Cell-Specific Transcription in Cancer Microenvironment in vitro and in vivo

体外和体内癌症微环境中的细胞特异性转录

• 批准号: 8236326
• 负责人: RICHARD A STEINMAN
• 金额: $ 32.38万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-09 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8236326
• 关键词: Bioreactors; Bone Marrow; Bone Marrow Cells; Breast Cancer Cell; Cancer cell line; Cell Line; Cell Separation; Cell Survival; Cells; Clinical; Coculture Techniques; Computer Simulation; Cues; Data; Disseminated Malignant Neoplasm; Environment; Enzymes; Fluorescence; Future; Gene Targeting; Genes; Genetic Transcription; Goals; Growth; Human; Imagery; Immunohistochemistry; Immunotherapy; In Vitro; Label; Learning; Luciferases; MCF7 cell; Malignant Neoplasms; Maps; Marrow; Measurement; Measures; Mediator of activation protein; Mesenchymal Stem Cells; Messenger RNA; Metabolic; Metastatic Neoplasm to the Bone; Methodology; Microdissection; Modeling; Molecular; Mus; NF-kappa B; NOD/SCID mouse; Neoplasm Metastasis; Paracrine Communication; Pathway interactions; Pharmaceutical Preparations; Pharmacotherapy; Phase; Radiation therapy; Resistance; Sampling; Signal Pathway; Signal Transduction; Site; Specimen; Staging; Stromal Cells; Stromal Neoplasm; Testing; Time; Touch sensation; Transcript; Variant; Workplace; Xenograft Model; bone; cancer cell; candidate marker; carcinogenesis; cell motility; cell type; in vivo; malignant breast neoplasm; novel; novel strategies; research study; response; small hairpin RNA; therapeutic development; three-dimensional modeling; tumor; tumorigenic

DESCRIPTION (provided by applicant): Our overall goal is to identify how breast cancer cells modulate transcription in surrounding bone marrow stroma to engender a supportive environment. Despite the importance of cancer cell-stromal interactions, current approaches are poorly suited to analyze the effect of cancer cells on their microenvironment (and vice versa) while within tumors. Our specific project uses a novel cell-specific metabolic labeling strategy to identify stromal genes that are necessary for cancer growth in vivo. We hypothesize that bone metastatic breast cancer growth requires reprogramming of specific bone marrow stromal transcripts that can be discovered through stromal-specific metabolic mRNA labeling, functionally validated in the intact tumor microenvironment and confirmed in clinical bone metastatic breast cancer samples. Specific Aims of this project are to: 1. Measure reprogramming of active transcription in human bone marrow stromal cells and in marrow-derived mesenchymal stem cells (MSCs) resulting from direct contact with breast cancer cells. The approach exploits a protozoan enzyme (UPRT) to enable cell-type specific transcript labeling without requiring microdissection or cell separation. Global reprogramming of stromal cells during direct contact with highly bone-metastatic and less metastatic cancer cells will be measured. 2. Generate stromal cell variants that are resistant to cancer-induced or -repressed transcription of key stromal pathways and test their effect on cancer cell survival, growth and motility in vitro. Suppression of the marrow stromal cell response to cancer cells is predicted to inhibit the growth or motility of breast cancer cells in vitro. We will target genes including cancer-induced stromal NF-?B and Stat1 and cancer-repressed stromal Wnt5a, which our preliminary data implicate as candidate mediators of tumorigenic crosstalk. 3. Determine whether reprogramming of select stromal genes are necessary and/or sufficient for stroma to promote carcinogenesis in vivo, and measure in vivo changes in cell-specific transcription in different tumor microenvironments. This aim tests the hypothesis that reactive changes in select bone marrow stromal genes are necessary for stroma to promote carcinogenesis in vivo. Orthotopic and intraosseous xenograft models combining cancer cells and manipulated stroma are used. 4. Validate stromal reprogramming and stromal effects on cancer in clinical samples. Stromal genes induced (or repressed) in Aims 1-3 are predicted to be induced (or repressed) in clinical samples of bone metastatic breast cancer. Completion of these aims will for the first time identify and functionally validate cancer induced changes in bone marrow stromal transcription as it occurs in vivo; conversely, this will also be the first analysis of stromal-induced changes in breast cancer cell transcription as it occurs in vivo. While this proposal focuses on bone metastases, these methodologies may be applied to other metastatic sites. Our work sets the stage for profiling of specific cells in the tumor environment as they respond to, or resist, pharmacotherapy, radiotherapy, or immunotherapy. PUBLIC HEALTH RELEVANCE: Bone metastases cause much of the suffering of advanced breast cancer. It is believed that the cancer cells change the bone marrow environment to make it more hospitable. This proposal seeks to measure for the first time which genes are turned on in normal bone marrow cells (stromal cells) by breast cancer cells that are touching them as this occurs. We will then identify how the cancer cells within growing tumors in mice change their own gene transcription in response to the molecular cues from adjacent bone marrow cells. We will test whether some of these signals have arisen in bone specimens containing breast cancer. Learning this information will guide us in selecting drugs to treat metastatic breast cancer that interrupt specific responses of the stromal cells to the cancer cells and vice versa.

描述(申请人提供)：我们的总体目标是确定乳腺癌细胞如何调节周围骨髓基质中的转录，以产生支持性环境。尽管癌细胞-间质相互作用很重要，但目前的方法不太适合分析肿瘤内癌细胞对其微环境的影响(反之亦然)。我们的具体项目使用一种新的细胞特异性代谢标记策略来识别体内癌症生长所必需的间质基因。我们假设，骨转移性乳腺癌的生长需要对特定的骨髓基质转录本重新编程，这些转录本可以通过基质特异性代谢mRNA标记发现，在完整的肿瘤微环境中进行功能验证，并在临床骨转移性乳腺癌样本中得到证实。该项目的具体目标是：1.测量人骨髓基质细胞和骨髓间充质干细胞(MSCs)中因直接接触乳腺癌细胞而产生的活性转录的重编程。该方法利用原生动物酶(UPRT)来实现细胞类型的特定转录物标记，而不需要显微解剖或细胞分离。在直接接触高转移性和低转移性癌细胞的过程中，将测量基质细胞的全局重新编程。2.产生抗癌诱导或抑制的关键基质通路转录的基质细胞变异体，并在体外检测它们对癌细胞存活、生长和运动的影响。抑制骨髓基质细胞对癌细胞的反应被预测为在体外抑制乳腺癌细胞的生长或运动。我们将针对包括癌症诱导的间质核因子-βB和STAT1以及癌症抑制的间质Wnt5a在内的基因，我们的初步数据表明这些基因是肿瘤发生串扰的候选介体。3.确定选择的间质基因重新编程是否对间质促进体内癌变是必要和/或充分的，并在体内测量不同肿瘤微环境中细胞特异性转录的变化。这一目的验证了一种假设，即选择的骨髓基质基因的反应性变化是基质在体内促进癌症发生所必需的。采用原位和骨内异种移植模型，结合癌细胞和操纵的基质。4.在临床样本中验证间质重编程和间质对癌症的影响。在AIMS 1-3中诱导(或抑制)的间质基因被预测在骨转移乳腺癌的临床样本中被诱导(或抑制)。这些目标的完成将首次识别并从功能上验证体内发生的癌症诱导的骨髓间质转录变化；相反，这也将是第一次分析体内发生的间质诱导的乳腺癌细胞转录变化。虽然这项建议侧重于骨转移，但这些方法也可以应用于其他转移部位。我们的工作为描述肿瘤环境中特定细胞对药物治疗、放射治疗或免疫治疗的反应或抵抗奠定了基础。 与公共卫生相关：骨转移是晚期乳腺癌的主要原因。据信，癌细胞会改变骨髓环境，使其更适宜生存。这项提议试图首次测量正常骨髓细胞(间质细胞)中哪些基因被接触的乳腺癌细胞激活。然后，我们将确定生长中的小鼠肿瘤内的癌细胞如何改变自己的基因转录，以响应来自邻近骨髓细胞的分子信号。我们将测试其中一些信号是否出现在含有乳腺癌的骨骼标本中。了解这些信息将指导我们选择治疗转移性乳腺癌的药物，这些药物可以干扰基质细胞对癌细胞的特定反应，反之亦然。