Molecular Pathology Research for Cancer Diagnostics and Biomarkers

癌症诊断和生物标志物的分子病理学研究

• 批准号: 8763738
• 负责人: Robert Simpson
• 金额: $ 98.09万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763738
• 关键词: 4-hydroxy-5-nitrophenyl acetic acid; Acute; Address; Affect; Affinity; Age; Aging; Algorithms; Animal Model; Antigens; Applied Research; Area; Automated Pattern Recognition; Automation; B-Lymphocytes; Bartonella; Biological Markers; Biological Models; Biopsy; Blood Vessels; Bovine Viral Diarrhea Viruses; Breast Cancer Cell; Cancer Diagnostics; Cancer Model; Canine Parvovirus; Cell Culture Techniques; Cell Lineage; Cell Maturation; Cell Proliferation; Cell division; Cell surface; Cells; Chickens; Chromogranins; Clinical; Collaborations; Collagen; Complication; Comprehension; Computer-Assisted Image Analysis; Cytokeratin; DNA Sequence Analysis; Data; Defect; Development; Diagnosis; Diagnostic; Diagnostic Neoplasm Staging; Disease; Embryo; Environment; Equipment; Evaluation; Exposure to; Feline Coronavirus; Female; Fibroblasts; Gamma globulin; Generations; Genes; Genetic Engineering; Genotype; Growth; Harvest; Heating; Hematopoietic Neoplasms; Histologic; Human; ITGB2 gene; Image; Image Analysis; Immune response; Immunization; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunohistochemistry; Immunological Diagnosis; In Vitro; Infectious Agent; Insulin; Investigation; KAI1 gene; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Life; Light; Lighting; Liquid substance; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Medical; Medicine; Mesenchymal; Methods; Microdissection; Modeling; Molecular; Molecular Analysis; Molecular Profiling; Mouse Strains; Mus; Neoplasm Metastasis; PECAM1 gene; PTPRC gene; Paraffin; Pathology; Phenotype; Play; Problem Solving; Process; Progesterone Receptors; Property; Proteins; Proto-Oncogene Proteins c-akt; RNA; Reaction; Reagent; Research; Research Design; Research Project Grants; Residual Tumors; Resources; Role; Signal Transduction; Site; Slide; Specimen; Streptococcus pneumoniae; Stress; Structure of germinal center of lymph node; Structure of parenchyma of lung; Sunlight; Surgical wound; Techniques; Technology; Temperature; Tissue Preservation; Tissue Procurements; Tissues; Training; Transcript; Tumor Tissue; Tumorigenicity; activation-induced cytidine deaminase; base; biobank; cancer type; carcinogenesis; computerized data processing; design; digital; human FRAP1 protein; human PHEMX protein; human disease; immunoreactivity; improved; killings; mTOR inhibition; male; malignant breast neoplasm; microorganism antigen; molecular imaging; molecular pathology; mortality; mouse model; novel; optical imaging; pneumococcal surface protein A; pre-clinical; quality assurance; research study; response; sarcoma; sex; small molecule; soft tissue; technology development; thyroid transcription factor 1; tissue resource; tool; tumor growth; wound

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.

在该项目中，研究的目的是表征和开发人类疾病的新动物模型，并开发更好地表征模型相关性的方法，解决研究进展的关键障碍。其他目标包括开发用于疾病生物标志物评估和应用的新研究技术。在开发癌症诊断方法以及可用于开发和表征人类癌症新模型以及正确开发和利用组织生物库的研究资源方面取得了进展。该研究项目包括开发癌症模型分子诊断能力、开发用于定量病理学的癌症样本自动形态测量图像分析方法、研究 S100 在癌症中的作用、开发用于活检或模型动物等有限组织的质谱新方法。在开发组织生物样本库质量保证方法方面也不断取得进展和应用。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测量图像分析来量化生物样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、肺和结缔间质组织（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预计将通过帮助指导选择适合研究的样本来增强生物标志物的发现。研究成果为转移抑制因子 KAI1/CD82 的新型动物模型的开发做出了贡献。 KAI1/CD82 四跨膜蛋白是一种广泛表达的细胞表面分子，被认为可以组织多种细胞信号传导过程。 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-/- 小鼠在这些过程中均未表现出明显的基因型相关缺陷，并且在 12 或 18 个月的生命后也未表现出组织病理学异常。非永生、野生型和 Kai1/Cd82-/- 小鼠胚胎成纤维细胞 (MEF) 的表达谱表明了不同的性别特异性和基因型特异性谱。这些数据分别根据女性和男性 MEF 中的 Kai1/CD82 基因型状态识别出 191 和 1,271 个差异表达转录本（P < 0.01 时两倍）。雄性 MEF 中差异表达的基因在细胞分裂相关过程中出人意料地丰富，表明 Kai1/Cd82 可能在功能上影响这些过程。这表明，当受到可能在适应新转移位点的新环境的挑战时，Kai/Cd82 在增殖和分裂的早期建立中发挥着未被重视的作用。其他研究对受伤组织中乳腺癌肿瘤生长的小鼠模型进行了表征。急性手术伤口附近残留肿瘤生长加快是一种公认​​的诊断和治疗临床并发症；然而，伤口促进肿瘤生长的机制仍不清楚。开发了一种同基因、原位乳腺癌小鼠模型。将转移性小鼠乳腺癌细胞 (4T1) 暴露于伤口液中含量增加的 SDF-1a 中，会导致肿瘤生长加快。 4T1 细胞受伤和暴露于 SDF-1a 都会增加肿瘤生长、细胞增殖率和基质胶原。相反，用小分子 AMD 3100 系统性抑制 SDF-1a 信号，消除了生长促进作用，使增殖、胶原蛋白和新血管生成降低至对照中观察到的水平。此外，使用不同的小鼠品系证实，受伤对肿瘤生长和 SDF-1a 水平的影响是品系依赖性的。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发现疾病生物标志物的研究。预分析变量和组织处理显着影响生物样本库组织资源的生物标志物发现和利用。研究得出的结论是，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。在连续石蜡切片中评估了温度和光照对 49 种细胞或微生物抗原的影响。将载玻片储存在室温 (RT) 黑暗中、4°C 黑​​暗中、RT 荧光灯下或 RT 窗玻璃暴露在阳光下。免疫组织化学在自动免疫染色仪中同时进行。任何免疫反应性（IR）的损失与组织切片的年龄成正比，并且在黑暗中保存的切片中损失最小。 CD45 的 IR 仅在曝光切片中完全消失，且最早在 1 个月后消失。 IR 完全丧失的其他标志物包括牛病毒性腹泻病毒、CD18（荧光）、CD31、CD68、犬细小病毒、嗜铬粒蛋白和甲状腺转录因子 1。无论治疗如何，八种标记物（巴尔通体属、CD11d、HMW 细胞角蛋白、猫冠状病毒、GATA-4、胰岛素、p63、黄体酮受体）的 IR 下降幅度很小。总之，光诱导的抗原衰变（组织切片老化）是抗原依赖性的，并且可以解释储存的石蜡切片中意外的微弱或阴性 IHC 反应。研究合作建立了一种新模型，可用于研究 mTOR 功能改变对体液免疫反应的影响。针对抗原/感染因子产生高亲和力 Abys 对于产生持久的免疫反应至关重要。 B 细胞成熟和 Aby 对抗原的反应需要 Ig 体细胞超突变 (SHM) 和类别转换重组 (CSR) 来实现高亲和力反应。使用与鸡丙种球蛋白赖氨酸 (NP-CGG) 缀合的 4-羟基-3-硝基苯乙酰基半抗原 (NP) 或热灭活的肺炎链球菌 14 型荚膜蛋白 (Pn14) 进行免疫后，mTOR 功能低效型的敲入 (KI) 小鼠产生高亲和力的能力下降 抗 NP 特异性或抗 Pn14 特异性 Abys，并执行 SHM/CSR。与野生型 (WT) (0%) 同窝小鼠相比，亚形性 mTOR 小鼠的死亡率也很高 (40%)，并且在用活肺炎链球菌免疫和攻击时，其肺炎球菌表面蛋白 A 特异性 aby 滴度较低。与 WT 同窝小鼠相比，B 细胞谱系中 mTOR 缺失（敲除 [KO]）的小鼠也产生了较少的脾生发中心，并降低了对 NP-CGG 的高亲和力 aby 反应。 mTOR 敲入 (KI) 和 KO 小鼠的 CSR 率较低，WT B 细胞中 mTOR 的药理学抑制导致离体 CSR 率降低。 KI 和 B 细胞特异性 KO 小鼠 B 细胞中激活诱导的胞苷脱氨酶 (AID)（SHM 和 CSR 必需的蛋白质）的 RNA 和蛋白质水平较低，同时磷酸化 AKT 和 FOXO1 增加。增加 KI B 细胞中 AID 表达的拯救实验使 CSR 水平恢复到野生型 B 细胞的水平。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 abys。本项目的重要材料、设备或方法包括重组DNA技术的使用、体外细胞培养、DNA序列分析、免疫诊断、分子成像、形态测量、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。