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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

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 ...

Our methods creates a reliable hypoxic hepatocellular carcinoma model for noninvasive imaging of tumor hypoxia to study HCC biology in vivo, which can better recapitulate cancer development in humans. Our reliable HCC model together with a highly-producible image method serves as an important platform for translational research into the pathophysiology of human HCC. Real-time monitoring of tumor hypoxia provides insights into molecular changes leading to tumor aggressiveness, allowing determining areas of hypoxia and predicting response to treatment.Although intraabdominal surgery can be technically challenging, adequate planning and animal care will improve the overall surgery success rate and the wellbeing of small animals. To begin, sterilize the dorsal region of an anesthetized 6 to 8-week-old mouse either at the left or right side of the lower flank with 70%ethanol. Then, use forceps to lift the skin and gently insert the injection needle underneath.To avoid accidental leakage of the injected cell suspension during needle withdrawal, advance the needle 4 to 5 millimeters from the puncture site along the subcutaneous plane. After releasing the forceps, carefully discharge the contents of the syringe without penetrating the opposite side. After euthanizing the subcutaneous tumor-bearing mouse, make an incision on the skin of the tumor site with a scalpel blade.Then, excise the tumor block and immediately transfer it to the culture media. Next, using sharp surgical scissors, cut the tumor block into small 1 cubic millimeter cubes, avoiding the core region of the tumor block. Keep all the cubes in culture media.For orthotopic liver implantation, extend the limbs of an anesthetized mouse and secure them with tape to maximally expose the ventral abdomen and thorax. Then, sterilize the entire abdominal skin with 10%povidone-iodine solution followed by 70%ethanol. Next, perform a laparotomy by first making a midline incision of 10 millimeters using sharp scissors to access the peritoneum.Then, clamp and pull the xiphoid process toward the head with forceps and open the surgical field using subcostal retractors. Using a wet gauze swab, retract the median and left liver lobes upward. Then, move the intestines outside the abdomen onto the left side of the mouse and cover them with a wet gauze swab.Next, under a magnifying lamp for optimum visualization, perform hepatic artery ligation on the common hepatic artery, which originates from the pancreatic head to its root in the hepatic pedicle. Expose the left lobe by retracting the median and left lobe's back with a wet gauze swab. Then, using a sterile scalpel blade, make an incision of about 2 millimeters long and deep in the left lobe, and immediately insert a tumor fragment into the liver incision with sterile forceps.Bury the tumor securely in the liver before applying a figure-eight suture, with a 6-0 nylon suture over the incision site to ensure proper tumor implantation and hemostasis. Then, close the abdominal incision with interrupted 5-0 sutures. Dispense an aliquot of 18-FMISO to a tube and dilute it with sterile saline to ensure a total activity concentration of 18 to 20 megabecquerel in 100 microliters.Next, draw the solution with a 1 milliliter syringe and record the radioactivity and time shown on the dose calibrator. After recording the body weight of the subject mouse, carefully inject the prepared radiotracer through the tail vein. To account for decay correction, record the injection time and residual activity in the syringe.To begin the PET acquisition, locate the mouse position in the scanner by performing a scout view. Adjust the mouse bed position to ensure that the whole mouse body with the torso region is at the center of the field of view of the MR.Next, select PET Acquisition in the study list window. For using the predetermined mouse bed positions from the scout view, choose Scan Range on Previous Acquisition.Then, click on Prepare to automatically move the mouse bed from MR to PET, followed by Go to start the acquisition. Select the appropriate radionucleotide under the Radiopharmaceutical Editor and enter the details of injection dose and time. Additionally, under the Subject Information menu, enter the weight of the mouse.Once the PET scan is complete, select Prepare to move the mouse from PET to MR, and click Go to perform the MR acquisition. Once all the scans are finished, move the imaging bed back to the default position by selecting Table Out. The PET imaging used in this study revealed an increase in the tumor uptake of 18-FMISO in mice with hepatic artery ligation but not in control mice.However, tumor uptake of the glycolytic marker 18F-FDG was similar in mice with and without hepatic artery ligation. Quantification using this standardized uptake value-based approach revealed a 2.3-fold higher 18-FMISO uptake by tumors in mice with hepatic artery ligation than that in control mice. Similarly, the 18-FMISO uptake was higher in the livers of mice with hepatic artery ligation than in the livers of control mice.Hepatic artery ligation also resulted in enhanced tumor hypoxia, as evidenced by a higher level of hypoxia-inducible factor 1-alpha expression in tumor secretions from mice with hepatic artery ligation than those from control mice. After 18-FMISO PET imaging, our sequencing can be performed to identify the roles of metabolic genes induced under hypoxia, leading to new treatment strategies. This technique can be used to monitor tumor hypoxia accurately, thereby allowing a rapid evaluation of treatment effectiveness and also the creation of new therapeutic strategy targeting the hypoxia pathway in HCC.

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Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

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 survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

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 ...

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

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

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

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

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 ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

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

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is typically performed via transfemoral access (TFA). However, transradial access (TRA) is preferred in coronary artery interventions due to decreased complications and mortality. Whether the advantages of TRA can be applied to TACE required investigation.
Patients receiving TRA TACE at a single center were retrospectively enrolled for study. Procedural details, technical success, radial artery occlusion (RAO) rate, and access site-related bleeding complications were evaluated. From October 2017 to October 2018, 112 patients underwent 160 TRA TACE procedures. The overall technical success rate was 95.0% (152/160). The rate of crossover from TRA to TFA was 1.9%. No access site-related bleeding complications were found in any cases. Asymptomatic RA occlusion occurred in three patients (2.7%). Compared with TFA, TRA can increase safety and patient satisfaction while decreasing access site-related bleeding complications. Moreover, TRA interventions can benefit patients with advanced age, obesity, or a high risk of bleeding complications.

Transarterial chemoembolization (TACE) is the standard therapy for patients in the intermediate stage of hepatocellular carcinoma and is typically performed through femoral artery access. Compared with transfemoral access, transradial access (TRA) can decrease the rate of bleeding complications and improve patient tolerance. A method is presented here to perform transarterial chemoembolization via radial artery access.

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is ...

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An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

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

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

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

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

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

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...