phosphatase and tensin homologue (PTEN) expression may be prognostic in colorectal cancer (CRC) and may have a correlation with vascular endothelial growth factor (VEGF) expression via hypoxia-inducible factor 1 (HIF-1) alpha, and the PI3K/mTOR pathways. We therefore have explored the prognostic association of PTEN loss and the potential that PTEN loss may be predictive of outcome with bevacizumab. Patients enrolled in the AGITG MAX trial, a randomized Phase III trial of capecitabine (C) +/− bevacizumab (B) (+/− mitomycin C [M]) with available tissues were analyzed for PTEN expression (loss vs. no loss) as assessed using a Taqman® copy number assay (CNA). Of the original 471 patients enrolled, tissues from 302 (64.1%) patients were analyzed. PTEN loss was observed in 38.7% of patients. There was no relationship between PTEN loss and KRAS or BRAF mutation. PTEN status was not prognostic for progression-free survival (PFS) or overall survival (OS) in multivariate analyses adjusting for other baseline factors; loss versus no loss PFS hazard ratio (HR) 0.9 (0.7–1.16), OS HR 1.04 (0.79–1.38). PTEN was not prognostic when assessed by KRAS and BRAF status. By using the comparison of C versus CB+CBM, PTEN status was not significantly predictive of the effectiveness of B for PFS or OS. PTEN status was not prognostic for survival in advanced colorectal cancer, irrespective of KRAS or BRAF status. PTEN status did not significantly predict different benefit with bevacizumb therapy.Timothy J. Price, Jennifer E. Hardingham, Chee K. Lee, Amanda R. Townsend, Joseph W. Wrin, Kate Wilson, Andrew Weickhardt, Robert J. Simes, Carmel Murone & Niall C. Tebbut
Discordance in 21-gene recurrence scores between paired breast cancer samples is inversely associated with patient age
No full textBackground: The Oncotype DX 21-gene Recurrence Score is a genomic-based algorithm that guides adjuvant chemotherapy treatment decisions for women with early-stage, oestrogen receptor (ER)-positive breast cancer. However, there are age-related differences in chemotherapy benefit for women with intermediate Oncotype DX Recurrence Scores that are not well understood. Menstrual cycling in younger women is associated with hormonal fluctuations that might affect the expression of genomic predictive biomarkers and alter Recurrence Scores. Here, we use paired human breast cancer samples to demonstrate that the clinically employed Oncotype DX algorithm is critically affected by patient age. Methods: RNA was extracted from 25 pairs of formalin-fixed paraffin-embedded, invasive ER-positive breast cancer samples that had been collected approximately 2 weeks apart. A 21-gene signature analogous to the Oncotype DX platform was assessed through quantitative real-time PCR, and experimental recurrence scores were calculated using the Oncotype DX algorithm. Results: There was a significant inverse association between patient age and discordance in the recurrence score. For every 1-year decrease in age, discordance in recurrence scores between paired samples increased by 0.08 units (95% CI − 0.14, − 0.01; p = 0.017). Discordance in recurrence scores for women under the age of 50 was driven primarily by proliferation- and HER2-associated genes. Conclusion: The Oncotype DX 21-gene Recurrence Score algorithm is critically affected by patient age. These findings emphasise the need for the consideration of patient age, particularly for women younger than 50, in the development and application of genomic-based algorithms for breast cancer care.Sarah M. Bernhardt, Pallave Dasari, Joseph Wrin, Wendy Raymond, Suzanne Edwards, David Walsh, Amanda R. Townsend, Timothy J. Price, and Wendy V. Ingma
Impact of KRAS and BRAF gene mutation status on outcomes from the phase III AGITG MAX Trial of capecitabine alone or in combination with Bevacizumab and Mitomycin in advanced colorectal cancer
No full textPurpose: Mutations affecting the KRAS gene are established predictive markers of outcome with anti–epithelial growth factor receptor (EGFR) antibodies in advanced colorectal cancer (CRC). The relevance of these markers for anti–vascular endothelial growth factor (VEGF) therapy is controversial. This analysis was performed to assess the predictive and prognostic impact of KRAS and BRAF gene mutation status in patients receiving capecitabine with bevacizumab (CG) or capecitabine without bevacizumab in the phase III AGITG MAX (Australasian Gastrointestinal Trials Group MAX) study. Patients and Methods: Mutation status was determined for 315 (66.9%) of the original 471 patients. Mutation status was correlated with efficacy outcomes (response rate, progression-free survival [PFS], and overall survival [OS]), and a predictive analyses was undertaken. Results: Mutations in KRAS and BRAF genes were observed in 28.8% and 10.6% of patients, respectively. KRAS gene mutation status (wild type [WT] v mutated [MT]) had no prognostic impact for PFS (hazard ratio [HR], 0.89; CI, 0.69 to 1.14) or OS (HR, 0.97; CI, 0.73 to 1.28). BRAF mutation status (WT v MT) was not prognostic for PFS (HR, 0.80; CI, 0.54 to 1.18) but was prognostic for OS (HR, 0.49; CI, 0.33 to 0.73; P = .001). By using the comparison of capecitabine versus capecitabine and bevacizumab (CB) and CB plus mitomycin (CBM), KRAS gene mutation status was not predictive of the effectiveness of bevacizumab for PFS or OS (test for interaction P = .95 and 0.43, respectively). Similarly, BRAF gene mutation status was not predictive of the effectiveness of bevacizumab for PFS or OS (test for interaction P = .46 and 0.32, respectively). Conclusion: KRAS gene mutation status was neither prognostic for OS nor predictive of bevacizumab outcome in patients with advanced CRC. BRAF gene mutation status was prognostic for OS but was not predictive of outcome with bevacizumab.Timothy J. Price, Jennifer E. Hardingham, Chee K. Lee, Andrew Weickhardt, Amanda R. Townsend, Joseph W. Wrin, Ann Chua, Aravind Shivasami, Michelle M. Cummins, Carmel Murone and Niall C. Tebbut
Pharmacological blockade of aquaporin-1 water channel by AqB013 restricts migration and invasiveness of colon cancer cells and prevents endothelial tube formation in vitro
No full textBACKGROUND Aquaporins (AQP) are water channel proteins that enable fluid fluxes across cell membranes, important for homeostasis of the tissue environment and for cell migration. AQP1 knockout mouse models of human cancers showed marked inhibition of tumor-induced angiogenesis, and in pre-clinical studies of colon adenocarcinomas, forced over-expression of AQP1 was shown to increase angiogenesis, invasion and metastasis. We have synthesized small molecule antagonists of AQP1. Our hypothesis is that inhibition of AQP1 will reduce migration and invasiveness of colon cancer cells, and the migration and tube-forming capacity of endothelial cells in vitro. METHODS Expression of AQP1 in cell lines was assessed by quantitative (q) PCR, western blot and immunofluorescence, while expression of AQP1 in human colon tumour tissue was assessed by immunohistochemistry. The effect of varying concentrations of the AQP1 inhibitor AqB013 was tested on human colon cancer cell lines expressing high versus low levels of AQP1, using wound closure (migration) assays, matrigel invasion assays, and proliferation assays. The effect of AqB013 on angiogenesis was tested using an endothelial cell tube-formation assay. RESULTS HT29 colon cancer cells with high AQP1 levels showed significant inhibition of migration compared to vehicle control of 27.9 % ± 2.6 % (p < 0.0001) and 41.2 % ± 2.7 (p <0.0001) treated with 160 or 320 μM AqB013 respectively, whereas there was no effect on migration of HCT-116 cells with low AQP1 expression. In an invasion assay, HT29 cells treated with 160 μM of AqB013, showed a 60.3 % ± 8.5 % decrease in invasion at 144 hours (p < 0.0001) and significantly decreased rate of invasion compared with the vehicle control (F-test, p = 0.001). Almost complete inhibition of endothelial tube formation (angiogenesis assay) was achieved at 80 μM AqB013 compared to vehicle control (p < 0.0001). CONCLUSION These data provide good evidence for further testing of the inhibitor as a therapeutic agent in colon cancer.Hilary S. Dorward, Alice Du, Maressa A. Bruhn, Joseph Wrin, Jinxin V. Pei, Andreas Evdokiou, Timothy J. Price, Andrea J. Yool, and Jennifer E. Hardingha
Estimating selection pressures on HIV-1 using phylogenetic likelihood models
No full textHuman immunodeficiency virus (HIV-1) can rapidly evolve due to selection pressures exerted by HIV-specific immune responses, antiviral agents, and to allow the virus to establish infection in different compartments in the body. Statistical models applied to HIV-1 sequence data can help to elucidate the nature of these selection pressures through comparisons of non-synonymous (or amino acid changing) and synonymous (or amino acid preserving) substitution rates. These models also need to take into account the non-independence of sequences due to their shared evolutionary history. We review how we have developed these methods and have applied them to characterize the evolution of HIV-1 in vivo. To illustrate our methods, we present an analysis of compartment-specific evolution of HIV-1 em) in blood and cerebrospinal fluid and of site-to-site variation in the gag gene of subtype C HIV-1. Copyright (C) 2008 John Wiley & Sons, Ltd
Fitness Ranking of Individual Mutants Drives Patterns of Epistatic Interactions in HIV-1
No full textThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Clinical deterioration during antituberculosis treatment in a high HIV-1 prevalence setting
No full textTheoretical studies of rolled-up and wrinkled nanomembranes
No full textTitle: Theoretical studies of rolled-up and wrinkled nanomembranes Author: Mgr. Peter Cendula Department: Department of Condensed Matter Physics Thesis Supervisors: Prof. Dr. Oliver G. Schmidt, Prof. RNDr. Václav Holý, CSc. Abstract : The thesis is devoted to three similar topics from the field of rolled-up and wrinkled nanomembranes. We start by recalling classical theory of thin plates, which will be used to describe deformation of nanomembranes. In the first topic, relaxation of internal strain is studied when a flat film is partially released from the substrate by etching the sacrificial layer underneath. Energetic competition of the tube and wrinkle shape is quantitatively investigated. Similar model is used to investigate the limiting maximum value of tube rotations. In the second topic, roll-up of initially wrinkled film is shown to favor tubes forming on the flat edge of rectangular wrinkled pattern, enabling precise control of tube position. Experiment is provided to justify our theoretical predictions. In the third topic, quantum well is assumed inside a wrin- kled nanomembrane. Shift of transition energy induced by lateral modulation due to bending strain is quantified, being of interest for strain-sensitive optical detectors and emitters. In addition, lateral localization of electron and hole due to..
