Systems Approaches to Understanding the Impact of Cell-Cell Fusion on Therapeutic Resistance

了解细胞间融合对治疗耐药性影响的系统方法

基本信息

批准号：
10607123
负责人：
Andrea L Gardner
金额：
$ 3.92万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10607123
关键词：
Aftercare Aneuploidy Bar Codes Breast Cancer Cell Breast Cancer Model Breast Cancer Patient Breast Cancer cell line Calibration Cell Culture Techniques Cell fusion Cell model Cells Chemoresistance Chromosomal Instability Clonality Coculture Techniques Color DNA Data Discrimination ERBB2 gene Effectiveness Estrogens Event Evolution Gene Expression Generations Genome Stability Genomic Instability Heterogeneity Human Individual Label Libraries Link Malignant Neoplasms Measurement Methods Modeling Molecular Nucleotides Patient-Focused Outcomes Patients Pattern Phenotype Play Ploidies Population Population Heterogeneity Prediction of Response to Therapy Progesterone Prognosis Recovery Relapse Resistance Resolution Role Sampling Schedule Selection for Treatments Series Solid Neoplasm Stains Stress System Techniques Testing Therapeutic Therapeutically Targetable Time Treatment Failure Update Visualization breast cancer diagnosis cancer cell cell determination chemotherapy clinically relevant driving force experimental study fluorophore forging hormone therapy improved in silico indexing live cell imaging malignant breast neoplasm mathematical model mortality multiple omics multiscale data novel overexpression predictive modeling receptor response single cell sequencing single-cell RNA sequencing standard of care targeted treatment therapy outcome therapy resistant transcriptomics treatment response treatment strategy triple-negative invasive breast carcinoma tumor tumor heterogeneity whole genome

项目摘要

PROJECT SUMMARY Ploidy altering mechanisms such as cell-cell fusion and whole genome doubling (WGD) can drive intratumoral heterogeneity and alter chemotherapeutic response. Understanding the causes and consequences of these mechanisms is especially important in triple negative breast cancer (TNBC) which accounts for 15-20% of diagnosed breast cancers and does not have targeted treatment options. Broad spectrum chemotherapies remain the standard of care for TNBC, but their efficacy is dampened by high cellular heterogeneity and 40% of treated TNBC patients will relapse. We hypothesize that ploidy altering mechanisms are a driving force behind intratumoral heterogeneity and can function as active escape or damage recovery mechanisms to allow cells to resist chemotherapy. An updated version of the ClonMapper barcoding system which includes nucleotide- indexed expressed DNA barcodes on GFP and mCherry will be used to enable parallel tracking of cell-cell fusion events in fluorescent live-cell imaging and determination of detailed transcriptomic and karyotypic clonal trajectories in ploidy-separated longitudinal single-cell sequencing. The ClonMapper expressed DNA barcodes will be added to TNBC cell lines and patient-derived cell cultures. In a series of controlled experiments we will (1) determine the rates and distinguish the effects of pre-existing aneuploidy and therapy-induced aneuploidy in clinically relevant TNBC models across different chemotherapeutic treatments, (2) follow subclonal transcriptomic and karyotypic trajectories in scRNA-seq and scDNA-seq under normal and chemotherapeutic conditions to determine if different chemotherapies select for or generate specific transcriptomic or karyotypic patterns, and (3) elucidate the molecular factors which activate cell-cell fusion, WGD, or other ploidy altering mechanisms. The results of these experiments will be rich in longitudinal single cell data on transcriptomic and chromosomal transitions utilized by TNBC cells across chemotherapeutic perturbation and recovery. As the degree of tumor aneuploidy can be easily determined from patient samples, we will experimentally parameterize and validate an agent-based model which includes aneuploidy fraction, spontaneous and stress-induced mechanisms of ploidy alteration, and genomic stability to predict tumor evolution under different therapeutic schedules. This study will be the first to systematically investigate spontaneous and chemotherapy-induced mechanisms of ploidy alteration in a longitudinal single-cell framework. In elucidating the cellular states which predispose cancer cells for ploidy alteration, identifying the phenotypic and chromosomal transitions induced by these events, and quantifying the effects of pre-existing and de novo generated aneuploidy on chemotherapeutic resistance, the causes and consequences of cell-cell fusion and WGD may be revealed as primary contributors to chemoresistance and inspire novel treatment strategies to improve TNBC patient outcomes.

项目摘要拼合的机制，例如细胞 - 细胞融合和整个基因组加倍（WGD）可以驱动肿瘤内异质性和改变化学治疗反应。了解这些原因和后果机制在三重阴性乳腺癌（TNBC）中尤其重要，该乳腺癌占15-20％被诊断出乳腺癌，没有针对性的治疗选择。广谱化学疗法仍然是TNBC的护理标准，但它们的功效受到高细胞异质性的抑制作用和40％治疗的TNBC患者将复发。我们假设拼倍改变机制是背后的驱动力肿瘤内异质性，可以用作主动逃生或损坏恢复机制，以使细胞得以实现抵抗化学疗法。 ClonMapper条形码系统的更新版本，其中包括核苷酸 - GFP和MCHERRY上的索引表达的DNA条形码将用于实现细胞融合的平行跟踪荧光活细胞成像中的事件以及确定详细的转录组和核型克隆的事件曲线分离的纵向单细胞测序中的轨迹。 clonmapper表示DNA条形码将添加到TNBC细胞系和患者衍生的细胞培养物中。在一系列受控实验中，我们将（1）确定速率并区分先前存在的非整倍性和治疗诱导的非整倍性的效果跨不同化学治疗的临床相关TNBC模型，（2）遵循亚克隆在正常和化学治疗中，在SCRNA-SEQ和SCDNA-SEQ中的转录组和核型轨迹确定不同化学疗法是否选择或生成特定转录组或核型的条件模式，（3）阐明激活细胞融合，WGD或其他倍性改变的分子因子机制。这些实验的结果将富含转录组和转录单细胞数据 TNBC细胞在化学治疗扰动和恢复中使用的染色体过渡。作为肿瘤性的程度可以轻松地从患者样品中确定，我们将在实验中进行参数化并验证基于代理的模型，该模型包括非整倍部分，自发和应力诱导倍改变的机制和基因组稳定性在不同的治疗中预测肿瘤的演变时间表。这项研究将是第一个系统地研究自发和化学疗法诱导的一项纵向单细胞框架中倍性改变的机制。在阐明细胞状态中易倍改变的易感性癌细胞，鉴定由表型和染色体转变这些事件，并量化了现有和从头疾病的影响对化学治疗的影响电阻，细胞融合和WGD的原因和后果可能被揭示为主要贡献者化学抗性并激发新的治疗策略，以改善TNBC患者的结果。