Tracing Intratumoral Cellular Heterogeneity using genetic barcoding in small cell lung cancer

使用遗传条形码追踪小细胞肺癌的瘤内细胞异质性

• 批准号: 10327313
• 负责人: Hannah G. Wollenzien
• 金额: $ 2.87万
• 依托单位: UNIVERSITY OF SOUTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2022-08-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327313
• 关键词: Adult; Allografting; Attention; Automobile Driving; Bar Codes; Biology; Bread; CRISPR/Cas technology; Cancer cell line; Candidate Disease Gene; Cell Culture Techniques; Cells; Characteristics; Chemoresistance; Clinical; Clonal Evolution; Data; Dependovirus; Development; Disease; Distal; Distant; Drug resistance; Embryonic Development; Exhibits; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetically Engineered Mouse; Growth; Guide RNA; Hematopoiesis; Heterogeneity; Human; In Vitro; Individual; Knowledge; Lead; Lentivirus; Life; Lung; Malignant Neoplasms; Measures; Mediating; Methods; Modeling; Mus; Nature; Neoplasm Metastasis; Nude Mice; Organ; Outcome; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Population; Primary Neoplasm; Process; Regulation; Research; Resistance; Role; Sampling; Site; Survival Rate; Technology; Testing; Therapeutic; Time; Trachea; Tumor-Associated Process; Work; Xenograft Model; Xenograft procedure; arm; cancer heterogeneity; cancer stem cell; cell type; chemotherapy; effective therapy; embryonic stem cell; experimental study; gene network; gene therapy; in vivo; innovation; knock-down; lung small cell carcinoma; molecular subtypes; mouse model; pluripotency; pluripotency factor; pressure; progenitor; single-cell RNA sequencing; stem; stem cells; targeted treatment; therapeutic target; therapy resistant; tool; transcription factor; transcriptome; transcriptomics; tumor; tumor growth; tumor initiation; tumor microenvironment; tumor progression

PROJECT SUMMARY Tumors are often highly heterogeneous, with a number of functionally different cells existing within a single tumor, giving some cells the ability to initiate new tumors, metastasize, and/or become resistant to chemotherapy. Small Cell Lung Cancer (SCLC) is often a heterogeneous tumor, and is characterized by a low 5- year survival rate, high metastatic rates, and rapid acquisition of chemoresistance. The heterogeneous nature of this tumor makes it difficult to study and to treat, as current attempts to understand tumor dynamics use a bulk approach that averages the contribution of all cells within a tumor. Commonly upregulated in cancer, the core pluripotency networks of Sox2, Oct4, and Nanog have a profound effect as master regulators of pluripotency, and their expression in cancers leads to poor clinical outcomes. Sox2 in particular is often highly expressed in SCLC, and is a marker of lung progenitor cells. Expression of these factors can reprogram cells to an induced pluripotent state, and may contribute to enhanced plasticity in cancer. Therefore, I hypothesize that the dysregulation of pluripotency factors imparts a high degree of cellular plasticity SCLC, driving its growth, metastasis, and chemoresistance. I will test this hypothesis in two aims using a cellular barcoding lineage tracing approach combined with single-cell RNA sequencing (scRNAseq). Aim 1 seeks to trace the contribution of individual cells towards tumor growth and chemoresistance in xenograft models of SCLC. I will use a lentivirus that will insert a unique genetic barcode, as well as a GFP tag in to SCLC cell lines that represent the two most common molecular subtypes of SCLC. The cells will be sampled prior to xenografting, and will then be xenografted in to nude mice. At the end of this aim, the endpoint xenograft, as well as the early tumor sample will be evaluated by scRNAseq, which will provide data to evaluate gene networks responsible for tumor initiation, progression, and chemoresistance. I will validate identified candidates by knockdown experiments in cell culture to elucidate the role of the gene candidates, with a particular focus on progenitor and stem pathways. Aim 2 will describe the genes responsible for growth, metastasis, and chemoresistance in a mouse model of SCLC. The well-characterized SCLC Rblox/lox, p53lox/lox, p130lox/lox mouse model, bread to an H11lox-stop-lox-Cas9 mouse will be used in combination with an adeno-associated virus containing a gRNA to Rosa26, and Rosa26 homology arms flanking a GFP and barcode sequence. Shortly after tumor initiation by intratracheal Cre expression, the barcoding-AAV will be injected into the trachea in order to barcode the newly-formed tumors. After tumors grow, metastasize, and acquire chemoresistance, they will be dissected and primary tumors, metastases, and early tumor samples collected by intratracheal brushing will undergo scRNA-seq to identify the genes responsible for growth, metastasis, and chemoresistance. The successful completion of this work will identify the genes responsible for tumor dynamics, develop the in vivo cellular barcoding approach and lead to the identification of potential therapeutic targets.

项目摘要 肿瘤通常是高度异质性的，在单个肿瘤细胞中存在许多功能不同的细胞。 肿瘤，使一些细胞能够启动新的肿瘤，转移，和/或成为耐药， 化疗小细胞肺癌（SCLC）通常是一种异质性肿瘤，其特征在于低分化。 5-年生存率、高转移率和快速获得耐药性。异质性 这种肿瘤的存在使得研究和治疗变得困难，因为目前理解肿瘤动力学的尝试使用了大量的 这种方法平均了肿瘤内所有细胞的贡献。通常在癌症中上调， Sox 2、Oct 4和Nanog的多能性网络作为多能性的主要调节剂具有深远的影响， 并且它们在癌症中的表达导致不良的临床结果。特别是Sox 2通常在哺乳动物中高度表达。 SCLC，并且是肺祖细胞的标志物。这些因子的表达可以将细胞重编程为诱导的 多能状态，并可能有助于增强癌症的可塑性。因此，我假设 多能性因子的失调赋予SCLC高度的细胞可塑性，驱动其生长， 转移和化学抗性。我将使用细胞条形码谱系追踪在两个目标中测试这个假设 方法结合单细胞RNA测序（scRNAseq）。目标1旨在追踪 在SCLC的异种移植模型中，单个细胞对肿瘤生长和化学抗性的影响。我会用慢病毒 这将插入一个独特的遗传条形码，以及一个GFP标签到SCLC细胞系，代表两个最 SCLC的常见分子亚型。细胞将在异种移植之前取样，然后将在移植后的第二天进行移植。 异种移植到裸鼠体内。在该目标结束时，终点异种移植物以及早期肿瘤样品 将通过scRNAseq进行评估，scRNAseq将提供数据以评估负责肿瘤起始的基因网络， 进展和耐药性。我将通过细胞培养中的敲除实验来验证已确定的候选人 阐明候选基因的作用，特别关注祖细胞和干细胞途径。目标2将 描述了在小细胞肺癌小鼠模型中负责生长、转移和化学抗性的基因。的 良好表征的SCLC Rblox/lox、p53 lox/lox、p130 lox/lox小鼠模型，将向H11 lox-stop-lox-Cas9小鼠提供面包。 与含有针对Rosa 26的gRNA和Rosa 26同源臂的腺相关病毒组合使用 侧接GFP和条形码序列。在肿瘤开始后不久，Cre表达， 将条形码-AAV注射到气管中，以便对新形成的肿瘤进行条形码化。肿瘤生长后， 转移，并获得化疗耐药性，他们将解剖和原发肿瘤，转移，和早期 通过气管内刷拭收集的肿瘤样品将进行scRNA-seq以鉴定负责以下的基因： 生长、转移和化学抗性。这项工作的成功完成将确定基因 负责肿瘤动力学，开发体内细胞条形码方法，并导致识别 潜在的治疗目标。