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: 8507617
• 负责人: RICHARD A STEINMAN
• 金额: $ 30.23万
• 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/8507617
• 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; tumor microenvironment; 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.

描述（由申请人提供）：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-基质相互作用很重要，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基质基因。 我们假设骨转移性乳腺癌的生长需要特定骨髓基质转录本的重编程，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直接接触乳腺癌细胞的骨髓间充质干细胞（MSC）中活性转录的重编程。 该方法利用原生动物酶（UPRT）来实现细胞类型特异性转录物标记，而不需要显微切割或细胞分离。 将测量在与高度骨转移性和转移性较小的癌细胞直接接触期间基质细胞的整体重编程。 2.产生对癌症诱导的或抑制的关键基质通路的转录具有抗性的基质细胞变体，并在体外测试它们对癌细胞存活、生长和运动性的影响。 据预测，抑制骨髓基质细胞对癌细胞的反应可抑制体外乳腺癌细胞的生长或运动。 我们将靶向基因，包括癌症诱导的基质NF-？B和Stat 1和癌症抑制的基质Wnt 5a，我们的初步数据暗示其为致瘤性串扰的候选介质。 3.确定选择的基质基因的重编程是否是必要的和/或足够的基质，以促进体内致癌，并测量在不同的肿瘤微环境中的细胞特异性转录的体内变化。 这一目的测试的假设，选择骨髓基质基因的反应性变化是必要的基质，以促进体内致癌。 使用结合癌细胞和操作基质的原位和骨内异种移植模型。 4.临床样本中的间质重编程和间质对癌症的影响。 预测在目的1-3中诱导（或抑制）的基质基因在骨转移性乳腺癌的临床样品中被诱导（或抑制）。 这些目标的完成将首次识别和功能验证癌症诱导的骨髓基质转录的变化，因为它发生在体内;相反，这也将是第一次分析基质诱导的乳腺癌细胞转录的变化，因为它发生在体内。 虽然该建议侧重于骨转移，但这些方法可应用于其他转移部位。 我们的工作为肿瘤环境中特定细胞的特征分析奠定了基础，因为它们对药物治疗，放射治疗或免疫治疗有反应或抵抗。