Is tumor adjacent histologically normal tissue primed for tumorigenesis?

 描述(申请人提供)：在乳房保乳疗法(BCT)8-13中，对于应该切除的正常周围乳房组织的数量存在相当大的争议，导致外科医生在再次切除率方面存在很大差异(0%-70%)8.理想情况下，手术边缘应该使用客观的生物学标准来定义，而不是任意的物理距离。为了实现这种个性化的利润率，有必要了解导致局部复发的所有因素。虽然局部复发被认为是由手术切缘14-16之外的残留癌细胞引起的，但我们的数据表明，距离乳腺肿瘤1厘米的组织学正常组织中的细胞也可能导致局部复发。上皮细胞表现出“癌症特征”改变的证据，而成纤维细胞与癌症相关成纤维细胞(CAF)具有共同的特征，CAF已知促进邻近细胞的肿瘤形成。由于国家指南建议进行2 mm的手术切缘肿块切除术12，而野生性癌变组织可以从切缘延伸1厘米，因此手术后通常仍留在女性体内。我们之前的研究已经确定转化生长因子相关的特性是肿瘤邻近的正常组织(TANN-1)和真正的正常乳腺组织之间的关键区别。例如，TANN-1组织中的上皮细胞表现出上皮向间充质转化(EMT)的标记和性质，这是转化生长因子18信号转导的关键终点。此外，基质表现出转化生长因子驱动的性质，如α-SMA阳性的肌成纤维细胞和致密的细胞外基质(ECM)，富含纤维状胶原和MMP218。有趣的是，我们发现，在原代培养的TANN-1成纤维细胞中，可以分泌能诱导上皮细胞发生EMT的外切体，这表明在TANN-1组织中观察到的与转化生长因子相关的特性可能是一种机制。基于这些观察，我们假设肿瘤周围组织中含有成纤维细胞，这些成纤维细胞分泌外切体，通过上皮细胞中的转化生长因子-β信号诱导内胚层转化。目标1和目标2将分别涉及干扰THN-1上皮细胞和外切体处理的MCF10A细胞中转化生长因子-β途径的单个分子。我们将通过利用siRNA多路阵列(96个井)来实现这一点。然后，我们将在3D培养和体内验证这些关键分子。我们将在tahn-1上皮细胞和外切体处理的MCF10A细胞中使用shRNA构建物来沉默这些关键分子。然后我们将在3D Matrigel培养中培养它们，并测量被破坏的根尖基极。为了在体内验证这些分子，我们将使用shRNA沉默THEN-1上皮细胞和外切体处理的MCF10A细胞中的这些关键分子，并将其与不同群体的成纤维细胞一起注射到人源化的乳腺脂肪垫中。目的3将涉及鉴定在tahn-1上皮细胞、成纤维细胞、外切体和外切体处理的上皮细胞中差异表达的转化生长因子相关分子。我们将对成纤维细胞和上皮细胞以及用外切体处理的MCF10A细胞进行RNAseq。我们还将使用miRNA分析和蛋白质组学来分析外切体的内容。

 DESCRIPTION (provided by applicant): There is considerable controversy about the amount of normal surrounding breast tissue that should be excised in Breast Conservation Therapy (BCT) 8-13, resulting substantial surgeon-specific variation in re-excision rates (0%-70%)8. Ideally, surgical margins would be defined using objective biological criteria as opposed to an arbitrary physical distance. To achieve this personalized margin, it is necessary to understand all factors contributing to local recurrence. While local recurrences are thought to be caused by residual cancer cells outside the surgical margin14-16, our data suggest that cells in histologicaly normal tissue up to 1 cm away from breast tumors may also have the potential to contribute to local recurrence. The epithelial cells demonstrate evidence of "Hallmarks of Cancer" alterations, and the fibroblasts share characteristics with Cancer Associated Fibroblasts (CAFs), which are known to promote tumorigenesis in adjacent cells. Since National Guidelines suggest a 2 mm surgical margin lumpectomy 12, and field cancerized tissue can extend 1 cm from the margin, it often remains in a woman after surgery. Our previous studies have identified TGF-ß- related properties as the key differences between Tumor Adjacent Histologically Normal tissue up to 1 cm from the tumor margin (TAHN-1) and truly normal breast tissue18. For example, the epithelial cells in TAHN-1 tissues exhibit markers and properties of epithelial to mesenchymal transition (EMT), a key endpoint of TGF-ß signaling 18. Additionally, the stroma exhibits TGF-ß-driven properties such as a-SMA-positive myofibroblasts and a dense extracellular matrix (ECM) enriched in fibrillar collagen and MMP218. Interestingly, we have shown that TAHN-1 fibroblasts, when grown in primary culture, secrete exosomes that induce EMT in epithelial cells, indicating a potential mechanism for the TGF-ß-related properties observed in TAHN-1 tissues. Based on these observations, we hypothesize that tumor adjacent tissues contain fibroblasts that secrete exosomes which induce EMT through TGF-ß signaling in epithelia. Aims 1 and 2 will involve interfering with individual molecules in the TGF-ß pathway in TAHN-1 epithelial cells and in exosome-treated MCF10a cells, respectively. We will do this by utilizing an siRNA multiplex array (96 well). We will then verify these key molecules in 3D culture as well as in vivo. We will silence these key molecules using shRNA constructs in TAHN-1 epithelial and exosome-treated MCF10a cells. We will then grow them in 3D Matrigel culture and measure for disrupted apicobasal polarity. To verify these molecules in vivo, we will use shRNA to silence these key molecules in TAHN-1 epithelial and exosome-treated MCF10a cells and inject into the humanized mammary fat pad together with different populations of fibroblasts. Aim 3 will involve identification of TGF-ß-related molecules differentially expressed in TAHN-1 epithelia, fibroblasts, exosomes, and exosome-treated epithelia. We will perform RNAseq on the fibroblast and epithelial populations, as well as MCF10a cells treated with exosomes. We will also analyze the contents of the exosomes using miRNA analysis and proteomics.