Lung metastases are a common severe outcome of many primary tumors. Combined treatment consisting of pulmonary metastasectomy and chemotherapy is often the patient’s best hope for cure, yet in most cases the 5 years survival rate is less than 50% [
2]. Thus, prevention of metastatic spread from primary tumors is of paramount importance.
Recently, a new treatment modality against cancerous cells was introduced—low intensity, intermediate frequency alternating electric fields or TTFields. These finely tuned electric fields are applied using a portable battery operated device (NovoTTF; NovoCure Ltd., Haifa, Israel) through insulated surface electrodes, and have been shown to inhibit the growth of primary solid tumors in both pre-clinical and clinical studies [
18,
20].
In a preliminary attempt to study the effect of TTFields on metastatic lesions we tested the fields’ effect when applied directly to the lung shortly after B16F10 melanoma cells were injected into the tail vein. After 7 days of TTFields application, the number of surface lung metastases in the TTFields treated mice was significantly reduced, compared to the sham control group. This result could be interpreted in several ways: the TTFields could have eliminated the B16F10 melanoma cells shortly after injection, or merely prevented their implantation in the lung. Alternatively, the implantation of the B16F10 melanoma cells in the lungs was not affected but the tumor progression was inhibited by the TTFields. The latter option is supported by the fact that 1 week after stopping treatment the number of lung metastases in the TTFields group was equal to the number observed in the sham control group. Furthermore, the different size distribution between the two groups suggests that the metastases progression was inhibited in the TTFields group during treatment, an effect which may have been attenuated or partially lost after 1 week recovery.
Inhibition of VX-2 carcinoma in rabbits’ kidneys
Previously we reported that TTFields treatment significantly reduced progression of malignant melanoma and adenocarcinoma tumors in mice as well as glioma cells inoculated intracranially in Fischer rats [
18,
19]. In the present study we demonstrate that TTFields can inhibit the growth of VX-2 carcinoma in the kidneys of New Zealand white rabbits. The MRI results illustrate that TTFields application significantly reduced tumor growth rate throughout treatment.
Though the inhibition of renal tumor growth was significant, it was not expected to result in such a noteworthy difference in the overall survival between the treatment group and the control. An alternative explanation was sought, that could account for the observed differences in the overall survival. Hence, we investigated the metastatic spread of the VX-2 tumors to the lungs [
21].
The TTFields intensity in the lungs was about 20% of the fields’ intensity in the kidney (data not shown). Such a 0.5 V/cm field intensity measured in the rabbit’ lungs is below the threshold required for cancerous cell growth inhibition [
19]. Since the TTFields application could not account for the decrease in the number of metastases, we searched for an alternative explanation for the results.
One such explanation relates to the inhibition of growth of the primary renal tumor. It is possible that by lowering the tumor load in the kidney, the metastatic potential of the tumor would decrease proportionately. Another possibility is based on the reported finding that there is an increase in metastatic spread of VX-2 tumors from the kidney between days 12 and 15 from implantation [
21]. Here we report that treating the primary tumor with TTFields after day 15 from implantation resulted in a smaller difference between treated and control rabbit in the number of lung metastases. Therefore, it is possible that the inhibition of metastatic spread is due to an inhibitory effect on the capability of the tumor cells to migrate into the circulation of the primary tumor. Such migration is dependant on the proper formation of a microtubule based processes in the migrating cells [
22]. Although we did not test this directly, the known inhibition of microtubule polymerization by TTFields during mitosis may have a similar effect on cancer cell migration and endothelial penetration. Future experiments are warranted to test this point directly.
An additional explanation for our findings is the enhancement of a systemic immune response to the tumor cells. This option is supported by the results of the immunostaining assay performed on treated and sham control lungs. It is becoming widely accepted that immune response can inhibit the proliferation of cancerous cell or even eliminate them (reviewed by Dunn et al. [
23]), and thus account for the reduction in the number of metastases. Naturally, the following question would be: How could TTFields activate the immune response in an organ distant from the location where the fields are applied? Zitvogel et al. [
14] reviewed several mechanisms through which conventional treatments could modulate the interaction between the tumor and the immune system: reduction of the tumor mass as a result of chemotherapy, surgery or radiation could reduce the tumor immunosuppressive properties; tumor cell destruction induced by chemotherapy or radiation could expose hidden tumor antigens; stimulation of the immune system through activation of immune effectors and regulatory mechanisms or by inducing lymphopenia followed by proliferation of immune effectors. Taken together, a self generated vaccination against cancer specific antigens may develop—leading to metastasis destruction by the immune system [
14]. In addition, we found that the cells infiltrating the lung metastases in the TTFields group were CD4, CD8 and CD45 positive. This milieu of immune markers indicates a T-cell mediated immune response; however, it is not yet sufficient to prove a tumor-antigen specific immune reaction. Interestingly, CD45 activation can induce the production of TNFα [
24] which in turn activates a family of cell-surface receptors that can mediate cell death.
Assuming that the activation of the immune response in the rabbits’ lungs was mediated by the TTFields treatment, a question arises regarding the TTFields effect on the immune system in the mice. The observation that the metastases renewed their growth in the lungs, once the TTFields application to the mice was terminated, does not support immune response activation in the treated group. It seems more likely that the inhibition of the metastases in the mice lungs was due to a direct inhibitory effect of the TTFields on the cancerous cells. Unlike the rabbits in which there was a substantial difference between the fields’ intensities in the lungs and the kidneys, in the mice lungs the TTFields intensity was sufficiently large to induce inhibition of the metastasis. Why wasn’t the immune system activated in the mice? The shorter treatment duration (1 week in mice compared to 5 weeks in rabbits) might not suffice for the induction of the immune response in mice. Differences between the cancerous cell lines used (VX-2 carcinoma in rabbits and B16F10 melanoma in mice) as well as differences between the animals’ immune system could also account for these results. In addition, the tumors subjected to TTFields application in the rabbits were established large tumors which provided a larger target for the immune system and possibly presenting more tumor specific antigens after the TTFields treatment.
In conclusion, we have shown that TTFields have the potential to inhibit the migration of metastases from a primary tumor, can inhibit the growth of metastases in the lungs once they have been seeded in the target organ, by the presence of the fields in the lungs themselves, and finally, TTFields may activate an anti-tumor antigen systemic immune response following treatment of a primary tumor. Therefore, TTFields may not only be clinically useful in treatment of locally advanced tumors, but in prevention and treatment of metastatic disease as well.