plasma is the 4th phase of substances following the solid , liquid , and gas phases , and it is constituted of gas containing electrons , cations , anions , neutral atoms , and neutral and charged molecules . plasma can be generated by dissociating molecules to atoms in gas by loading energy to the gas and then ionizing the atoms into ions and electrons . previous plasma is termed low - pressure plasma and its generation was limited to that in a vacuum container , but non - equilibrium atmospheric pressure plasma generated under atmospheric pressure at a low temperature has recently been developed , and it is now applied not only in the manufacturing and industrial fields but also the medical field , and plasma coagulation of the mucosa are performed . various therapeutic effects , such as inactivation of pathogens wound healing , blood coagulation , and tissue sterilization , and the ablation of cultured cancer cells , are also expected . basic studies on application of plasma for cancer therapy have just started over the world . to supplement it from standpoints of cancer researchers and clinicians , the current reviews makes referral to the study results of plasma therapy clarified so far , the possibility of its application in the future , and problems to be solved . it is said that currently , approximately one in three people die of cancer in japan . however , it has not yet been fully understood why self - destroying neoplasm emerges in one s own body . normal cells start to proliferate when they repair an injury made in the body or cell proliferation is switched on in response to its necessity as a part of metabolism . when active proliferation becomes unnecessary , the switch is turned off and cells enter a quiescence state . excluding some cells , such as the embryonic , blood , and bone marrow cells , basically , cells do not move to a site very far from the organ where they are originally present . on the other hand , how do cancer cells behave in these states ? cancer cells may also temporarily enter a dormant state , but their proliferation switch is basically on . cancer cells metastasize to multiple organs and uncontrollable proliferation continues while gradually undermining physical strength of the body , that s why cancer is malignant. generally , cancer is treated with surgery , chemotherapy with anticancer drugs , and radiotherapy , and these are said to be the three major treatment methods of cancer . regarding merits and demerits of the three major treatment modalities , when cancer is localized and resectable without markedly impairing the surrounding organs , cancer removal by however , it is accompanied by surgical stress and may be difficult to perform when physical strength is markedly reduced . in contrast , when lesions are present in multiple organs throughout the body or diffusely scattered on the surface of the body cavities , such as abdominal and thoracic cavities , chemotherapy with anticancer drugs is performed . recently ,  molecular target drugs targeting specific intracellular molecules helping cancer cell proliferation appeared in chemotherapy . anti - cancer drugs exhibit adverse effects damaging normal cells , in addition to cancer cells . its indication is considered when a tumor is localized to some extent but surgical removal en bloc is difficult . however , irradiation is not necessarily effective for all cancers , and there is variation in sensitivity ( fig .  1 ) . moreover , the same region can not be irradiated at a dose exceeding a specific dose , termed the maximum exposure dose . we fight against cancer by making full use of the  3 major treatment methods , but cancer is difficult to control because it is the why is intrinsic eradication of cancer not possible ? except for cancer which widely expands in the body from the beginning and does not respond to treatment , many types of cancer disappear from the body after a series of treatments as if they were cured ( remission ) . recurrence. cancer remits only for a short time and it recurs after a specific time and then is treated again , repeating recurrence and remission , like a cat - and - mouse game . cancer responds to anticancer drugs to some extent at the beginning , but the drugs gradually become ineffective and the cancer becomes highly malignant resistant cancer spreading ( metastasizing ) throughout the body , i.e. ,  recurrence and accompanying  resistance are the hardest obstacles against overcoming cancer . when cancer therapy is performed , how to induce remission and when remission can be induced , how to prevent recurrence and how to deal with cancer which unfortunately recurred are important therapeutic strategies across various cancer types . the effect of plasma irradiation on mammalian cells was initially reported by kieft et al . in 2004 , in which cell adhesion was actually inhibited by irradiation and resulted in induction of cell death . since then , apoptotic cell death induced by plasma irradiation has been reported over the last decade in not only normal cells , such as fibroblasts and mammary gland epithelial cells , but also various cancer cells , such as brain tumor and colorectal , skin , lung , pancreatic , and ovarian cancer cells . the therapeutic effect of plasma is not limited to a specific cancer type , and it is constant for all types of tumor . unlike necrosis , which is passive cell death in response to external stimulation , apoptosis is active cell death . apoptotic cell death is not accompanied by inflammation of the surrounding tissue , being considered as a mechanism managed and adjusted in the proliferation control system . why does plasma influence cell proliferation activity ? it has been confirmed that various ions , radical species , and particle types with biologically strong actions are generated in gas - phase plasma . inhibition of cell proliferation or action causing cell death is considered due to impairment of redox equilibrium of cells caused by reactive nitrogen oxide species ( rnos ) including free radicals released from plasma . when oxidative stress increases in cancer cells , gene mutation occurs , cell proliferation is promoted , and resistance to apoptosis is induced , increasing treatment resistance of cancer cells . however , when they are exposed to oxidative stress exceeding a specific threshold , the cell function is negatively controlled by oxidative damage , resulting in arrest of proliferation followed by induction of apoptosis . actually , rnos production increases in a manner dependent on the plasma irradiation time , and cancellation of the plasma - induced antitumor effect by the addition of oxidative stress scavenger to the culture system has been reported . however , considering that it is a treatment for the body , a selective antitumor effect acting on only cancer cells with less toxicity to normal cells is desirable . generally , anticancer drugs and radiotherapy are also toxic to normal cells , and these treatments utilize differences in the sensitivity between tumor and normal cells . the action is exhibited as an antitumor effect on cancer cells and an adverse effect on normal cells . therefore , if treatment can be applied within a range in which tumor cells are sensitive but normal cells are less influenced , adverse effects can be reduced . according to several previous studies , interestingly , there is a specific selectivity in the cell proliferation - inhibitory effect of plasma : the inhibitory effect on tumor cells is stronger than that on normal cells . zucker et al . reported that when highly metastatic melanoma cells ( skin cancer cells ) and normal skin keratinocytes were directly irradiated with plasma for 10  s , apoptosis was 4.9 times more frequently observed in the former than in the latter after 24  h. in addition , tumor cell selectivity of plasma compared with that for normal cells was clarified by comparison between lung cancer and normal cells ( embryonic fibroblasts and pulmonary epithelial cells ) ( panngom et al . ) , between glioma cells ( a type of brain tumor ) and astrocytes ( tanaka et al . ) , and between ovarian cancer cells and fibroblasts ( iseki et al . ) . although it is still hypothetic , these study results suggest that although high - energy plasma irradiation is also toxic to normal cells , a specific optimum treatment range ( treatment window ) is present between normal and tumor cells ( fig .  2 ) . if there is a difference in plasma sensitivity between normal and cancer cells , what is the underlying mechanism ? the fundamental difference between cancer and normal cells may be based on the proliferation activity . for example , generally , when a space between normal cells is wide , cells proliferate to fill the space , and when cells become dense and closely contacted , the proliferation signal is turned off to stop further proliferation . in contrast , this control system does not act on cancer cells and they infinitely proliferate . in culture systems , when cancer cells proliferate and fill the dish , they repeat proliferation lying on top of one another and die due to malnutrition because the proliferation mechanism is constantly turned on in cancer cells , as described above . it is possible that the presence of these differences in the proliferation activity and apoptosis - inhibitory control system between cancer and normal cells influences changes in plasma sensitivity . clarified that plasma - irradiated solution selectively inhibits cultured glioma cells of brain tumor on comparison of proliferation activity with that of astrocytes , and this mechanism is due to inhibition of akt molecule , which serves as a hub of proliferation / survival signal transmission . various molecular mechanisms may be identified in the future , in which normal cells represent all cells other than cancer cells . in previous studies , only 12 types of normal cells were picked up and a more marked inhibitory effect of plasma irradiation at a specific energy on proliferation of cancer cells was observed , i.e. , normal cell death occurs when the 
 energy is increased , and it is possible that some normal cells are more readily influenced by plasma than cancer cells . to apply plasma for treatment in consideration of this point , attention should be paid to the necessity of careful re - investigation . is the proliferation - inhibitory effect via apoptosis the only influence of plasma irradiation on cells ? not only active proliferative ability but also various cell functions , such as abilities to infiltrate the surrounding tissue ( invasive capacity ) , actively move for metastasis ( motility ) , and tightly adhere to other cells to fix scaffolds and form metastatic lesions ( adhesiveness ) , are used as indices of malignant transformation of cancer . wang et al . performed cell invasion assay using breast cancer cells and a transwell chamber . this assay applies the property of cells that cancer cells cultured on a membrane with small 8-m pores can not pass through the pores because the cell size is far larger than the pore size , but some cells with metastatic motility alter their morphology to an elongated shape and pass through the pores . the membrane is coated with a specific matrix ( matrigel ) , and cells can not reach the undersurface of the membrane unless they secrete a specific enzyme and dissolve the matrix , i.e. , not only motility but also tissue - invasive capacity are observed by counting cells which moved to the undersurface of the membrane . these potentials can be compared by counting cells which could pass through the membrane out of a specific number of cells seeded on the membrane . the invasive capacity of plasma - irradiated cells decreased in a irradiation time ( 3090  s)-dependent manner ( decreased to about 30% by 90-s irradiation ) compared with that of non - irradiated control cells . kim et al . also investigated the invasive capacity of colorectal cancer cells using a similar assay system and observed almost the same results . in these assays , the influence on cell motility was presented without strongly reflecting differences in proliferative ability because the time to assay after exposure to plasma was relatively short . the findings suggested that exposure to plasma at an energy level not inducing apoptosis reduces metastatic invasive capacity of cancer . active species in the gas phase generated by plasma irradiation are incorporated into fluid around tissue before they directly contact the biological tissue and affect the cells and tissue . some points are still unclear with regard to how active species are generated in the liquid phase , but there is one new method to deal with it , termed in - liquid plasma ( not a formal name , but i call it so ) . when a solution is irradiated with plasma , rnos is produced . this is an indirect plasma treatment method in which plasma - irradiated solution ( aqueous plasma ) is added to the culture supernatant of separately cultured cells to expose them to rnos without direct plasma irradiation ( fig . this indirect plasma irradiation has been suggested to exhibit an effect comparable to that of direct irradiation and be sufficiently applicable for treatment . in the gynecological oncology field , ovarian cancer is one of the cancer types with the poorest prognosis among gynecological malignant tumors . drug - resistant recurrence occurs in most cases of progressive ovarian cancer within 2 years even though remission was achieved by multidisciplinary treatment . nagoya university collaborative study group developed an original non - equilibrium atmospheric pressure plasma generator . when culture medium irradiated with plasma generated by this device was added to ovarian cancer cells , the irradiated culture medium exhibited an antitumor effect in a manner dependent on the exposure time . the fluid was less toxic to normal cells ( human mesothelial cells ) and a selective antitumor effect was observed in only cancer cells , as described above ( fig .  4 ) . in addition , signals of rnos - induced reduction products were detected in ovarian cancer cells exposed to aqueous plasma , suggesting that apoptosis induction by increased intracellular rnos is a cause of the toxicity . interestingly , the proliferation - inhibitory effect of in - liquid plasma on an anticancer drug - resistant cell line derived from the original ovarian cancer cells was stronger than that on the parent cells . therefore , in - liquid plasma therapy is expected to exhibit a therapeutic effect on ovarian cancer cells which acquired resistance to anticancer drugs . a treatment , intraperitoneal administration of aqueous plasma against ovarian cancer which expanded over the abdominal cavity and became resistant to anticancer drugs , the influence of plasma on cells appears to be a constant proliferation - inhibitory effect via apoptosis , although the application method differs : direct plasma irradiation and aqueous plasma . however , only a few studies on the tumor - inhibitory effect in animal models , which is a preliminary step before clinical application have been reported . regarding the antitumor effect of plasma in animal models , tumor growth inhibition was reported by vandamme in 2010 . they subcutaneously transplanted brain tumor cells into mice and applied dbd irradiation for 5 days to the ectopic tumor formed under the skin . the tumor size did not change at the beginning of irradiation compared with that in the non - irradiated control group , but the converted tumor volume was reduced to below 1/2 at the completion of irradiation . this difference increased with time , resulting in prolongation of the survival time by more than 30% in the treatment group compared with that in the control group . investigated the combined effect of dbd plasma and an anticancer drug , gemcitabine , on pancreatic cancer cells in a mouse orthotopic graft model . they applied plasma irradiation using a plasma gun to the tumor on days 14 and 24 after transplantation and administered concomitant gemcitabine 5 times during this period . the tumor weight decreased by about 20% in the combination group compared with that in the group treated with gemcitabine alone . the tumor volume was also reduced to about 50% in the combination group compared with that in the group treated with plasma alone . keidar et al . investigated the antitumor effect of plasma using plasma jet in a mouse subcutaneous ectopic tumor model of urinary bladder cancer , and observed that the tumor markedly shrank and the mean survival time extended by about 9 days in the plasma treatment group compared with that in the untreated group ( plasma treatment group : 33.5 days vs untreated group : 24.5 days ) . other than the effect of direct plasma irradiation , does local injection of plasma - irradiated solution exhibit an effect ? we prepared a nude mouse model of subcutaneous tumor formation in the thigh using anticancer drug - resistant ovarian cancer cells to investigate the antitumor effect of plasma - irradiated culture medium in vivo . the weight of anticancer drug - sensitive ovarian cancer cell tumor excised on day 28 after treatment initiation was reduced to 66% in the in - liquid plasma treatment group compared with that in the control group and that of anticancer drug - resistant ovarian cancer cells was also reduced to 52% compared with that in the control group . although the administration method was different : direct plasma irradiation and local administration of in - liquid plasma , the therapeutic tumor - shrinking effect on localized tumor was observed in the animal experimental model . although it is necessary to accumulate various basic study results , plasma irradiation of localized superficial tumor present on the body or body cavity surface is sufficiently assumable treatment . however , it is practically difficult to apply plasma irradiation to many tumors diffusely spreading all over the body cavity . the problem in actual clinical practice is the presence of many small lesions widely spreading over the body cavity , such as peritoneal metastasis of ovarian cancer , and either surgery or radiation is unlikely to be useful treatment . anticancer drugs may be used , but when the tumor has acquired resistance , the efficacy is not satisfactory . there are various cavities in the body , and the abdominal cavity containing the stomach , intestine , liver , gall bladder , spleen , uterus , and ovary is the largest . the abdominal cavity is covered with the peritoneum , which often serves as a tumor bed of diffusely spreading digestive organ - derived and ovarian cancers . there is also the thoracic cavity holding the lung and medullary cavity holding the brain and spinal cord . the pathologies of cancer extensively spreading in these body cavities are termed peritoneal , pleural , and intramedullary dissemination , respectively . intra - cavity treatment with aqueous plasma is thought to be one of the most effective modalities of plasma cancer therapy . if plasma is indicated for cancer therapy , for what type of cancer is it effective and what are the problems ? direct plasma irradiation for local treatment is relatively similar to radiotherapy among the three major cancer therapies . irradiation induces cell activation when the dose is very low , but it induces cell death at a dose higher than a specific level . utilizing the fact that cancer cells are more sensitive to radiation compared with normal cells , the therapeutic dose range in which the therapeutic effect can be obtained while minimizing damage of normal cells is established . these properties are common to the apoptosis - inductivity of plasma , as described above . in - liquid plasma has a point in common with intra - cavity drug administration capable of extensively exposing cancer to drugs , such as intraperitoneal administration against ovarian cancer . in addition , there may be a cell death - inducing mechanism different from the conventional mechanism of anticancer drugs , for which an effect on anticancer drug - resistant cancer cells may be expected . furthermore , previous basic studies clarified that plasma inhibits the motility and invasive capacity of cancer cells . although it is still hypothetical , it may inhibit secondary metastasis of cancer cells which escaped from cell death . the mechanism of the proliferation - inhibitory effect of plasma is rnos production in cells , as described , but reactive oxygen species ( ros ) scavengers , such as glutathione , are abundantly present in body tissue . oxidative stress is constantly generated in the body and biologics would not have advanced to this level without the development of the system removing the stress and protecting the body . considering this , it is understood that the presence of scavengers is deep - rooted . generally , the influence of plasma tends to decrease in animal experiments although a marked effect was observed in cell studies , and this may be due to the removal of plasma - induced ros by ros scavengers in vivo . solution of this problem by improving the plasma irradiator and developing a method to temporarily inhibit scavengers is continuously searched for . multiple applications and intra - cavity perfusion of in - liquid plasma are also being investigated on the assumption that the effect of single administration will be scavenged . another problem is the extent of tumor infiltration of plasma . according to a previous report since the penetration capability of rnos generated by the current experimental plasma generator is shallow , it can not readily reach the inner region of tumors which have already grown to a certain size . can it be actually solved by increasing the plasma intensity ? to apply high - energy plasma irradiation or in - liquid plasma , it is necessary to sufficiently confirm the absence of administration - related adverse events at the animal experiment level . in any case , it is necessary to further improve the plasma irradiation device through information exchange among physicians , physicists , and engineers . the prognosis of metastatic and recurrent cancers is still poor despite advances in medicine , as described above . the environment to promote  plasma medical science will be developing from now on , and early creation of the new interdisciplinary area and preparation of a systematic research structure are important . plasma medicine , which may cause a stir in the cancer therapy field with a sense of stagnation , is promising in the future .
the therapeutic effects of non - thermal plasma are expected in the medical fields , including hemostasis , vascularization , prevention of organ adhesion , and cell proliferation . 
 cancer is an internal enemy arising from normal tissue in the body . 
 the prognosis of metastatic and recurrent cancers is still poor despite advances in medicine . to apply non - thermal plasma in cancer treatment is now on going . 
 the mechanism of the proliferation - inhibitory effect of plasma is reactive nitrogen oxide species / reactive oxygen species production in cells . 
 there are a number of problems to be overcome , such as existence of intrinsic reactive oxygen species / reactive nitrogen species scavengers and the shallow infiltration of plasma on tumor surface . 
 the current reviews makes referral to the study results of plasma therapy clarified so far , the possibility of its application in the future .