浸润性膀胱移行细胞癌中的完整的人MUC1转基因小鼠免疫诱导免疫治疗发展模型
Summary
的N-丁基-N-（4 – 羟丁基）亚硝胺的人粘蛋白1（MUC1）测试的目的MUC1基因定向免疫治疗的转基因小鼠中诱导的膀胱癌模型的开发。施用MUC1的目标肽疫苗后，MUC1的细胞毒性T淋巴细胞反应证实，通过测定血清中的细胞因子水平和T细胞的比活性。
Abstract
浸润性膀胱癌的临床前模型开发的人粘蛋白1（MUC1）转基因小鼠（MUC1.Tg）的评估免疫治疗和/或细胞毒性化疗的目的。为了诱发膀胱癌，C57BL / 6小鼠（MUC1.Tg和野生型）与致癌物质N-丁基-N-（4 – 羟丁基）亚硝胺（OH-BBN）口服3.0毫克/天，5天/周12周。 OH-BBN血清细胞因子在肿瘤发展的影响进行评估，采集全血，通过颌下出血治疗前，每四个星期。另外，MUC1的定位的肽疫苗和安慰剂给药组小鼠每周八周。多重荧光微珠immunoanalyses的血清细胞因子在肿瘤的发展和疫苗接种后进行。终止时，干扰素γ（IFN-γ）/白细胞介素4（IL-4）的酶联免疫斑点法分析MUC1基因特异性T细胞免疫反应和肿瘤类型的病理组织学评价和档次进行。结果表明：（1）在既MUC1.Tg和野生型小鼠的膀胱癌的发病率是67％，（2）以2:1的比例在开发移行细胞癌（TCC）相比，鳞状细胞癌（SCC） （3）炎性细胞因子随时间而增加肿瘤的发展过程中，及（4）给药的肽疫苗诱导的Th1偏振光血清细胞因子的档案中和MUC1的特定的T细胞应答。所有MUC1.Tg小鼠的肿瘤MUC1表达阳性，并侵入所有肿瘤在MUC1.Tg和野生型小鼠的一半。总之，用团队的方式通过协调药理学家，免疫学家，病理学家和分子生物学家的努力，我们已经开发了一个完整的转基因小鼠模型免疫膀胱癌表达hMUC1。
Introduction
膀胱癌是最常见的一种癌症第四和第八，在美国男性癌症死亡的首要原因。在美国，膀胱癌新发病例估计有72,500和15,000人死亡，预计在2013年1相结合的男性和女性。膀胱癌的发病率是3倍左右高的妇女相比，在人。在美国超过90％的情况下，移行细胞癌（TCC）帐户，而鳞状细胞癌（SCC）的发生率小于2％2。乳头状台泥整体相对5年生存率为91.5％相比，只有30.9％为鳞状细胞癌2。虽然非侵入性乳 头状的部队派遣国占约75％的病例在诊断时，即使处理超过50％的患者会经历复发5年之内，多达30％的患者进展到肌层浸润性疾病3,4 。典型的治疗方案非肌肉INV意味，疾病，包括经尿道前列腺切除术（TUR），然后通过膀胱内灌注化疗。高档Ta或T1肿瘤患者，可能会重复TUR 3,4化疗前。 TUR对于低同类钽复发的或高档的Ta或T1病变的患者，其次是辅助化疗或免疫治疗中的形式，卡介苗（BCG），可以使用3,4。膀胱内BCG已被证明优于膀胱内丝裂霉素C相对于时间复发5。对于T2肌层浸润性疾病，根治性膀胱切除术，化疗或不推荐的过程中，治疗3。鳞状细胞癌的患者，根治性膀胱切除似乎是最有效的治疗6。给予了很高的复发率，尽管最好的治疗，显然是有一个新的，更有效的治疗膀胱癌的需求。
拓展新的免疫疗法bladdER癌是一种可能的方法，可能有希望延长无病生存。从历史上看，BCG一直是唯一有效的免疫治疗膀胱癌。其作用机制被认为涉及的非特异性的T辅助细胞1（Th1细胞）型免疫应答的诱导，通过增加白细胞介素-2（IL-2）和干扰素-γ（IFN-γ）4。蜂窝或Th1免疫，体液，或Th2癌症免疫疗法中是至关重要的，免疫从来没有被证明是有效的抗实体肿瘤，对生长因子受体7抗体的异常。在试图改善后的好处，单用卡介苗，IFN-α2B/BCG组合的免疫进行了评估，第二阶段的临床试验与不确定的结果8。膀胱癌的免疫治疗的另一种方法可能是针对肿瘤相关抗原（塔斯），鉴定，其中的癌症免疫疗法。具体7 </s高达>。
一个这样的TAA是粘蛋白1（MUC1的），它是一种细胞表面糖蛋白的过度表达在许多上皮细胞癌，膀胱癌，乳腺癌，肺癌，胰腺癌9,10。癌变过程中MUC1的表达和修改也被大大地改变，使得underglycosylation公开的氨基酸被称为可变数目的串联重复序列（VNTR）上的肽核心抗原序列。 MUC1的是一个自分子，这些免疫VNTR区域通常不暴露，由于广泛的糖基化作用，因此它们被视为由免疫系统的外国11,12。已分离出的肿瘤引流淋巴结的乳腺癌患者13，以及14,15骨髓瘤患者的血液和骨髓，MUC1的一个潜在的目标为细胞毒性T淋巴细胞（CTL）的特异性识别MUC1的抗原决定簇的细胞免疫应答。本的underglycosylate免疫优势的VNTRCTL识别MUC1的D型，从而导致肿瘤细胞的破坏16-19。癌MUC1的原住民蜂窝网络和/或体液免疫反应，但是，没有强大到足以消除肿瘤。为了加强已经存在的免疫反应弱MUC1，合成免疫肽可以通过接种疫苗，以产生足够强大的CTL反应，临床受益18,20。 ÂMUC1脂质体疫苗已经表明，提高肺癌患者的生存21,22，产生的CTL能杀死MUC1阳性肿瘤细胞，并产生Th1细胞极化的细胞因子反应23,24。 MUC1表达高水平的9,11,25，膀胱癌是检测MUC1定向免疫26,27的一个合乎逻辑的候选。此外，MUC1有潜力的舞台和档次明显伴有膀胱癌28，MUC1表达台泥作为预后因子，转移性TCC已被证明，继续表达MUC1的29。
为了评估MUC1定向免疫治疗膀胱癌的潜在效用，我们开发了免疫完好的人MUC1（hMUC1）表达的转基因小鼠模型（MUC1.Tg）膀胱癌同源C57BL / 6的背景30。人MUC1表示为一个自它自己的启动子控制下的蛋白质，从而在组织中的表达模式与观察人类30,31一致。在诱导小鼠膀胱已知的致癌物质N-丁基-N-（4 -羟丁基）亚硝胺（OH-BBN）32，然后所产生的肿瘤进行了评价的hMUC1的表达与肿瘤的类型和等级。 Th1/Th2型细胞因子水平的肿瘤的发生发展过程中的致癌物质的效果进行评估，定期收集血清样品进行多重分析。然后将小鼠治疗与MUC1的定位的肽疫苗，血清中的细胞因子和免疫反应的评价方法的特德多重荧光微珠免疫分析法和酶联免疫斑点。
Protocol
Representative Results
Discussion
浸润性移行膀胱癌和鳞状细胞在人类MUC1.Tg小鼠成功诱导免疫发展提供了临床前模型。免疫治疗的研究需要使用一个自发的，免疫的完整的模型，以评估随着时间的推移，以及免疫治疗的免疫反应的肿瘤进展的炎症反应。在自发肿瘤的发展模式，在肿瘤微环境保持完好，肿瘤发展一个更具代表性的增长率，允许的抗肿瘤治疗的效果进行评估。此外，免疫系统可以测量和监测通过的生物标志物，可以…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者想感谢加州大学戴维斯分校的鼠标老鼠繁殖生物学计划。这项研究是从默克公司，德国达姆施塔特的赠款支持。
Materials
| Reagent | |||
| N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) | TCI America | B0938 | |
| 20 G Gavage Needles | Popper & Sons, Inc. | 7921 | Stainless steel |
| Peptide Vaccine | N/A | N/A | investigational agent |
| BD Microtainers | BD | 365957 | |
| Tissue Cassettes | Simport | M490-12 | |
| 10% Neutral Buffered Formalin | Fisher Scientific | SF100-4 | |
| Lysis Buffer | Pierce | 87787 | |
| Halt Protease & Phosphatase inhibitor cocktail | Thermo Scientific | 78444 | |
| Pierce BCA Protein Assay Kit | Pierce | 23225 | |
| Mouse Cytokine 20plex Kit | Invitrogen | LMC006 | |
| Magnetic Microsphere Beads | Luminex | MC100xx-01 | xx is the bead region |
| Anti-mouse TNF- Capture Antibody | BD Pharmingen | 551225 | |
| Anti-mouse TNF- Detection Antibody | BD Pharmingen | 554415 | |
| Anti-mouse IFN- Capture Antibody | Abcam | ab10742 | |
| Anti-mouse IFN- Detection Antibody | Abcam | ab83136 | |
| PBS, pH 7.4 | Sigma | P3813-10PAK | |
| Tween-20 | Fisher | BP337-500 | |
| Assay Buffer | Millipore | L-MAB | |
| Cytokine Standard | Millipore | MXM8070 | |
| Multi-screen HTS 96well filter plates | Millipore | MSBVN1210 | |
| SA-PE | Invitrogen | SA10044 | |
| 100 m Nylon Tissue Sieves | BD | 352360 | |
| Splenocyte Separation Media | Lonza | 17-829E | |
| TNF- /IL-4 ELISpot plates | R&D Systems | ELD5217 | |
| Rabbit Anti-MUC1 monoclonal antibody | Epitomics | 2900-1 | |
| Goat Anti-actin monoclonal antibody | Sigma | A1978 | |
| Anti-rabbit HRP antibody | Promega | W401B | |
| Goat anti-mouse HRP antibody | Santa Cruz Biotechnology, Inc. | SC-2005 | |
| PVDF membrane | BioRad | 162-0174 | |
| Mini Protean TGX Precast Gels | BioRad | 456-1083 | |
| Muse Count & Viability Kit | Millipore | MCH100104 | |
| MUC1 Antibody | BD Pharmingen | 550486 | IHC antibody |
| Animal Research Peroxidase Kit | Dako | K3954 | IHC staining |
| [header] | |||
| Equipment and Software | |||
| Millipore plate vaccum apparatus | Millipore | MSVMHTS00 | |
| Luminex Lx200 | Millipore / Luminex | 40-013 | Manufactured by Luminex, distributed by Millipore |
| Luminex Xponent Software | Millipore / Luminex | N/A | Version 3.1; included with Luminex Lx200 |
| Milliple Analyst Software | Milliplex / VigeneTech | 40-086 | Version 5.1 |
| Muse Cell Analyzer | Millipore | 0500-3115 | |
| Muse Software | Millipore | N/A | Version 1.1.0.0; included with Analyzer |
| Dissecting Microscope | Unitron | Z730 | |
| Graphpad Prism Software | Graphpad Software Inc. | N/A | Version 5.1 |
| Mini Protean Tetra Cell Gel apparatus | BioRad | 165-8001 | |
| Trans Blot SD Cell and PowerPac | BioRad | 170-3849 |
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