Name: orthotopic implantation of patient-derived cancer cells in mice recapitulates advanced colorectal cancer
Uploaded: 2023-02-10T13:05:00
Description: Watch this Scientific Journal Video about Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer at JoVE.com

我们感谢 Cellex 基金会、CIBERONC 网络和 Instituto de Salud Carlos III 的支持。此外，我们还要感谢进行实验的Vall d'Hebron研究所（VHIR）的临床前成像平台。

获得所有患者的书面知情同意书。该项目已获得西班牙巴塞罗那Vall d'Hebron大学医院研究伦理委员会的批准（批准编号：PR（IR）79/2009 PR（AG）114/2014，PR（AG）18/2018）。人类结肠组织样本包括来自原发性腺癌或肝转移的非坏死区域的活检，对应于接受肿瘤切除的结肠癌和直肠癌患者。实验按照欧盟的动物护理指令（86/609/EEC）进行，并得到了VHIR-Vall d'Hebron研究所动物实验伦理委员会的批准（ID：40/08 C...

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	<li>Sung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Cancer+Statistics+2020:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: a Cancer Journal for Clinicians. 71 (3), 209-249 (2021).</li><li>Puig, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+personalized+preclinical+model+to+evaluate+the+metastatic+potential+of+patient-derived+colon+cancer+initiating+cells.">A personalized preclinical model to evaluate the metastatic potential of patient-derived colon cancer initiating cells.</a> Clinical Cancer Research. 19 (24), 6787-6801 (2013).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+development+and+disease+with+organoids.">Modeling development and disease with organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>Byrne, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+open+issues+in+cancer+precision+medicine+with+patient-derived+xenografts.">Interrogating open issues in cancer precision medicine with patient-derived xenografts.</a> Nature Reviews. Cancer. 17 (4), 254-268 (2017).</li><li>Vatandoust, S., Price, T. J., Karapetis, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+cancer:+Metastases+to+a+single+organ.">Colorectal cancer: Metastases to a single organ.</a> World Journal of Gastroenterology. 21 (41), 11767-11776 (2015).</li><li>Cespedes, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+microinjection+of+human+colon+cancer+cells+in+nude+mice+induces+tumor+foci+in+all+clinically+relevant+metastatic+sites.">Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.</a> The American Journal of Pathology. 170 (3), 1077-1085 (2007).</li><li>Durkee, B. Y., Weichert, J. P., Halberg, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+micro-CT+colonography.">Small animal micro-CT colonography.</a> Methods. 50 (1), 36-41 (2010).</li><li>Boll, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-contrast+micro-CT+colonoscopy+in+live+mice.">Double-contrast micro-CT colonoscopy in live mice.</a> International Journal of Colorectal Disease. 26 (6), 721-727 (2011).</li><li>O'Brien, C. A., Pollett, A., Gallinger, S., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+colon+cancer+cell+capable+of+initiating+tumour+growth+in+immunodeficient+mice.">A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.</a> Nature. 445 (7123), 106-110 (2007).</li><li>Jensen, M. M., Jorgensen, J. T., Binderup, T., Kjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+volume+in+subcutaneous+mouse+xenografts+measured+by+microCT+is+more+accurate+and+reproducible+than+determined+by+18F-FDG-microPET+or+external+caliper.">Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper.</a> BMC Medical Imaging. 8, 16 (2008).</li><li>Herpers, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+patient-derived+organoid+screenings+identify+MCLA-158+as+a+therapeutic+EGFR+x+LGR5+bispecific+antibody+with+efficacy+in+epithelial+tumors.">Functional patient-derived organoid screenings identify MCLA-158 as a therapeutic EGFR x LGR5 bispecific antibody with efficacy in epithelial tumors.</a> Nature Cancer. 3 (4), 418-436 (2022).</li><li>Chicote, I., Camara, J. A., Palmer, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+colorectal+cancer+orthotopic+patient-derived+xenograft+models+for+cancer+and+stem+cell+research.">Advanced colorectal cancer orthotopic patient-derived xenograft models for cancer and stem cell research.</a> Methods in Molecular Biology. 2171, 321-329 (2020).</li></ol>

在过去的几十年里，许多新的抗癌疗法已经在不同肿瘤类型的患者中开发和测试，包括结直肠癌（CRC）。尽管在许多病例中已经观察到临床前模型的有希望的结果，但晚期转移性结直肠癌患者的治疗效果经常受到限制。因此，迫切需要临床前模型，以测试新治疗药物在临床相关转移情况下的疗效。
该手稿详细描述了一种先进的CRC原位PDX模型，该模型基于将患者肿瘤细胞植入?...

结直肠癌 （CRC） 是全球癌症死亡的第二大原因1.从个体患者肿瘤细胞中生成体外或体内肿瘤模型的能力推动了肿瘤学的精准医学发展。在过去的十年中，患者来源的类器官 （PDO） 或异种移植物 （PDX） 已被世界各地的许多研究小组使用2。PDO是多细胞体外结构，类似于原始肿瘤组织的特征，可以自组织和自我更新3。这些有前途的体外模型可以成功地用于药物筛选和促进转化研究。另一方面，PDX 模型忠实地概括了所有相关水平的原始 CRC，从组织学到分子性状和药物反应 2,4。
体内研究 PDX模型主要在免疫缺陷小鼠中作为皮下肿瘤生长。使用这种方法，PDX已成为癌症研究的金标准，特别是用于研究药物敏感性或耐药性。然而，结直肠癌相关死亡主要与肝脏、肺或腹膜腔中存在转移性病变有关，两种方法（PDO或PDX）都不能概括晚期临床情况。此外，肿瘤生长的特定部位已被证明可以确定对药物疗效和疾病预后有影响的重要生物学特征2.因此，迫切需要建立临床前模型，用于评估抗癌药物在临床相关转移环境中的疗效6。
显微计算机断层扫描 （μCT） 扫描仪可以用作缩小的临床 CT 扫描仪，以与癌症患者 CT 图像成正比的缩放图像分辨率提供小鼠的原发肿瘤和转移成像7.为了抵消μCT技术的软组织对比度差，可以使用放射性碘造影剂来改善造影剂和评估肿瘤负荷。使用双重造影剂方法，在不同的时间口服和腹膜内给药碘。口服造影剂有助于确定肿瘤组织和肠内盲肠含量之间的界限。 另一方面，腹膜内给药的造影剂可以识别肿瘤肿块的外部限制，肿瘤肿块经常生长并侵入腹膜8。
该手稿描述了在免疫缺陷小鼠盲肠壁中进行患者来源的癌细胞原位植入的方案，以及使用μCT扫描监测肠道肿瘤生长的方法。本手稿表明，该模型概括了 CRC 患者晚期肠道肿瘤和转移性疾病的临床情况，这些情况无法使用 PDO 或 PDXO 模型进行研究。由于CRC的原位PDX模型概括了CRC患者的临床情况，因此我们得出结论，它们是迄今为止测试抗肿瘤药物在晚期肠道肿瘤和转移性疾病中的疗效的最佳模型。

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orthotopic implantation of patient-derived cancer cells in mice recapitulates advanced colorectal cancer

在过去的十年中，已经使用患者来源的癌细胞和 3D 类肿瘤建立了更复杂的临床前结直肠癌 （CRC） 模型。由于患者来源的肿瘤类器官可以保留原始肿瘤的特征，因此这些可靠的临床前模型能够进行癌症药物筛选和耐药机制研究。然而，患者中与结直肠癌相关的死亡主要与转移性疾病的存在有关。因此，在相关 体内 模型中评估抗癌疗法的疗效至关重要，这些模型真正概括了人类癌症转移的关键分子特征。我们建立了一个基于将CRC患者来源的癌细胞直接注射到小鼠盲肠壁中的原位模型。这些肿瘤细胞在盲肠中形成原发性肿瘤，并转移到肝脏和肺部，这在晚期结直肠癌患者中很常见。该CRC小鼠模型可用于评估显微计算机断层扫描（μCT）监测的药物反应，这是一种临床相关的小规模成像方法，可以轻松识别患者的原发性肿瘤或转移。在这里，我们描述了将患者来源的癌细胞植入免疫缺陷小鼠盲肠壁的外科手术和所需方法。

每周通过μCT扫描监测原位植入患者来源的癌细胞的小鼠。在实验结束时，动物被安乐死。收集肠、盲肠（图1A，B）、肝脏、肺和任何其他可能的病变，包括在盒中，并用4%福尔马林固定过夜。使用盲肠中没有肿瘤的小鼠的肠道组织作为对照（图1C）。最后，将盒更换为70%乙醇至少3小时并包埋石蜡。使用组织病理学设施标准方案对盲肠、肝?...

Watch this Scientific Journal Video about Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer at JoVE.com

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

该方案描述了在免疫缺陷小鼠盲肠壁中将患者来源的癌细胞原位植入。该模型概括了晚期结直肠癌转移性疾病，并允许在肺和肝转移的临床相关情况下评估新的治疗药物。

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

该方案描述了在免疫缺陷小鼠盲肠壁中将患者来源的癌细胞原位植入。该模型概括了晚期结直肠癌转移性疾病。这里介绍的原位结直肠癌模型概括了结直肠癌患者晚期肠道肿瘤和转移性疾病的临床情况，这些情况无法使用患者来源的类器官或患者来源的异种移植模型进行研究。从安乐死的小鼠中，使用剪刀和镊子小心地将其从皮肤和周围的非肿瘤组织中取出肿瘤。将收获的肿瘤置于PBS中，温度为4摄氏度。使用刀片，在含有1毫米培养基的10厘米培养板中解离肿瘤。将均匀的解离样品放入 15 毫升锥形管中。然后，将培养基加入到最终体积为 5 毫升。然后，将消化培养基添加到试管中。将试管置于细胞培养箱内的 45 度位置，在 37 摄氏度下放置一小时。在一小时的孵育期间，每 15 分钟用 5 毫升移液管混合一次溶液。孵育后，加入 5 毫升培养基并充分混合。然后，使用无菌 100 毫升管用 50 微米细胞过滤器对溶液进行分选。在室温下以 500g 离心分选的细胞 10 分钟。吸出上清液并将沉淀重悬于三毫升1x RBC裂解缓冲液中。在室温下孵育 10 分钟。然后，加入三毫升培养基并混合样品。在室温下以 500g 离心 10 分钟并吸出上清液。用 5 至 10 毫升培养基重悬沉淀，并使用细胞计数器计算细胞总数。在室温下以 500g 离心细胞 10 分钟后，重悬于 10 毫升 PBS 中。重复离心并用10毫升PBS洗涤。重悬沉淀以获得每毫升 6 个细胞的 20 乘以 10 至 6 个细胞的浓度，并充分混合以获得均匀的细胞悬液。接下来，准备一个29g注射器进行盲肠注射。将 50 微升肿瘤细胞悬液装入注射器中，并确保注射器中没有气泡残留。将注射器放在冰上。首先，通过喷洒消毒清洁剂并擦拭对手术部位进行消毒。然后，使用鼠标脱毛机对腹部进行脱毛。将鼠标置于仰卧位，并用洗必泰或聚维酮碘擦洗腹部。使用手术剪刀在下腹部做一个一厘米的纵向切口。小心地将皮肤分开到两侧，露出腹膜。然后，在腹膜上做一个 0.5 到 1 厘米的切口，足以使盲肠外化。使用预先切割的无菌纱布，小心地将盲肠与小鼠隔离。在整个手术过程中用生理盐水润湿盲肠。用镊子小心地握住盲肠，固定盲肠，然后将针头浅浅地引入盲肠壁。慢慢地，注射整个50微升体积的肿瘤细胞悬液。此过程大约需要 10 秒。注射后，慢慢将针头从盲肠中取出，并用棉尖涂抹器轻轻按压注射部位。用生理盐水清洁盲肠以去除任何碎屑，并将盲肠放回动物的腹部。使用 5/0 缝合线闭合腹膜，然后使用 5/0 缝合线闭合腹部皮肤。在植入患者来源的癌细胞的小鼠肠道中观察到原位 CRC-PDX 肿瘤。通过苏木精和盲肠上的伊红染色进行组织学分析显示存在肿瘤细胞。组织学分析还显示植入小鼠存在肺转移和肝转移。携带原位肿瘤的小鼠用载体、测试药物或化疗药物伊立替康治疗。显微CT扫描图像分析表明，与载体相比，试验药物诱导肿瘤体积减少，与伊立替康治疗联合治疗进一步减少。该模型将有助于研究结直肠癌肿瘤转移的生物学。

Research

JoVE Journal

Cancer Research

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

肝大肠癌转移的高级动物模型：成像技术和转移性克隆性质

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

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 ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

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 and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Translational Orthotopic Models of Glioblastoma Multiforme

多形性胶质母细胞瘤的转化原位模型

Genetically engineered mouse (GEM) models for human glioblastoma multiforme (GBM) are critical to understanding the development and progression of brain ...