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

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肿瘤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异性骨髓基质转录物的重编程，这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得到功能验证，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。测量直接接触乳腺癌细胞的人骨髓基质细胞和骨髓间充质干细胞（MSCs）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，而不需要显微解剖或细胞分离。在与高骨转移性和低转移性癌细胞直接接触时，将测量基质细胞的全局重编程。2. 生成能够抵抗癌症诱导或抑制关键基质通路转录的基质细胞变体，并在体外测试其对癌细胞存活、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抑制体外乳腺癌细胞的生长或运动。我们的目标基因包括癌症诱导的基质NF-？B和Stat1以及癌症抑制基质Wnt5a，我们的初步数据表明它们是致瘤性串扰的候选介质。3. 确定基质基因重编程是否为基质促进体内癌变的必要和/或充分条件，并测量不同肿瘤微环境下细胞特异性转录的体内变化。本研究旨在验证骨髓基质基因的反应性改变是基质促进体内癌变所必需的假设。采用原位和骨内异种移植结合癌细胞和操纵基质的模型。4. 在临床样本中验证基质重编程和基质对癌症的影响。Aims 1-3诱导（或抑制）的基质基因预计会在骨转移性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和功能验证癌症诱导的骨髓基质转录变化，因为它发生在体内；相反，这也将是首次分析基质诱导的乳腺癌细胞转录变化，因为它发生在体内。虽然这一建议侧重于骨转移，但这些方法可能适用于其他转移部位。我们的工作为肿瘤环境中特定细胞的谱分析奠定了基础，因为它们对药物治疗、放疗或免疫治疗有反应或抵抗。

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.