TGF-betas in breast cancer progression

在2020财年，我们继续使用我们开发的一种新的功能成像方法来研究转化生长因子-β在调节癌症干细胞(CSC)隔间中的作用，该方法允许实时和原位显示这一少数细胞群体。我们的慢病毒CSC报道使用了一种合成启动子，其中荧光蛋白的表达是由干细胞主转录因子Oct4和Sox2驱动的。使用这种方法，我们已经开发出了在体外和体内扩展单个CSC的细胞命运图谱的方法，包括与阿尔伯特·爱因斯坦医学院John Condeelis博士的实验室持续合作，对原发乳腺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，CSCs是原发肿瘤中移动缓慢的侵袭性细胞，优先与驱动肿瘤细胞进入血流的显微解剖结构(TMEM)相关。重要的是，我们已经在体外和体内显示了通过Notch依赖的机制在非干细胞肿瘤细胞与巨噬细胞接触时诱导干细胞表型。我们已经描述了在整个转移过程中CSC表达的变化，显示了早期到达转移部位的CSC数量的峰值。W已经表明，在干细胞和非干细胞中，转化生长因子-β的作用和转化生长因子-β的信号转导是不同的，而且转化生长因子-β对CSC的调节也不同，这取决于它是作为肿瘤抑制因子还是促进进展因子发挥作用。这些结果对正在进行的转化生长因子-β途径拮抗剂的临床开发具有重要意义。了解调节癌症干细胞群体大小的详细机制的研究正在进行中。通过对主干转录因子表达的表观遗传调控，我们发现染色质修饰酶PADI4对CSC群体具有新的肿瘤抑制活性。我们已经开发了命运映射方法来解决转化生长因子-β对表型可塑性和自我更新与分化细胞分裂的影响。我们还将这些分析与基因组和单细胞方法相结合，以解决潜在的分子机制。了解CSCs在体内是如何调控的，对于开发更有效的癌症治疗方法至关重要，因为这些细胞在很大程度上对现有的治疗方法具有抵抗力。

In FY20, 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 vitro and in vivo, including 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 primary breast 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. Importantly, we have shown induction of a stem phenotype in non-stem tumor cells on contact with macrophages, via a Notch-dependent mechanism in vitro and in vivo. We have characterized changes in CSC representation across the entire metastatic process, demonstrating peak CSC numbers on early arrival at the metastatic site. W have shown that TGF-beta effects and TGF-beta signal transduction are different in stem vs. non-stem cells, and also that TGF-beta regulates the CSC compartment differently depending on whether it is functioning as tumor suppressor or pro-progression factor. These results have important implications for the ongoing clinical development of TGF-beta pathway antagonists. Studies to understand the detailed mechanisms regulating the size of the cancer stem cell population are ongoing. We have identified the chromatin-modifying enzyme PADI4 as having novel tumor suppressive activity on the CSC population through epigenetic regulation of the expression of master transcription factors of stemness. We have developed fate-mapping approaches to address the effects of TGF-beta on phenotypic plasticity and on self-renewing vs differentiating cell divisions. We are also integrating these analyses with genomic and single cell approaches to address underlying molecular mechanisms. 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.