In this project research is conducted to characterize and develop new animal models of human disease and to develop the means to better characterize a model's relevance, addressing critical barriers to research progress. Additional aims include the development of new research technologies for the evaluation and application of disease biomarkers. Progress was made in developing cancer diagnostics and in research resources useful in developing and characterizing new models of human cancer and for the proper development and utilization of tissue biobanks. This research project included developing capabilities in molecular diagnostics for cancer models, developing methods for automated morphmetric image analysis of cancer specimens for quantitative pathology, investigating the role of S100 in cancer, developing new methods in mass spectrometry for limited tissue such as biopsies or model animals. Continued advances and applications in developing quality assurance methods for tissue biobanking were also continued. Unique spatial-spectral image analysis algorithms were developed for applying automated pattern recognition morphometric image analysis to quantify histologic tumor and non-tumor tissue areas in biospecimen tissue sections. Additional progress was made in developing and validating algorithms for cancers of the blood and vascular tissues, lung, and connective mesenchymal tissues (soft tissue sarcoma). Quantitative image analysis automation is anticipated to enhance biomarker discovery by helping to guide the selection of study-appropriate specimens. Research resulted in contributions to development of a novel animal model of the metastasis suppressor KAI1/CD82. The KAI1/CD82 tetraspanin is a widely expressed cell surface molecule thought to organize diverse cellular signaling processes. KAI1/CD82 suppresses metastasis but not tumorigenicity. Phenotypic properties of Kai1/Cd82 deleted mice, were characterized. Kai1/Cd82-/- mice showed no obvious genotype associated defects in any of these processes and displayed no histopathologic abnormalities after 12 or 18 months of life. Expression profiles of non-immortal, wild-type and Kai1/Cd82-/- mouse embryo fibroblast (MEFs) indicated distinct sex-specific and genotype-specific profiles. These data identify 191 and 1,271 differentially expressed transcripts (by twofold at P 0.01) based on Kai1/CD82 genotype status in female and male MEFs, respectively. Differentially expressed genes in male MEFs were surprisingly enriched for cell division related processes, suggesting that Kai1/Cd82 may functionally affect these processes. This suggests that Kai/Cd82 has an unappreciated role in the early establishment of proliferation and division when challenged with a new environment that might play a role in adaptability to new metastatic sites. Additional research resulted in the characterization of a mouse model of breast cancer tumor growth in wounded tissue. Increased growth of residual tumors in the proximity of acute surgical wounds is a well-recognized clinical complication of diagnosis and treatment;however, the mechanisms of wound-promoted tumor growth remain unknown. A syngeneic, orthotopic mouse model of breast cancer was developed. Exposure of metastatic mouse breast cancer cells (4T1) to SDF-1a, which is increased in wound fluid, results in increased tumor growth. Both, wounding and exposure of 4T1 cells to SDF-1a increased tumor growth, cell proliferation rate and stromal collagen. Conversely, systemic inhibition of SDF-1a signaling with the small molecule AMD 3100 abolished the growth promoting effects, decreased proliferation, collagen, and neoangiogenesis to the levels observed in controls. Furthermore, using different mouse strains established that the effect of wounding on tumor growth and SDF-1a levels is strain dependent. Wound-promoted tumor growth is mediated by elevated SDF-1a levels. The MPU conducts research designed to advance the field of tissue diagnoses and for discovery of disease biomarkers. Pre-analytic variables and tissue handling significantly influence biomarker discovery and utilization of biobank tissue resources. Research led to the conclusion that environmental stresses can alter immunoreactivity of biomarkers in stored tissue sections. The effect of temperature and lighting on 49 cellular or microbial antigens was evaluated in serial paraffin sections. Slides were stored at room temperature (RT) in the dark, at 4C in the dark, at RT under fluorescent light, or at RT with windowpane exposure to sunlight. Immunohistochemistry was performed simultaneously in an automated immunostainer. Any loss of immunoreactivity (IR) was proportional to the tissue section age and was least in sections stored in the dark. IR was only completely lost in light-exposed sections and as early as 1 month for CD45. Other markers with complete loss of IR were bovine viral diarrhea virus, CD18 (fluorescent light), CD31, CD68, canine parvovirus, chromogranins, and thyroid transcription factor-1. Eight markers (Bartonella spp, CD11d, HMW cytokeratins, feline coronavirus, GATA-4, insulin, p63, progesterone receptor) had minimal decrease in IR, regardless of treatment. In conclusion, light-induced antigen decay (tissue section aging) is antigen dependent and could explain unexpectedly weak or negative IHC reactions in stored paraffin sections. Research collaboration established a novel model useful for study of the influence of altered mTOR function on humoral immune response. Generation of high-affinity Abys in response to antigens/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Aby responses to antigens require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken gamma globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to develop high-affinity anti-NP-specific or anti-Pn14-specific Abys, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates, and had lower pneumococcal surface protein A-specific aby titers when immunized and challenged with live S. pneumoniae. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity aby responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR knock-in (KI) and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B-cell-specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in wild type B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity abys. The significant materials, equipment or methods in this project include use of recombinant DNA technology, in vitro cell culture, DNA sequence analysis, immunodiagnostics, molecular imaging, morphometrics, computer assisted image analysis, optical imaging, mass spectrometry, molecular pathology, and veterinary medical diagnosis.
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