A novel preclinical model of spontaneous metastasis

一种新型的自发转移临床前模型

基本信息

批准号：
9153804
负责人：
GLENN T. MERLINO
金额：
$ 63.89万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9153804
关键词：
Adjuvant Chemotherapy Adjuvant Therapy Angiogenesis Inhibitors Animal Model Anterior Pituitary Gland Antineoplastic Agents BRAF gene Biological Markers Bioluminescence C57BL/6 Mouse CCR Cancer Model Cell Culture Techniques Cell Cycle Cells Cessation of life Cisplatin Clinic Clinical Collaborations Cutaneous Melanoma Data Development Disease Disease Progression Distant Drug Monitoring Drug Targeting Drug resistance Excision Exhibits Family suidae Fluorescence Genetically Engineered Mouse Goals Growth Head Housing Human Cell Line Immune Immune system Immunocompetent Immunocompromised Host Immunotherapeutic agent Immunotherapy Kinetics Label Laboratories Lesion Lewis Lung Carcinoma Luciferases Lung Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of lung Manuscripts Melanoma Cell Memorial Sloan-Kettering Cancer Center Metastatic Lesion Metastatic Melanoma Modeling Molecular Monitor Mus Neoplasm Metastasis Non-Small-Cell Lung Carcinoma Operative Surgical Procedures Organ Paclitaxel Patients Pharmaceutical Preparations Phosphotransferases Pre-Clinical Model Primary Neoplasm Process Proteins Publishing Rattus Reaction Time Reagent Recurrence Recurrent disease Reporter Reporting Research Personnel Resected Resistance Resources Science Signal Transduction Site Somatotropin Staging Subfamily lentivirinae System Technology Testing Therapeutic Time Tissues Transgenic Mice Transplantation Treatment Efficacy Work Writing Xenobiotics antibody inhibitor base chemotherapeutic agent chemotherapy clinically relevant clinically significant design fusion gene host neoplasm interaction improved in vivo inhibitor/antagonist insight meetings melanoma mortality mouse model mutant neoplastic cell new therapeutic target novel novel strategies pre-clinical pre-clinical research preclinical study prevent programs promoter protein expression response small molecule subcutaneous success targeted treatment tool tumor tumor microenvironment tumor progression

项目摘要

The major goal of this project is to develop and exploit a tractable, preclinical mouse models of metastatic melanoma that recapitulate the clinical progression of surgical removal of primary tumors, treatment with adjuvant therapy and clinical recurrence at distant sites. We see a translational opportunity to devise a novel strategy to prevent the clinical recurrence of metastatic disease. The models will be designed to incorporate normal immune system function to maintain an appropriate tumor microenvironment and provide superior tumor-host interactions, an approach that is significantly more likely to yield biologically and clinically relevant data. Since metastasis often targets internal organs and the timing of its recurrence may vary greatly, the preclinical model will allow for the non-invasive, long-term monitoring of disease progression within the immunocompetent mouse. To meet these criteria, we originally developed as a proof-of-concept a syngeneic immunocompetent mouse model of Lewis Lung Carcinoma (LLC) using tissue that was never adapted to cell culture and labeled using a high-titer lentivirus encoding a luciferase/GFP fusion reporter protein, developed by this laboratory in collaboration with Dom Esposito at the Protein Expression Laboratory, Advanced Technology Program (Day et al., Pig Cell Melanoma Res 22:283-295, 2009). Our decision to employ the NSCLC mouse LLC model was well aligned with the new NCI Lung Cancer Program, and provided a robust, well-characterized system to validate our preclinical approach. LLC exhibits highly favorable growth kinetics and virtually 100% take rate of lung metastases in C57BL/6 mice, and was instrumental in the successful development of early conventional chemotherapies that are still used in the clinical today. For this preclinical model we employed an in vivo-maintained LLC stock that had never been adapted to cell culture, an approach that had been reported to yield more reliable preclinical data. In full collaboration with Dr. Melinda Hollingshead (DTP, NCI), we have established a stably labeled bank of highly metastatic LLC tissue (LLC-Luc/GFP) through multiple rounds of subcutaneous transplantation, resection, and selection of Luc/GFP+ metastatic clones in syngeneic C57BL/6 mice. Our preclinical approach was to subcutaneously inoculate Luc/GFP-labeled LLC cells into albino C57BL/6 mice and resect at a selected tumor size, at which time drug treatment was initiated. Recurrence of clinically significant LLC macrometastases was evident in the lungs as Luc/GFP+ lesions within a week, and entire drug studies were completed in a month. The BL signal allowed the monitoring of disease after resection of the primary tumor, the progression of metastatic growth over time, and the response of the tumor to therapy. We have found that this resection/recurrence metastasis model fulfilled multiple requirements for a tractable and robust syngeneic preclinical model of advanced stage disease, one that we anticipate may more accurately predict therapeutic response. We treated mice in a setting akin to post-surgical first-line adjuvant chemotherapy using cisplatin, paclitaxel and/or antiangiogenic agents. As in the clinic these drugs were found to be most effective against progression when used in combination. However, the response of metastases to agents could not be predicted from, and often opposed, their effects on subcutaneous tumors. Moreover, time to macrometastasis onset, rather than growth, correlated with both mouse survival and treatment efficacy. This work has been published (Day et al., Int. J. Cancer 130:190-9, 2011). We have noted that even low expression of the xenobiotic reporters GFP and/or luciferase can render growth of labeled metastatic tumors unpredictable in syngeneic mice and severely limit the utility of any immunocompetent preclinical model. We have therefore developed transgenic mice in both FVB/N and C57BL/6 backgrounds that express the Luc/GFP fusion gene at an irrelevant site (in this case the anterior pituitary through use of a rat growth hormone promoter) (Day et al., PLoS One. 2014 Nov 4;9(11):e109956). These "glowing head" mice are thus pre-tolerized to both foreign markers, and we note that clearly labeled metastatic lesions arise much more regularly, consistently, and with a stronger signal in the lungs of inoculated, resected host mice. These mice are currently being employed in our ever improving preclinical models (Day et al., manuscript submitted). The glowing head mice have already been sent to multiple investigators and are now being distributed by Jackson Laboratories. The primary goal now is to combine all the tools and reagents we have developed over the last few years to study the mechanisms by which BRAF mutant, NRAS mutant, and wildtype BRAF/NRAS mutant melanomas recur at metastatic sites when patients fail targeted and immunotherapeutic drug treatment. This extensive preclinical effort is being performed in full collaboration with Dr. Terry Van Dyke and CAPR, and in collaboration with Drs. Nick Restifo (CCR), Marcus Bosenberg (Yale), Martin McMahon (UCSF), Jedd Wolchok (MSKCC) and Jennifer Wargo (MD Anderson). We have already developed a wildtype BRAF/NRAS metastatic melanoma model using our UV-initiated HGF transgenic mouse. BRAF mutant tumors have been generated and are being characterized, and BRAF mutant melanomas are under construction. Preclinical trials with immunotherapy (e.g., anti-CTLA-4, anti-PD-1, anti-PD-L1) and targeted therapy (e.g., BRAFi, MEKi, METi) are already underway and providing novel insights into why some melanomas but not others respond to immune checkpoint inhibitors. We have written a PRIMER for Cell that reviews optimal use of preclinical cancer models (Day, Merlino, Van Dyke, CELL, in press).

该项目的主要目的是开发和利用一种可进行的临床前小鼠模型的转移性黑色素瘤模型，该模型概括了原发性肿瘤外科手术去除的临床进展，辅助治疗和遥远部位的临床复发。我们看到了一个翻译的机会，可以制定一种新的策略，以防止转移性疾病的临床复发。这些模型将旨在纳入正常的免疫系统功能，以保持适当的肿瘤微环境并提供优质的肿瘤宿主相互作用，这种方法更有可能在生物学和临床上相关的数据产生。由于转移通常靶向内部器官，并且其复发的时间可能有很大变化，因此临床前模型将允许对免疫能力小鼠内疾病进展的无创，长期监测。为了符合这些标准，我们最初是作为概念证明的刘易斯肺癌（LLC）的合成性免疫能力的小鼠模型（LLC），它使用从未适应细胞培养的组织，并使用高介质的慢病毒标记，并使用编码Locifife蛋白酶/GFP融合蛋白质的蛋白质的高素质慢病毒，该蛋白质与DOUMETER ESTORITON与DOUMENTORATION一起，与DOMSSSERATION相关表演，以与DOUMENTORIEN一起，与DOMS的协作相结合，该方案是协作的。（Day等人，猪细胞黑色素瘤Res 22：283-295，2009）。我们采用NSCLC小鼠LLC模型的决定与新的NCI肺癌计划很好地对准，并提供了一个健壮，表征良好的系统来验证我们的临床前方法。 LLC在C57BL/6小鼠中表现出高度有利的生长动力学和几乎100％的肺转移率，并且在当今仍在临床中仍然使用的早期常规化学疗法的成功开发中发挥了作用。对于这种临床前模型，我们采用了从未适应细胞培养的体内维护良好的LLC股票，这种方法据报道产生更可靠的临床前数据。在与Melinda Hollingshead博士（DTP，NCI）的完全合作中，我们通过皮下移植，切除，LUC/GFP+转移性Clones的选择，通过多轮移植，切除和选择合成C57Bl/6 mices来建立了稳定的高度转移性LLC组织（LLC-LUC/GFP）库。我们的临床前方法是皮下接种LUC/GFP标记的LLC细胞中的白化病C57BL/6小鼠，并以选定的肿瘤大小恢复，此时开始药物治疗。在肺部的LUC/GFP+病变中，临床上显着的LLC宏观变体的复发是明显的，整个药物研究在一个月内完成。 BL信号允许切除原发性肿瘤后监测疾病，随着时间的流逝，转移性生长的进展以及肿瘤对治疗的反应。我们发现，这种切除/复发转移模型满足了晚期疾病的可触犯和稳健的临床前临床前模型的多个要求，我们预计这种疾病可能会更准确地预测治疗反应。我们在使用顺铂，紫杉醇和/或抗血管生成剂的情况下治疗了类似于手术后一线佐剂化学疗法的小鼠。与诊所一样，发现这些药物在组合使用时最有效地抵抗进展。但是，转移对剂的反应不能从它们对皮下肿瘤的影响中预测，并且通常反对。此外，大量开始出现而不是生长的时间与小鼠的存活和治疗效果相关。这项工作已经发表（Day等，Int。J.Cancer 130：190-9，2011）。我们已经注意到，即使是异种生物记者GFP和/或荧光素酶的低表达也可以使标记的转移性肿瘤在合成小鼠中无法预测，并严重限制任何免疫能力的临床前模型的实用性。因此，我们已经在FVB/N和C57BL/6背景中开发了转基因小鼠，这些背景在无关的位点表达Luc/GFP融合基因（在这种情况下，通过使用大鼠生长激素启动子）（Day等人，Plos One，2014年11月4日； 9（11）：9（11）：E1099556）。因此，这些“发光的头”小鼠被预先耐受到两个异物标记物，我们注意到，在接种，切除的宿主小鼠的肺中，明显更定期，一致地出现了更定期，始终如一的信号，并具有更强的信号。这些小鼠目前正在我们不断改进的临床前模型中使用（Day等人，提交的手稿）。发光的头鼠已经发送给多个调查人员，现在由杰克逊实验室分发。现在的主要目的是将过去几年中我们开发的所有工具和试剂结合起来，以研究BRAF突变体，NRAS突变体和WildType BRAF/NRAS突变突变型黑色素瘤在转移性部位复发的机制，而患者的靶向和免疫治疗药物治疗失败和免疫治疗。这项广泛的临床前努力正在与Terry Van Dyke和CAPR博士完全合作，并与Drs合作。 Nick Restifo（CCR），Marcus Bosenberg（Yale），Martin McMahon（UCSF），Jedd Wolchok（MSKCC）和Jennifer Wargo（MD Anderson）。我们已经使用UV引起的HGF转基因小鼠开发了Wildtype BRAF/NRAS转移性黑色素瘤模型。 BRAF突变肿瘤已经产生并正在表征，BRAF突变型黑色素瘤正在建设中。免疫疗法（例如抗CTLA-4，抗PD-1，抗PD-L1）和靶向疗法（例如Brafi，Meki，Meti）进行免疫疗法（例如，抗CTLA-4，抗PD-1，抗L1）的临床前试验，并提供了一些新颖的见解，并提供了为什么有些黑色素瘤但没有其他对免疫检查点抑制剂的反应。我们为细胞写了一份底漆，该底漆回顾了临床前癌模型的最佳使用（Day，Merlino，Van Dyke，Cell，Press）。