All mice tolerated the treatment well. Following treatment mice were monitored for signs of pain or distress as indicated by: changes in posture (hunched or sleeping appearance), piloerection, dehydration, changes in behavior (increased timidity or aggression, isolation), changes in activity (reflex withdrawal, biting at the treated area), and changes in locomotion (unsteady gait, lameness). After recovering from anesthesia, no signs of pain were noted for the duration of the study. Growth and regression of the tumors was normalized to their initial volume by dividing their volume, V, by their volume at the time of treatment, V0. The relative growth curves of all tumors may be found in , where the relative volumes of the individual tumors are shown versus time.
Fig. 2 Tumor growth curves. The growth of individual tumors over time is presented relative to the volume measured immediately prior to treatment. Closed circles represent tumors in mice that received IRE treatment while open circles represent the tumors in (more ...)
The bioluminescent images seen in are from the second experimental trial and show that the fully regressed physical tumors contained no sign of viable cancerous cells at 4 weeks post-treatment. The tumors from the treatment group that did not regress showed continued growth, though no evidence of tumor metastasis was observed.
Fig. 3 Bioluminescence of IRE-treated tumors: The five mice of the second IRE treatment group with bioluminescence from the tagged cancerous cells from the tumors may be seen in location and density. The time the images were taken were at a 1 h prior to treatment, (more ...)
Histological examination of the tissue taken from the site of tumor implantation was done in a blinded fashion by a board certified anatomic veterinary pathologist (NDK). Three control tumors and five IRE-treated tumors, three of which regressed, were examined. As shown in , the tumors from the control group had central necrotic cores surrounded by sheets of viable tumor cells, among which many mitoses were noted. Thick bands of plasma cells, and fewer lymphocytes, were present along the outer margins, interpreted as host reaction to the tumor (). The two IRE-treated tumors that did not regress had a similar appearance (not shown). For the three tumors that regressed after treatment, the neoplastic cells were absent (), apart from one of the three cases, where a few cells (mostly degenerate) were present (). Mitoses were not noted in this lesion and the interpretation was that the tumor was near the final stage of regression. Reactive fibroplasia and neovascularization, with lymphoplasmacytic infiltration were present in the dermis and subcutis along the margins of the lesions.
Fig. 4 Histology of the mammary fat pad of tumor-bearing mice 30 days following IRE treatment. a Mock treatment showing densely packed, viable cancer cells in the subcutis and dense inflammatory cell infiltrate along the margin. Inset: higher magnification of (more ...)
By compiling the data from the tumor growth as measured by calipers, bioluminescent imaging of tagged cancer cells from the tumor, and histological examination, it may be determined that tumor regression occurred for five out of the seven treated tumors (both treated tumors from the first group, and three out of five from the second group). All control tumors showed continued growth over the experimental period. Analysis of the tumor growth from caliper measurements (continued growth against regression) was performed using Pearson’s test with JMP software (JMP, Cary, NC), where it was found that treatment is statistically significant in regressing the tumors (P = 0.0221).
The numerical models of electric field distribution may be seen in , where each color represents an electric isofield contour, and the volume within each color is at least that respective electric field. The black borders represent the physical domains in the models, including the tumor and all peripheral tissues. The distribution from the treatment around the average tumor dimensions may be seen in , where it appears that the tumor was entirely covered by an electric field of approximately 1000 V/cm. This suggests that this electric field may be treated as the minimum threshold required to induce IRE in these tumors in vivo. This finding is consistent with previous in vitro experiments that used a similar protocol on the same cell line while in suspension and determined the electric field threshold to be 1000 V/cm [18
]. The second numerical model output of shows how the electric field distribution would change in a clinical setting, where the tumor would likely be located deep within the breast. The results indicate that in deep tumors, the applied voltage should be slightly increased relative to that used in the mouse model to ensure complete treatment of a similarly sized tumor.
Typical surgical resection procedures remove a 0.5 cm margin around the tumor to ensure removal of any potentially infiltrative cells beyond the tumor borders [8
]. Incorporating treatment margins would likely be employed in IRE ablation therapies as well by expanding the electric field so that the IRE threshold found to be 1000 V/cm from the numerical model in reaches 0.5 cm beyond the tumor. Conventional focal ablation therapies, such as radiofrequency (RF) ablation and cryosurgery, rely on thermal energy [15
], which present complications including inconsistencies between the predicted or visualized heated/cooled zone and true cell death regions [14
] and trouble with thermal dissipative properties (the blood perfusion “heat sink” or “cold sink” effect) of vascularized tissue [13
]. Additionally, hyperthermic techniques such as RF ablation can induce charring at the electrode interface [15
], require minimum treatment depth of at least 1 cm to prevent skin injury [23
], and produce significant scar tissue [14
], reducing accurate follow-up. IREs benefits, including rapid lesion creation and resolution, as well as minimal scarring [19
], means that larger treatment regions including a margin would likely have fewer negatives than conventional surgical and ablative therapies.
Expanding and shaping the electric field to treat larger tumors and margins beyond the tumor may be done using several techniques to ensure complete treatment of the targeted area while minimizing effects on healthy tissue. One method includes using customized electrode geometries, something easily done with the electrode design used. For reasonably simple geometries, such as spheres and ellipsoids, the layer diameters and lengths of the electrode could be altered. For instance, longer and further separated cathodes and anodes will axially extend the treatment margins, while shortening them will produce a more spherical distribution. The diameters may be made larger to increase treatment volume, or narrower to reduce invasiveness or improve insertion for stiffer tumors that thicker needles may have trouble penetrating.
For very large or highly irregular lesion geometries that cannot reasonably be treated with a single insertion of the electrode, multiple insertions of electrodes allow treatment volume to be more effectively shaped, as is often done with thermal focal ablation therapies [16
]. When applying combinations of treatments, each pulse application will have its own electric field distribution and thus region of IRE cell death, which may be superimposed. In this way, each portion of the tumor may be independently treated until the entire mass has been ablated. The electrodes used for such an approach may be very small or of a complex geometry to keep invasiveness low. This versatility can facilitate the treatment of many different cancers located throughout the body while minimizing secondary effects from physical invasiveness. Additional analysis of electrode selection and treatment planning to shape lesions may be found in [3
]. Furthermore, electric pulse parameters may be adjusted to treat different volumes. Because IRE lesions follow the electric field strength, larger volumes may be simply treated by increasing the applied voltage.
Most previous in vivo investigations of IRE used healthy tissue, and do not account for the potential differences in IRE related to the structure and organization of cancerous tissue [7
]. Previously published studies using IRE to treat tumors used plate electrodes placed on the tumor to deliver IRE pulses [1
]. This required that the entire tumor be physically exposed to allow for electrode placement. We present the first investigation to treat solid tumors with a clinically relevant electrode.
Understanding why two tumors continued to grow provides insight into improving upon the findings of this initial study. Both tumors that did not regress were the widest, one of which being the largest (9.7 × 8.1 mm); suggesting that the size or shape of the tumor may have exceeded the treatment region for the protocol used. This is supported by the bioluminescent image of the largest tumor taken 3 days after treatment (not shown), showing no living cancer cells near where the electrode was placed, but some remained at the outer edges of the tumor, where the electric field would be the weakest. In the image 4 weeks after treatment (), the largest tumor is the one furthest to the right, and the greatest concentrations of cancer cells are in these same regions, suggesting that the surviving cells were responsible for the continued growth of the tumor. To prevent this in future studies and treatments, a maximum treatment region should be found for a given protocol, such as through numerical modeling, and the treatment region should be adjusted as previously discussed for targeted volumes larger than this maximum.
In addition to preventing the errors that may have led to the two tumors that did not regress, the findings of this study can be improved in future studies and treatments. This experiment used a standardized electrode geometry and protocol, which did not allow for variations in tumor size or shape. In clinical settings, the electrode dimensions and protocol used may be adjusted according to treatment demands. Furthermore, tumor response was obtained in immunodepressed mice without the combinatorial approach typical of breast conserving therapies. Additional treatment modalities, such as electrochemotherapy [17
], can selectively kill cancer cells experiencing electric fields above the reversible electroporation threshold (~250 V/cm). This as well as an immune response, which is promoted by electroporation [17
], will improve the effectiveness and reliability of these treatments by killing cancer cells which may otherwise survive IRE alone.