间变性甲状腺癌的自发性鼠模型
Summary
在这里，我们提出了一种通过自发基因工程小鼠模型获得鼠ATC肿瘤的标准管道。此外，我们呈现有关原发性和转移性病变的肿瘤动力学和病理信息。该模型将帮助研究人员了解肿瘤发生并促进药物发现。
Abstract
间变性甲状腺癌（ATC）是一种罕见但致命的恶性肿瘤，预后令人沮丧。迫切需要对ATC的致癌作用和发展以及治疗方法进行更深入的研究，因为ATC患者的标准治疗基本上已经耗尽。然而，低患病率阻碍了彻底的临床研究和组织样本的收集，因此在创造有效治疗方法方面进展甚微。我们使用基因工程在C57BL / 6背景中创建条件诱导的ATC小鼠模型（mATC）。ATC小鼠模型通过TPO-cre/ERT2进行基因分型;布拉夫CA/wt;Trp53ex2-10 / ex2-10并通过腹腔注射他莫昔芬诱导。使用小鼠模型，我们研究了肿瘤动力学（诱导4个月后肿瘤大小范围为12.4mm 2至32.5mm2），生存（中位生存期为130天）和转移（91.6%的小鼠发生肺转移）曲线和病理特征（以Cd8，Foxp3，F4 / 80，Cd206，Ki67和Caspase-3免疫组织化学染色为特征）。结果表明，自发性mATC具有与人ATC肿瘤高度相似的肿瘤动力学和免疫微环境。综上所述，mATC模型具有较高的病理生理特征相似性和统一的基因型，在一定程度上解决了临床ATC组织和样本异质性的不足。因此，它将有助于ATC的机制和转化研究，并为研究ATC的小分子药物和免疫治疗剂的治疗潜力提供途径。
Introduction
甲状腺癌是最常见的内分泌恶性肿瘤之一1，起源于甲状腺上皮。近年来，全球甲状腺癌的发病率迅速上升2。甲状腺癌根据肿瘤细胞分化程度可分为不同的类型。根据临床行为和组织学，甲状腺癌分为高分化癌，包括甲状腺状癌（PTC）和滤泡性甲状腺癌（FTC）、低分化癌（PDTC）和未分化或间变性甲状腺癌（ATC）3。PTC是一种行为温和、预后较好的常见类型4，而PTC是一种罕见且高度侵袭性的恶性肿瘤，占所有甲状腺肿瘤的2%至3%5。虽然ATC很少见，但它导致大约50%的甲状腺癌相关死亡，生存率低（6-8个月）6,7。超过 50% 的 ATC 病例被诊断为肺转移8.除了ATC的侵袭性外，临床上还开发了有限的有效治疗方法。因此，ATC患者的预后为9,10,11。这表明迫切需要对ATC发展和治疗的分子机制进行进一步深入研究。
ATC的肿瘤发生是一个动态的去分化过程。临床研究每个阶段收集人类肿瘤样本的困难阻碍了对从高分化到未分化癌的发展机制的理解。相比之下，小鼠ATC模型（mATC）的使用有利于在整个肿瘤发生过程中收集mATC样本。因此，通过分析动态去分化过程，我们可以更好地了解肿瘤形成的机制。此外，临床ATC样本的异质性也导致了分子机制理解的困难。然而，小鼠具有相同的遗传背景，并保持在相似的生活环境中，确保每个肿瘤的一致性。这有助于探索ATC发展的普遍作用12,13,14。此外，mATC是一种原位肿瘤模型，可以恢复解剖位置和组织特异性微环境的影响。因此，与常用的免疫缺陷小鼠相比，mATC是一种自发的小鼠模型，具有完整的免疫系统和免疫微环境。
因此，我们用C57BL / 6菌株构建了条件诱导的mATC，这是一种能够再现去分化甲状腺癌病理特征的小鼠模型。基于该模型，本文简要概述了mATC的分子基础、构建思路、病理特征和应用。此外，我们观察并报告了mATC的肿瘤生长，存活时间，转移和病理特征。我们相信这将是一个信息概述，以帮助其他研究人员更轻松地使用该模型。
我们构建了一个条件诱导mATC模型，如McFadden15首次报道的那样;最初，我们构建了小鼠：TPO-cre / ERT2，Braf flox / wt和Trp53flox / wt。 具体而言，TPO-cre / ERT2小鼠包括人甲状腺过氧化物酶（TPO）启动子（甲状腺特异性启动子），驱动cre-ERT2融合基因（融合到人雌激素受体配体结合域的cre重组酶）的表达。Cre-ERT2通常局限于细胞质中，只有在暴露于他莫昔芬时才进入细胞核，他莫昔芬诱导cre发挥重组酶活性。当小鼠与携带loxP侧翼序列的小鼠杂交时，他莫昔芬诱导后，cre介导的重组删除甲状腺细胞中的絮状序列，以达到敲除或敲除特定基因的目的。
此外，Brafflox/wt小鼠是基于cre-loxP系统的人类Braf的敲入等位基因。Brafflox/wt 鼠转录本由内源性外显子 1-14 和 loxP 侧翼人外显子 15-18 编码。在Cre介导的絮状区域切除后，突变外显子15（用与人类癌症中组成活性Braf V600E连接的V600E氨基酸取代物修饰）和内源性外显子16-18用于生成转录本。此外，Trp53 flox/wt小鼠是人类Trp53的敲除等位基因，并且在Trp53的外显子2-10侧翼具有loxP位点。当与具有cre重组酶的小鼠杂交时，cre介导的重组删除絮状序列以敲除Trp53。然后，将TPO-cre/ERT2、Braf flox/w和Trp53flox/wt小鼠杂交以获得TB（TPO-cre/ERT2;布拉夫弗洛克斯/重量）小鼠和TBP（TPO-cre/ERT2;布拉夫絮絮/重量;Trp53flox/wt）小鼠，可用于产生PTC和ATC。大约8周后，通过腹膜内（ip）施用溶解在玉米油中的150mg / kg他莫昔芬诱导小鼠两次给药。肿瘤生长可以通过高频超声检查监测（超声检查的第一个时间点记录在第0天）。引入他莫昔芬后 40 天进行初始超声检查。
Protocol
Representative Results
Discussion
甲状腺肿瘤清扫方案中的关键步骤
在解剖过程中，需要正确了解甲状腺的解剖位置。甲状腺是位于下颌下腺背侧靠近甲状腺软骨和气管的蝴蝶状腺体。在手术过程中，小心翼翼地避免切断颈部两侧的血动脉。
mATC品种的修改和故障排除
ATC是一种罕见且高度侵袭性的恶性肿瘤。mATC和ATC患者的临床特征有一定的相似之处。具体而言，mATC死亡的主要?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家重点研究发展计划（2021YFA1301203）的支持;国家自然科学基金（82103031,82103918,81973408,82272933）;四川大学华西医院临床研究孵化项目（22HXFH019）;成都市科技局国际合作项目（2020-GH02-00017-HZ）;四川省自然科学基金， 2022NSFSC1314;“四川大学华西医院优秀学科1.3.5项目”（ZYJC18035、ZYJC18025、ZYYC20003、ZYJC18003）;四川省科技计划（2023YFS0098）。
Materials
| 100x Citrate antigen retrieval solution (PH 6.0) | MXB | Cat# MVS-0101 | |
| 50x EDTA antigen retrieval solution(pH 9.5) | ZSGB-GIO | Cat# ZLI-9071 | |
| Brafflox/wt mice | Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China | ||
| Caspase-3 | Beyotime | Cat# AC033 | |
| CD8 | Cell Signaling Technology | Cat# 98941; RRID:AB_2756376 | |
| CD206 | Cell Signaling Technology | Cat# 24595; RRID:AB_2892682 | |
| Chamber for anesthesia induction | RWDlifescience | ||
| Enhanced DAB chromogenic kit | MXB | Cat# DAB-2031 | |
| Eosin staining solution | ZSGB-GIO | Cat# ZLI-9613 | |
| F4/80 | Abcam | Cat# 100790; RRID:AB_10675322 | |
| Foxp3 | Cell Signaling Technology | Cat# 12653; RRID:AB_2797979 | |
| Fully enclosed tissue dehydrator | Leica Biosystems | ASP300S | |
| Hematoxylin staining solution | ZSGB-GIO | Cat# ZLI-9610 | |
| HistoCore Arcadia fully automatic tissue embedding machine | Leica Biosystems | ||
| Ki67 | Beyotime | Cat# AF1738 | |
| Rotating Slicer | RWDlifescience | Minux S700 | |
| SPlink detection kits (Biotin-Streptavidin HRP Detection Systems) | ZSGB-GIO | Cat# SP-9001 | |
| TPO-cre/ERT2 mice | Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China | ||
| Trp53flox/wt mice | Collaboration with Institute of Life Science, eBond Pharmaceutical Technology Ltd, Chengdu, China | ||
| Ultrasonic cell crusher | Ningbo Xinyi Ultrasound Equipment Co., Ltd | JY92-IIN | |
| Ultrasound gel | Keppler | KL-250 | |
| Ultrasound system | VisualSonics | Vevo 3100 |
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