Is tumor adjacent histologically normal tissue primed for tumorigenesis?

肿瘤邻近的组织学正常组织是否已做好肿瘤发生的准备？

基本信息

批准号：
9070718
负责人：
Kristina Antonia Trujillo
金额：
$ 34.65万
依托单位：
UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-01 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9070718
关键词：
Biological Breast Breast-Conserving Surgery Cell Cycle Kinetics Cells Characteristics Conditioned Culture Media Data Dependence Ductal Epithelial Epithelial Cells Epithelium Excision Exhibits Extracellular Matrix Family Fatty acid glycerol esters Fibrillar Collagen Fibroblasts Gene Expression Profiling Goals Guidelines Health Histologic Human Image Immunohistochemistry In Vitro Individual Injection of therapeutic agent Life MCF10A cells Malignant Neoplasms Mammaplasty Mammary Gland Parenchyma Mammary Neoplasms Mammary gland Measures Mediator of activation protein MicroRNAs Molecular Mutate Myofibroblast National Comprehensive Cancer Network Normal tissue morphology Operative Surgical Procedures Pathway interactions Patients Population Process Production Property Proteomics Public Health RNA Splicing Radiation Recurrence Residual Cancers Role Sampling Signal Transduction Signaling Molecule Small Interfering RNA Staining method Stains Structure Surgeon Surgical margins Survival Rate Testing Tissues Transcript Transforming Growth Factor beta Transforming Growth Factor beta Receptors Tumor Tissue United States Variant Vimentin Woman base breast lumpectomy cancer cell differential expression epithelial to mesenchymal transition exosome in vivo insight matrigel migration prevent protein profiling research study small hairpin RNA three dimensional cell culture transcription factor transcriptome sequencing tumor tumorigenesis

项目摘要

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.

描述（由适用提供）：关于乳房保存疗法（BCT）8-13中应超过的正常乳房组织的数量存在很大争议，导致重新分配率（0％-70％）8的外科医生特异性变化。理想情况下，将使用客观生物学标准而不是任意物理距离来定义手术边缘。为了达到这个个性化的利润，有必要了解导致本地复发的所有因素。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，与乳腺肿瘤相距不高1厘米的组织学正常组织中的细胞也可能有可能有助于局部复发。上皮细胞显示出“癌症的标志”改变的证据，成纤维细胞与癌症相关的成纤维细胞（CAF）具有特征，这些（CAF）已知可以促进相邻细胞中的肿瘤发生。由于国家指南表明12毫米手术边缘肿块切除术12，而现场癌组织可以从边缘延伸1 cm，因此在手术后通常保留在女性中。我们以前的研究已将TGF-ß-相关特性确定为距肿瘤边缘（TAHN-1）和真正正常乳腺组织的组织学正常组织的肿瘤之间的关键差异18。例如，TAHN-1组织中的上皮细胞表现出上皮和间质转化（EMT）的标志性和特性，这是TGF-β信号传导的关键终点18。其他，基质还表现出TGF-β驱动的特性，例如A-SMAMMASMAS驱动的特性，例如A-SMA阳性的浓度纤维细胞和浓度的凝胶纤维纤维凝集（浓缩）（浓缩）（浓缩）（浓缩）（浓度） MMP218。有趣的是，我们已经表明，TAHN-1成纤维细胞在原发性培养物中生长时会影响上皮细胞中EMT的秘密外泌体，表明在THN-1组织中观察到的TGF-β相关特性的潜在机制。基于这些观察结果，我们假设肿瘤相邻组织中包含成纤维细胞，这些成纤维细胞秘密外泌体，这些外泌体在上皮中通过TGF-ß信号传导影响EMT。目标1和2将涉及分别在TAHN-1上皮细胞和外部处理的MCF10A细胞中干扰TGF-ß途径中的单个分子。我们将利用siRNA多重阵列（96井）来做到这一点。然后，我们将在3D培养物以及体内验证这些关键分子。我们将使用TAHN-1上皮和外部处理过的MCF10A细胞中的shRNA构建体沉默这些关键分子。然后，我们将在3D母质培养物中种植它们，并测量破坏的apicobasal极性。为了在体内验证这些分子，我们将使用shRNA在TAHN-1上皮和外部处理的MCF10A细胞中沉默这些关键分子，并将其注入人源化的乳腺脂肪垫中，以及不同的成纤维细胞。 AIM 3将涉及鉴定在TAHN-1上皮，成纤维细胞，外泌体和外泌体治疗的上皮菌中表达不同表达的TGF-β相关分子。我们将在成纤维细胞和上皮种群以及用外泌体处理的MCF10A细胞上执行RNASEQ。我们还将使用miRNA分析和蛋白质组学分析外泌体的含量。