Name: an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis
Uploaded: 2019-09-12T13:00:00
Description: Watch this Scientific Journal Video about An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis at JoVE.com

这项工作由NIH/NCI R01 CA164335-01A1（K.F.斯塔维利-奥卡罗尔，PI）和NIH/NCI R01CA208396（马克·凯斯特、李广富、凯文·斯塔维利-奥卡罗尔）支持。

注：所有程序，包括动物科目，都已获得密苏里大学IACUC的批准。所有小鼠均按照《实验室动物护理和使用指南》中概述的标准得到人道照顾。以下细胞分离和接种过程应在罩内执行。所有表演者应佩戴标准个人防护设备，以处理小鼠和组织。
1. 肝纤维化和肝硬化与四氯化碳IP注射 （CCl4）
注：参见图1。（CCl4是高度危险的试剂...

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通过该协议，我们建立了一个可靠和可重复的HCC模型，模仿人类HCC的启动和进展。临床上，许多危险因素相继诱发肝损伤、肝纤维化、肝硬化和HCC的最后阶段。在我们的协议中，CCl4的 IP 注射用于在野生型小鼠中首先产生肝纤维化，这允许随后的致癌肝细胞在肝纤维化设置中形成肿瘤。我们发现，肿瘤形成最成功地发生在CCl4治疗两周后接受肝细胞接种的小鼠身上，而在其他时间点...

肝细胞癌（HCC）是美国（美国）1、2、3类癌症中增加速度最快的类型。每年约有85万新病例被诊断为4，5和70万患者死于这种致命的疾病6，7，8，9，10，使其成为全球癌症相关死亡的第二高原因。HCC的管理包括手术切除，移植，消融，化疗，或全身疗法，如索拉菲尼布11。早期诊断和管理与手术切除或移植有最高的整体生存效益4。不幸的是，大多数患者在后期出现，需要管理与消融，化学代谢或索拉菲尼布12。Sorafenib，一种受体酪氨酸激酶抑制剂（RTKI），于2008年被美国食品和药物管理局批准为治疗不可分离的HCC的唯一系统药物治疗。虽然这种药物只提供了一个适度的增加整体存活率，从7.9个月到10.7个月13，它提供了一个新的治疗策略，可以用来管理HCC。
操纵免疫系统来消除已建立的癌症是癌症研究中一个迅速成长的领域。免疫检查点研究大大推进了癌症治疗中的免疫治疗药物开发15、16。FDA批准使用抗体（Abs）对抗细胞毒性T淋巴细胞抗原4（CTLA-4），程序化细胞死亡蛋白1（PD-1），及其配体PD-L1用于治疗黑色素瘤、肺癌、头颈部癌症和膀胱癌1718,19,20.使用一种或多种抗体治疗高级HCC的单一疗法或联合疗法的临床试验正在进行中，21、22、23和一些试验已显示出良好的结果。2017年，FDA批准加速批准抗PD-1抗体治疗HCC患者，这些患者对索拉菲尼具有抗药性，但该疗法的总体反应率仅为14.3%。其他策略尚未转化为临床实践在这个时候24，25。克服肿瘤诱发的深层免疫耐受性，改善免疫检查点治疗26;预测免疫检查点治疗的疗效;预防免疫相关不良事件;优化管理路线、剂量和频率;并找到有效的治疗方法组合27，28，29都仍然是极具挑战性的任务。
目前，在小鼠模型中，有几种用于诱导HCC的常规方法，根据研究者的特定研究问题30加以利用。化学诱导的HCC小鼠模型与基因毒性化合物模仿损伤引起的恶性。通过HCC细胞系的异位或正位植入的异种移植模型适用于药物筛选。一些转基因小鼠被设计用于研究HCC的病理生理学。表达病毒基因、肿瘤基因和/或生长因子的转基因小鼠能够识别与肝癌有关的途径。由于固有的局限性，这些模型不重述在人类HCC中看到的典型免疫特征，这大大阻碍了阐明潜在的机制和开发创新的免疫治疗策略14 ， 15.我们最近创建了一个临床相关的鼠模型。这种新颖的模型不仅模仿人类HCC的启动和进展，而且反映了人类疾病的最典型特征，包括免疫功能障碍。我们具有其生物学和免疫学特征。利用这一新颖的模式，我们探索了治疗HCC31、32、33、34、35、36的各种免疫治疗策略。 37.这个独特的平台使我们能够研究肿瘤诱导免疫耐受机制，并为HCC制定概念验证治疗策略，最终实现临床翻译。

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="847">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Anesthesia machine</td>
			<td style="width:123px;">VETEQUIP</td>
			<td style="width:344px;">IMPAC6</td>
			<td style="width:167px;">anesthesia machine for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butterfly needle</td>
			<td>BD</td>
			<td>8122963</td>
			<td>Needle used for liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice</td>
			<td>Jackson Lab</td>
			<td>000664&#160;</td>
			<td>mice used in prototol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td>CRESCENT CHEMICAL</td>
			<td>20402</td>
			<td>carprofen for pain release</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer&#160;</td>
			<td>CORNING</td>
			<td>REF 431751</td>
			<td>Cell strainer, 70&#181;m, for hepatocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-30R</td>
			<td>centrifuge for cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clips&#160;</td>
			<td>Teleflex Medical</td>
			<td>REF 523700</td>
			<td>Titanium Clips for spleen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td>Primovert&#160;</td>
			<td>microscope for cell observation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Mtd2 mice</td>
			<td>N/A</td>
			<td></td>
			<td>Gift from Dr. William A Held at roswell Park Cancer Institute in 2002, maintained in our lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle</td>
			<td>BD</td>
			<td>REF 305109</td>
			<td>BD precisionglide needle, 27G x 1/2 (0.4mm x 13mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture</td>
			<td>ETHICON</td>
			<td>J303H</td>
			<td>coated VICRYL suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SV40 T Ag antibody</td>
			<td>Abcam</td>
			<td>ab16879</td>
			<td>anti-SV40 T-antigen antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe</td>
			<td>BD</td>
			<td>REF 309626</td>
			<td>1 mL TB syringe for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td>SIGMA</td>
			<td>T 8154</td>
			<td>Trypan blue solution for cell viability test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wound clips</td>
			<td>Reflex</td>
			<td>reflex9, Part. No. 201-1000</td>
			<td>stainless steel wound clips for wound close</td>
		</tr>
	</tbody>
</table>

an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis

缺乏临床相关动物模型，解决肝细胞癌（HCC）的典型免疫特征，大大阻碍了阐明其基本机制和开发创新的免疫治疗策略。为了开发一种理想的动物模型来概括人类HCC，免疫能力雄性C57BL/6J小鼠首先接受四氯化碳（CCl4）注射以诱导肝纤维化，然后从年轻雄性小鼠获得组织学正常的致癌肝细胞SV40 T抗原（TAg）-转基因小鼠（MTD2）通过精内（ISPL）接种。在青春期接受的雄性小鼠中产生的雄激素在肝脏特异性启动子的控制下启动TAg表达。因此，转移的肝细胞成为癌细胞，在肝纤维化/肝硬化的设置中形成肿瘤质量。这一新颖的模型模仿了人类HCC在肝纤维化/肝硬化背景下的启动和进展，反映了人类HCC的最典型特征，包括免疫功能障碍。

从TAg转基因小鼠中分离的致基因肝细胞（图2）通过内注射在野生型小鼠的肝脏中播种（图3）。移植的肝细胞在肝炎症和纤维化设置中成功可靠地生长出具有肿瘤特异性抗原SV40 TAg的正交HCC肿瘤（图4）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59368/59368fig1.jpg"> 图1：用于准备HC...

Watch this Scientific Journal Video about An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis at JoVE.com

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

在这里，我们描述了一个临床相关的肝癌小鼠模型的发展，概括了肝细胞癌（HCC）的典型免疫特征。

在肝炎症和纤维化环境中产生的肝细胞癌的致癌肝细胞诱导正交小鼠模型

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

仅仅使用一个模型来开发一个肝细胞癌质量模型可以帮助人们阐明他们的肿瘤诱导免疫容忍和特定治疗诱导，任何肿瘤免疫反应。肝纤维化技术发展起来的正畸肿瘤模仿了人类肝癌的临床特征。该方法可为肝细胞癌免疫学研究提供有用的工具。此方法的可视化演示可以说明技术上更具挑战性的步骤，如静脉内注入，这些步骤对于成功执行此协议至关重要。接下来，手动约束一个雄性，六到八周大的C57黑色6鼠标后侧向上。使用备用 70%乙醇和贝塔丁磨砂清洁注射部位三次。然后通过内丙酮注射将160微升的四氯化碳送到小鼠，然后将动物送回其家笼。在确认对手趾捏缺乏反应后，在一只5个月大的西米安病毒40T抗原转基因小鼠中，将小鼠放在超生位置，用胶带固定四肢。使中线腹腔切除术切口沿线阿尔巴的长度，大到足以提供足够的肝脏暴露，并取代肠道左侧。在肝脏上方解剖以暴露劣质的维纳卡瓦。诱导动脉夹将容器进行起立。使用蝴蝶针静脉注射入口静脉，并手动固定导管。仔细切换每个溶液的注射器，成功交付15毫升溶液一，15毫升0.75%胶原酶溶液2，15毫升溶液2，不用导管胶酶，以每分钟约8.9毫升溶液的流量。在灌注结束时，将肿瘤质量收获成含有 10 到 15 毫升 PBS 的 50 毫升锥形管中。正如刚刚证明的，从下一个MTD2小鼠切除肿瘤。用剪刀把肝脏切成小块。将组织转移到新鲜 PBS 的新容器中，以从组织中去除其余血液，然后再将组织片段转移到含有 5 毫升完整 RPMI 介质的 50 毫升圆锥形管中。切碎洗净的肝脏样本，直到碎片可以很容易地通过五毫升移液器的尖端。向管子添加足够的介质，以达到30毫升的最终体积。使用五毫升移液器将组织碎片三分液，然后通过 70 微米滤网将溶液过滤到新的 50 毫升管中。用完整的介质清洗过滤器几次。使用额外的完整介质将最终体积调整为 50 毫升。在停止离心机并 decing 上一代之前，通过离心快速旋转悬架至最大 50 倍 g。然后将颗粒重新在 20 毫升 PBS 中重新暂停，以进行计数。对于MTD2肝细胞的静脉注射，每只接受动物装一根毫升注射器，每只接受动物装有27针，每只小鼠有220微升的肝细胞。确认麻醉、四氯化碳处理动物对手趾捏缺乏反应。从脊柱肌肉的正下方开始，接下来，在左侧侧翼做一厘米的切口，与背部极端的第十三根肋骨平行，并使用钝点钳将脾脏外化。在脾动脉和静脉之间用两个中等大小的钛夹住脾脏，将200微升细胞送到脾脏的劣极中。用一个中等大小的夹子夹住小柱的劣质分支，并在两个最初放置的夹子之间切割脾脏。取出直接注射肿瘤细胞的脾脏的劣质极点，使用3-0多糖蛋白910中断，以关闭内肌层。然后使用灭菌的钢伤口夹关闭外皮层，皮下施用每公斤卡普罗芬5毫克。与T抗原转基因小鼠分离后，可以观察到肝细胞和肿瘤渗透细胞。细胞通常表现出接近100%的可行性后处理。在静脉接种到野生型碳四氯化处理动物后，移植的肝细胞成功可靠地生长正向细胞癌肿瘤，表达肿瘤特异性西米安病毒40T抗原。从MTD2质量中制备我们可行和纯净的肝细胞，并准确管理宫内注射剂，对于手术的成功至关重要。虽然我们的团队已经通过血管内注射和内腹膜注射对接受小鼠进行了肝细胞接种测试，但这些其他方法无法诱导正交细胞癌。该模型提供了一个独特的平台，使研究免疫监测和耐受性在其早期和后期的肿瘤进展阶段。使用四氯化碳时请注意，因为四氯化碳有毒。

Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

胰腺癌的荧光原位小鼠模型

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

科研

癌症研究

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

毛尿胰腺腺腺腺癌超声引导正射植入

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

胰腺癌矫形小鼠模型

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

纯化的DEC-205定向抗体与全长蛋白的化学偶联，用于在体外和体内靶向小鼠树突状细胞

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...