In vivo models in breast cancer research: progress, challenges and future directions.

Research using animal model systems has been instrumental in delivering improved therapies for breast cancer, as well as in generating new insights into the mechanisms that underpin development of the disease. A large number of different models are now available, reflecting different types and stages of the disease; choosing which one to use depends on the specific research question(s) to be investigated. Based on presentations and discussions from leading experts who attended a recent workshop focused on in vivo models of breast cancer, this article provides a perspective on the many varied uses of these models in breast cancer research, their strengths, associated challenges and future directions. Among the questions discussed were: how well do models represent the different stages of human disease; how can we model the involvement of the human immune system and microenvironment in breast cancer; what are the appropriate models of metastatic disease; can we use models to carry out preclinical drug trials and identify pathways responsible for drug resistance; and what are the limitations of patient-derived xenograft models? We briefly outline the areas where the existing breast cancer models require improvement in light of the increased understanding of the disease process, reflecting the drive towards more personalised therapies and identification of mechanisms of drug resistance.

The Sharing Experimental Animal Resources, Coordinating Holdings (SEARCH) Framework: Encouraging Reduction, Replacement, and Refinement in Animal Research.

While significant medical breakthroughs have been achieved through using animal models, our experience shows that often there is surplus material remaining that is frequently never revisited but could be put to good use by other scientists. Recognising that most scientists are willing to share this material on a collaborative basis, it makes economic, ethical, and academic sense to explore the option to utilise this precious resource before generating new/additional animal models and associated samples. To bring together those requiring animal tissue and those holding this type of archival material, we have devised a framework called Sharing Experimental Animal Resources, Coordinating Holdings (SEARCH) with the aim of making remaining material derived from animal studies in biomedical research more visible and accessible to the scientific community. We encourage journals, funding bodies, and scientists to unite in promoting a new way of approaching animal research by adopting the SEARCH framework.

SEARCHBreast: a new resource to locate and share surplus archival material from breast cancer animal models to help address the 3Rs.

Animal models have contributed to our understanding of breast cancer, with publication of results in high-impact journals almost invariably requiring extensive in vivo experimentation. As such, many laboratories hold large collections of surplus animal material, with only a fraction being used in publications relating to the original projects. Despite being developed at considerable cost, this material is an invisible and hence an underutilised resource, which often ends up being discarded. Within the breast cancer research community there is both a need and desire to make this valuable material available for researchers. Lack of a coordinated system for visualisation and localisation of this has prevented progress. To fulfil this unmet need, we have developed a novel initiative called Sharing Experimental Animal Resources: Coordinating Holdings-Breast (SEARCHBreast) which facilitates sharing of archival tissue between researchers on a collaborative basis and, de facto will reduce overall usage of animal models in breast cancer research. A secure searchable database has been developed where researchers can find, share, or upload materials related to animal models of breast cancer, including genetic and transplant models. SEARCHBreast is a virtual compendium where the physical material remains with the original laboratory. A bioanalysis pipeline is being developed for the analysis of transcriptomics data associated with mouse models, allowing comparative study with human and cell line data. Additionally, SEARCHBreast is committed to promoting the use of humanised breast tissue models as replacement alternatives to animals. Access to this unique resource is freely available to all academic researchers following registration at https://searchbreast.org.

Introducing SEARCHBreast: a virtual resource to facilitate sharing of surplus animal material developed for breast cancer research.

Animals studies have made significant contribution to expanding our knowledge of breast cancer. Often material is leftover and archived. SEARCHBreast provides a platform for collaborative sharing of archived material via a dedicated on-line database whereby users can both share and search available tissue. The SEARCHBreast database has information on over 50 different mouse models, including tissue from PDX models, available to share. With thousands of samples freely available, SEARCHBreast should be the first point of call for any researcher looking for animal material to aid their breast cancer research.

SEARCHBreast Workshop Proceedings: 3D Modelling of Breast Cancer.

SEARCHBreast, a UK initiative supported by the NC3Rs, organised a workshop entitled 3D Modelling of Breast Cancer. The workshop focused on providing researchers with solutions to overcome some of the perceived barriers to working with human-derived tumour cells, cell lines and tissues, namely: a) the limited access to human-derived material; and b) the difficulty in working with these samples. The workshop presentations provided constructive advice and information on how to best prepare human cells or tissues for further downstream applications. Techniques in developing primary cultures from patient samples, and considerations when preserving tissue slices, were discussed. A common theme throughout the workshop was the importance of ensuring that the cells are grown in conditions as similar to the in vivo microenvironment as possible. Comparisons of the advantages of several in vitro options, such as primary cell cultures, cell line cultures, explants or tissue slices, suggest that all offer great potential applications for breast cancer research, and highlight that it need not be a case of choosing one over the other. The workshop also offered cutting-edge examples of on-chip technologies and 3-D tumour modelling by using virtual pathology, which can contribute to clinically relevant studies and provide insights into breast cancer metastatic mechanisms.

The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS.

The PapC usher is a ß-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species.

Detection and structural features of the betaB2-B3-crystallin heterodimer by radical probe mass spectrometry (RP-MS).

The predilection of the beta-crystallin B2 subunit to interact with the betaB3 subunit rather than self associate is evident by the detection of the betaB2-B3-crystallin heterodimer by native gel electrophoresis and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47,450 +/- 1 Da. Radical probe mass spectrometry (RP-MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the betaB2 subunit and six of the betaB3 subunit were found to oxidise, with far greater oxidation observed within the betaB3 versus the betaB2 subunit. This, and the observation that the oxidation data of betaB2 subunit is inconsistent with the structure of the betaB2 monomer, demonstrates that the protection of betaB2 is conferred by its association with betaB3 subunit within the heterodimer where only the residues of, and towards, its N-terminal domain remain exposed to solvent. The results suggest that the betaB2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the betaB3 subunit within the heterodimer.

Mass spectrometry analysis of the influenza virus.

The role of mass spectrometry to probe characteristics of the influenza virus, and vaccine and antiviral drugs that target the virus, are reviewed. Genetic and proteomic approaches have been applied which incorporate high resolution mass spectrometry and mass mapping to genotype the virus and establish its evolution in terms of the primary structure of the surface protein antigens. A mass spectrometric immunoassay has been developed and applied to assess the structure and antigenicity of the virus in terms of the hemagglutinin antigen. The quantitation of the hemagglutinin antigen in vaccine preparations has also been conducted that is of importance to their efficacy. Finally, the characterization and quantitation of antiviral drugs against the virus, and their metabolites, have been monitored in blood, serum, and urine. The combined approaches demonstrate the strengths of modern mass spectrometric methods for the characterization of this killer virus. [This article was published online 10 September 2008. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 7 November 2008.]

Kinetics of antigen-antibody interactions employing a MALDI mass spectrometry immunoassay.

Time-course MALDI mass spectrometry immunoassays have been shown to be able to detect differences in the relative rates of binding of peptides, both from within and across epitopic domains, with antibodies in non-competitive and competitive experiments. A monoclonal antibody raised to target the HA1 subunit of the hemagglutinin antigen of type A H3N2 influenza strains is found to recognize two epitopic peptides comprising residues 109-125 and 158-166 that likely form part of an extended discontinuous domain. Time-course experiments show the smaller peptide binds antibody at a rate that is 5-fold faster than that for the larger peptide. A shorter segment of this larger peptide, comprised of residues 119-125, is also found to bind at twice the rate of the extended peptide. Studies of modified peptide variants and synthetic variants of HA peptide 119-125 has enabled important contact residues to be identified whose accessibilities in the native protein are in accord with the mass spectrometry results.

Antigenic characterisation of H3N2 subtypes of the influenza virus by mass spectrometry.

The antigenic characterisation of three H3N2 type A influenza strains by mass spectrometry is described. The approach, developed in this laboratory, employs matrix-assisted laser desorption ionisation (MALDI) mass spectrometry to analyse gel-resolved antigens, post their proteolysis and treatment with monoclonal antibodies. The primary structure and antigenicity of the component antigens of the virus can be determined in a single step. Four antigenic domains of hemagglutinin have been identified and these are localised at residues 109-125, 158-170 and 316-326 of the HA1 subunit and to residues 159-183 of the HA2 subunit. The results demonstrate the applicability of the approach for identifying antigenic determinants across various H3N2 strains with high throughput and at low sample levels. Comparative rates of antibody binding between two of the antigenic peptides have also been reported.

Fingerprinting a killer: surveillance of the influenza virus by mass spectrometry.

Influenza is a deadly virus that continues to kill and inflict illness and suffering the world over. Despite a global surveillance strategy, an annual response to vaccine preparation and the development of new anti-viral drugs to treat the virus ahead of, or after, infection, no cure exists. Future pandemics are a very real threat and countries have mobilised efforts to stockpile treatments and prepare for outbreaks. A new surveillance approach in which the structure and antigenicity of the virus can be rapidly screened by mass spectrometry is expected to have a greater role in the characterisation of emerging influenza strains, even at the site of an outbreak.

A computer algorithm for the identification of protein interactions from the spectra of masses (PRISM).

A new algorithm is reported to assist with the identification of protein interaction domains by comparing pairs of MALDI mass spectra recorded for protein digests treated with a binding partner versus an untreated control. Known as PRISM, for protein interactions from the spectra of masses, the algorithm imports m/z versus peak area data directly from a pair of MALDI mass spectra recorded for the control and reaction sample. The algorithm is shown to be able to successfully identify antigenic determinants for protein antigens within mixed protein digests. The algorithm has general utility for the comparative analysis of differences within any two mass spectra of any type and is easily implemented using a simple, intuitive graphical user interface (GUI).

A proteomics approach to survey the antigenicity of the influenza virus by mass spectrometry.

A proteomics-based approach is described that combines gel electrophoresis and MS in order to identify protein interactions and the nature of the interaction interface with high-sample throughput and sensitivity. Results for protein antigens of the influenza virus have demonstrated that the approach can be successfully employed to detect determinants within the hemagglutinin antigen of two divergent type A forms of the virus in present circulation. The determinants are localised to residues 206-224 following tryptic digestion of the hemagglutinin antigen. Specific peptide-antibody complexes formed after treatment of gel-recovered antigen are shown to be able to be preserved on the MALDI target array as has been previously demonstrated in this laboratory for whole virus. The approach has broad applicability for the analysis of a wide array of protein complexes with identification of the interaction interface in a single step with high-sample throughput and at low sample levels.