Is tumor adjacent histologically normal tissue primed for tumorigenesis?

 描述（由申请人提供）：关于在乳房保留治疗（BCT）中应切除的正常周围乳房组织的量存在相当大的争议8-13，导致再次切除率（0%-70%）8的外科医生特异性差异。理想情况下，手术切缘将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边界，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边缘外的残留癌细胞引起的14 -16，但我们的数据表明，距离乳腺肿瘤1 cm以内的组织学正常组织中的细胞也可能导致局部复发。上皮细胞显示出“癌症标志”改变的证据，并且成纤维细胞与癌症相关成纤维细胞（CAF）共享特征，已知其促进邻近细胞中的肿瘤发生。由于国家指南建议2 mm的手术边缘肿块切除术12，并且野癌变组织可以从边缘延伸1 cm，因此手术后通常仍留在女性体内。我们先前的研究已经将TGF-β相关性质确定为肿瘤邻近组织学正常组织（距肿瘤边缘最多1cm）（TAHN-1）与真正正常乳腺组织之间的关键差异18。例如，TAHN-1组织中的上皮细胞表现出上皮向间充质转化（EMT）的标志物和性质，EMT是TGF-β 1信号传导的关键终点18。此外，基质表现出TGF-β驱动的特性，例如α-SMA阳性肌成纤维细胞和富含纤维胶原和MMP 218的致密细胞外基质（ECM）。有趣的是，我们已经表明，当在原代培养物中生长时，TAHN-1成纤维细胞分泌在上皮细胞中诱导EMT的外来体，这表明在TAHN-1组织中观察到的TGF-β相关性质的潜在机制。基于这些观察，我们假设肿瘤邻近组织含有分泌外泌体的成纤维细胞，所述外泌体通过上皮中的TGF-β信号传导诱导EMT。目的1和2将分别涉及干扰TAHN-1上皮细胞和外来体处理的MCF 10a细胞中TGF-β通路中的单个分子。我们将通过利用siRNA多重阵列（96孔）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上皮细胞和外泌体处理的MCF 10a细胞中使用shRNA构建体沉默这些关键分子。然后，我们将在3D Matrigel培养中培养它们，并测量破坏的顶基极性。为了在体内验证这些分子，我们将使用shRNA在TAHN-1上皮和外泌体处理的MCF 10a细胞中沉默这些关键分子，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、外来体和外来体处理的上皮细胞中差异表达的TGF-β相关分子。我们将对成纤维细胞和上皮细胞群以及用外泌体处理的MCF 10a细胞进行RNAseq。我们还将使用miRNA分析和蛋白质组学分析外泌体的内容物。

 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.