TGF-betas in breast cancer progression

在2019财年，我们继续使用我们开发的一种新的功能成像方法来解决TGF-β在调节癌症干细胞（CSC）区室中的作用，该方法允许在真实的时间和原位可视化这种少数细胞群体。我们基于慢病毒的CSC报告基因使用合成启动子，其中荧光蛋白的表达由干细胞主转录因子Oct 4和Sox 2驱动。使用这种方法，我们已经开发出了允许在2D和3D培养系统中扩展单个CSC的细胞命运映射的方法，并且我们正在与阿尔伯特爱因斯坦医学院的John Condeelis博士的实验室进行合作，以对原发性肿瘤和肺转移部位的CSC群体进行活体成像。活体成像方法使我们能够证明CSC是原发性肿瘤中移动缓慢的侵入性细胞，其优先与驱动肿瘤细胞内渗到血流中的微解剖结构（TMEM）相关。我们已经显示了与巨噬细胞接触的非干细胞肿瘤细胞中干细胞表型的诱导，并且已经表征了整个转移过程中CSC代表性的变化，证明了早期到达转移部位时的峰值CSC代表性。我们现在正在研究TGF-β对乳腺癌模型系统中CSC生物学的影响，这些模型系统显示对TGF-β的肿瘤抑制或促进展反应。重要的是，我们已经表明，在体内用中和性抗TGF-β抗体治疗将增加两种异种移植乳腺癌模型中的癌症干细胞群体，其中TGF-β作为肿瘤抑制因子发挥作用，而在TGF-β具有促进展作用的模型中减少癌症干细胞群体。此外，我们有证据表明TGF-β信号转导在干细胞与非干细胞中是不同的。这些结果对正在进行的TGF-β通路拮抗剂的临床开发具有重要意义。了解TGF-β诱导癌症干细胞群体扩增或减少的详细机制的研究正在进行中。我们专注于TGF-β对表型可塑性的影响以及对自我更新与分化细胞分裂的差异影响。我们通过对到达肺转移部位后早期的干细胞和非干细胞成像以及体外3D培养来实现这一点。我们还采用整合的基因组和单细胞方法来解决TGF-β在疾病过程的早期和晚期对CSC和非CSC隔室的差异作用的分子机制。了解CSC在体内的调节方式对于开发更有效的癌症疗法至关重要，因为这些细胞在很大程度上对现有的治疗方法具有抗性。

In FY19, we have continued to address the role of TGF-beta in regulating the cancer stem cell (CSC) compartment using a novel functional imaging approach that we developed to allow visualization of this minority cell population in real time and in situ. Our lentiviral-based CSC reporter uses a synthetic promoter in which expression of a fluorescent protein is driven by the stem cell master transcription factors Oct4 and Sox2. Using this approach, we have developed methods that allow extended cell fate mapping of individual CSCs in 2D and 3D culture systems and we have an ongoing collaboration with the lab of Dr. John Condeelis at the Albert Einstein College of Medicine to perform intravital imaging of the CSC population in the primary tumor and at the lung metastatic site. The intravital imaging approach has allowed us to demonstrate that the CSCs are slow-moving, invasive cells in the primary tumor that are preferentially associated with the microanatomical structure (TMEM) that drives intravasation of tumor cells into the bloodstream. We have shown induction of a stem phenotype in non-stem tumor cells on contact with macrophages, and have characterized changes in CSC representation across the entire metastatic process, demonstrating peak CSC representation on early arrival at the metastatic site. We are now addressing the effect of TGF-beta on CSC biology in breast cancer model systems that show either tumor suppressor or pro-progression responses to TGF-beta. Importantly, we have shown that treatment with neutralizing anti-TGF-beta antibodies in vivo will increase the cancer stem cell population in two xenograft breast cancer models in which TGF-beta functions as a tumor suppressor, while decreasing it in a model in which TGF-beta has pro-progression effects. Furthermore, we have evidence that TGF-beta signal transduction is different in stem vs. non-stem cells. These results have important implications for the ongoing clinical development of TGF-beta pathway antagonists. Studies to understand the detailed mechanisms by which TGF-beta induces either an expansion or a reduction in the cancer stem cell population are ongoing. We are focusing on effects of TGF-beta on phenotypic plasticity and differential effects on self-renewing vs differentiating cell divisions. We do this through imaging of stem and non-stem cells early after arrival at the metastatic site in the lung, as well as in 3D culture in vitro. We are also employing integrated genomic and single cell approaches to address the molecular mechanisms underlying differential effects of TGF-beta on CSC and non-CSC compartments at early and late stages of the disease process. Understanding how CSCs are regulated in vivo will be critical to development of more effective cancer therapies, as these cells are largely resistant to existing therapeutic approaches.