Evaluating the impact of obesity-associated inflammation on breast cancer heterogeneity and metastasis using single-cell RNA-seq

项目摘要 乳腺癌仍然是美国癌症相关死亡的第二大原因。肥胖是一个既定的 是几种侵袭性乳腺癌亚型的危险因素，也与乳腺癌的增加有关。 转移和死亡率。在细胞水平上，肥胖导致炎症增加和瘦素失调 信号传导，这两者都已被独立地证明会增加癌细胞的迁移和侵袭。 然而，这些驱动因素将肥胖与乳腺癌转移增加联系起来的确切机制是 不太了解。由于肥胖在美国和许多其他国家的流行率很高， 大多数乳腺癌相关的死亡是由于转移性疾病，干预策略，打破 肥胖和乳腺癌转移之间的联系，迫切需要降低乳腺癌死亡率。 几个系列的转移性乳腺癌细胞系（每个系列由不同的遗传改变驱动， 转移潜能不同），其可用于原位移植模型以研究 肥胖对转移性乳腺癌的影响我们在其中一个模型中的初步发现支持了 肥胖-转移联系，并表明肥胖引起的炎症可能是这种联系的基础。该提案将使用 肥胖和转移性乳腺癌的鼠模型与几种最先进的机制一致， 方法，包括炎症标志物的多重测定和高通量单细胞RNA 用于评估肿瘤微环境的测序方法，以严格检验肥胖- 相关的炎症和瘦素信号传导共同作用，通过驱动一种新的细胞因子来增强乳腺癌转移。 癌症干细胞表型。这一假设将通过两个综合的具体目标进行检验： 目标1.量化肥胖和抗炎治疗对肿瘤生长、转移、代谢的影响 表型，以及炎性和干细胞样标志物在三个单独的转移性小鼠乳腺肿瘤系。 目标二。明确肥胖和抗炎治疗对乳腺肿瘤基因表达的影响， 使用高通量、无偏的单细胞RNA测序方法来评估转移潜力。利用这些数据， 将鉴定一种转移性细胞系中的潜在驱动因子，使用CRISPR进行操作，在体外进行确认， 在另外两个细胞系中验证。在验证之后，将在体内测定改变的细胞系以测试细胞的生物学特性。 转移潜能操纵线在试点小鼠研究中比较瘦和肥胖动物。这项建议 目的是确定乳腺癌转移中富集的分子特征，并最终确定 通过限制转移来帮助降低乳腺癌死亡率的治疗靶点，特别是在肥胖患者中 患者人群。再加上卓越的培训环境， Drs.赫斯特和安德斯，并有针对性的培训计划，这项奖学金将提供一个关键的基础， 发展我未来的职业生涯作为一个独立资助的医生，科学家在癌症转移领域。

Project Abstract Breast cancer remains the second-leading cause of cancer-related death in the US. Obesity is an established risk factor for several aggressive breast cancer subtypes, and is also associated with increased breast cancer metastasis and mortality. On a cellular level, obesity drives increased inflammation and dysregulated leptin signaling, both of which have been independently shown to increase cancer cell migration and invasion. However, the exact mechanisms by which these drivers link obesity to increased breast cancer metastasis are poorly understood. Since the prevalence of obesity in the US and many other countries is very high, and the vast majority of breast cancer-related deaths are due to metastatic disease, intervention strategies for breaking the link between obesity and breast cancer metastasis are urgently needed to reduce breast cancer mortality. Several series of metastatic breast cancer cell lines (each series driven by a different genetic alteration and varying in metastatic potential) have been developed that can be used in orthotopic transplant models to study the effects of obesity on metastatic breast cancer. Our preliminary findings in one of these models support the obesity-metastasis link and suggest obesity-induced inflammation may underlie this link. This proposal will use murine models of obesity and metastatic mammary cancer in concert with several state-of-the-art mechanistic approaches, including multiplexed assays of inflammatory markers and a high-throughput single-cell RNA sequencing approach for assessing the tumor microenvironment, to rigorously test the hypothesis that obesity- associated inflammation and leptin signaling act together to enhance breast cancer metastasis by driving a cancer stem-cell phenotype. This hypothesis will be tested with two integrated specific aims: Aim 1. Quantify the impact of obesity and anti-inflammatory treatment on tumor growth, metastasis, metabolic phenotype, and inflammatory and stem-like markers in three separate metastatic murine mammary tumor lines. Aim 2. Define the impact of obesity and anti-inflammatory treatment on mammary tumor gene expression and metastatic potential using a high-throughput, unbiased single-cell RNA-sequencing approach. Using this data, potential drivers in one metastatic cell line will be identified, manipulated using CRISPR, confirmed in vitro and validated in the other two cell lines. Following validation, the altered cell line will be assayed in vivo to test the metastatic potential manipulated line in a pilot mouse study comparing lean and obese animals. This proposal aims to define molecular characteristics enriched in breast cancer metastasis and ultimately to identify therapeutic targets to help decrease breast cancer mortality by limiting metastasis, particularly in the obese patient population. Combined with the exceptional training environment at UNC, comprehensive mentoring from Drs. Hursting and Anders, and a focused training plan, this fellowship will provide a critical foundation for developing my future career as an independently-funded physician-scientist in the field of cancer metastasis.