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

• 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
• 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

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 倍性改变机制，如细胞-细胞融合和全基因组加倍（WGD），可以驱动肿瘤内 异质性和改变化疗反应。了解这些问题的原因和后果 在三阴性乳腺癌（TNBC）中，这种机制尤其重要，TNBC占乳腺癌的15-20%。 诊断为乳腺癌，没有针对性的治疗选择。广谱化疗 仍然是TNBC的护理标准，但它们的疗效受到高细胞异质性和40%的 治疗的TNBC患者会复发。我们假设倍性改变机制是 肿瘤内异质性，并可作为主动逃逸或损伤恢复机制，使细胞 抵抗化疗。ClonMapper条形码系统的更新版本，包括核苷酸- GFP和mCherry上的索引表达DNA条形码将用于实现细胞-细胞融合的平行跟踪 荧光活细胞成像中的事件以及详细的转录组和核型克隆 倍性分离的纵向单细胞测序中的轨迹。ClonMapper表达DNA条形码 将被添加到TNBC细胞系和患者来源的细胞培养物中。在一系列受控实验中，我们将 (1)确定比率并区分预先存在的非整倍体和治疗诱导的非整倍体的影响， 不同化疗治疗中的临床相关TNBC模型，（2）遵循亚克隆 正常和化疗条件下scRNA-seq和scDNA-seq中的转录组学和核型轨迹 确定不同的化疗是否选择或产生特定的转录组或核型 模式，和（3）阐明激活细胞-细胞融合，WGD，或其他倍性改变的分子因子 机制等这些实验的结果将丰富关于转录组学和细胞生物学的纵向单细胞数据。 TNBC细胞在化疗干扰和恢复中利用的染色体转换。为 肿瘤非整倍体的程度可以很容易地从患者样本中确定，我们将通过实验参数化 并验证了一个基于代理的模型，其中包括非整倍体分数，自发和应力诱导 倍性改变的机制和基因组稳定性来预测不同治疗下的肿瘤演变 时间表。这项研究将是第一个系统地研究自发和化疗诱导的 纵向单细胞框架中倍性改变的机制。在阐明细胞状态时， 使癌细胞易于发生倍性改变，鉴定 这些事件，并量化预先存在的和从头产生的非整倍体对化疗药物的影响。 耐药性、细胞-细胞融合和WGD的原因和后果可能是主要的贡献者 并激发新的治疗策略，以改善TNBC患者的结果。