Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.
Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.
Pancreatic ductal adenocarcinoma (PC) is projected to become the second leading cause of cancer deaths in the US around 20201. The vast majority of patients diagnosed with PC will eventually die from distant metastatic disease2. The PC microenvironment is notoriously immunosuppressive and chemoresistant. Its desmoplastic stroma contains a scarcity of effector (anti-tumor) T cells and a prominence of immunosuppressive leukocytes, including tumor-associated macrophages (TAMs), myeloid derived suppressor cells (MDSCs), and regulatory T cells (Tregs)3. These underlie the need to develop multimodal strategies that counteract these effects of the microenvironment.
IRE has been developed as a non-thermal method of tumor ablation. Unlike thermal ablation techniques, IRE does not cause rapid coagulative necrosis but instead results in gradual apoptotic cell death4. Importantly for pancreatic tumors, IRE is not vulnerable to “heat sink” effects and can be performed right next to blood vessels5. This technology has 510(k) clearance from the FDA6 and is currently being used clinically, for selected patients with locally advanced or borderline resectable pancreatic cancer. In the largest published series of IRE for PC7, the median survival of patients undergoing IRE was approximately double the survival of patients treated with modern chemotherapy alone without resection8,9.
Several studies have demonstrated that thermal ablation induces a systemic immune response in other tumor types (reviewed in Chu et al.10). Radiofrequency ablation (RFA) in animal tumor models leads to increased T cell infiltrates11,12, including an increase in activated natural killer (NK) cells in hepatocellular cancer patients13,14, and a decrease in immunosuppressive Tregs in lung cancer patients15. A much smaller number of studies have examined immune, microenvironmental, and injury responses to IRE16. IRE has been shown to stimulate a systemic immune response in immunocompetent mouse models in which the growth of secondary (contralateral) renal cell allografts was reduced or prevented by IRE of a primary tumor two weeks earlier17. They also observed that immunocompetent mice required less voltage for complete regression than did immunocompromised mice. It has been hypothesized that IRE may result in improved antigen presentation compared to the coagulative necrosis of thermal ablation, but this has not been specifically studied.
We have developed a syngeneic mouse model of PC from the KPC-Luc 4580 cell line (gift from J.J. Yeh at University of North Carolina), which was derived from a tumor that developed in a male LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1Cre/+; LSL-ROSA26 Luc/+ mouse, to study the local and systemic effects of IRE18,19. This luciferase-expressing cell line is immunogenic and also tumorigenic in immunocompetent C57BL/6 mice when injected SQ or orthotopically and reliably produces liver metastases when injected into the spleen. We have utilized a programmable square wave pulse generator to deliver 100-µs pulses of electricity at a voltage/distance ratio of 1,500 V/cm using a two-needle array probe (separated by 5 mm) or platinum tweezer-trodes to SQ or orthotopic tumors, respectively, in mice to model the effects of IRE in a small animal.
All animal experiments performed following this protocol must be approved by the respective Institutional Animal Care and Use Committee (IACUC). All procedures described here have been approved by IACUC UCSD.
1. Procure Recipient Animals
NOTE: The KPC-Luc 4580 cell line was established from a tumor arising in a LSL-KrasG12D/+; LSL-Trp53R172H/+; PDX1Cre/+; LSL-ROSA26 Luc/+ mouse (KPC), which is a genetically engineered model of PC on a C57BL/6 background. The advantage of this cell line is that it constitutively expresses luciferase-enabling tumor monitoring in orthotopic models. However, other cell lines may also be used as long as they are compatible with the genetic background of the recipient mice.
2. Culture Cells
3. Induction of SQ Tumors
4. Induction of Orthotopic Tumors
5. IRE of SQ Tumors
6. IRE of Orthotopic Tumors
NOTE: IRE of orthotopic tumors involves a second survival surgery on the same mouse thus requiring special approval from local IACUC before beginning.
We followed the procedure described above and generated SQ tumors on 5 – 6 week old wild type C57BL/6 mice inoculated with 1 x 106 cells with 50% BMM. When the tumor size reached 5 – 6 mm in diameter, a few of the mice were euthanized, their tumors were excised, and implanted orthotopically in a recipient C57BL/6 mice. IRE was performed 10 days post implantation as shown in the timeline on Figure 1. IRE was performed on the remaining mice bearing SQ tumors.
In SQ tumors, the IRE voltage and pulse duration were kept constant at 1,500 V/cm and 100 µs, respectively, but the number of pulses varied. Figure 2 shows that the SQ tumors in a few mice regressed completely after 150 pulses of IRE but not as well with 75 pulses. In total, 4 out of 9 mice showed complete tumor regression after 150 pulses of IRE. Histological analysis of tumor tissue 1 week post IRE showed large regions of central tumor necrosis that were not seen in control untreated tumors. This necrotic core was flanked by live tumor tissue in cases of incomplete tumor regression (Figure 3). Successful implantation of orthotopic tumor was also achieved, and the growth rate was monitored using in vivo bioluminescence imaging showing tumor at day 10 and day 15 (Figure 4) after implantation. IRE at 150 pulses also proved to be effective in orthotopic tumors (Figure 5) showing reduced tumor volumes. Overall, these results demonstrate the ability of this model to simulate effects of IRE in an immunocompetent mouse model, thereby providing a platform to test various IRE conditions and combinations to treat PC.
Figure 1: Schematic representation shows the time course of tumor implantation and IRE. For SQ tumors, IRE is performed as soon as the tumor reaches 5 – 6 mm in diameter. Please click here to view a larger version of this figure.
Figure 2: Luciferase-bioluminescence imaging shows a reduction in tumor growth post IRE in SQ model. KPC-Luc cell line constitutively expresses luciferase making it feasible to monitor tumor growth in response to IRE in real-time. Day 14 bioluminescence imaging post IRE shows the complete regression of tumor in one of the mice treated with 150 pulses of IRE, whereas incomplete regression was seen with 75 pulses of IRE when compared to the untreated control. Please click here to view a larger version of this figure.
Figure 3: Hematoxylin and eosin staining of tumor tissue shows the changes in the tissue architecture post IRE. SQ tumors exposed to IRE showed large regions of necrosis centrally, surrounded by regions of live tissue indicating inadequate IRE coverage in some cases. Please click here to view a larger version of this figure.
Figure 4: Bioluminescence imaging shows the successful implantation of orthotopic tumor and its growth over time. Images were taken using a commercial bioluminescence imaging instrument with the luminescence captured at 1 min exposure. The mice were injected with 30 mg/kg of D-luciferin solution intraperitoneally 10 min before imaging. Mice were kept anaesthetized using 2% isoflurane during the procedure. Please click here to view a larger version of this figure.
Figure 5: IRE induces tumor growth reduction in orthotopic PC tumors. (A) Bioluminescecnce images of mice harboring orthotopic PC showed reduced luciferase signal 7 days post-IRE compared to sham surgery suggesting reduced live tumor burden as a result of IRE. (B) Mean volume of excised orthotopic tumors (+ standard error) in control mice vs. mice that underwent IRE. Please click here to view a larger version of this figure.
In this study, we have demonstrated an immunocompetent mouse model for PC that can be used to study the effects of IRE on the tumor growth. Currently, IRE is being used as a non-thermal ablation technique only in highly selected locally advanced PC patients who do not have distant disease progression after months of preoperative therapy. Its use has therefore been limited because most patients with locally advanced PC develop distant metastatic disease20. This model will serve as a basis for studies to evaluate the effects of IRE on PC using various extended treatment parameters and combinations.
The most important thing to consider during the protocol is the size of the tumor at which IRE is performed. The commercially available probes for mice models are limited by their electrode distance (5 - 10 mm). Hence, in tumors significantly larger than the electrode distance, incomplete ablation takes place. For SQ tumors, the use of 2-needle array electrodes over tweezer-trodes is recommended as the skin surrounding the tumor increases resistance to the flow of current in tweezer-trodes. The size limitation of the 2-needle array electrodes can be overcome by performing the IRE at various depths and angles in the same tumor; however, this approach makes it difficult to treat tumors uniformly. Alternatively, for larger tumors (up to 10 mm), an incision can be made on the skin below the tumor to a length slightly longer than the tumor diameter. The tumor can then be externalized through the incision by inverting the overlying skin using forceps. The tumor can then be treated with tweezer-trodes, similar to the orthotopic tumors described above. The electroporated tumor can then be reinserted under the skin, and the skin sutured with a 4-0 non-absorbable suture as an interrupted stitch. However, the effects of the incision may confound the effects of IRE, and we have found that performing IRE when the tumors are within the working distance of the two-needle array electrodes (~ 5 – 6 mm) provides the best standardization.
Regardless of the approach, the number of pulses needs to be determined empirically depending on the tumor type. Published studies have utilized between 150 - 300 pulses at this voltage17. However, we determined the optimal number of pulses in a preliminary dose-response experiment. There are models to predict the zone of complete ablation based on voltage and electrode distance, but tumor types can vary greatly in vascularity and fibrosis, which may affect the response to electroporation21.
Each set of 10 pulses was separated by 10 s in order to avoid the heating effects that occur if too many pulses are delivered too quickly. Over-treatment can result in thermal ablation, which may prevent accurate characterization of the effects of non-thermal IRE. The 10 s interval also allows us to frequently confirm electrode positioning accurately since muscle contractions can cause electrode displacement. Our IACUC has not yet allowed the use of paralytic agents in small animals, which can greatly reduce muscle contractions during IRE. In case of orthotopic tumors, the tweezer-trodes allows us to hold the tumors tightly and have a larger electrode surface area compared to the needle electrode.
Although IRE is currently only used as an ablative procedure in the clinical setting, electroporation in general has a wide spectrum of applications ranging from nerve and muscle activations to delivery of various drugs and oligonucleotides. By carefully analyzing the pathophysiological changes occurring during and after IRE using this model, it is possible to develop countless therapeutic strategies for PC.
The authors have nothing to disclose.
RRW has received support for this work from a Collaborative Translational Research Grant funded by the San Diego C3 Padres Pedal the Cause (#PTC2017).
ECM 830 square wave electroporator | Harvard Apparatus | BTX # 45-0002 ( 58018-004 ) | |
2 needle array electrode | Harvard Apparatus | 45-0167 | |
Safety foot switch | Harvard Apparatus | 45-0211 | |
Platinum Tweezer-trode | Harvard Apparatus | 45-0486 | |
DMEM-F12 media | ThermoFisher Scientific | 11320-033 | |
Fetal Bovine Serum | ThermoFisher Scientific | 10437028 | |
Trypsin | ThermoFisher Scientific | 25200056 | |
Matrigel | Corning | 354230 | |
Isoflurane | Sigma-Aldrich, Inc. | 792632 | |
Lacrilube | Fisher Scientific | 19090646 | |
Buprenorphine | Fisher Scientific | NC1292810 | |
D-luciferrin | Perkin Elmer | 122799 | |
IVIS Spectrum In Vivo Imaging System | Perkin Elmer | 124262 | |
Mouse strain C57BL/6J | The Jackson Laboratory | 000664/Black 6 | |
Cell line (KPC-Luc 4580) | J.J. Yeh Lab at University of North Carolina | ||
BD Precisionglide syringe needles | Sigma-Aldrich, Inc. | Z192406 | |
Alcohol Swab(70% isopropyl alcohol ) | BD | 326895 | |
Digital calipers | ThermoFisher Scientific | 14-648-17 | |
Disposable Scalpels, Sterile | VWR | 21909 | |
Cotton Tipped Applicators | VWR | 89198 | |
Suture Needle, 45 cm, Size 6-0 | Harvard Apparatus | 72-3308 | |
Suture Needle, 45 cm, Size 4-0 | Harvard Apparatus | 72-3314 | |
Povidone-iodine 10% | BD | 29900-404 | |
Disposable Warming Pad | KENT SCIENTIFIC CORP. | TP-3E | |
Mouse Hair Clipper | KENT SCIENTIFIC CORP. | CL8787 | |
Surgical Drape | Harvard Apparatus | 59-7421 | |
Phosphate-buffered Saline | ThermoFisher Scientific | 10010023 |