Name: an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis
Uploaded: 2019-09-12T13:00:00
Description: 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

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...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma+-+United+States.">Hepatocellular carcinoma - United States.</a> Morbidity and Mortality Weekly Report. 59, 517-520 (2010).</li><li>Petrick, J., Kelly, S., Altekruse, S., McGlynn, K., Rosenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+of+Hepatocellular+Carcinoma+Incidence+in+the+United+States+Forecast+Through+2030.">Future of Hepatocellular Carcinoma Incidence in the United States Forecast Through 2030.</a> Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 34, 1787-1794 (2016).</li><li>Greten, T., Lai, C., Li, G., Staveley-O'Carroll, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+and+Immune-based+Therapies+for+Hepatocellular+Carcinoma.">Targeted and Immune-based Therapies for Hepatocellular Carcinoma.</a> Gastroenterology. 156, 510-524 (2019).</li><li>Llovet, J., Zucman-Rossi, J., Pikarsky, E., Sangro, B., Schwartz, M., Sherman, M., Gores, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+Carcinoma.">Hepatocellular Carcinoma.</a> Nature reviews. 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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">
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		<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

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

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

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

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

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

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

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

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

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

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

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

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

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

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

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

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 ...