This work was supported by the clinical research special fund from Zhongshan Hospital, Fudan University (2016ZSLC17). The authors are very grateful to Dr. Xianglin Hu in Zhongshan Hospital of Fudan University for his very professional suggestions to English writing.

This single-center retrospective study was approved by the local Institutional Review Board of Zhongshan Hospital, Fudan University.
1. Obtaining informed consent
<ol>
	<li>Before the TRA interventions, have interventional radiologists (IRs) explain the benefits and potential complications of TRA to the patients.</li>
</ol>
2. Patient evaluation
<ol>
	<li>After obtaining informed consent, evaluate the RA for the feasibility of puncture and cannulation.</li>
	<li>Perform a comprehensive review of the patient&#39;s medical history. Confirm if patients had severe vascular tortuosity, severe peripheral vascular disease, a fistula for dialysis, or preparation for RA dialysis operation. These are relative contraindications for patients receiving TRA interventions.</li>
	<li>Evaluate the visibility of the RA.
	<ol>
		<li>Perform a Barbeau test with the use of pulse oximetry to evaluate how visible the hand collateral arteries are before intervention11. The Barbeau D waveform is considered an absolute contraindication for RA cannulation.</li>
		<li>For patients tested as Barbeau C waveform, apply a Doppler ultrasound examination to provide more reliable information about the amount of collateral circulation in the forearm and hand. An inner diameter smaller than 2 mm is considered a contraindication. 
		NOTE: After medical history evaluation and the RA evaluation, patients with contraindications should abstain from a puncture via the ipsilateral RA. The contralateral RA might serve as an ideal supply if it is found to be patent through Barbeau test evaluation. The left RA is initially chosen as a preferred access route. The right RA might serve as an alternative choice if the left RA was found unsuitable.</li>
	</ol>
	</li>
</ol>
3. Radial artery access
<ol>
	<li>Place the patient in a supine position on the angiography table. Then, place the left arm parallel to the patient&#8217;s body and close to the left waist, allowing easy placement of the catheter and wire and enabling operator positioning comparable to that with the TFA.</li>
	<li>Mark the distal RA pulse by palpation. Clean the skin surface with 10% povidone-iodine surgical scrub solution and allow the solution to air dry. Cover the left arm with a surgical drape.
	<ol>
		<li>In case of potential left RA puncture failure, prepare an alternative access route by sterilizing and draping the right arm or right inguinal region.</li>
	</ol>
	</li>
	<li>Apply local anesthesia (i.e., 1 mL of lidocaine 2%) proximal to the styloid process along the axis of the most powerful pulsation of the left RA.</li>
	<li>Extend the wrist, and puncture the RA with a 20 G needle using the modified Seldinger technique. When pulsatile arterial blood return isobserved , introduce a 0.025 inch hydrophilic guidewire.
	<ol>
		<li>Retract the guidewire and readjust the needle if resistance is encountered. Do not force the insertion of the guidewire. With the assistance of of digital subtraction angiography (DSA), inject about 1 to 2 mL of contrast to highlight the RA and help insert the hydrophilic guidewire.</li>
	</ol>
	</li>
	<li>Once access is obtained, remove the needle and introduce a 4-French hydrophilic sheath with the guidewire. After sheath insertion, gently pump back a small amount of arterial blood with a syringe to confirm that the sheath tip is within the vessel.</li>
</ol>
4. Anticoagulation and vascular dilation
<ol>
	<li>Prepare 10 mL of a vasodilation cocktail solution (3,000 IU of unfractionated heparin, 0.1 mg of nitroglycerin, and 20 mg of lidocaine).</li>
	<li>Administer 8 mL of the vasodilation cocktail solution through the sheath at a speed of 0.5 mL/s (Figure 1). 
	NOTE: Reduce or stop the dose of heparin for patients with moderate or high bleeding risk.</li>
</ol>
5. Catheter selection
<ol>
	<li>Use a 4-French, 125 cm common catheter and a standard 0.035 inch x 180 cm hydrophilic wire to traverse the subclavian artery and engage the descending aorta. Use DSA fluoroscopy to visualize the proximal axillary artery during navigation within the arm to avoid potential lesions to an artery loop or vascular tortuosity. 
	NOTE: The subclavian artery has many arterial branches. Angiographic guidance prevents guidewire catheters from entering collateral vessels during retrograde catheterization. A few cases have an artery loop in the radial artery. If the standard wire cannot pass the loop, use of a microcatheter and angled 0.016 inch or 0.018 inch microwire is recommended.</li>
	<li>Use the 4-French, 125 cm common catheter in combination with a standard 0.035-inch wire to negotiate the transverse arch to direct the guidewire toward the descending aorta. 
	NOTE: If the angle between the aorta and left subclavian artery is very acute, a Cobra 2-shaped catheter (e.g., Simmons I or Simmons II catheter) is recommended to accomplish this turn.</li>
	<li>After catheterization of the descending aorta, replace the common catheter via a coaxial technique. Once the common catheter is inserted into the descending aorta, steer the catheter tip ventrally for catheterization of the celiac trunk under the guidance of DSA fluoroscopy. In most cases, it is easy to catheterize and perform angiography of the celiac trunk, the hepatic artery, and superior mesenteric artery. 
	NOTE: If the angle between the celiac artery and the descending aorta is very acute, use a Cobra catheter to complete the procedure.</li>
	<li>For hepatic embolization procedures, perform super-selective catheterization and chemoembolization using a coaxial technique and place a 2.8-French 150 cm microcatheter into the targeted branch of the hepatic artery feeding the tumors (Figure 2). Perform TACE according to the burden of disease and patient preference.</li>
	<li>Perform an angiogram through the common catheter using a high-pressure injector to confirm adequate embolization. The catheter tip is usually located in the common hepatic artery. Inject 9&#8211;12 mL of the contrast agent at a rate of 3&#8211;4 mL per s, with a fluoroscopy time of ~15 s. Then, remove the catheter over a guidewire to avoid damage to the RA.&#160;</li>
</ol>
6. Radial artery hemostasis
NOTE: Nonocclusive hemostasis is performed using a special tourniquet to maintain RA patency (Figure 3).
<ol>
	<li>Administer the remaining 2 mL of vasodilation cocktail solutions (section 4) through the RA sheath. Immediately after, retrieve the sheath about 5 cm.</li>
	<li>Place a tourniquet over the radial access site on the left wrist, and adequately inflate the tourniquet air bag of with air using the accompanying syringe. Then completely remove the sheath, and slowly deflate the air bag. When leaking is observed at the access site, add 1 mL of air back to the cuff. Typically, 10&#8211;15 mL of air is added to the air bag to keep hemostasis.</li>
	<li>Confirm that there is no bleeding or leaking. At the same time, ensure that the distal radial artery pulse is palpable during hemostasis. Use the pulse oximeter waveform to confirm the arterial waveform on the left thumb.</li>
	<li>Slowly inflate the air sac at ~2 mL every 2 h for no longer than 6 h. Reconfirm that hemostasis is accomplished once the tourniquet is removed 6 h after operation. 
	NOTE: If bleeding or leaking from the puncture site is observed during deflation, air is added back to the air sac for 30 min and the process is repeated.</li>
	<li>Before discharge, conduct Barbeau test to confirm the patency of the RA and record patients with radial artery occlusion and closely follow up.</li>
</ol>
7. Follow up
<ol>
	<li>About 1 month after intervention, give TRA patients a thorough physical examination, including inspection of the left wrist and pulse examination. For patients with potentially occluded RAs, perform subsequent evaluations of hand blood supply using forearm Doppler ultrasound or pulse oximetry.</li>
	<li>Closely follow up all patients after TACE. If new tumor nodules were evident on CT scans and the initial lesions seemed to revascularize, perform another TACE treatment.</li>
</ol>

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Study concept and design by WZ and ZPY; acquisition of data by ND, ZHZ and MJY; obtained funding by ZPY. The authors have no relevant financial disclosures.

TRA interventional therapy has grown significantly worldwide in recent years, especially in diagnostic and Interventional cardiology procedures12. Moreover, there has been increasing attention to peripheral vascular disease intervention. Without compromising procedural success rates, TRA to cardiac intervention can effectively reduce the rates of bleeding and vascular complications compared with TFA13,14. Compared with TFA, TRA is superior...

Hepatocellular carcinoma (HCC) is a very common malignancy, with the sixth highest incidence rate worldwide. It is also the second leading cause of cancer mortality around the world1. Because only 5%&#8211;20% of patients can receive curative therapy, transarterial chemoembolization (TACE) is the most popular palliative treatment for patients with unresectable HCC2. TACE has been recognized as the most commonly used and effective treatment approach for HCC patients at the intermediate stage3. Transfemoral access (TFA) chemoembolization is the most common approach for TACE4. However, there are risks associated with TFA intervention, including bleeding at the access site and major vascular complications5. These complications lead to prolonged hospitalization and increased costs. Moreover, TFA requires immobilization for at least 6 h, which increases discomfort and dissatisfaction for the patients.&#160;
Transradial access (TRA) is an alternative approach that has been used in percutaneous coronary intervention (PCI) for more than two decades5,6. TRA PCI has several advantages: increased procedure comfort, decreased access site-related bleeding, decreased major vascular complications, and decreased mortality7,8. The radial artery (RA) is easy to access and puncture because of its superficial location7. Hemostasis is easy to conduct after intervention and there is no strict immoblization9. Despite encouraging evidence for TRA intervention in cardiac catheterization, to date only a few studies used TRA in peripheral disease intervention. TRA interventions for malignant liver tumors are even rarer. Here, the clinical feasibility and safety of TRA hepatic embolization is analyzed. One institution&#8217;s experience with the step-by-step TRA protocol provided is also described.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Reagents</td>
			<td style="width:127px;"></td>
			<td style="width:123px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Embosphere</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">20173776165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Gelfoam</td>
			<td style="width:127px;">Alicon</td>
			<td style="width:123px;">20143771056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Heparin</td>
			<td style="width:127px;">Hepatunn</td>
			<td style="width:123px;">H51021209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Injection syringe</td>
			<td style="width:127px;">KDL</td>
			<td style="width:123px;">20163150518</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:141px;">Iodinated oil</td>
			<td style="width:127px;">Yantai Luyin Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H37022398</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:141px;">Lidocaine</td>
			<td style="width:127px;">Shandong Hualu Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H37022147</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:141px;">Lobaplatin</td>
			<td style="width:127px;">Hainan Changan International Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H20050308</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:141px;">Nitroglycerin</td>
			<td style="width:127px;">Brijing Yimin Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H11020289</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:141px;">Normal saline</td>
			<td style="width:127px;">Anhui Shuanghe Pharmaceutical Co.Ltd</td>
			<td style="width:123px;">H34023609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Pharmorubicin</td>
			<td style="width:127px;">Pfizer</td>
			<td style="width:123px;">H20000496</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Ultravist 370</td>
			<td style="width:127px;">Bayer</td>
			<td style="width:123px;">H20171333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:141px;">Hydrophilic Guide Wire</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">LWSTDA38180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Injection syringe</td>
			<td style="width:127px;">KDL</td>
			<td style="width:123px;">20163150518</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:141px;">Maestro Microcatheter</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">28MC24150SN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPA1 (I) catheter</td>
			<td style="width:127px;">Cordis</td>
			<td style="width:123px;">451-406P0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Sheath Introducer</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">PSI-4F-11-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Steerable Guidewire</td>
			<td style="width:127px;">Merit</td>
			<td style="width:123px;">TNR2411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">TR Band</td>
			<td style="width:127px;">Terumo</td>
			<td style="width:123px;">XX*RF06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">DSA</td>
			<td style="width:127px;">Toshiba</td>
			<td style="width:123px;">INFX-9000V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:141px;">Ultrasonic machine</td>
			<td style="width:127px;">SonoScape</td>
			<td style="width:123px;">20172231180</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

From October 2017 to October 2018, 112 patients underwent 160 TRA TACE procedures, and the overall technical success rate was 95.0% (152/160). Eight cases were met with technical failure. Of these, five cases were caused by left RA puncture failure and subsequently underwent successful TACE with right RA access. The other three cases were caused by cannulation failure, and underwent subsequent successful intervention by crossover to right FA access. The crossover rate of RA access to FA access was only 1.9%. No access si...

Watch this Scientific Journal Video about Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients at JoVE.com

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

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 ...

transradial-access-chemoembolization-for-hepatocellular-carcinoma

Research

JoVE Journal

Cancer Research

5.3K Views.  Fudan University. 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.

Video: Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

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 ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

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 ...

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.

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 ...

Establishment and Characterization of Patient-Derived Xenograft Models of Anaplastic Thyroid Carcinoma and Head and Neck Squamous Cell Carcinoma

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its heterogeneity. Pharmacodynamic results based on PDX models are highly correlated with clinical practice. Anaplastic thyroid carcinoma (ATC) is the most malignant subtype of thyroid cancer, with strong invasiveness, poor prognosis, and limited treatment. Although the incidence rate of ATC accounts for only 2%-5% of thyroid cancer, its mortality rate is as high as 15%-50%. Head and neck squamous cell carcinoma (HNSCC) is one of the most common head and neck malignancies, with over 600,000 new cases worldwide each year. Herein, detailed protocols are presented to establish PDX models of ATC and HNSCC. In this work, the key factors influencing the success rate of model construction were analyzed, and the histopathological features were compared between the PDX model and the primary tumor. Furthermore, the clinical relevance of the model was validated by evaluating the in vivo therapeutic efficacy of representative clinically used drugs in the successfully constructed PDX models.

The present protocol establishes and characterizes a patient-derived xenograft (PDX) model of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), as PDX models are rapidly becoming the standard in the field of translational oncology.

Patient-derived xenograft (PDX) models faithfully preserve the histological and genetic characteristics of the primary tumor and maintain its ...

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...

Prognostic Significance of Immune Cell Infiltration in Hepatocellular Carcinoma

Mast Cells in the Microenvironment of Hepatocellular Carcinoma Confer Favorable Prognosis: A Retrospective Study using QuPath Image Analysis Software

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies investigating the expression and localization of immune cells within tumor tissues are associated with several challenges, including a lack of precise annotation for tumor regions and random selection of microscopic fields of view. QuPath is an open-source, user-friendly software that could meet the growing need for digital pathology in whole-slide image (WSI) analysis.
The infiltration of HCC and adjacent tissues by CD1a+ immature dendritic cells (iDCs), CD117+ mast cells, and NKp46+ natural killer cells (NKs) cells was assessed immunohistochemically in representative specimens of 67 patients with HCC who underwent curative resection. The area fraction (AF) of positively stained cells was assessed automatically in WSIs using QuPath in the tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells was evaluated for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS).
The AF of mast cells was significantly greater than the AF of NKs, and the AF of iDCs was significantly lower compared to NKs in each region of interest. High AFs of mast cells in the IM and PT areas were associated with longer DFS. In addition, high AF of mast cells in IM was associated with longer OS.
Computer-assisted analysis using this software is a suitable tool for obtaining prognostic information for tumor-infiltrating immune cells (iDCs, mast cells, and NKs) in different regions of HCC after resection. Mast cells displayed the greatest AF in all regions of interest (ROIs). Mast cells in the peritumor region and IM showed a positive prognostic significance.

Author Spotlight: Investigating Immune Cell Dynamics in the Tumor Microenvironment &#8212; Challenges and Innovations in Cancer Prognosis

The presence of mast cells in the inner margin and peritumor areas of hepatocellular carcinoma after resection confers a favorable prognosis. This study endorses QuPath image analysis software as a promising platform that could meet the need for reproducibility, consistency, and accuracy in digital pathology.

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies ...