This work is supported by NIH/NCI R01 CA164335-01A1 (K. F. Staveley-O&#8217;Carroll, PI) and NIH/NCI R01CA208396 (Mark Kester, Guangfu Li, Kevin F. Staveley-O&#8217;Carroll).

NOTE: All the procedure including animal subjects have been approved by the IACUC at the University of Missouri. All mice received humane care according to the criteria outlined in the &#34;Guide for the Care and Use of Laboratory Animals&#34;. The following procedure for cell isolation and inoculation should be performed in a hood. All performers should wear the standard personal protective equipment for handling of the mice and tissue.
1. Induction of Liver Fibrosis and Cirrhosis with IP Injection of Carbon Tetrachloride (CCl4)
NOTE: See Figure 1. (CCl4 is highly hazardous reagent, it should be handled carefully and with wearing chemical-resistant gloves)
<ol>
	<li>Obtain male C57BL/6J mice that are six to eight weeks old (see Table of Materials).</li>
	<li>Prepare 10% CCl4 (v/v) solution in corn oil in a centrifuge tube. Determine total volume based on number of mice to be injected (see step 1.6).</li>
	<li>Use appropriate mice handling technique to select one mouse for injection.</li>
	<li>Manually restrain the mouse with its dorsal (abdomen) side up.</li>
	<li>Clean the injection site on abdominal wall of the mouse by scrubbing with 70% alcohol.</li>
	<li>Inject male C57BL/6J mice with 160 &#181;L of 10% CCl4 solution by intraperitoneal (IP) injection using a 25-gauge disposable needle.</li>
	<li>Ensure that the needle penetrates just through the abdominal wall (approximately 4-5 mm) with bevel-side up and slightly angled at 15-20 degrees.</li>
	<li>Inject mice twice a week for a total of four weeks-each mouse will receive a total of eight injections. 
	NOTE: Two weeks after the last injection, treated mice are ready for ISPL inoculation of oncogenic hepatocytes from MTD2 mice.</li>
</ol>
2. Isolating Tag-transgenic Hepatocytes from Line MTD2 mice
NOTE: See Table 1 for solution recipes.
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>10x Earle&#8217;s Balanced Salt Solution without Ca or Mg (EBSS without Ca or Mg)</td>
			<td>4 g KCl 
			68 g NaCl 
			1.4 g NaH2PO4&#183;H2O 
			10 g dextrose 
			Add water to 1 liter, pH to 4.32 
			Pass through filter</td>
		</tr>
		<tr>
			<td>Solution 1</td>
			<td>20 mL 10x EBSS without Ca or Mg 
			44 g NaHCO3 
			1.33 mL 1.5M Hepes 
			10 mL of 10 mL EGTA 
			Add water to 200 mL</td>
		</tr>
		<tr>
			<td>Solution 2</td>
			<td>100 mL 10x EBSS 
			2.2 g NaHCO3 
			6.67 mL 1.5 M Hepes 
			Add water to 1 liter</td>
		</tr>
		<tr>
			<td>0.75% collagenase solution</td>
			<td>15 mg collagenase type 1 
			20 mL of solution 2</td>
		</tr>
		<tr>
			<td>Complete Medium</td>
			<td>2 RPMI 
			50 mL FBS 
			5 mL 100x Penicillin-Streptomycin</td>
		</tr>
	</tbody>
</table>
Table 1: Solution recipes.
<ol>
	<li>Obtain line MTD2 mice38 to serve as the source of oncogenic hepatocytes.</li>
	<li>Anesthetize 5-week-old MTD2 mice using 2.5% isoflurane. 
	NOTE: Proper anesthetization will be checked by toe pinch method. In brief, using two fingers, give the mouse toe/foot a good squeeze. If there is no withdrawal reaction, the animal is judged deep enough to commence surgery.</li>
	<li>When adequately sedated, place mice in a supine position and fix the extremities with tape to provide adequate exposure of abdominal surface.</li>
	<li>Perform a midline laparotomy incision using scissors along the length of the linea alba large enough to provide an adequate exposure of the liver.</li>
	<li>Displace the intestines to the left to provide better exposure of the liver and portal triad.</li>
	<li>Dissect above the liver to expose the inferior vena cava (IVC).</li>
	<li>Ligate the IVC above the liver using an artery clamp.</li>
	<li>Returning to the inferior border of the liver, use a butterfly needle (see materials) to gain IV access to the portal vein. Fix catheter by hand.</li>
	<li>Successively perfuse the mouse liver using an injection syringe at 8.9 mL/min with with 15 mL of solution 1, 15 mL of 0.75% collagenase solution 2, and 15 mL of solution 2 via the catheter.</li>
	<li>Harvest the perfused liver by cutting and taking the tumor mass from MTD2 mice in a 50 mL conical tube with 10-15 mL of PBS.</li>
	<li>Remove PBS and wash an additional time with PBS; do not centrifuge at this step.</li>
	<li>Cut the liver into smaller pieces using scissors and then wash again with PBS &#160;2x to remove remaining blood.</li>
	<li>Add 5 mL of complete RPMI medium to the conical tube and continuously mince the liver with scissors to small pieces (&#60;3 mm)-tissue should smoothly go through a 5 mL pipette.</li>
	<li>Add complete RPMI to a final volume of 30 mL and suspend liver using a 5 mL pipette.</li>
	<li>Filter the mixed solution with a 70 &#181;m strainer into a 50 mL conical tube.</li>
	<li>Wash the strainer several times with complete RPMI and adjust the final volume to 50 mL by adding additional RPMI medium.</li>
	<li>Quickly spin the suspension by centrifuge to a maximum of 500 rpm; once the speed is accelerated to 50 x g, the centrifuge should be stopped.</li>
	<li>Decant the supernatant and suspend pellets in 20 mL of PBS.</li>
	<li>Count cells using Trypan blue exclusion and a hemocytometer, then adjust the cell concentration to 2.5 x 106/mL for the following cell inoculation. 
	NOTE: The expected yield from 5 grams of tumor tissue is 80 million hepatocytes with viability &#62;95%.</li>
</ol>
3. Inoculating the hepatocytes from MTD2 mice to the liver of wild type C57BL/6J mice by ISPL injection
<ol>
	<li>The aseptic technique should be used in all the procedures</li>
	<li>Anesthetize the CCl4-treated male C57BL/6J mice with 2.5% isoflurane, the mice should be treated with eye lube to prevent eyes from drying out.</li>
	<li>Prepare syringes with 200 &#181;L of hepatocytes for injection.</li>
	<li>When adequately sedated, position mice with left side up.</li>
	<li>Shave the entire left flank of the mice, then scrub the area, alternating between 70% alcohol and betadine three times.</li>
	<li>Administer 5 mg/kg of carprofen subcutaneouly prior to surgical incision.</li>
	<li>Make a 1 cm incision on the left flank parallel to the 13th rib from the dorsal extreme beginning just below the spine muscle.</li>
	<li>Identify the spleen, then exteriorize it using blunt-pointed forceps.</li>
	<li>Clip the spleen with two medium-sized titanium clips. Place both clips between the splenic artery and vein, leave room between the clips to cut later after inoculation. 
	NOTE: The goal is to isolate the inferior pole of the spleen to reduce risk of seeding.</li>
	<li>Inject 200 &#181;L (0.5 million) of the prepared hepatocytes into the inferior pole of the spleen using a 27 G needle.</li>
	<li>Clip the inferior branch of the pedicle (inferior splenic pole vessels) with one medium-sized clip.</li>
	<li>Cut the spleen between the two initially placed clips.</li>
	<li>Remove the inferior pole of the spleen that was directly injected with tumor cells.</li>
	<li>Use 3-0 polyglactin 910 interrupted suturing to close the inner muscle layer.</li>
	<li>Use sterilized steel wound clips to close the outer skin layer. 
	NOTE: Steel clips are preferred over sutures to avoid animals chewing out sutures, leaving a gaping wound.</li>
	<li>Administer 5 mg/kg of carprofen subcutaneously after suture.</li>
	<li>Place all recovering animals on a temperature-controlled heating pad and monitor closely until fully recovered from anesthesia.</li>
	<li>Give mice free access to water after surgery. If the mouse becomes dehydrated during surgery, administer subcutaneous fluids (&#60;1 mL).</li>
	<li>Remove skin clips at 7-10 days post-operatively.</li>
</ol>

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</ol>

There are none to declare.

With this protocol, we have established a reliable and reproducible murine model of HCC that mimics human HCC initiation and progression. Clinically, many risk factors successively induce liver injury, liver fibrosis, cirrhosis and the final stage of HCC. In our protocol, IP injection of CCl4 is used to first produce liver fibrosis in wild type mice, which allows the subsequent oncogenic hepatocytes to form the tumors in the setting of liver fibrosis. We found that tumor formation occurred most successfully in...

Hepatocellular cancer (HCC) is the most rapidly increasing type of cancer in the United States (US)1,2,3. Every year, approximately 850,000 new cases are diagnosed4,5 and 700,000 patients die from this lethal disease6,7,8,9,10, making it the second-highest cause of cancer-related death worldwide. Management of HCC includes surgical resection, transplantation, ablation, chemoembolization, or systemic therapies, such as sorafenib11. Early diagnosis and management with surgical resection or transplantation have the highest overall survival benefit4. Unfortunately, the majority of patients present at a later stage and require management with ablation, chemoembolization or sorafenib12. Sorafenib, a receptor tyrosine kinase inhibitor (RTKI), was approved by the Food and Drug Administration in 2008 as the only systemic drug therapy available for treating unresectable HCC. Although the drug only provides a modest increase in overall survival, from 7.9 to 10.7 months13, it provided a new therapeutic strategy that could be utilized to manage HCC.
Manipulating the immune system to eliminate established cancers is a rapidly growing field in cancer research14. Immune checkpoint studies have considerably advanced immunotherapeutic drug development in cancer treatment15,16. The FDA approved the use of antibodies (Abs) against cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and its ligand PD-L1 for the treatment of melanoma, lung cancer, head and neck cancer, and bladder cancer17,18,19,20. Clinical trials of monotherapy or combination therapy using one or multiple antibodies against PD-1, PD-L1, or CTLA-4 for the treatment of advanced HCC are ongoing21,22,23, and some trials have shown favorable results. In 2017, the FDA granted accelerated approval for anti-PD-1 antibody to treat HCC patients, who are resistance to sorafenib, but the overall response rate of this therapy is only 14.3%. Other strategies have not been translated into clinical practice at this time24,25. Overcoming tumor-induced profound immune tolerance to improve immune checkpoint therapy26; predicting efficacy of immune checkpoint therapy; preventing immune-related adverse events; optimizing administration route, dosage, and frequency; and finding effective combinations of therapies27,28,29 all remain extremely challenging tasks.
There are several conventional approaches used to induce HCC in mouse models currently and are utilized depending on the investigator&#39;s particular research question30. Chemically-induced HCC mouse models with genotoxic compounds mimic injury-induced malignancy. Xenograft models through either ectopic or orthotopic implantation of HCC cell lines are suitable for drug screening. A number of genetically modified mice have been engineered to investigate the pathophysiology of HCC. Transgenic mice expressing viral genes, oncogenes and/or growth factors allow the identification of pathways involved in hepatocarcinogenesis. Due to inherent limitations, these models do not recapitulate the typical immune characteristics seen in human HCC, which has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies14,15. We recently created a clinically relevant murine model. This novel model not only mimics human HCC initiation and progression but also reflects most typical features of human disease including immune dysfunction. We have characterized its biological and immunological characteristics. Leveraging this novel model, we have explored various immunotherapeutic strategies to treat HCC31,32,33,34,35,36,37. This unique platform allows us to study mechanisms of tumor-induced immunotolerance and to develop proof-of-concept therapeutic strategies for HCC toward eventual clinical translation.

<table><tbody><tr><td>Anesthesia machine</td><td>VETEQUIP</td><td>IMPAC6</td><td>anesthesia machine for surgery</td></tr><tr><td>Butterfly needle</td><td>BD</td><td>8122963</td><td>Needle used for liver perfusion</td></tr><tr><td>C57BL/6 mice</td><td>Jackson Lab</td><td>000664&amp;nbsp;</td><td>mice used in prototol</td></tr><tr><td>Carprofen</td><td>CRESCENT CHEMICAL</td><td>20402</td><td>carprofen for pain release</td></tr><tr><td>Cell Strainer&amp;nbsp;</td><td>CORNING</td><td>REF 431751</td><td>Cell strainer, 70&amp;micro;m, for hepatocytes isolation</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Allegra X-30R</td><td>centrifuge for cell isolation</td></tr><tr><td>Clips&amp;nbsp;</td><td>Teleflex Medical</td><td>REF 523700</td><td>Titanium Clips for spleen</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Primovert&amp;nbsp;</td><td>microscope for cell observation</td></tr><tr><td>Mtd2 mice</td><td>N/A</td><td></td><td>Gift from Dr. William A Held at roswell Park Cancer Institute in 2002, maintained in our lab</td></tr><tr><td>Needle</td><td>BD</td><td>REF 305109</td><td>BD precisionglide needle, 27G x 1/2 (0.4mm x 13mm)</td></tr><tr><td>Suture</td><td>ETHICON</td><td>J303H</td><td>coated VICRYL suture</td></tr><tr><td>SV40 T Ag antibody</td><td>Abcam</td><td>ab16879</td><td>anti-SV40 T-antigen antibody for IHC</td></tr><tr><td>Syringe</td><td>BD</td><td>REF 309626</td><td>1 mL TB syringe for cell injection</td></tr><tr><td>Trypan blue</td><td>SIGMA</td><td>T 8154</td><td>Trypan blue solution for cell viability test</td></tr><tr><td>Wound clips</td><td>Reflex</td><td>reflex9, Part. No. 201-1000</td><td>stainless steel wound clips for wound close</td></tr></tbody></table>

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.

Oncogenic hepatocytes isolated from TAg-transgenic mice (Figure 2) were seeded in the liver of wild type mice by intra-splenic injection (Figure 3). The transplanted hepatocytes successfully and reliably grew orthotopic HCC tumors (Figure 4) with tumor specific antigen SV40 TAg (Figure 5) in the setting of hepatic inflammation and fibrosis (Figure 1).
<img alt="Figure 1" class=...

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

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 ...

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JoVE Journal

Cancer Research

8.8K Views.  University of Missouri-Columbia. Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

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

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

Medicine

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

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 ...

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 ...

Patient&#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC.
Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation.
In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

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

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

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

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

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Immunology and Infection

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

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

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

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

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

Mouse Extended Hepatectomy Model for Liver Regeneration Studies

Extended 78&#37; Hepatectomy in a Mouse Surgical Model

Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of disease models. In the classical partial 2/3 hepatectomy in mice, two of the five liver lobes, namely the left and median lobes representing approximately 66% of the liver mass, are resected en bloc with an expected postoperative survival of 100%. More aggressive partial hepatectomies are technically more challenging and hence, have seldom been used in mice. Our group has developed a mouse model of an extended hepatectomy technique in which three of the five liver lobes, including the left, median, and right upper lobes, are resected separately to remove approximately 78% of the total liver mass. This extended resection, in otherwise healthy mice, leaves a remnant liver that cannot always sustain adequate and timely regeneration. Failure to regenerate ultimately results in 50% postoperative lethality within 1 week due to fulminant hepatic failure. This procedure of extended 78% hepatectomy in mice represents a unique surgical model for the study of small-for-size syndrome and the evaluation of therapeutic strategies to improve liver regeneration and outcomes in the setting of liver transplantation or extended liver resection for cancer.

Author Spotlight: Evaluating Therapeutic Strategies to Enhance Liver Regeneration

The mouse model of partial 2/3 (66%) hepatectomy is well described in the literature, but more extended hepatectomies mimicking small-for-size syndrome after liver transplantation have seldom been used. We describe an extended 78% hepatectomy procedure in a mouse model that results in approximately 50% postoperative lethality in healthy mice.

Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of ...

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

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