Name: non-invasive pet/mr imaging in an orthotopic mouse model of hepatocellular carcinoma
Uploaded: 2022-08-31T13:40:00
Description: Watch this Scientific Journal Video about Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma at JoVE.com

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.

All animal studies were carried out in accordance with the Committee on the Use of Live Animals in Teaching and Research (CULATR) in the Centre for Comparative Medicine Research (CCMR) at the University of Hong Kong, a program accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The animals used in the study were female BALB/cAnN-nu (Nude) mice at the age of 6-8 weeks, weighted at 20 g &#177; 2 g. Food and water were provided ad libitum.
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<ol>
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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.

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	<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&#8217;s modified Eagle&#8217;s medium)</td>
			<td>Gibco</td>
			<td>10566024</td>
			<td>high glucose, GlutaMAX&#8482; 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 CO2 incubator</td>
			<td>Techcomp Limited</td>
			<td>NU5841</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Dose calibrator</td>
			<td>Biodex</td>
			<td>&#160;N/A</td>
			<td>Atomlab 500</td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#8217;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>&#160;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 &#38; 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>&#160;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&#160;</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>&#160;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 &#38; 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 &#181;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 (Video) | JoVE 

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).

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 ...

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