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

基本信息

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
项目类别：
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

项目摘要

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（由适用提供）：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的环境。尽管癌细胞质相互作用的重要性，但目前的方法不适合分析癌细胞在肿瘤内的微环境（反之亦然）的影响。我们的特定项目使用一种新型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重新编程特定的骨髓基质转录物，这些转录物可以通过基质特异性的代谢mRNA标记来发现，并在完整的肿瘤微环境中进行了功能验证，并在临床骨转移性乳腺癌样品中得到了证实。该项目的具体目的是：1。测量人骨髓基质细胞和骨髓来源的间充质干细胞（MSC）的重新编程，这是直接与乳腺癌细胞直接接触而导致的。该方法利用原生动物酶（UPRT）启用细胞类型的特定转录本标记，而无需微分解或细胞分离。将测量与高度骨转移性和较少转移性癌细胞直接接触过程中基质细胞的全球重编程。 2。产生基质细胞变异，对关键基质途径的抗癌症诱导或抑制转录具有抗性，并测试其对癌细胞存活，生长和体外运动的影响。预测抑制骨髓基质细胞对癌细胞的反应将抑制体外乳腺癌细胞的生长或运动。我们将针对包括癌症诱导的基因NF-κB和STAT1以及癌症抑制的基质Wnt5a的基因，我们的初步数据将其视为肿瘤性串扰的候选介体。 3。确定重新编程是否需要重新编程和/或足以使基质在体内促进癌变，并在不同肿瘤微环境中测量细胞特异性转录的体内变化。该目标检验了以下假设：基质中，必要的骨髓基因基因的反应性变化是促进体内致癌作用的必要假设。使用了结合癌细胞和操纵基质的原位和骨内异种移植模型。 4。验证基质重编程和基质对临床样品中癌症的影响。预计在AIM 1-3中诱导的基因基因（或再现）将在骨转移性乳腺癌的临床样本中诱导（或再现）。这些目标的完成将首次确定并在功能上验证癌症引起的骨髓基质转录的变化，因为它发生在体内。相反，这也将是基质诱导的乳腺癌细胞转录变化时的首次分析。尽管该提案的重点是骨转移，但这些方法可以应用于其他转移部位。我们的工作为在肿瘤环境中对特定细胞的反应或抵抗，药物疗法，放疗或免疫疗法时为特定细胞分析奠定了基础。