Genomic dissection of tumor heterogeneity and progression

肿瘤异质性和进展的基因组解剖

• 批准号: 10262426
• 负责人: John Shern
• 金额: $ 74.38万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10262426
• 关键词: Bar Codes; Biological Assay; Biological Models; Biopsy; Biopsy Specimen; Bromodomain; Cell Communication; Cells; Cellular Assay; Childhood Solid Neoplasm; Clinical Trials; Coupled; DNA; DNA Sequence; DNA sequencing; Data; Data Set; Disease; Disease Progression; Dissection; EWS-FLI1 fusion protein; Early Diagnosis; Early treatment; Emerging Technologies; Enrollment; Epigenetic Process; Evolution; Ewings sarcoma; Expression Profiling; FOXO1A gene; Foundations; Fusion Oncogene Proteins; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic; Genetic Heterogeneity; Genetic Transcription; Genomics; Goals; Heterogeneity; Histologic; Image; Immune; Individual; MEKs; Methods; Modeling; NF1 gene; Nerve; Neurofibrosarcoma; Operative Surgical Procedures; PAX3 gene; Patients; Pediatric Neoplasm; Plexiform Neurofibroma; Poly A; Population; Pre-Clinical Model; Procedures; Recurrent disease; Refractory Disease; Resistance; Rhabdomyosarcoma; Sampling; Sirolimus; Solid Neoplasm; Specimen; Staging; System; Time; Topoisomerase Inhibitors; Work; cell free DNA; cell type; deep sequencing; evidence base; experimental study; genome sequencing; immunosuppressive macrophages; in vivo Model; inhibitor/antagonist; mTOR Inhibitor; neoplastic cell; neurofibroma; new therapeutic target; novel; pressure; programs; resistance mechanism; response; single cell sequencing; single-cell RNA sequencing; small molecule; therapy resistant; tumor; tumor DNA; tumor heterogeneity; tumor progression; vector; whole genome

The first aim of this project is to use single cell sequencing to understand the complexities of cell types and cell to cell heterogeneity that is present within pediatric solid tumors. In this work, we are focusing on generating comprehensive gene expression profiling of the cells present within tumors that occur in patients with NF1. To date we have collected and analyzed surgical specimens from Plexiform Neurofibromas (PN), Atypical Neurofibromas (AN) and Malignant Peripheral Nerve Sheath Tumors (MPNST). Over the past year, we have generated high gene coverage sequencing on 350,000 single cells from 12 patients with NF1 nerve tumors including histologically validated PN, ANF and MPNST. Our largest dataset generated to date includes single-cell sequencing of PN and ANF. From these experiments, we capture the landscape of cellular heterogeneity within these tumors. Within PNs we have identified at least 21 unique cell populations including a variety of stromal and immune cell types. This rich dataset details the transcriptional profile of each of these populations and highlights both known and novel cell types. Following the same procedure, we have generated scRNAseq data from 300,000 cells from three MPNST tumors and 10 PDX MPNST models. Important findings from this project are multiple clusters of transcriptionally distinct cells that compose the tumor cluster as well as a large population of immune suppressive macrophages. Currently, we are using this dataset to describe the multitude of cell to cell interactions within these tumors to dissect potential tumor specific vulnerabilities. Given the difficulty in obtaining multiple sequential biopsy specimens from patients with solid tumors, in the past year we have undertaken a project to assay cell free DNA to assess disease status in NF1 patients. In this work we have developed an assay that marries low pass whole genome sequencing with NF1 specific targeted capture deep sequencing of selected genes. Currently we are evaluating our assays value using samples collected on the actively enrolling clinical trial SARC031 (NCT03433183) "MEK Inhibitor Selumetinib (AZD6244) in Combination with the mTOR Inhibitor Sirolimus for Patients With MPNST". Initial studies correlating the changes observed in the circulating tumor DNA with the changes observed on re-staging imaging are underway. Secondary efforts will pair the circulating tumor DNA with an on-treatment tumor biopsy to observe correlation and description of the global genomic changes in the circulating tumor DNA to discover mechanisms of tumor evolution. The second aim of this work is to develop novel barcoding strategies married with single cell sequencing that can be used in preclinical model systems to model tumor cell resistance and survival. Single cell sequencing has the ability to dissect the gene expression profile of thousands of cells in parallel but is limited in its ability to track populations of cells under a selective pressure. Within the current year we completed pilot experiments that highlighted the need to add a feature to our experimental system which enabled tracking a particular cell over time and as it replicated. To accomplish this, we have incorporated a unique sequence "DNA barcode" into each cell within a pool of cells (greater than 10 million unique barcodes in 10 separate pools). Importantly, our vector generates a polyadenylated expressed gene that is compatible with capture using single cell RNA sequencing methods. Experiments have been completed which demonstrated that we are able to detect the expressed DNA barcodes within the scRNAseq data over time and with cell population expansion. Current experiments are employing the single cell barcoding strategy to understand the heterogenous tumor cell responses to topoisomerase inhibitors and small molecules targeting epigenetic modifiers. We anticipate that this will enable discernment of the expression profiles of individual surviving cells and definition of new therapeutic targets.

该项目的第一个目的是使用单细胞测序来了解儿科实体瘤中存在的细胞类型和细胞间异质性的复杂性。在这项工作中，我们的重点是产生全面的基因表达谱的细胞内发生在NF1患者的肿瘤。到目前为止，我们收集和分析了丛状神经纤维瘤（PN），非典型神经纤维瘤（AN）和恶性外周神经鞘瘤（MPNST）的手术标本。在过去的一年中，我们对来自12名NF1神经肿瘤患者的350，000个单细胞进行了高基因覆盖率测序，包括组织学验证的PN，ANF和MPNST。我们迄今为止生成的最大数据集包括PN和ANF的单细胞测序。从这些实验中，我们捕捉到了这些肿瘤内细胞异质性的景观。在PN中，我们已经鉴定了至少21种独特的细胞群，包括各种基质细胞和免疫细胞类型。这个丰富的数据集详细介绍了每个群体的转录谱，并强调了已知和新型细胞类型。按照相同的程序，我们已经从来自三个MPNST肿瘤和10个PDX MPNST模型的300，000个细胞中生成了scRNAseq数据。该项目的重要发现是组成肿瘤簇的多个转录不同细胞簇以及大量免疫抑制性巨噬细胞。目前，我们正在使用这个数据集来描述这些肿瘤内的细胞与细胞之间的相互作用，以剖析潜在的肿瘤特异性脆弱性。鉴于从实体瘤患者中获得多个连续活检标本的困难，在过去的一年中，我们进行了一个项目，以测定无细胞DNA来评估NF 1患者的疾病状态。在这项工作中，我们开发了一种将低通全基因组测序与选定基因的NF 1特异性靶向捕获深度测序结合的测定法。目前，我们正在使用积极招募的临床试验SARC 031（NCT 03433183）“MEK抑制剂司美替尼（AZD 6244）联合mTOR抑制剂西罗莫司治疗MPNST患者”中收集的样本评估我们的检测价值。将循环肿瘤DNA中观察到的变化与再分期成像中观察到的变化相关联的初步研究正在进行中。二次努力将循环肿瘤DNA与治疗中的肿瘤活检配对，以观察循环肿瘤DNA中全局基因组变化的相关性和描述，从而发现肿瘤演变的机制。这项工作的第二个目的是开发与单细胞测序结合的新型条形码策略，其可用于临床前模型系统以模拟肿瘤细胞抗性和存活。单细胞测序具有平行剖析数千个细胞的基因表达谱的能力，但其在选择压力下追踪细胞群体的能力有限。在本年度内，我们完成了试点实验，强调需要为我们的实验系统添加一个功能，使其能够随着时间的推移跟踪特定的细胞并复制。为了实现这一点，我们将独特的序列“DNA条形码”掺入细胞池内的每个细胞中（10个单独的池中超过1000万个独特的条形码）。重要的是，我们的载体产生多聚腺苷酸化的表达基因，其与使用单细胞RNA测序方法的捕获相容。已经完成的实验表明，我们能够随着时间的推移和细胞群体扩增检测scRNAseq数据中表达的DNA条形码。目前的实验采用单细胞条形码策略来理解对拓扑异构酶抑制剂和靶向表观遗传修饰剂的小分子的异质性肿瘤细胞应答。我们预计，这将使个体存活细胞的表达谱的识别和新的治疗靶点的定义。