Molecular Pathology Research for Cancer Diagnostics and Biomarkers

在这个项目中，进行研究是为了确定和开发新的人类疾病动物模型，并开发更好地确定模型相关性的手段，解决研究进展的关键障碍。其他目标包括开发新的研究技术，以评估和应用疾病生物标志物。在开发癌症诊断方法和研究资源方面取得了进展，这些资源有助于开发和表征新的人类癌症模型，并有助于适当开发和利用组织生物库。该研究项目包括开发癌症模型的分子诊断能力，开发用于定量病理的癌症标本的自动形态图像分析方法，研究S100在癌症中的作用，开发用于有限组织（如活组织检查或模型动物）的质谱新方法。在组织生物库质量保证方法开发方面的持续进展和应用也在继续。开发了独特的空间光谱图像分析算法，用于应用自动模式识别形态学图像分析来量化生物标本组织切片中的组织学肿瘤和非肿瘤组织区域。在开发和验证血液和血管组织、肺和结缔组织间充质组织（软组织肉瘤）癌症的算法方面取得了额外进展。定量图像分析自动化有望通过帮助指导选择适合研究的标本来增强生物标志物的发现。研究结果有助于开发一种新的转移抑制因子KAI1/CD82的动物模型。KAI1/CD82四跨蛋白是广泛表达的细胞表面分子，被认为组织多种细胞信号转导过程。KAI1/CD82抑制转移，但不抑制致瘤性。对Kai1/Cd82缺失小鼠的表型特性进行了表征。Kai1/Cd82-/-小鼠在这些过程中没有明显的基因型相关缺陷，并且在12或18个月后没有出现组织病理学异常。非不朽型、野生型和Kai1/Cd82-/-小鼠胚胎成纤维细胞（MEFs）的表达谱显示出不同的性别特异性和基因型特异性。这些数据分别在女性和男性mef中发现了191个和1271个基于Kai1/CD82基因型的差异表达转录物（P < 0.01）。男性mef中差异表达基因在细胞分裂相关过程中富集，表明Kai1/Cd82可能在功能上影响这些过程。这表明Kai/Cd82在受到新环境挑战时，在增殖和分裂的早期建立中具有未被认识到的作用，可能在对新转移部位的适应性中发挥作用。进一步的研究导致了乳腺癌肿瘤在损伤组织中生长的小鼠模型的特征。急性外科伤口附近残留肿瘤的增加生长是公认的诊断和治疗的临床并发症；然而，伤口促进肿瘤生长的机制尚不清楚。建立了一种同基因的原位乳腺癌小鼠模型。转移性小鼠乳腺癌细胞（4T1）暴露于伤口液中增加的SDF-1a，导致肿瘤生长增加。4T1细胞损伤和暴露于SDF-1a均可增加肿瘤生长、细胞增殖率和基质胶原。相反，用小分子AMD 3100对SDF-1a信号的系统性抑制消除了促进生长的作用，减少了增殖、胶原蛋白和新生血管生成，达到了对照组的水平。此外，使用不同的小鼠品系证实，损伤对肿瘤生长和SDF-1a水平的影响是品系依赖的。伤口促进的肿瘤生长是由SDF-1a水平升高介导的。MPU开展旨在推进组织诊断领域和疾病生物标志物发现的研究。分析前变量和组织处理显著影响生物标志物的发现和生物库组织资源的利用。研究得出结论，环境压力可以改变储存组织切片中生物标志物的免疫反应性。温度和光照对49种细胞或微生物抗原的影响。载玻片分别保存在室温暗处、4℃暗处、荧光灯下的室温或有窗玻璃暴露在阳光下的室温下。在自动免疫染色机上同时进行免疫组化。任何免疫反应性（IR）的损失与组织切片年龄成正比，在黑暗中保存的切片中最小。仅在光暴露的切片中IR完全消失，CD45早在1个月就消失了。其他IR完全丧失的标志物有牛病毒性腹泻病毒、CD18（荧光）、CD31、CD68、犬细小病毒、嗜铬颗粒蛋白和甲状腺转录因子-1。8种标志物（巴尔通体、CD11d、HMW细胞角蛋白、猫冠状病毒、GATA-4、胰岛素、p63、孕酮受体）在IR中均有最小的下降，无论治疗方式如何。总之，光诱导的抗原衰变（组织切片老化）是抗原依赖的，可以解释储存的石蜡切片中意想不到的弱或阴性免疫组化反应。研究合作建立了一个新的模型，有助于研究mTOR功能改变对体液免疫反应的影响。在抗原/感染因子应答中产生高亲和力的Abys对于产生持久的免疫应答至关重要。B细胞成熟和Aby对抗原的反应需要Ig体细胞超突变（SHM）和类转换重组（CSR）来实现高亲和力反应。用鸡γ球蛋白赖氨酸（NP- cgg）或热杀肺炎链球菌荚膜14型蛋白（Pn14）偶联的4-羟基-3-硝基苯乙酰半抗原（NP）免疫后，mTOR功能低下的敲入（KI）小鼠产生高亲和力抗NP特异性或抗Pn14特异性Abys的能力下降，并进行SHM/CSR。与野生型（WT）小鼠（0%）相比，半胚型mTOR小鼠的死亡率（40%）也较高，并且在接种活肺炎链球菌后，其肺炎球菌表面蛋白a特异性的婴儿滴度较低。B细胞谱系中mTOR缺失的小鼠（敲除[KO]）产生的脾生发中心也减少，对NP-CGG的高亲和力婴儿反应也减少。mTOR敲入（KI）和KO小鼠的CSR率较低，WT B细胞中mTOR的药物抑制导致体外CSR率降低。激活诱导胞苷脱氨酶（一种SHM和CSR必需的蛋白）的RNA和蛋白水平在KI和B细胞特异性KO小鼠的B细胞中都较低，同时磷酸化AKT和fox01的水平升高。援救实验增加KI型B细胞AID表达，使CSR水平恢复到野生型B细胞水平。因此，mTOR在生发中心发挥重要的免疫调节作用，至少部分通过AID信号传导，产生高亲和力的abys。本项目使用的重要材料、设备或方法包括重组DNA技术、体外细胞培养、DNA序列分析、免疫诊断、分子成像、形态测量学、计算机辅助图像分析、光学成像、质谱、分子病理学和兽医医学诊断。

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.