We acknowledge the support of the Hong Kong Anticancer Trust Fund, Hong Kong Research Grants Council Collaborative Research Fund (CRF C7018-14E) for the small animal imaging experiments. We also thank the support of the Molecular Imaging and Medical Cyclotron Center (MIMCC) at The University of Hong Kong for the provision of [18F]FMISO and [18F]FDG.

<ol>
	<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>Chen, C., Lou, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+inducible+factors+in+hepatocellular+carcinoma.">Hypoxia inducible factors in hepatocellular carcinoma.</a> Oncotarget. 8 (28), 46691-46703 (2017).</li><li>Lu, R. -. C., 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=Positron-emission+tomography+for+hepatocellular+carcinoma:+Current+status+and+future+prospects.">Positron-emission tomography for hepatocellular carcinoma: Current status and future prospects.</a> World Journal of Gastroenterology. 25 (32), 4682-4695 (2019).</li><li>Larsson, P., 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=Adding+11C-acetate+to+18F-FDG+at+PET+examination+has+an+incremental+value+in+the+diagnosis+of+hepatocellular+carcinoma.">Adding 11C-acetate to 18F-FDG at PET examination has an incremental value in the diagnosis of hepatocellular carcinoma.</a> Molecular Imaging and Radionuclide Therapy. 21 (1), 6-12 (2012).</li><li>Huo, L., 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=Kinetic+analysis+of+dynamic+11C-acetate+PET/CT+imaging+as+a+potential+method+for+differentiation+of+hepatocellular+carcinoma+and+benign+liver+lesions.">Kinetic analysis of dynamic 11C-acetate PET/CT imaging as a potential method for differentiation of hepatocellular carcinoma and benign liver lesions.</a> Theranostics. 5 (4), 371-377 (2015).</li><li>Lopci, E., 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=PET+radiopharmaceuticals+for+imaging+of+tumor+hypoxia:+A+review+of+the+evidence.">PET radiopharmaceuticals for imaging of tumor hypoxia: A review of the evidence.</a> American Journal of Nuclear Medicine and Molecular Imaging. 4 (4), 365-384 (2014).</li><li>Mao, X., 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=Mechanisms+through+which+hypoxia-induced+caveolin-1+drives+tumorigenesis+and+metastasis+in+hepatocellular+carcinoma.">Mechanisms through which hypoxia-induced caveolin-1 drives tumorigenesis and metastasis in hepatocellular carcinoma.</a> Cancer Research. 76 (24), 7242-7253 (2016).</li><li>Kung-Chun Chiu, D., 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=Hypoxia+regulates+the+mitochondrial+activity+of+hepatocellular+carcinoma+cells+through+HIF/HEY1/PINK1+pathway.">Hypoxia regulates the mitochondrial activity of hepatocellular carcinoma cells through HIF/HEY1/PINK1 pathway.</a> Cell Death &amp; Disease. 10 (12), 934 (2019).</li><li>Li, Y., 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=Establishment+of+cell+clones+with+different+metastatic+potential+from+the+metastatic+hepatocellular+carcinoma+cell+line+MHCC97.">Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97.</a> World Journal of Gastroenterology. 7 (5), 630-636 (2001).</li><li>Faustino-Rocha, A., 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=Estimation+of+rat+mammary+tumor+volume+using+caliper+and+ultrasonography+measurements.">Estimation of rat mammary tumor volume using caliper and ultrasonography measurements.</a> Lab Animal. 42 (6), 217-224 (2013).</li><li>Liu, Q., Tan, K. V., Chang, H. C., Khong, P. L., Hui, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+quantification+of+brown+and+beige+adipose+tissues+in+mice+using+[18F]+FDG+micro-PET/MR+imaging.">Visualization and quantification of brown and beige adipose tissues in mice using [18F] FDG micro-PET/MR imaging.</a> Journal of Visualized Experiments: JoVE. (173), e62460 (2021).</li><li>Lin, W. -. 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=Hypoxia-activated+cytotoxic+agent+tirapazamine+enhances+hepatic+artery+ligation-induced+killing+of+liver+tumor+in+HBx+transgenic+mice.">Hypoxia-activated cytotoxic agent tirapazamine enhances hepatic artery ligation-induced killing of liver tumor in HBx transgenic mice.</a> Proceedings of the National Academy of Sciences. 113 (42), 11937-11942 (2016).</li><li>Wong, T. L., 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=CRAF+methylation+by+PRMT6+regulates+aerobic+glycolysis-driven+hepatocarcinogenesis+via+ERK-dependent+PKM2+nuclear+relocalization+and+activation.">CRAF methylation by PRMT6 regulates aerobic glycolysis-driven hepatocarcinogenesis via ERK-dependent PKM2 nuclear relocalization and activation.</a> Hepatology. 71 (4), 1279-1296 (2020).</li><li>Yang, X., 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=Development+of+cisplatin-loaded+hydrogels+for+trans-portal+vein+chemoembolization+in+an+orthotopic+liver+cancer+mouse+model.">Development of cisplatin-loaded hydrogels for trans-portal vein chemoembolization in an orthotopic liver cancer mouse model.</a> Drug Delivery. 28 (1), 520-529 (2021).</li><li>Shi, J., 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=Longitudinal+evaluation+of+five+nasopharyngeal+carcinoma+animal+models+on+the+microPET/MR+platform.">Longitudinal evaluation of five nasopharyngeal carcinoma animal models on the microPET/MR platform.</a> European Journal of Nuclear Medicine and Molecular Imaging. 49 (5), 1497-1507 (2021).</li><li>Kilian, K., 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=Imaging+of+hypoxia+in+small+animals+with+F+fluoromisonidasole.">Imaging of hypoxia in small animals with F fluoromisonidasole.</a> Nukleonika. 61 (2), 219-223 (2016).</li><li>Kawamura, M., 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=Evaluation+of+optimal+post-injection+timing+of+hypoxic+imaging+with+18F-Fluoromisonidazole-PET/CT.">Evaluation of optimal post-injection timing of hypoxic imaging with 18F-Fluoromisonidazole-PET/CT.</a> Molecular Imaging and Biology. 23 (4), 597-603 (2021).</li></ol>

The authors have no conflicts of interest to disclose.

In this study, we described the procedures to perform HAL on liver orthotopic HCC xenografts using subcutaneous tumors, along with methods for the non-invasive monitoring of tumor hypoxia in orthotopic xenografts using [18F]FMISO and [18F]FDG PET/MR. Our interest lies in the metabolic imaging of various cancer and disease models for early diagnosis and treatment response evaluation11,13,14,<sup c...

Hepatocellular carcinoma (HCC) is the sixth most diagnosed cancer and the third most common cause of death from cancer worldwide, with more than 900,000 new cases and 800,000 deaths in 20201. The major risk factor is cirrhosis, which occurs as a result of viral infections (hepatitis B and C viruses), alcohol abuse, diabetes, and non-alcoholic steatohepatitis2. The management of HCC is rather complex, and several treatment options are available, including surgical resection, thermal or chemical ablation, transplantation, transarterial chemoembolization, radiation, and chemotherapy, depending on the disease staging2,3. HCC is a chemotherapy-refractory tumor with disease recurrence in up to 70% of patients following curative-intent therapy2.
Despite the high degree of tumor heterogeneity, HCC is associated with two common outcomes: (i) HCC is very hypoxic, and (ii) tumor hypoxia is linked to greater tumor aggressiveness and treatment failure. The uncontrolled proliferation of HCC cells results in a high oxygen consumption rate that precedes vascularization, thus creating a hypoxic microenvironment. Low intra-tumoral oxygen levels then trigger a range of biological responses that influence tumor aggressiveness and treatment response. Hypoxia-inducible factors (HIFs) are often recognized as the essential transcriptional regulators in the response to hypoxia2,3. Hence, the ability to detect hypoxia is crucial to visualize neoplastic tissues and identify the inaccessible sites, which require invasive procedures. It also helps to better understand the molecular changes that lead to tumor aggressiveness and improve patient treatment outcomes.
Molecular imaging using positron emission tomography (PET) is commonly used in the diagnosis and staging of many cancers, including HCC. In particular, the combined use of dual-tracer PET imaging involving [18F]Fluorodeoxyglucose ([18F]FDG) and [11C]Acetate can significantly increase overall sensitivity in HCC diagnosis4,5. Imaging of hypoxia, on the other hand, can be achieved by using the commonly used hypoxic marker [18F]Fluoromisonidazole ([18F]FMISO). In clinical practice, the non-invasive assessment of hypoxia is important to differentiate between various types of tumors and regions for radiation therapy planning6.
Preclinical imaging has become an indispensable tool for the non-invasive and longitudinal evaluation of mouse models for different diseases. A robust and highly reproducible HCC model represents an important platform for preclinical and translational research into the pathophysiology of human HCC and the assessment of novel therapies. Together with PET imaging, in vivo behaviors can be elucidated to provide important insights at the molecular level for any given timepoint. Here, we describe a protocol for the generation of hepatic artery ligation (HAL) orthotopic HCC xenografts and analysis of their in vivo tumor metabolism using [18F]FMISO and [18F]FDG PET/MR. The incorporation of HAL makes a suitable model of transgenic or chemically induced HCC mice xenografts to study tumor hypoxia in vivo, as HAL can effectively block the arterial blood supply to induce intra-tumoral hypoxia7,8. In addition, unlike ex vivo immunohistochemical staining using pimonidazole, changes in tumor metabolism as a result of hypoxia can be readily visualized and accurately quantified non-invasively using PET imaging, enabling longitudinal assessment of treatment response or gauging of the emergence of resistance3,7,8. Our method shown here allows the creation of a robust hypoxic HCC model together with non-invasive monitoring of tumor hypoxia using PET/MR imaging to study HCC biology in vivo.

<table><tbody><tr><td>0.9% sterile saline</td><td>BBraun</td><td>N/A</td><td>0.9% sodium chloride intravenous infusion, 500 mL</td></tr><tr><td>10# Scalpel blade</td><td>RWD Life Science Co.,ltd</td><td>S31010-01</td><td>Animal surgery tool</td></tr><tr><td>10% povidone-iodine solution</td><td>Banitore</td><td>6.425.678</td><td>For disinfection</td></tr><tr><td>25G needle with a 1 mL syringe</td><td>BD PrecisionGlide</td><td>N/A</td><td>1 mL syringe with 25G needle for cell suspensions injections</td></tr><tr><td>5 mL syringe</td><td>Terumo</td><td>SS05L</td><td>5 mL syringe Luer Lock</td></tr><tr><td>70% Ethanol</td><td>Merck</td><td>1.07017</td><td>For disinfection</td></tr><tr><td>Automated Cell Counter</td><td>Invitrogen</td><td>AMQAF2000</td><td>For automated cell counting</td></tr><tr><td>Buprenorphine</td><td>HealthDirect</td><td>N/A</td><td>Subcutaneous injection (0.05-0.2 mg/kg/12 hours) for analgesic after surgery</td></tr><tr><td>Cell Culture Dish (60 mm diameter)</td><td>Thermo Scientific</td><td>150462</td><td>For tumor tissue processing</td></tr><tr><td>Centrifuge</td><td>Sigma</td><td>3-16KL, fixed-angle rotor 12311</td><td>For cell suspensions collection</td></tr><tr><td>Centrifuge Conical Tube</td><td>Eppendorf</td><td>EP0030122151</td><td>For cell suspensions collection</td></tr><tr><td>Culture media (Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium)</td><td>Gibco</td><td>10566024</td><td>high glucose, GlutaMAX&amp;trade; Supplement</td></tr><tr><td>Digital Caliper</td><td>RS PRO</td><td>841-2518</td><td>For subcutaneous tumor size measurement</td></tr><tr><td>Direct heat CO&lt;sub&gt;2 &lt;/sub&gt;incubator</td><td>Techcomp Limited</td><td>NU5841</td><td>For cell culture</td></tr><tr><td>Dose calibrator</td><td>Biodex</td><td>&amp;nbsp;N/A</td><td>Atomlab 500</td></tr><tr><td>DPBS (Dulbecco&amp;rsquo;s phosphate-buffered saline)</td><td>Gibco</td><td>14287072</td><td>For cell wash and injection</td></tr><tr><td>Eye lubricant</td><td>Alcon Duratears</td><td>&amp;nbsp;N/A</td><td>Sterile ocular lubricant ointment, 3.5 g</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>A4766801</td><td>Used for a broad range of cell types, especially sensitive cell lines</td></tr><tr><td>Forceps (curved fine and straight blunt)</td><td>RWD Life Science Co.,ltd</td><td>F12012-10 &amp;amp; F12011-13</td><td>Animal surgery tool</td></tr><tr><td>Heating pad</td><td>ALA Scientific Instruments</td><td>N/A</td><td>Heat pad for mice during surgery</td></tr><tr><td>Insulin syringe</td><td>Terumo</td><td>10ME2913</td><td>1 mL insulin syringe with needle for radiotracer injections</td></tr><tr><td>InterView fusion software</td><td>Mediso</td><td>Version 3.03</td><td>Post-processing and image analysis software</td></tr><tr><td>Inverted microscope</td><td>Yu Lung Scientific Co., Ltd</td><td>BM-209G</td><td>For cells morphology visualization</td></tr><tr><td>Isoflurane</td><td>Chanelle Pharma</td><td>&amp;nbsp;N/A</td><td>Iso-Vet, inhalation anesthetic, 250 mL</td></tr><tr><td>Ketamine</td><td>Alfasan International B.V.</td><td>HK-37715</td><td>Ketamine 10% injection solution, 10 mL&amp;nbsp;</td></tr><tr><td>Medical oxygen</td><td>Linde HKO</td><td>101-HR</td><td>compressed gas, 99.5% purity</td></tr><tr><td>nanoScan PET/MR Scanner</td><td>Mediso</td><td>&amp;nbsp;N/A</td><td>3 Tesla MR</td></tr><tr><td>Needle holder</td><td>RWD Life Science Co.,ltd</td><td>F31026-12</td><td>Animal surgery tool</td></tr><tr><td>Nucline nanoScan software</td><td>Mediso</td><td>Version 3.0</td><td>Scanner operating software</td></tr><tr><td>Nylon Suture (6/0 and 5/0)</td><td>Healthy Medical Company Ltd</td><td>000524 &amp;amp; 000526</td><td>Animal surgery tool</td></tr><tr><td>Penicillin- Streptomycin</td><td>Gibco</td><td>15140122</td><td>Culture media for a final concentration of 50 to 100 I.U./mL penicillin and 50 to 100 &amp;micro;g/mL streptomycin.</td></tr><tr><td>Pentabarbital</td><td>AlfaMedic</td><td>13003</td><td>Intraperitoneal injection (330 mg/kg) to induce cessation of breathing of mice</td></tr><tr><td>Sharp scissors</td><td>RWD Life Science Co.,ltd</td><td>S14014-10</td><td>Animal surgery tool</td></tr><tr><td>Spring Scissors</td><td>RWD Life Science Co.,ltd</td><td>S11005-09</td><td>Animal surgery tool</td></tr><tr><td>Trypan Blue Solution, 0,4%</td><td>Gibco</td><td>15250061</td><td>For cell counting</td></tr><tr><td>Trypsin-ethylenediaminetetraacetic acid (EDTA, 0.25%), phenol red.</td><td>Gibco</td><td>25200072</td><td>For cell digestion</td></tr><tr><td>Xylazine</td><td>Alfasan International B.V.</td><td>HK-56179</td><td>Xylazine 2% injection solution, 30 mL</td></tr></tbody></table>

non-invasive pet/mr imaging in an orthotopic mouse model of hepatocellular carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

To obtain a suitable tumor block for successive orthotopic implantation, stable clones were first generated by subcutaneous injection of 200 &#956;L of cell suspension in DPBS (containing MHCC97L cells) into the lower flank of nude mice (Figure 1A). Tumor growth was monitored and, when tumor size reached 800-1000 mm3 (around 4 weeks post injection), mice were euthanized, and the resulting tumor block was cut into approximately 1 mm3 fragments for subsequent liver orthot...

Watch this Scientific Journal Video about Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma at JoVE.com

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

non-invasive-petmr-imaging-an-orthotopic-mouse-model-hepatocellular

Research

JoVE Journal

Cancer Research

2.1K Views.  The University of Hong Kong. Onze methoden creëren een betrouwbaar hypoxisch hepatocellulair carcinoommodel voor niet-invasieve beeldvorming van tumorhypoxie om HCC-biologie in vivo te bestuderen, die de ontwikkeling van kanker bij mensen beter kan samenvatten. Ons betrouwbare HCC-model samen met een zeer produceerbare beeldmethode dient als een belangrijk platform voor translationeel onderzoek naar de pathofysiologie van menselijke HCC. Real-time monitoring van tumorhypoxie biedt inzicht in moleculaire veranderingen di...

Video: Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biology

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

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

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

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

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Medicine

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Immunology and Infection

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Author Spotlight: Enhancing Transplantation Research Through MicroCT Angiography in Murine Models 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video