Elsevier

Free Radical Biology and Medicine

Volume 115, 1 February 2018, Pages 43-56
Free Radical Biology and Medicine

Original article
Anti-tumor effects of cold atmospheric pressure plasma on vestibular schwannoma demonstrate its feasibility as an intra-operative adjuvant treatment

https://doi.org/10.1016/j.freeradbiomed.2017.11.011Get rights and content

Highlights

  • Cold atmospheric pressure plasma (CAP) efficiently induces cell death in benign vestibular schwannoma (VS) cells and tissues.

  • Exposure to CAP induces activation of the cell death pathways of apoptosis, necroptosis, and necrosis.

  • In VS-grafted mouse models, intra-operative CAP treatment after the surgical removal of a tumor inhibited tumor recurrence.

Abstract

Vestibular schwannoma (VS), although a benign intracranial tumor, causes morbidities by brainstem compression. Since chemotherapy is not very effective in most Nf2-negative schwannomas, surgical removal or radiation therapy is required. However, depending on the size and site of the tumor, these approaches may cause loss of auditory or vestibular functions, and severely decrease the post-surgical wellbeing. Here, we examined the feasibility of cold atmospheric pressure plasma (CAP) as an intra-operative adjuvant treatment for VS after surgery. Cell death was efficiently induced in both human HEI-193 and mouse SC4 VS cell lines upon exposure to CAP for seven minutes. Interestingly, both apoptosis and necroptosis were simultaneously induced by CAP treatment, and cell death was not completely inhibited by pan-caspase and receptor-interacting serine/threonine-protein kinase 1 (RIK1) inhibitors. Upon CAP exposure, cell death phenotype was similarly observed in patient-derived primary VS cells and tumor mass. In addition, CAP exposure after the surgical removal of primary tumor efficiently inhibited tumor recurrence in SC4-grafted mouse models. Collectively, these results strongly suggest that CAP should be developed as an efficient adjuvant treatment for VS after surgery to eliminate the possible remnant tumor cells, and to minimize the surgical area in the brain for post-surgical wellbeing.

Introduction

Vestibular schwannoma (VS), although a benign intracranial tumor, can cause morbidities including hearing loss, tinnitus, dizziness, and possible mortality by brainstem compression [1], [2]. The treatment strategies of VS are conservative, because both microsurgery and stereotactic radiation therapy pose risks of morbidity [3], [4]. During the microsurgery of tumors with large volume, attempting the complete removal can increase the damage of cranial nerves, thereby leading to possible facial palsy or hearing loss. Minimizing the damage of cranial nerves during surgery may have a positive impact on the patient's wellbeing. On the other hand, incomplete resection can cause tumor recurrence [5], [6]. Thus, a modified approach such as the subtotal removal followed by stereotactic radiosurgery is usually performed to prevent both neural damage complication and recurrence.

Unfortunately, there are several limitations of applying stereotactic radiosurgery to VS after surgery. Stereotactic radiosurgery has been reported to have risk of radiation hazard such as intracranial tumor swelling and to develop occasionally a new primary tumor in the intracranial region with the risk of radiation exposure [7], [8], [9], [10]. Anti-cancer therapies such as chemo- and radio-therapy are based on the observation that malignant cancer cells tend to respond more sensitively to oxidative stress than normal cells [11], [12], [13]. However, unlike malignant cancer cells, benign tumors of the head and neck including VS express high levels of survivin, which enable tumor cells to be resistant to chemo- and radio-therapy [14], [15], [16], [17]. Also, chemotherapy is not very effective in Nf2-negative schwannomas [18]. Thus, stereotactic radiosurgery to VS after surgery is not a preferred treatment approach and there is an increased need to develop an adjunct treatment for VS using new medical tools to overcome these obstacles. A novel adjunct therapy that efficiently removes the possible remnant tumor cells around cranial nerves would be helpful to minimize the surgical area to reduce the morbidity due to damage and the risk of recurrence.

Cold atmospheric pressure plasma (CAP) is an ionized gas generated by electrical discharges in the atmospheric pressure at room temperature (for a review, [19]). Currently, CAP has been extensively studied for clinical applications since it can be easily generated, causes no thermal damage to cells, and can be controlled by adding gases and/or adjusting the electric field (for a review, see [20]). CAP has been reported to induce cell death in various types of cancer cells via increasing the intracellular ROS (for a review, see [20]). However, CAP has been mostly applied to induce cell death in malignant tumors, and its effect on benign tumors has not yet been studied [21], [22], [23], [24], [25].

In this study, in order to verify the potential of CAP as an intra-operative adjunctive therapy of VS after surgery, we examined the effect of CAP on Nf2-deficienct human and mouse VS cell lines, primary VS cells, and tumor tissues derived from human patients. We also demonstrated the effect of CAP on recurrent tumor growth, when CAP was applied to the areas of surgical removal of a primary tumor in SC4-grafted mouse models.

Section snippets

Air-based cold atmospheric generators for exposure of cell lines and mice

For cell exposure in vitro, CAP was generated by a micro dielectric barrier discharge (μ-DBD) plasma device (Fig. S1A), which is similar to the device reported by previously [26], [27]. Air was used as the source of gas supply. The pump in the control device could pump out air to the CAP generator at 2 standard liters per minute (SLM) (Fig. S1B). Voltage and current were supplied by a circuit located inside the control device. The CAP-generating area covered an entire 35-mm culture dish (Fig.

CAP shows highly efficient anti-proliferative effects in schwannoma cell lines

As the first step to validate the feasibility of CAP as an adjuvant treatment for VS after surgery, we examined the cellular effects of CAP exposure in both human HEI-193 and mouse SC4 VS cell lines that have been reported as Nf2-deficient [30], [31]. We determined the optimal conditions of CAP treatment for anti-proliferative effects in HEI-193 and SC4 cells, by adjusting the exposure time and the volume of medium. HEI-193 and SC4 cells were exposed to single continuous air-based plasma of 2

Discussion

Vestibular schwannoma (VS) is an intracranial benign tumor, and sometimes needs to be surgically removed because of its possible risk of brain stem compression [1], [2]. However, its complete removal without cranial nerve damage is difficult, particularly in the case of a huge tumor [63]. In addition, most VSs with Nf2 deletion do not respond well to conventional chemotherapy [18], and may arise at multiple sites, making radiotherapy less successful [18], [64]. Thus, an adjuvant treatment that

Conclusions

We assessed “the feasibility of cold atmospheric pressure plasma (CAP) as an intra-operative adjuvant treatment modality for vestibular schwannoma after surgery” by investigating the anti-proliferative effect of CAP on human and mouse VS cells, human VS tissues, and VS mouse models after surgery in vivo. CAP treatment efficiently induced apoptotic, necroptotic, and necrotic cell death in human VS cells and tissues, without triggering resistance to the subsequent treatment. Our results strongly

Acknowledgements

None.

Funding

This research was supported by a Grant from the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT & Future Planning (Grant number NRF-016M3A9C6918). Yeo Jun Yoon was supported in part by the BK21 and BK21 PLUS program of the NRF of Korea.

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