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 Injec...

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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 border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="847">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Anesthesia machine</td>
			<td style="width:123px;">VETEQUIP</td>
			<td style="width:344px;">IMPAC6</td>
			<td style="width:167px;">anesthesia machine for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butterfly needle</td>
			<td>BD</td>
			<td>8122963</td>
			<td>Needle used for liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice</td>
			<td>Jackson Lab</td>
			<td>000664&#160;</td>
			<td>mice used in prototol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td>CRESCENT CHEMICAL</td>
			<td>20402</td>
			<td>carprofen for pain release</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer&#160;</td>
			<td>CORNING</td>
			<td>REF 431751</td>
			<td>Cell strainer, 70&#181;m, for hepatocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-30R</td>
			<td>centrifuge for cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clips&#160;</td>
			<td>Teleflex Medical</td>
			<td>REF 523700</td>
			<td>Titanium Clips for spleen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td>Primovert&#160;</td>
			<td>microscope for cell observation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">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 height="21">
			<td height="21" style="height:21px;">Needle</td>
			<td>BD</td>
			<td>REF 305109</td>
			<td>BD precisionglide needle, 27G x 1/2 (0.4mm x 13mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture</td>
			<td>ETHICON</td>
			<td>J303H</td>
			<td>coated VICRYL suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SV40 T Ag antibody</td>
			<td>Abcam</td>
			<td>ab16879</td>
			<td>anti-SV40 T-antigen antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe</td>
			<td>BD</td>
			<td>REF 309626</td>
			<td>1 mL TB syringe for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td>SIGMA</td>
			<td>T 8154</td>
			<td>Trypan blue solution for cell viability test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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=...

Watch this Scientific Journal Video about An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis at JoVE.com

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 ...

Using just a model to develop a hepatocellular cancer mass model could help people elucidate their tumor-induced immunotolerance and specific treatment-induced, any tumor immunoresponses. The orthotopic tumor that develops with the liver fibrosis technique mimics the clinical features of human liver cancer. This method could provide a useful tool in hepatocellular cancer immunological study.Visual demonstration of this method can illustrate the more technically challenging steps, such as the intrasplenic injection, that are essential for a successful execution of this protocol. Next, manually restrain a male, six to eight-week old C57 black 6 mouse dorsal-side up. Use alternate 70%ethanol and Betadine scrubs to clean the injection site three times.Then deliver 160 microliters of carbon tetrachloride to the mouse by intraperitoneal injection and return the animal to its home cage. After confirming a lack of response to toe pinch, in a five-month old simian virus 40 T antigen transgenic mouse, place the mouse in the supine position and secure the limbs with tape. Make a midline laparotomy incision along the length of the linea alba, large enough to provide an adequate exposure of the liver, and displace the intestines to the left.Dissect above the liver to exposure the inferior vena cava. Induce an artery clamp to ligate the vessel. Use a butterfly needle to gain intravenous access to the portal vein and manually secure the catheter.Carefully switching syringes for each solution, successfully deliver 15 milliliters of solution one, 15 milliliters of 0.75%collagenase solution two, and 15 milliliters of solution two without collagenase via catheter, at an approximate 8.9 milliliters of solution per minute flow rate. At the end of the perfusion, harvest the tumor mass into a 50-milliliter conical tube containing 10 to 15 milliliters of PBS. Excise the tumor from the next MTD2 mouse as just demonstrated.Use scissors to cut the livers into smaller pieces. Transfer the tissues into a new container of fresh PBS to remove the rest of the blood from the tissue before transferring the tissue pieces into a 50-milliliter conical tube containing five milliliters of complete RPMI medium. Mince the washed liver samples until the pieces can fit easily through the tip of a five-milliliter pipette.Add enough medium to the tube to reach a final volume of 30 milliliters. Use a five-milliliter pipette to triturate the tissue fragments, before filtering the solution through a 70-micrometer strainer into a new 50-milliliter tube. Wash the strainer several times with complete medium.Adjust the final volume to 50 milliliters with additional complete medium. Quickly spin the suspension by centrifugation to a maximum of 50 times g before stopping the centrifuge and decanting the supernatant. Then resuspend the pellet in 20 milliliters of PBS for counting.For intrasplenic injection of the MTD2 hepatocytes, load a one-milliliter syringe equipped with a 27-gauge needle per each recipient animal with 220 microliters of hepatocytes per mouse. Confirm a lack of response to toe pinch in an anesthetized, carbon tetrachloride-treated animal. Beginning just below the spine muscle, next, make a one-centimeter incision on the left flank parallel to the thirteenth rib from the dorsal extreme and use blunt-pointed forceps to exteriorize the spleen.Clip the spleen with two medium-sized titanium clips between the splenic artery and vein, and deliver 200 microliters of cells into the inferior pole of the spleen. Clip the inferior branch of the pedicle with one medium-sized clip and cut the spleen between the two initially placed clips. Remove the inferior pole of the spleen that was directly injected with tumor cells and use 3-0 polyglactin 910 interrupted suturing to close the inner muscle layer.Then use sterilized steel wound clips to close the outer skin layer and subcutaneously administer five milligrams per kilogram of carprofen. After isolation from T antigen transgenic mice as demonstrated, both hepatocytes and tumor infiltrating cells can be observed. The cells typically demonstrate an almost 100%viability after processing.After intrasplenic inoculation into wild-type carbon tetrachloride-treated animals, the transplanted hepatocytes successfully and reliably grow orthotopic hepatocellular carcinoma tumors that express the tumor-specific simian virus 40 T antigen. The preparation of our viable and pure population of hepatocytes from MTD2 mass and accurate administration of the intrasplenic injection are important to the success of the procedure. Although our team has tested hepatocyte inoculation into recipient mice via intravascular and intraperitoneal injection, these other methods weren't able to induce orthotopic hepatocellular carcinoma.This model provides a unique platform enabling the investigation for immune surveillance and the tolerance in its early and the later stages of tumor progression. Please be careful when using the carbon tetrachloride, as it is toxic.

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Research

JoVE Journal

Cancer Research

8.6K Views.  University of Missouri-Columbia. Using just a model to develop a hepatocellular cancer mass model could help people elucidate their tumor-induced immunotolerance and specific treatment-induced, any tumor immunoresponses. The orthotopic tumor that develops with the liver fibrosis technique mimics the clinical features of human liver cancer. This method could provide a useful tool in hepatocellular cancer immunological study.Visual demonstration of this method can illustrate the more technically challenging steps, such ...