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: 9035368
• 负责人: RICHARD A STEINMAN
• 金额: $ 31.96万
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9035368
• 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; MCF10A cells; MCF7 cell; Malignant Neoplasms; Maps; Marrow; Measurement; Measures; Mediator of activation protein; Mesenchymal Stem Cells; Messenger RNA; Metabolic; Metastatic Neoplasm to the Bone; Metastatic breast cancer; 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; collaborative environment; 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-κB 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颖的细胞特异性代谢标记策略来识别体内癌症生长所需的基质基因。 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进行重新编程，这些转录本可以通过基质特异性代谢 mRNA 标记来发现，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 (MSC) 中因与乳腺癌细胞直接接触而产生的活性转录的重编程。 该方法利用原生动物酶（UPRT）来实现细胞类型特异性转录物标记，而不需要显微解剖或细胞分离。 将测量在与高骨转移和较少转移的癌细胞直接接触期间基质细胞的整体重编程。 2. 生成对癌症诱导或关键基质途径转录抑制具有抵抗力的基质细胞变体，并在体外测试它们对癌细胞存活、生长和运动的影响。 抑制骨髓基质细胞对癌细胞的反应预计会在体外抑制乳腺癌细胞的生长或运动。 我们将靶向包括癌症诱导的基质 NF-κB 和 Stat1 以及癌症抑制的基质 Wnt5a 在内的基因，我们的初步数据表明它们是致瘤串扰的候选介质。 3.确定所选基质基因的重编程对于基质促进体内致癌作用是否是必要和/或充分的，并测量不同肿瘤微环境中细胞特异性转录的体内变化。 该目的检验了这样的假设：特定骨髓基质基因的反应性变化对于基质促进体内致癌作用是必要的。 使用结合癌细胞和操纵基质的原位和骨内异种移植模型。 4. 在临床样本中验证基质重编程和基质对癌症的影响。 目标 1-3 中诱导（或抑制）的基质基因预计在骨转移性乳腺癌的临床样本中也会被诱导（或抑制）。 这些目标的完成将首次识别并在功能上验证癌症在体内发生的骨髓基质转录变化；相反，这也将是首次对基质诱导的体内乳腺癌细胞转录变化进行分析。 虽然该提案侧重于骨转移，但这些方法可能适用于其他转移部位。 我们的工作为分析肿瘤环境中的特定细胞对药物疗法、放射疗法或免疫疗法的反应或抵抗奠定了基础。