Mechanisms of stromal fibroblast activation in esophageal tumors

食管肿瘤基质成纤维细胞活化机制

• 批准号: 8201863
• 负责人: Todd Joseph Waldron
• 金额: $ 4.84万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-26 至 2013-07-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8201863
• 关键词: American; Attention; Attenuated; Biological; Buffers; Carcinogens; Cells; Characteristics; Coculture Techniques; Conditioned Culture Media; Development; Dexamethasone; Diagnosis; Disease; Endothelial Cells; Esophageal; Esophageal Neoplasms; Esophagus; Exhibits; Fibroblasts; Formalin; Fostering; Frequencies; Generations; Genetic; Goals; Growth Factor; Histamine; Histidine; Histidine Decarboxylase; ITGAM gene; Immune; Immunohistochemistry; In Vitro; Interleukin-6; Investigation; Knockout Mice; Laboratories; Malignant Neoplasms; Malignant Squamous Cell Neoplasm; Malignant neoplasm of esophagus; Mediating; Modeling; Mus; Myelogenous; Myeloid Cells; Nerve; Nude Mice; Pathogenesis; Pericytes; Phenotype; Play; Property; RNA Interference; Recombinants; Research; Role; Signal Transduction; Source; Staging; Suppressor-Effector T-Lymphocytes; Testing; Transgenic Mice; Transgenic Organisms; Translating; Tumor Cell Line; Tumor-Derived; Tumorigenicity; United States; Work; angiogenesis; cancer diagnosis; cancer initiation; cell type; combinatorial; cytokine; density; in vivo; innovation; insight; mouse model; neoplastic cell; neutralizing antibody; new therapeutic target; novel; oral tissue; outcome forecast; prevent; promoter; research study; stem; tumor; tumor growth; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): The goal of this proposal is to study mechanisms by which non-tumor cells within the tumor microenvironment (TME) promote the development and progression of esophageal cancer. This proposal will test the hypothesis that GR1+CD11b+ myeloid derived suppressor cells (MDSCs) play an essential role in promoting esophageal cancer through activation of stromal fibroblasts. Previous work has demonstrated the capacity of MDSCs to activate fibroblasts. The first aim of this proposal is to elucidate the mechanism whereby tumor-conditioned MDSCs activate fibroblasts and characterize the role of this activity on tumor progression. Co-culture experiments will be used to determine whether the capacity to activate fibroblasts is a characteristic of all MDSCs or a unique property of MDSCs conditioned by tumor-derived factors. Activation of quiescent fibroblasts will be determined using immunohistochemistry (IHC) for a-SMA and Fsp-1, established markers of fibroblast activation. Cytokine array analyses of conditioned media from the MDSC-fibroblasts co-cultures will allow investigation into the mechanism of MDSC-mediated activation of fibroblasts. Candidate factors will be evaluated using neutralizing antibodies and RNAi. Finally, a panel of fibroblasts activated in vitro with recombinant TGF-b, recombinant IL-6, or conditioned media from various sources, including MDSCs and esophageal tumor cell lines will be co-injected with esophageal tumor cells into nude mice to determine whether the source of fibroblast activation influences the capacity to promote tumorigenesis. The second aim of this study will be to investigate the requirement of MDSCs initiation and progression of esophageal tumors. This aim will be undertaken using Histidine Decarboxylase-null (HDC-/-) mice, which exhibit elevated MDSC activity, resulting in increased tumorigenicity when exposed to carcinogens. L2-Cre;p120flox/flox mice serve as a model of esophageal cancer. HDC-/- mice will be used to generate HDC-/-;L2-Cre;p120flox/flox mice to study the effect of enhanced MDSC activity on the tumor phenotype in this mouse model of esophageal cancer. In a complementary approach, we will generate transgenic mice expressing HDC under the control of the CD11b promoter (CD11b-HDC). MDSCs in these mice will maintain expression of HDC in the tumor microenvironment, promoting differentiation and preventing the accumulation of the immune suppressive cells in the tumor stroma. Transgenic HDC mice will be used to generate CD11b-HDC;L2-Cre;p120flox/flox mice to determine the impact of MDSC inhibition on esophageal cancer initiation and progression with particular attention paid to the effect on activated fibroblasts in the TME. Esophagi, forestomachs, and oral tissues containing tumors will be fixed in buffered formalin, and histological analyses performed. Furthermore, IHC for a-SMA and Fsp-1 will be employed to determine the activation status of cancer-associated fibroblasts (CAFs). In aggregate, fulfillment of the aims described above will provide insight into mechanisms of activation of CAFs by MDSCs, and determine whether MDSCs are required for intiation and progression of esophageal cancer. PUBLIC HEALTH RELEVANCE: This year over 16,000 Americans will be diagnosed with esophageal cancer, a disease that carries a very poor prognosis. Our research involves the study of ways in which non-tumor cells aid the development and progression of esophageal cancer with the goal of providing new therapeutic targets.

描述（由申请人提供）：该建议的目的是研究肿瘤微环境（TME）中非肿瘤细胞促进食管癌的发育和进展的机制。该建议将检验以下假设：GR1+ CD11b+髓样衍生的抑制细胞（MDSC）在通过激活基质成纤维细胞激活促进食管癌中起着至关重要的作用。先前的工作证明了MDSC激活成纤维细胞的能力。该提案的第一个目的是阐明肿瘤条件的MDSC激活成纤维细胞并表征该活性在肿瘤进展中的作用的机制。共培养实验将用于确定激活成纤维细胞的能力是所有MDSC的特征还是由肿瘤衍生因子调节的MDSC的独特特性。静止成纤维细胞的激活将使用免疫组织化学（IHC）确定为A-SMA和FSP-1，这是成纤维细胞激活的标记。来自MDSC纤维细胞共培养的条件培养基的细胞因子阵列分析将允许研究MDSC介导的成纤维细胞激活的机理。将使用中和抗体和RNAi评估候选因素。最后，由重组TGF-B，重组IL-6或来自各种来源的条件培养基在体外激活的成纤维细胞，包括MDSC和食管食管肿瘤细胞系，将食管肿瘤细胞共同注入裸小鼠中，以确定能力促进fibroblast Activation促进Tamorigensease的能力。这项研究的第二个目的是研究食管肿瘤的MDSC启动和进展的需求。这种目标将使用组氨酸脱羧酶无（HDC - / - ）小鼠进行，后者表现出升高的MDSC活性，从而导致暴露于致癌物时的肿瘤性增加。 L2-CRE; P120Flox/Flox小鼠是食管癌的模型。 HDC - / - 小鼠将用于产生HDC - / - ; L2-CRE; P120Flox/Flox小鼠在这种小鼠食管癌模型中研究增强MDSC活性对肿瘤表型的影响。在互补方法中，我们将在CD11b启动子（CD11b-HDC）控制下生成表达HDC的转基因小鼠。这些小鼠中的MDSC将在肿瘤微环境中保持HDC的表达，从而促进分化并防止免疫抑制细胞在肿瘤基质中的积累。转基因HDC小鼠将用于产生CD11b-HDC; L2-CRE; P120FLOX/FLOX小鼠，以确定MDSC抑制对食管癌开始和进展的影响，并特别注意TME中对激活的成纤维细胞的影响。食管，Forestomachs和含有肿瘤的口腔组织将用缓冲福尔马林进行固定，并进行组织学分析。此外，将采用用于A-SMA和FSP-1的IHC来确定与癌症相关的成纤维细胞（CAF）的激活状态。总体而言，上述目的的实现将提供对MDSC激活CAF的机制，并确定是否需要MDSC来进行食管癌的直觉和进展。 公共卫生相关性：今年将有16,000多名美国人被诊断出患有食道癌，这种疾病的预后较差。我们的研究涉及对非肿瘤细胞有助于食管癌发展和进展的方式的研究，目的是提供新的治疗靶标。