Cell density effects on therapeutic sensitivity

细胞密度对治疗敏感性的影响

基本信息

批准号：
10014681
负责人：
Jayne Stommel
金额：
$ 20.76万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10014681
关键词：
Bioinformatics Candidate Disease Gene Carmustine Cell Communication Cell Culture Techniques Cell Cycle Progression Cell Death Induction Cell Death Inhibition Cell Density Cells Cisplatin Combined Modality Therapy Developmental Therapeutics Program Drug Screening Gene Cluster Gene Expression Genes Glioma Goals Growth Human Implant In Situ In Vitro Lead Molecular Mus Oncoproteins Paclitaxel Pathway interactions Patients Performance Pharmaceutical Preparations Phosphoproteins Protein Tyrosine Kinase Proteins Resistance Sampling Signal Pathway Signal Transduction Stromal Cells Testing Therapeutic Tyrosine Kinase Inhibitor Tyrosine Phosphorylation Work Xenograft procedure cancer cell chemotherapeutic agent density drug development drug discovery glioma cell line high throughput screening in vivo knock-down novel therapeutics oncology tumor tumor microenvironment

项目摘要

Within the tumor microenvironment, cancer cells receive a multitude of signals through direct cell-cell interactions with normal stromal cells, other cancer cells, and cells comprising the tumor vasculature via juxtacrine signaling pathways. Chemotherapeutic agents that are dependent on cell cycle progression for the induction of cell death, such as cisplatin, BCNU, and taxol, are known to be less effective in densely grown cultures with abundant cell-cell contacts, though this is widely attributed to growth arrest rather than pro-survival juxtacrine signaling. We observed an inhibition of cell death in densely-grown cells treated with tyrosine kinase inhibitors, a class of agents not known to be dependent on cell cycle progression for the induction of cell death. Therefore, this project aims to uncover the mechanisms by which the effects of tyrosine kinase inhibition are circumvented in dense cultures, a growth mode that is likely to be more reflective of tumors in situ. Because high-throughput screens for drug discovery typically use low density cultures not subject to juxtacrine survival signaling, this work may lead to the development of drugs with better in vivo efficacy. Plans and goals: 1. Define the molecular mechanisms of TKI resistance in high density cell cultures. Using primary glioma cell lines, we will profile gene expression, protein tyrosine phosphorylation, and oncoprotein abundance in cells grown at low and high density. Candidate pro-survival genes will then be validated by knocking down expression in the appropriate growth mode, then determining which RNAis restore TKI sensitivity in dense cultures. Bioinformatics analyses of the candidate genes will also be used to determine which pathways are essential for cell-cell contact-dependent TKI resistance. 2. Screen for drugs that cooperate with TKIs in high density cell cultures. Using the Approved Oncology Drugs set from the NCI Developmental Therapeutics Program, we will screen for drugs that restore TKI sensitivity in high density cell cultures. We will then determine the mechanism by which these new drugs impact TKI sensitivity by examining the effect of these drugs on the candidate genes and phospho-proteins revealed in Aim 1. 3. Validate the drug or pathway for use in TKI combination therapy in vivo. We predict that the pathways that confer TKI resistance in high density cultures may also contribute to poor performance of these drugs in vivo. To test this hypothesis, we will compare the survival of mice orthotopically implanted with human primary glioma cells treated with a single TKI to those concurrently treated with the TKI plus a drug revealed in Aim 2. We will also examine whether knocking down the genes or pathways induced by high density growth prolongs survival of the TKI-treated xenograft-implanted mice. Finally, we will perform hierarchical clustering of genes expressed in low and high density in vitro cultures to orthotopic implants and patient samples to determine whether high density cultures more closely recapitulate in vivo tumors and are thus more suited for high-throughput drug screens. The work on this project concluded in April 2019.

在肿瘤微环境中，癌细胞通过与正常基质细胞，其他癌细胞和包含肿瘤脉管结构的细胞 - 细胞相互作用通过近距离分析信号通路接收多种信号。众所周知，依赖于细胞死亡的细胞周期进展的化学治疗剂，例如顺铂，BCNU和紫杉醇，在具有丰富细胞细胞接触的密集生长的培养物中的有效性较低，尽管这广泛归因于生长滞留而不是促含促阳离子的浓度。我们观察到用酪氨酸激酶抑制剂处理的密集生长的细胞中细胞死亡的抑制作用，这类剂量不知道依赖细胞周期进程来诱导细胞死亡。因此，该项目旨在揭示酪氨酸激酶抑制作用的作用在密集培养物中绕过的机制，这种生长模式可能会更反映原位的肿瘤。由于用于药物发现的高通量筛选通常使用不受近去二分酸存活信号的低密度培养物，因此这项工作可能导致在体内疗效更好的药物中发展。计划和目标：1。定义高密度细胞培养物中TKI抗性的分子机制。使用原发性神经胶质瘤细胞系，我们将在低密度和高密度生长的细胞中介绍基因表达，蛋白酪氨酸磷酸化和癌蛋白丰度。然后，将通过在适当的生长模式下击倒表达，然后确定哪些RNAI恢复了密集培养物中的TKI敏感性，从而验证了候选生存基因。候选基因的生物信息学分析还将用于确定哪些途径对于细胞 - 细胞接触依赖性TKI抗性至关重要。 2。在高密度细胞培养物中与TKIS合作的药物的筛选。使用NCI发育疗法计划的批准的肿瘤药物，我们将筛选出在高密度细胞培养物中恢复TKI敏感性的药物。然后，我们将通过检查这些药物对AIM1。3中揭示的候选基因和磷酸蛋白的影响来确定这些新药物对TKI敏感性的影响。我们预测，在高密度培养物中赋予TKI耐药性的途径也可能导致这些药物在体内的性能不佳。为了检验这一假设，我们将比较用单个TKI处理的人类原代胶质瘤细胞原位植入的小鼠的存活与与TKI同时处理的tKI以及AIM 2中揭示的药物的生存。我们还将检查敲击高密度诱导的基因或高密度诱导的途径是由高密度诱导的tki-wreateated Xenoveraft-immim imim imim imim imim imim imim imim imim imim imim imim imim imim imim immimmimmim m mim的生存。最后，我们将对在原位植入物和患者样品中以低密度和高密度培养表达的基因进行分层聚类，以确定在体内肿瘤中是否更紧密地概括了高密度培养物，因此更适合于高通量药物筛查。该项目的工作于2019年4月结束。