Transglutaminase Modulated Tumor-Stroma Interaction in Pancreatic Cancer

描述（由申请人提供）：癌细胞和胰腺基质之间的相互作用与胰腺癌进展以及对化疗和放疗的耐药性有关。基于我们的初步结果，证明了胰腺肿瘤微环境中显著的组织转氨酶（TG 2）表达和活性，我们假设胰腺癌细胞分泌的TG 2调节基质，促进癌细胞存活，并损害化疗递送。我们提出通过三个具体目标来检验该假设：目标1：测量胰腺基质细胞响应于胰腺癌细胞分泌的TG 2的增殖和活化。 目的2：证明TG 2敲低或其酶抑制调节胰腺肿瘤中的基质组成。 目的3：证明TG 2敲低或其酶抑制增加吉西他滨递送到胰腺环境中。目的1通过共培养实验，检测分泌型TG 2对成纤维细胞和内皮细胞增殖、存活和活化的影响。通过使用多重ELISA，在基质细胞和癌细胞调节的培养基中测量成纤维细胞分泌的细胞因子和生长因子。目的2和3将使用由胰腺癌细胞产生的原位胰腺异种移植物，其中通过使用靶向TG 2的sh-RNA或反义构建体的稳定转染来敲除TG 2，或者通过使用特异性酶抑制剂KCC 009来抑制TG 2。实验的终点是测量和比较：纤维化（胶原蛋白和三色染色的免疫染色）、基质细胞（肌成纤维细胞和胰腺星状细胞）的数量和增殖、血管生成（微血管密度）、胰腺癌细胞的存活和增殖以及化疗进入胰腺组织的转移率。对于后一个终点，我们将通过使用HPLC质谱法测量源自表达正常或低TG 2水平的癌细胞的胰腺组织中吉西他滨的浓度。总之，成功证明组织转氨酶在胰腺基质调节中的作用将为进一步研究TG 2抑制作为抗癌策略或作为使胰腺肿瘤对化疗敏感的手段提供强有力的理论基础。这些研究与胰腺癌患者有直接的临床相关性。

DESCRIPTION (provided by applicant): The interaction between cancer cells and the pancreatic stroma has been implicated in pancreatic cancer progression and resistance to chemotherapy and radiation. Based on our preliminary results demonstrating prominent tissue transglutaminase (TG2) expression and activity in the pancreatic tumor millieu, we hypothesized that TG2 secreted by pancreatic cancer cells modulates stroma, promotes cancer cell survival, and impairs chemotherapy delivery. We propose to test the hypothesis by three specific aims: Aim 1: Measure proliferation and activation of pancreatic stromal cells in response to TG2 secreted by pancreatic cancer cells. Aim 2: Demonstrate that TG2 knockdown or its enzymatic inhibition regulate stroma composition in pancreatic tumors. Aim 3: Demonstrate that TG2 knockdown or its enzymatic inhibition increases gemcitabine delivery into the pancreatic milieu. Aim 1 will use co-culture experiments to measure the effects of secreted TG2 on proliferation, survival and activation of fibroblasts and endothelial cells. Cytokines and growth factors secreted by fibroblasts will be measured in media conditioned by stromal and cancer cells by using Multiplex ELISA. Aims 2 and 3 will use orthotopic pancreatic xenografts generated from pancreatic cancer cells in which TG2 was knocked down by using stable transfection of sh-RNA or antisense construct targeting TG2, or TG2 will be inhibited by using KCC009, a specific enzymatic inhibitor. Endpoints of the experiments are measurement and comparison of: fibrosis (immunostaining for collagen and trichrome stain), number and proliferation of stromal cells (myofibroblasts and pancreatic stellate cells), angiogenesis (microvessel density), survival and proliferation of pancreatic cancer cells, and penetrance of chemotherapy into pancreatic tissue. For the latter endpoint, we will measure the concentration of gemcitabine within pancreatic tissue derived from cancer cells expressing normal or low TG2 levels by using HPLC mass spectrometry. In summary, successful demonstration of the role of tissue transglutaminase in modulation of the pancreatic stroma will provide a strong rationale for investigating further TG2 inhibition as an anti-cancer strategy or as means to sensitize pancreatic tumors to chemotherapy. These studies have direct clinical relevance to pancreatic cancer patients.