Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.
Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.
Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide, and the third leading cause of cancer-related death, making it a significant health concern1,2. For patients who are not eligible for tumor resection, or those awaiting liver transplantation, locoregional therapy involving transarterial embolization (TAE) or transarterial chemoembolization (TACE) are applied as standard palliative care3,4. These therapies exploit the unique feature of dual blood supply in the liver whereby tumors are fed almost exclusively by hepatic arterial blood flow, while the surrounding liver receives the majority of its blood supply from the portal vein5,6.
Due to the extremely limited efficacies of established therapies for HCC, oncolytic viruses have emerged as promising alternative therapeutics. JX-594, recently renamed Pexa-Vec, is a thymidine kinase-deleted vaccinia vector, armed with granulocyte-macrophage colony-stimulating factor (GM-CSF), which has completed phase II clinical trial for HCC7. More recently, a recombinant vesicular stomatitis virus vector (VSV) expressing human interferon-beta has entered a phase I clinical trial for sorafenib-refractory HCC (NCT01628640). As oncolytic viruses move closer to obtaining approval for clinical application for HCC in patients, the need for an effective administration route to target multifocal disease is evident. While systemic delivery is largely ineffective due to inefficient tumor transduction, intratumoral applications could limit the efficacy of the therapy to the injected tumor, leaving uninjectable microscopic lesions susceptible to disease progression.
We have established a method of isolating the hepatic artery in rats to administer oncolytic virus therapy in a locoregional manner to target orthotopic HCC. We have demonstrated that this administration route results in safe and effective transduction of multifocal HCC nodules, resulting in significant survival prolongation in immune competent rats8-10. Here, we describe the method of accessing, dissecting, and injecting into the hepatic artery in rats. A scheme of the procedure is shown in Figure 1 (previously published9)
Note: The following steps have been performed in accordance with the guidelines of our institution and the local government of Bavaria, Germany. All attempts to reproduce this protocol must be made in adherence with the local guidelines for the humane treatment of animals, as dictated by local animal care and use committee. For example, many institutes require that sterile gloves are worn during rodent surgery. Furthermore, work with viruses must be performed according to local regulations, taking care for personal and environmental safety. Care must be taken to properly dispose of contaminated waste and to clean instruments and workspace (i.e., by autoclaving and cleaning with an appropriate antiviral disinfectant according to the manufacturer's instructions).
1. Preparations Before Beginning Surgery
2. Preparation of the Rat
3. Laparotomy
4. Isolation of the Hepatic Artery
5. Preparation of the Artery for Injection
6. Closing
With experience, a nearly 100% success rate (meaning that the artery was successfully dissected and injected, and the animal survived surgery) can be achieved. However, the health status of the rat prior to surgery (degree of tumor burden, underlying liver function, etc.) will obviously play a role in the outcome of the surgical intervention. Following surgery, rats can be expected to experience transient weight loss and a slight, transient elevation of liver enzymes, even if buffer alone, without virus, was injected.
We have previously demonstrated that trans-hepatic arterial administration of oncolytic vesicular stomatitis virus (VSV) or Newcastle disease virus (NDV) results in efficient tumor transduction and tumor-specific virus replication, in an orthotopic, multifocal HCC model8,9. Although the two viruses differ in their intratumoral kinetics of virus replication, at the respective time-points of maximal virus replication, numerous foci of virus propagation, as evidenced by positive enzymatic staining for the LacZ reporter gene, could be observed exclusively within HCC nodules, while the surrounding livers showed no evidence of virus replication (Figure 3 and 4, previously published data8,9). Additionally, morphometric analysis of randomly selected VSV-treated tumor nodules demonstrated that tumor transduction efficiency does not correlate with tumor size (Figure 3).
Furthermore, the ability to deliver therapeutic agents through the hepatic artery also offers the possibility to combine therapy with an embolization agent as an alternative to TACE. We have demonstrated that, by mixing oncolytic VSV at a 1:1 ratio with a degradable starch microsphere (DSM) embolization agent and infusing the suspension through the hepatic artery, we could achieve massive tumor necrosis and very significant survival prolongation in rats bearing multifocal HCC nodules11. Trans-hepatic arterial administration of the DSM resulted in a nearly complete embolization of tumor nodules, while having a minimal effect on the perfusion of normal liver tissue, as demonstrated by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using a gadolinium contrast agent (Figure 5, previously published data11).
Figure 1. Schematic representation of hepatic arterial infusion procedure. The hepatic vessels (common hepatic artery, proper hepatic artery, and gastroduodenal artery) are dissected with the aid of a dissecting microscope. After ligation of the gastroduodenal artery and temporal block of the common hepatic artery, 1 ml of PBS or VSV vector are administered via the gastroduodenal artery through the proper hepatic artery. After injection, the proximal site of the gastroduodenal artery is ligated to prevent bleeding, the block of the common hepatic artery is released, and the recommencement of hepatic blood flow is confirmed. This figure has been previously published9. Please click here to view a larger version of this figure.
Figure 2. Photographs of the hepatic arterial infusion procedure. A laparotomized rat demonstrates the placement of the abdominal wall retractor and gauze swabs (A). After lifting out the hepatic lobes, the common hepatic artery (designated CHA) and gastroduodenal artery (designated GA) are identified and dissected (B). After ligating the distal end of the gastroduodenal artery, the common hepatic artery is temporarily clamped (C). The gastroduodenal artery is now ready for injection. Please click here to view a larger version of this figure.
Figure 3. Transduction and tumor-selective replication of VSV after hepatic artery administration in rats bearing multifocal HCC. Sets of animals (n = 3/time point) were sacrificed (A) at 30 min and (B) at day 1 post-virus infusion, and frozen liver sections were stained for β-gal expression. Representative sections are shown at low (left) and higher magnifications (right). Arrows indicate the borders between tumor lesions and hepatic parenchyma. (C) Relationship between tumor size and transduction efficiency. HCC-containing liver sections were analyzed by morphometric analysis to quantify total tumor and X-gal-positive areas. This figure has been previously published9. Please click here to view a larger version of this figure.
Figure 4. NDV/F3aa(L289A) treatment results in tumor-specific virus replication in hepatocellular carcinoma (HCC)-bearing rats. Male Buffalo rats bearing multifocal orthotopic HCC nodules were treated with rNDV/F3aa(L289A) (108 TCID50) by hepatic arterial infusion, and killed at the indicated time points post-treatment (n = 3). Tumor-containing liver sections were subjected to β-gal staining. Representative sections are shown at 5X (overview) and 20X (magnification of tumor and liver sections) magnification. Viral titers were quantified by TCID50 analysis of liver and tumor lysate, and are expressed as the mean ± SD. NDV, Newcastle disease virus; TCID50, 50% tissue culture infectious dose. Note, this Figure has been modified from its original form8. This figure has been previously published8. Please click here to view a larger version of this figure.
Figure 5. Dynamic contrast-enhanced magnetic resonance imaging of embolized HCC. McA-RH7777 tumor-bearing rats, either nonembolized or 30 min after hepatic arterial embolization with DSM, were imaged with DCE-MRI. (A) Axial T1-weighed precontrast (a, c) and postcontrast (b, d) images show lack of contrast in embolized tumor nodules (d). (B) Data were quantitatively analyzed by measuring gray-scale signal intensities in equal-sized regions of interest of tumor nodules and adjacent normal liver tissue. One representative data set is shown. This figure has been previously published11. Please click here to view a larger version of this figure.
Although direct intratumoral injection is undoubtedly the simplest method to result in efficient tumor transduction of a single tumor nodule, hepatic arterial infusion represents an ideal administration route to target multifocal, orthotopic HCC. This method has proven to be both safe and effective for treating HCC in immune competent rats with oncolytic viruses. Furthermore, since HCC patients are routinely treated by transarterial application of chemoembolization, the method described here is readily translatable to a clinical setting.
An obvious disadvantage of hepatic arterial delivery of tumor therapies in preclinical rodent models is the invasiveness of the procedure and the time required, both for the surgical intervention and the post-operative care of the rats. Researchers wishing to establish this technique must invest a substantial amount of time in becoming proficient at the procedure to produce reproducible results and a high survival rate. Furthermore, due to the extremely small diameter of the hepatic artery, to date there have been no reports of hepatic arterial infusions in mice. This is unfortunate, since the genetic models of HCC are primarily generated in mice, and we are therefore limited to testing hepatic arterially applied therapies in implantation or chemically induced HCC models in rats. Chemical induction of HCC is also associated with general liver toxicity, which would potentially preclude the rats from eligibility for receiving this therapeutic intervention due to safety concerns.
As an alternative to the invasive laparotomy approach to hepatic arterial virus delivery, it is theoretically feasible to feed a catheter through the femoral artery and up through the hepatic aorta and into the hepatic artery in rats, which is the standard procedure in patients. Although this approach would provide the huge benefit of avoiding surgical intervention, it is also technically challenging to establish and would require the aid of an experienced interventional radiologist.
Finally, it is noteworthy to mention that the procedure described here, although established for the purpose of efficient delivery of oncolytic viruses to orthotopic HCC, can be applied to any therapeutic agent for locoregional delivery to hepatic lesions, such as chemotherapy, immune-modulatory drugs, or small molecules. This method allows for a sophisticated pre-clinical model for testing novel therapies for clinical translation.
The authors have nothing to disclose.
This work is supported by the SFB 824 subprojects C6 and C7 (DFG Sonderforschungsbereich 824), German Research Foundation, Bonn, Germany.
Veterinary clippers | Aesculap | GT415 | Small, cordless trimmer ideal for removing fur from surgical area |
Stereomicroscope | Zeiss | Stemi SV6 | |
30G Needles | Braun | 4656300 | 30G x ½” |
1ml syringes | Braun | 9161406V | Tuberculin syringe |
Disposable scalpel | Feather | 2975#15 | #15 blade |
Standard surgical scissors | Fine Science Tools | 14001-13 | Sharp/blunt, for opening skin and muscle |
Adson forcep | Fine Science Tools | 1101-12 | With teeth, for grasping skin and muscle |
Alm retractor | Fine Science Tools | 17008-07 | With blunt teeth, for spreading abdominal cavity open during surgery |
Gauze swabs | Lohmann & Rauscher | 18504 | 7.5 x 7.5 cm, should be autoclaved prior to use |
Cotton-tipped applicator swabs | Lohmann & Rauscher | 11970 | Sterile |
Fine-tipped foreceps | Fine Science Tools | 11063-07 | 0.4mm, angled tip, for dissecting hepatic artery |
Vannas spring scissors | Fine Science Tools | 91500-09 | For delicate cutting |
Micro-needle holder | Fine Science Tools | 12076-12 | For ligating gastroduodenal artery |
Needle holder | Fine Science Tools | 12005-15 | Tungsten carbide jaws |
7-0 Prolene sutures | Ethicon | 8648H | Polypropylene suture with curved needle, for ligating gastroduodenal artery |
4-0 Vicryl sutures | Ethicon | V3040H | With curved needle attached |
Infrared warming lamp | Beurer | IL11 | For maintaining body temperature post-operatively |