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

Summary

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.

Abstract

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.

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the most rapidly increasing cause of cancer death in North America. The most prevalent risk factor for HCC is liver cirrhosis, most frequently occurring due to chronic viral hepatitis, alcohol misuse, autoimmune disease, or hereditary metabolic disorders ¹.

Despite the heavy disease burden imposed by HCC on populations worldwide, the pathophysiology of HCC is relatively poorly understood in comparison to other common cancers such as colorectal, breast, or prostate cancer. For example, specific molecular and cellular events driving tumorigenesis remain to be clearly defined ². Like most other solid epithelial cancers, genomic approaches have revealed heterogeneity in the aberrations associated with HCC ³. A number of studies have revealed disordered activity of a variety of signaling pathways involved in cell proliferation, survival, differentiation, and angiogenesis ⁴. In addition, the role of cancer stem cells in HCC pathobiology remains to be clarified ⁵.

With a limited understanding of HCC pathophysiology, the armamentarium of effective therapies for HCC has also remained relatively limited. Early-stage patients with tumors confined to the liver are candidates for curative therapy using tumor ablation or surgical resection, though recurrence is common. For patients with more advanced disease, chemotherapy and radiation are of limited efficacy and are used primarily for disease control with palliative intent ⁶.

High quality in vivo experimental models of human HCC thus provide a valuable platform for much needed basic research into the pathophysiology of human HCC as well as for evaluation of novel therapeutic approaches. As compared with the use of cell lines or highly defined mouse models, xenografts of primary human tumors in immunodeficient mice have emerged as valuable tools for such studies since they are capable of recapitulating the human disease with high fidelity while also capturing the heterogeneity that is present within and between different patients ^7,8. To this end, we have developed a variety of methods to establish human HCC xenografts in immunodeficient mice. While the majority of published studies involving HCC xenografts describe the use of well-established human HCC cell lines for this purpose, we have focused on optimizing our assays to generate xenografts from primary HCC specimens obtained immediately after surgical resection from patients.

Different xenografting techniques may be required for different research applications. For example, subcutaneous xenografts generated from tumor fragments are generated rapidly, are easily monitored, and may be more appropriate for local administration of novel therapeutics with convenient monitoring of tumor response. Intrahepatic xenografts may be more relevant for studies pertaining to the role of the hepatic microenvironment in HCC biology. Xenografts generated from tumor cell suspensions are necessary for the identification and characterization of tumor-initiating cell subsets or for experiments requiring in vitro manipulations of tumor cells prior to xenotransplantation. We have thus developed and validated the following protocols to establish subcutaneous or intrahepatic xenografts from cell suspensions or tumor fragments derived from primary human HCC specimens.

Protocol

A schematic overview of the protocol is presented in Figure 1.

1. Processing of Human HCC Samples

Obtain primary human HCC specimens with written patient consent and with the approval of the institutional research ethics board. These protocols have been carried out at our institution with approval from the University Health Network Research Ethics Board in compliance with all institutional, national, and international guidelines for human welfare.

Collect fresh HCC specimens as soon as possible following the surgical procedure once appropriate samples have been taken for clinical purposes. Ideally this should take place within 30 min after removal of the tissue from the patient. As illustrated in Figure 2, a sample of at least 1 cm³ obtained from the periphery of the tumor is optimal, as the central portion of the tumor may be necrotic. Tumors that have not received any treatment prior to resection such as radiation, chemotherapy, or ablation are preferred in order to maximize the chance that tumor cells are viable. Handle primary human tissues in accordance with standard personal protective protocols for biohazardous material. Perform all laboratory manipulations of tumor tissues and cell preparations in a class II biosafety cabinet using aseptic techniques.

1. Place the fresh HCC sample in 10-25 ml serum-free Dulbecco's Modified Eagle Medium/Ham's F12 at 4 °C and transfer on ice to the laboratory for immediate processing and preparation of tissue fragments and/or cells for xenografting into mice.
2. Using sterile forceps, place the tumor sample in a 100 mm x 20 mm Petri dish or other suitable sterile working surface. Divide the tumor sample into fragments of approximately 2-3 mm³ using a No.10 surgical scalpel blade. At this point, consider snap-freezing or formalin-fixing some tumor fragments for other experiments or analyses as required.
3. For xenografting of tumor fragments, place some of the HCC fragments into one or more microcentrifuge tubes containing enough Matrigel to allow the fragments to remain submerged. Keep these tubes on ice.
4. For preparation of a tumor cell suspension, use the surgical scalpel blade to mince the remaining HCC tissue as much as possible and mix with 5-10 ml of DMEM-F12 in a 50 ml conical tube depending on the volume of the minced tissue.
5. Add collagenase type IV and dispase II at final concentrations of 200 units/ml and 0.8 units/ml respectively. Pipette the mixture up and down well using a 25 ml pipette.
6. Seal the tube and incubate the mixture from step 1.6 at 37 °C in a 5% carbon dioxide incubator for 30-60 min depending on the softness of the tumor tissue. Pipette the mixture up and down a few times every 10 min to assess the progress of the enzymatic digestion.
7. After digestion is complete, pass the tumor solution through a 100 μm cell strainer. Gently mash the remaining tissue on the cell strainer using a 25 ml pipette tip to enable the maximum number of tumor cells to pass through. Collect the strained cell suspension in a 15 ml conical tube.
8. Centrifuge the tumor cell suspension at 1,200 rpm for 5 min at 4 °C.
9. Gently decant the supernatant. Depending on the size of the pellet, add 2-5 ml of ice cold 1x red blood cell (RBC) lysis buffer and gently pipette up and down to resuspend the pellet. Keep on ice for 5 min.
10. Add DMEM-F12 to a total volume of 15 ml and centrifuge at 1,000 rpm for 5 min at 4 °C to wash out the RBC lysis buffer.
11. Gently decant the supernatant and resuspend the RBC-free tumor cell pellet in DMEM-F12.
12. Count the viable cells using trypan blue exclusion either manually or with an automated cell counter.
13. Centrifuge tumor cell aliquots containing the desired number of cells for injection as described above, resuspend the resulting cell pellet in 30 μl of ice-cold Matrigel, and store on ice.

Optional: After step 1.11, we routinely deplete human CD45+ cells (leukocytes) from the bulk tumor cell suspension and/or purify subsets of tumor cells using flow cytometry or immunomagnetic beads. Detailed protocols for these techniques are well described by the manufacturers of the relevant antibodies, beads, and flow cytometers.

Note: The protocol described above can also be used to process human tumor xenografts harvested from mice in order to perform serial transplantation, substituting the xenograft tissue for the primary human HCC tissue in step 1.1. In this situation, after step 1.11, we routinely deplete infiltrating murine cells from the cell suspension using an antibody against the mouse histocompatibility antigen H2k.

2. Xenografting

Conduct all animal procedures in compliance with protocols approved by the institutional animal care committee. The procedures described herein have been completed under a specific Animal Use Protocol approved by the University Health Network Animal Care Committee in accordance and compliance with all relevant regulatory and institutional agencies, regulations, and guidelines.

Equipment for delivery of inhalational volatile anesthetic agents to small animals should be utilized according to standard operating procedures of the animal facility and research institute. Carry out all surgical procedures using aseptic technique and sterile instruments in a class II biosafety cabinet. Utilize non-obese diabetic severe combined immunodeficiency (NOD/SCID) or NOD/SCID/interleukin 2 receptor gamma chain null (NSG) strains of mice of either sex at 6-8 weeks of age (The Jackson Laboratory, Bar Harbor, ME) ^9,10. These mice must be housed and maintained in a facility capable of providing pathogen-free conditions suitable for immunodeficient animals.

Prepare mice for surgery in a chamber supplying 5% (v/v) inhaled Isoflurane in 1 L/min of oxygen. Maintain anesthesia until there is loss of corneal and toe reflex in the animal(s). For subcutaneous xenografting, shave one or more small areas on the dorsum of the animal(s) and cleanse the skin with 70% ethanol. For intrahepatic xenografting, shave the ventral thorax and abdomen of the animal(s) from the axillae down to the inguinal region and cleanse the skin with 70% ethanol.

2.1 Subcutaneous Implantation of Tumor Fragments

1. Place the anesthetized mouse prone, maintaining inhalational Isoflurane anesthesia (2% (v/v) in 1 L/min O₂) with a mouthpiece. Apply Tear-Gel to protect the animal's eyes from traumatic injury.
2. Sterilize the dorsal shaved area(s) with Betadine surgical scrub followed by 70% ethanol and lastly with povidone-iodine solution.
3. Make a 5 mm skin incision using sterile sharp scissors.
4. Insert a closed blunt scissor gently into the subcutaneous space and spread gently to develop a pocket large enough to accommodate a tumor fragment.
5. Insert the tumor fragment prepared in step 1.3 into the subcutaneous pocket using sterile fine forceps.
6. Close the skin incision using sutures or clips.
7. Provide postoperative care to the mouse as described below.

2.2 Subcutaneous injection of tumor cells

1. Prepare the animal as described in steps 2.1.1 and 2.1.2.
2. Load the suspension of tumor cells in Matrigel prepared in step 1.13 into an insulin syringe with a 29 G 1/2 in needle.
3. Insert the needle into the subcutaneous space and discharge the contents of the syringe. Advancing the needle several millimetres along the subcutaneous plane away from the skin puncture site prevents leakage of the tumor cell suspension out of the puncture site upon withdrawal of the needle.
4. Provide postoperative care to the mouse as described below.

2.3 Intrahepatic Implantation of Tumor Fragments

1. Using a 27 G 1/2 in needle on a 1 ml syringe, administer 350 μl of sterile normal saline solution subcutaneously in the dorsum of the anesthetized animal's neck to compensate for intraoperative fluid losses. For analgesia, administer 350 μl of sterile normal saline containing buprenorphine (0.1 mg/kg) subcutaneously on the animal's flank, using a 27 G 1/2-in needle on a 1 ml syringe.
2. Place the mouse supine on a pre-heated pad with the nose and mouth positioned inside the mouthpiece to deliver maintenance inhalational Isoflurane anesthesia (2% (v/v) in 1 L/min O₂).
3. Extend the limbs and secure them with tape to the operating surface to optimize exposure of the ventral abdomen and thorax.
4. Perform the procedure under a magnifying lamp to optimize visualization.
5. Sterilize the shaved skin on the ventral abdomen and thorax with Betadine surgical scrub followed by 70% ethanol and lastly with povidone-iodine solution.
6. Using sterile sharp scissors, make a transverse bilateral subcostal skin incision and divide the muscle layers to enter the peritoneal cavity to allow adequate exposure of the entire liver.
7. Place a stitch in the skin above the xiphoid process and secure it to the mouthpiece with tape in order to allow better exposure of the liver and surrounding structures.
8. Use two cotton-tipped applicators adjacent and posterior to the liver to stabilize it.
9. Make an incision 3 mm in length and depth on the surface of the liver using a sterile No. 10 scalpel blade.
10. Immediately apply Surgicel and gentle pressure to the incision site to achieve hemostasis; remove after 60-90 sec and proceed only if complete hemostasis has been achieved.
11. Place a tumor fragment step 1.3 into the liver incision with sterile fine forceps or with an 18 G needle.
12. Apply a small piece of Surgicel over the liver incision to prevent displacement of the tumor fragment and ensure continued hemostasis.
13. Close the incision with sutures or clips.
14. Provide postoperative care as described below

2.4 Intrahepatic Implantation of Tumor Cells via Direct Injection into the Liver

1. Prepare the mouse as described in Steps 2.3.1 to 2.3.7 above.
2. Load the tumor cell suspension (prepared in step 1.13) into an insulin syringe using a 29 G 1/2 in needle.
3. With the liver stabilized using a cotton-tip applicator, insert the insulin syringe needle into the liver and advance the tip a few millimetres beyond the puncture site along a subcapsular plane.
4. Gently discharge the contents of the syringe and then remove the needle from the liver.
5. Place Surgicel over the puncture site and apply gentle pressure with a cotton-tipped applicator to prevent leakage of the tumor cell suspension and to achieve complete hemostasis.
6. Close the incision with sutures or clips and provide postoperative care as described below.

2.5 Intrahepatic Xenografting of Tumor Cells via Injection into the Spleen

1. Prepare the mouse as described in steps 2.3.1 to 2.3.5 above.
2. Using sterile sharp scissors, make a 1 cm left subcostal incision to enter the peritoneal cavity.
3. Use a cotton-tipped applicator to reflect the stomach cranially and to the animal's right side in order to expose the spleen.
4. By handling the surrounding adipose tissues with sterile fine atraumatic forceps, deliver the spleen into the incision and place a cotton-tipped applicator behind the spleen to stabilize it.
5. Using 5-0 silk suture, place a loose pre-tied knot around the spleen above the lower pole.
6. Load the tumor cell suspension (prepared in step 1.13) into an insulin syringe using a 29 G 1/2 in needle.
7. Insert the insulin syringe needle into the lower pole of the spleen and advance it past the level of the loose pre-tied knot.
8. Slowly discharge the contents of the syringe, remove the needle from the spleen, and tighten the knot to prevent any leakage of the injected tumor cell suspension.
9. Replace the spleen into the peritoneal cavity.
10. Close the incision with sutures or clips and provide postoperative care as described below.

2.6 Partial Hepatectomy to Facilitate Intrahepatic Engraftment of Human Tumor Tissue

1. Prepare the mouse as described in steps 2.3.1 to 2.3.7.
2. With sterile sharp scissors, divide the falciform ligament attached to the median lobe of the liver.
3. Using a cotton-tipped applicator, mobilize the left lobe of the liver and position a loose pre-tied knot of 5-0 silk suture around the left lobe. Advance the loose knot as close as possible towards the bilio-vascular pedicle of the left lobe and tighten the knot.
4. Excise the left lobe distal to the ligature using sterile scissors, leaving a small stump to prevent slippage of the knot and subsequent hemorrhage.
5. Using a similar technique, ligate and resect the majority of the median lobe of the liver, avoiding the gallbladder.
6. Use Surgicel and gentle pressure with a cotton-tipped applicator along the cut surfaces of the liver parenchyma to achieve complete hemostasis.
7. For intrahepatic xenografting of a tumor fragment or tumor cells, proceed with steps 2.3.8 to 2.3.11 or 2.4.2 to 2.4.5 as described above.
8. For intrahepatic xenografting of tumor cells via splenic injection, utilize cotton-tipped applicators to gently reflect the stomach cranially and to the animal's right side, exposing the spleen. Proceed with steps 2.5.4 to 2.5.9 as described above.
9. Close the incision with sutures or clips and provide postoperative care as described below.

2.7 Postoperative Care

1. Remove the mouse from the inhalational anesthesia mouthpiece.
2. Place the mouse in a cage under a heat lamp for approximately 20 min until recovered from anesthesia and mobilizing fully.
3. Repeat the buprenorphine dose every 8-12 hr during the first 2-3 postoperative days.

Results

Figure 3 demonstrates the typical appearance of a subcutaneous human HCC xenograft and the corresponding histopathological appearance of the tumor. The development and growth of subcutaneous xenografts can be readily monitored by daily examination of recipient mice. The time interval between xenografting and development of a tumor may vary greatly depending on the type of tissue (tumor fragment vs. cell suspension), source of tissue (primary patient sample, passaged xenograft, or cell line), and quantity...

Discussion

We have described a variety of techniques to establish subcutaneous and intrahepatic human HCC xenografts in immunodeficient mice that can be applied to a wide variety of experimental questions and assays. While subcutaneous xenografts have been widely used to study various aspects of HCC biology, intrahepatic xenografts are rarely described in the literature. Furthermore, the majority of studies describing the use of xenografts have generated these from well established cell lines. Given the limitations of cancer cell l...

Materials

Name	Company	Catalog Number	Comments
Dulbecco’s Mod. Eagle Medium/Ham’s F12 50/50 Mix x1(DMEM-F12)	WISENT Bioproducts	319-075-CL
Collagenase TypeIV	Sigma-Aldrich	C5138
Dispase II	Stemcell Technologies	7923
Matrigel Matrix	Becton-Dickinson Biosciences	354234
10 % Buffered Formalin solution	Sigma-Aldrich	HT501128
0.9 % Saline Solution (NaCl), sterile	House Brand	1011-L8001
Betadine surgical scrub	Purdue Pharma	NPN 00158313
Buprenorphine (Temegesic) NR 0.3 mg/ml	Reckitt Benckiser
Isoflurane USP, 99.9 %, inhalation anesthetic	Pharmaceutical Partners of Canada Inc.	M60302
Tear-Gel	Novartis Pharmaceuticals
Frozen section compound	VWR	95057-838
Cryomold, Tissue -Tek	Sakura Finetek	4566
Precision Glide Needle 18G 1 ½	Becton-Dickinson Biosciences	305196
Precision Glide Needle 27G ½	Becton-Dickinson Biosciences	305109
Insulin syringe, 3/10 cc U-100, 29G½	Becton-Dickinson Biosciences	309301
Surgical blade No.10	Feather Safety Razor Co.	08-916-5A
#5-0 Soft silk surgical suture, 3/8" taper point needle	Syneture	VS-880
Transpore surgical tape	3M Health care	1577-1
Cotton applicator	Medpro	018-425
Surgicel, oxidized regenerated cellulose	Ethicon	1951
Cell strainer 100 μm nylon	Becton-Dickinson Biosciences	352360
Magnification lighting with mobile base	Benson medical Industries Inc.	model: RLM-CLT-120V
Petridish sterile 100x20 mm	Sarstedt	821474
Tissue forcep, 1x2 teeth, 4-1/2"	Almedic	A10-302
Adson dressing forcep 4-3/4"	Almedic	A10-220
Eye dressing forcep, serrated, straight, 4"	Almedic	A19-560
Hartman Hemostatic Forceps, curved, 3-1/2"	Almedic	A12-142
Iris scissor, curved, 4-1/4"	Almedic	A8-690
Iris scissor, straight, 4-1/2"	Almedic	A8-684
Olsen-Hegan needle driver, 5-1/2"	Almedic	A17-228