JP7696559B2 - Device for inducing immune cells into tumor tissue - Google Patents

Description

The present invention relates to a device for inducing immune cells into tumor tissue, which can be used to treat, prevent, or prevent tumors by injecting low-temperature atmospheric pressure plasma into the tumor tissue to non-invasively induce immune cells into the tumor and activate local immunity.

Tumors, especially malignant tumors that are prone to metastasis, such as stomach cancer, lung cancer, colon cancer, and breast cancer, are the leading cause of death among Japanese people. Treatment for such tumors includes surgical resection of the primary tumor site or lymph nodes, such as sentinel lymph nodes to which microtumor cells have metastasized or are metastasizing from the primary tumor site of breast cancer, as well as the primary tumor site and other organs to which the tumor has metastasized; chemotherapy using anticancer drugs or molecular targeted agents; endocrine therapy such as hormone therapy; radiation therapy using radiation such as X-rays, electron beams, proton beams, heavy particle beams, alpha beams, beta beams, and gamma beams; or multidisciplinary treatment combining drug therapy with surgery.

Surgical resection therapy causes great physical and psychological damage, chemotherapy is highly likely to cause serious side effects such as nausea, diarrhea, and hair loss due to damage not only to tumor cells but also to normal cells, endocrine therapy is likely to cause side effects such as hot flashes and joint and muscle disorders, and radiation therapy can cause acute disorders such as bone marrow suppression and rashes, as well as late-stage disorders such as taste disorders, respiratory disorders, bloody stools, and lymphedema.

In recent years, low-temperature atmospheric pressure plasma technology has been attempted as a new tumor treatment method. For example, Patent Document 1 discloses an ovarian cancer treatment device having a pair of opposing electrodes with recesses formed on the opposing surfaces of the electrodes, a housing covering a plasma generation area between the pair of opposing electrodes, a gas inlet for introducing a gas that generates plasma into the housing, and an irradiation unit for irradiating the plasma generated by the pair of opposing electrodes to the outside of the housing, the pair of opposing electrodes generating plasma with a plasma density in the range of 1×10 ¹⁴ cm ⁻³ to 1×10 ¹⁷ cm ⁻³ , and the irradiation unit irradiating the plasma to ovarian tumor cells. In this ovarian cancer treatment device, ovarian cancer cells are killed exclusively at the site irradiated with the low-temperature atmospheric pressure plasma by direct apoptosis caused by the low-temperature atmospheric pressure plasma.

In addition, rather than directly irradiating tumor cells with plasma, an antitumor effect has been found in the irradiated solution. For example, Patent Document 2 discloses an antitumor aqueous solution that selectively kills tumor cells, in which a first aqueous solution containing at least one solute selected from the group consisting of disodium hydrogen phosphate, sodium bicarbonate, L-glutamine, L-histidine, and L-tyrosine disodium dihydrate dissolved in water is irradiated with atmospheric pressure plasma to form a second aqueous solution, and the second aqueous solution is then frozen.

Furthermore, Patent Document 3 discloses a pharmaceutical composition for enhancing the therapeutic effect of cancer or tumor by plasma therapy, which contains a compound represented by R ¹ -NHCH ₂ COCH ₂ CH ₂ COOR ² or a salt or ester thereof. Patent Document 3 discloses a direct cell-killing effect on tumors by direct plasma irradiation using this pharmaceutical composition.

However, it is not known whether plasma irradiation induces local immunity in tumors located locally or at a slightly deeper site, especially in the primary tumor site or in the metastatic site from the primary tumor.

In addition, Non-Patent Document 1 analyzes the immunity of the surface skin due to exposure to low-temperature atmospheric pressure plasma. This Non-Patent Document 1 mainly examines skin damage due to low-temperature atmospheric pressure plasma exposure, and finds that inflammatory cell infiltration is observed in the skin at the irradiated site, and that a direct inflammatory reaction is induced in the skin at the irradiated site as an adverse event. However, this is not a tumor immunity induction or immune induction that has a therapeutic effect, but rather an inflammatory reaction induced by a burn (96°C) at the irradiated site.

JP 2013-153995 A JP 2016-3184 A JP 2019-123682 A

PLOS ONE, 12(4): e0174966.: https://doi.org/10.1371/journal.pone.0174966

The present invention has been made to solve the above problems, and aims to provide a simple and versatile device for inducing immune cells into tumor tissue that can be used to treat, prevent the progression of, and prevent tumors easily, quickly, and effectively by injecting low-temperature atmospheric pressure plasma onto a tumor, non-invasively inducing immune cells to activate local immunity, and indirectly inducing apoptosis in tumor lesions over a wide area not only at the site of low-temperature atmospheric pressure plasma irradiation but also in the vicinity.

The device for inducing immune cells into tumor tissue, which has been made to achieve the above-mentioned object, comprises a gas flow path for conducting gas within a tubular dielectric such as a glass tube or quartz tube, a tubular middle electrode surrounding the outside of the tubular dielectric, an upstream ground electrode arranged 70 mm upstream of the middle electrode, and a downstream ground electrode arranged 40 mm downstream of the middle electrode, a power source applied to generate plasma at room temperature under atmospheric pressure while the gas is conducted between these electrodes, and a single or multiple plasma airflow nozzles for spraying the plasma airflow generated by the plasma directly and/or indirectly into tumor tissue at the tip of the gas flow path , thereby inducing immune cells into the tumor tissue with the plasma.

This immune cell induction device for tumor tissue is preferably one in which the diameter of the plasma airflow nozzle is 1 mm or less, thereby increasing the efficiency of generating reactive oxygen and nitrogen species and inducing the immune cells to activate local immunity. In previous experiments, high effectiveness was obtained when the diameter of the plasma airflow nozzle was 200 to 700 μm.

This device for inducing immune cells into tumor tissue sets the flow rate of the gas at 1.5 L/min or less, preferably 0.5 to 1.5 L/min, so as not to dry out the biological tissue. For example, if the plasma airflow nozzle has a diameter of 650 μm, the flow rate is 1 L/min.

This immune cell induction device for tumor tissue limits the average current flowing from the plasma to the tumor tissue to, for example, 400 μA or less.

The gases used in this device for inducing immune cells into tumor tissue include, for example, helium gas, argon gas, nitrogen gas, oxygen gas, or a mixture of any of these gases.

It is preferable that this immune cell induction device for tumor tissue has a tapered tip and/or that the plasma airflow outlet has a smaller diameter than the tube diameter of the tubular dielectric, and further that the average current flowing from the plasma airflow to the tumor tissue is limited to 400 μA or less.

This immune cell induction device for tumor tissue is a dielectric barrier discharge in which plasma is generated in a glass or quartz tube. The dielectric generates plasma intermittently, like pulsed discharge plasma. By providing a complete dielectric barrier (a structure in which no electrodes are provided in the discharge space) in which all electrodes are installed outside the dielectric, it is even more preferable that the plasma density is relatively low, but the effects of heat can be minimized.

This immune cell induction device for tumor tissue may be one in which the plasma airflow outlet is provided at the tip of an endoscope, the tip of a catheter, or the tip of a probe.

This device for inducing immune cells into tumor tissue injects a plasma stream into the tumor tissue in one direction or in multiple directions with one shot or multiple shots.
This device for inducing immune cells into tumor tissue ejects the one-shot or multi-shot by using a switching device to switch the output of a high-voltage power supply.
This device for inducing immune cells into tumor tissue is such that the tumor tissue is a primary tumor focus, tumor cells, microtumor cells, superficial lymph nodes, sentinel lymph nodes, lymph nodes, blood vessels, disseminated tumor tissue, and/or metastatic tissue in vivo.
This immune cell induction device for tumor tissue is intended to locally inject the plasma stream in order to increase B lymphocytes, macrophages, neutrophils, T cells, NK cells, dendritic cells, and/or plasma cells in the tumor tissue.
The method of using the immune cell induction device to tumor tissue, which has been made to achieve the above-mentioned object, involves spraying the plasma airflow into the tumor tissue in one direction or in multiple directions with one shot or multiple shots.

The method of using this immune cell induction device for tumor tissue involves, for example, using a switching device to switch the output of a high-voltage power supply so that the one-shot or multi-shot is sprayed for a period of several seconds to several tens of minutes per shot.

In the method of using this immune cell induction device in tumor tissue, the tumor tissue is preferably a primary tumor focus, tumor cells, microtumor cells, superficial lymph nodes, sentinel lymph nodes, lymph nodes, blood vessels, disseminated tumor tissue, and/or metastatic tissue in vivo.

The method of using this immune cell induction device in tumor tissue involves locally injecting the plasma stream into the tumor tissue to increase B lymphocytes, macrophages, neutrophils, T cells, NK cells, dendritic cells, and/or plasma cells.

The device for inducing immune cells into tumor tissue of the present invention can non-invasively induce immune cells into tumor tissue and activate local immunity by injecting a plasma stream (direct plasma irradiation) generated by low-temperature atmospheric pressure plasma at room temperature.

By using this immune cell induction device for tumor tissue, the plasma airflow produced by low-temperature atmospheric pressure plasma can be sprayed onto tumor tissue in vivo, i.e., in vivo, and instead of the direct sterilization effect or direct apoptosis induction of tumor cells by direct plasma irradiation as in the past, it is possible to exert an action mechanism that is completely different from that of conventional plasma devices by indirectly inducing apoptosis through an immune response via immune cell induction.

This device for inducing immune cells into tumor tissue can be used to treat, prevent the progression of, and prevent tumors easily, quickly, safely, and effectively without damaging normal tissues, in tumor tissues, particularly primary tumor lesions and metastatic microtumor cells, as well as metastatic tissues such as surface lymph nodes, sentinel lymph nodes, lymph nodes, blood vessels, disseminated tumor tissues that have metastasized or are metastasizing, and metastatic tissues such as metastatic organs.

This device for inducing immune cells into tumor tissue can increase immune cells, particularly B lymphocytes, macrophages, neutrophils, T cells, NK cells, dendritic cells, and/or plasma cells, in the tumor tissue by locally spraying the plasma stream onto the tumor tissue, thereby inducing necrosis of tumor cells in the tumor tissue and thereby treating, preventing the progression of, and preventing tumors.

With this device for inducing immune cells into tumor tissue, it is possible to induce immune cells and activate local immunity by simply irradiating a portion of the tumor tissue with the plasma airflow for a specified period of time, without having to spray the plasma airflow over the entire tumor tissue, thereby enhancing the patient's own immune function and inducing necrosis of tumor cells throughout the tumor tissue.

This device for inducing immune cells into tumor tissue can induce necrosis in not only tumor cells in epidermal tumor tissue, but also tumor cells in subcutaneous tumor tissue, allowing for non-invasive treatment.

This device for inducing immune cells into tumor tissue is not only simple and versatile, but also small and portable, so it can be easily used in a variety of medical settings without the need for complex, large-scale equipment.

This device for inducing immune cells into tumor tissue has a simple structure and can be manufactured cheaply and easily.

This method of using an immune cell induction device in tumor tissue can induce immune cells and indirectly induce necrosis in tumor tissue, making it possible to use the device in tumor treatment with almost no side effects.

FIG. 1 is a schematic diagram showing an overview of an apparatus for inducing immune cells into tumor tissue according to the present invention. FIG. 11 is a schematic cross-sectional view showing another embodiment of a part of the device for inducing immune cells into tumor tissue to which the present invention is applied. FIG. 11 is a perspective view showing another embodiment of a part of the device for inducing immune cells into tumor tissue to which the present invention is applied. FIG. 13 is a diagram showing the current waveform of the plasma airflow of the immune cell inducing device for tumor tissue according to the present invention and the current waveform flowing into the tumor tissue. FIG. 11 is a perspective view showing another embodiment of a part of the device for inducing immune cells into tumor tissue to which the present invention is applied. This figure shows the results of luminol staining when tumor tissue from a mouse was prepared by heterotopic transplantation into the back of a healthy mouse, and was irradiated with a plasma airflow using a device for inducing immune cells into tumor tissue to which the present invention is applied, and when it was simply irradiated with a helium airflow using a device to which the present invention is not applied. This figure shows the temperature distribution on the skin surface when a plasma airflow was irradiated onto tumor tissue prepared by heterotopic transplantation into the back of a healthy mouse using a device for inducing immune cells to tumor tissue according to the present invention. FIG. 13 is a diagram showing the results of luminol staining, HE staining, and TUNEL staining when a plasma airflow is irradiated onto tumor tissue using an immune cell induction device for tumor tissue according to the present invention. 1 shows the results of estimating the numbers of B lymphocytes, macrophages, and neutrophils in the subcutaneous tissue and tumor tissue when a tumor tissue was prepared by heterotopic transplantation into the back of a healthy mouse and irradiated with a plasma airflow using an immune cell induction device for tumor tissue to which the present invention is applied.

The following describes in detail the embodiments of the present invention, but the scope of the present invention is not limited to these embodiments.

As shown in the schematic diagram of FIG. 1, the device 1 for inducing immune cells into tumor tissue of the present invention includes a low-temperature atmospheric pressure plasma generator 10, a gas supply device 20 having a gas cylinder 23, a voltage application device 30 for generating plasma 52, and a plasma generation control device 40 which is a personal computer (PC) for controlling the supply of carrier gas 51 and the generation of plasma 52.

This immune cell induction device 1 for tumor tissue can prevent the tumor tissue from drying out by narrowing the plasma airflow nozzle 15 and lowering the gas flow rate to the tumor tissue to 0.5 to 1.5 L/min. Furthermore, narrowing the nozzle can also suppress the current flowing to the tumor tissue. As a result, thermal damage to the tumor tissue and the surrounding normal tissue due to Joule heat can be suppressed.

The low-temperature atmospheric pressure plasma generator 10 is for ejecting the plasma airflow 53 generated by the generation of plasma 52. The low-temperature atmospheric pressure plasma generator 10 is provided with a tubular dielectric 13 made of glass or quartz as an inner tube dielectric forming a gas flow path 11, and the base side of the tubular dielectric 13 is connected to a gas cylinder 23 via a flexible gas pipe 21, an on-off valve 22a, a pressure regulator 22b, and a gas flow regulator 22c, and the other end side of the tubular dielectric 13 is surrounded and covered by a nozzle 16 made of a dielectric material such as polytetrafluoroethylene, which is cylindrical and has only the plasma airflow nozzle 15 open, so as to be blocked. The nozzle 16 has a plasma airflow nozzle 15 that ejects the plasma airflow 53 with a diameter smaller than the diameter of the tubular dielectric 13.

The tubular dielectric 13 is provided with an outer tube case 14, which is a cylindrical outer tube dielectric made of polytetrafluoroethylene (Teflon; registered trademark) with a gap therebetween so as to surround the gas flow path 11. A cylindrical coaxial inner electrode 12b surrounds the tubular dielectric 13 so as to be in contact with it. A cylindrical coaxial upstream electrode 12a surrounds the upstream side of the tubular dielectric 13 so as to be in contact with the outer tube case 14. A cylindrical coaxial downstream electrode 12c surrounds the downstream side of the tubular dielectric 13 so as to be in contact with the nozzle 16. The distance between 12a and 12b is 70 mm, and the distance between 12b and 12c is 40 mm. The thickness of the glass tube is 1 mm.

The electrode 12, which is composed of the middle electrode 12b, the upstream ground electrode 12a, and the downstream ground electrode 12c, is connected to a high-voltage power supply 31. The upstream ground electrode 12a and the downstream ground electrode 12c are grounded.

When an AC or pulse voltage is applied between the electrodes 12 by the high-voltage power supply 31, a dielectric barrier discharge occurs, generating plasma 52. The plasma 52 causes a plasma airflow 53 to be ejected from the plasma airflow nozzle 15 toward the tumor area 60 of the patient by the flow of carrier gas 51 in the gas flow passage 11.

This immune cell induction device 1 for tumor tissue has a bifurcated structure that separates the downstream side (between the middle electrode 12b and the downstream ground electrode 12c) from the upstream side that generates stable plasma between the upstream ground electrode 12a and the middle electrode 12b, and supplies the plasma airflow. Therefore, it can generate and maintain stable plasma even under a low gas flow rate, compared to conventional devices that generate plasma with two electrodes and a single electrode. Furthermore, by providing a plasma airflow nozzle 15, the supply amount of charged particles and excited species contained in the plasma airflow can be controlled.

The tubular dielectric 13 is a tube made of dielectric material, and may be, for example, a glass tube or a quartz tube. The plasma airflow nozzle 15 is, for example, a small-diameter nozzle provided coaxially with the tubular dielectric 13 in the center of the nozzle 16 while being blocked by the nozzle 16 at the tip side of the tubular dielectric 13. The plasma airflow nozzle 15 has a diameter of 650 μm. The appropriate diameter for preventing the living tissue from drying out with plasma irradiation for a maximum of about 15 minutes is about several hundred μm. If the diameter is larger than this range, a large amount of carrier gas is required to maintain stable plasma, and the living tissue is likely to dry out. Furthermore, as the plasma airflow nozzle 15 becomes larger, the current flowing to the living tissue also increases. As a result, the living tissue is damaged by Joule heat.

In FIG. 1, the plasma airflow nozzle 15 is shown as being opened in a nozzle 16 that closes the tip of the tubular dielectric 13, but as shown in FIG. 2(a), a nozzle may not be used and the tip of the tubular dielectric 13 of the same diameter may also serve as the plasma airflow nozzle 15. As shown in FIG. 2(b), the plasma airflow nozzle 15 may be a single hole in which the tip of the tubular dielectric 13 is closed in a dome shape or tapered. As shown in FIG. 2(c), the plasma airflow nozzle 15 may be multiple holes opened in the nozzle 16. As shown in FIG. 2(d), the plasma airflow nozzle 15 may be opened in the tapered tip of a conical or dome-shaped nozzle 16 that closes the tip of the tubular dielectric 13.

As shown in FIG. 2(e), the nozzle 16 may be provided with a supporter 16a to maintain a predetermined distance from the plasma airflow nozzle 15 to the surface of the tumor region 60. Specifically, as shown in FIG. 3, four claw-shaped supporters 16a may extend from a polytetrafluoroethylene nozzle, with the tips of the four supporters 16a bent inward. It is preferable that the supporters 16a are adjusted so that the distance from the plasma airflow nozzle 15 to the surface of the tumor region 60 is 10 to 20 mm depending on the gas flow rate and the length of the plasma jet.

The nozzles 16 shown in Figures 2(c) to (e) can be replaced as needed. For example, as shown in Figure 3, the diameter and shape of the nozzle 16, the aperture of the plasma airflow nozzle 15, and the length of the supporter 16a can be adjusted as needed.

Although not shown in the figure, the tubular dielectric 13 may have a constant outer diameter and only the inner diameter tapers to form a single hole with an open tip, the tubular dielectric 13 may taper to form a single hole with an open tip, the tubular dielectric 13 itself may be a thin tube of the same diameter and thus have a single hole with an open tip, the tubular dielectric 13 may have multiple holes at the tip, or may have an elliptical or groove shape within the above-mentioned diameter range.

The voltage application device 30 has an AC power supply or a pulse power supply as a high voltage power supply 31, and applies a pulse voltage of 1 to 10 kV, preferably several to 10 kV, at a low frequency of several to several tens of kHz, preferably about 10 kHz, to the electrode 12. When a carrier gas is flowed through the tubular dielectric 13 while a voltage is applied to the electrode 12, a plasma airflow 53 is generated and ejected as a non-equilibrium plasma jet in which the electron and molecular temperatures are different.

By using the dielectric barrier discharge method, plasma generation can be made intermittent. Figure 4 shows the current (discharge current) of the plasma airflow being blown out from the plasma airflow nozzle 15. Intermittent plasma with a quiescent period reacts with oxygen, nitrogen, and moisture in the air to generate active oxygen-nitrogen species, which can induce immune cells to tumor tissue. Furthermore, by using intermittent plasma with a quiescent period, an increase in the temperature of the plasma airflow can be suppressed. Even if the dielectric barrier discharge method is used, the quiescent period of the plasma is insufficient at frequencies of 100 kHz or more, making it difficult to suppress overheating of the plasma airflow.

The voltage application device 30 can generate the plasma at a high voltage (10 kV) and low current (1 mA or less) using a dielectric barrier discharge method. The average current values calculated from the current values shown in FIG. 4 are about 400 μA for both positive and negative currents. The maximum current value is 1 mA or less. As a result, the average power input is about 1 W. The small power input for plasma generation and the small current flowing to the surface of the tumor region 60 prevent overheating of the plasma airflow and Joule heating of the tumor region, thereby avoiding thermal damage.

The gas supply device 20 supplies a carrier gas 51 from a gas cylinder 23. The carrier gas 51 may be helium gas, argon gas, nitrogen gas, oxygen gas, and/or a mixture of these gases. Helium is more preferable because it is more likely to involve the surrounding atmosphere (oxygen, nitrogen, moisture) and cause a chemical reaction, and because less power is required to generate the plasma. Highly reactive active species (oxygen-nitrogen species) generated from the atmospheric pressure plasma are transported to the surface of the tumor region 60 by the plasma airflow 53.

The gas supply device 20 is controlled by the plasma generation control device 40, and the on-off valve 22a, pressure regulator 22b, and gas flow regulator 22c adjust the flow rate of the carrier gas from the gas cylinder 23 to the tubular dielectric 13. The flow rate of the carrier gas is preferably 0.5 to 1.5 L/min. If it is less than this range, the flow rate of the plasma airflow 53 is too low and the generated active oxygen and nitrogen species cannot be sufficiently transported to the surface of the tumor area. If it is more than this range, the surface of the tumor area will be excessively dried by the plasma airflow 53, making it difficult to induce immune cells.

The plasma generation control device 40 has a central control unit (CPU) and an input terminal to control the supply (opening/closing and flow rate) of carrier gas 51 from the gas cylinder 23 to the tubular dielectric 13 and the application of the high voltage power supply 31 of the AC power supply or pulse power supply of the voltage application device 30 to the electrode 12 for generating plasma 52. The CPU is for control in response to a signal from the input terminal, and has a carrier gas control circuit that controls the supply of carrier gas 51 and a voltage application control circuit that controls the voltage, current, and pulse of the high voltage power supply 31 of the AC power supply or pulse power supply of the voltage application device 20 (not shown).

The immune cell induction device 1 for tumor tissue may be one that directly grasps the tubular dielectric 13 and ejects the plasma airflow 53 directly to the tumor tissue 62 or indirectly to the tumor tissue 62 via the epidermis 61, as shown in FIG. 1, but instead of directly grasping the thin tubular dielectric 13, it may be provided at the tip of an endoscope 17a as shown in FIG. 5(a), at the tip of a catheter 17b as shown in FIG. 5(b), or at the tip of a probe 18 having a plurality of aligned circular or groove-shaped plasma airflow nozzles 15 at its tip as shown in FIG. 5(c), and the endoscope or catheter may be provided with a plastic housing case 19 for easy gripping as shown in FIG. 5(a-2).

In addition, the method of generating atmospheric pressure plasma in the immune cell induction device 1 for tumor tissue is to generate a stable dielectric barrier discharge inside the tubular dielectric 13 using the electrode 12 provided on the outside of the tubular dielectric 13, and to blow out a glow-like discharge plasma airflow 53 from the plasma airflow nozzle 15.

This immune cell induction device 1 for tumor tissue is used as follows:

When the immune cell induction device 1 for tumor tissue is driven and a predetermined voltage is applied between the electrodes 12 in the form of a predetermined sine wave or pulse wave while flowing the carrier gas 51, plasma 52 is generated by dielectric barrier discharge, and the plasma airflow 53 generated by the generation of the plasma 52 is ejected from the plasma airflow nozzle 15. This plasma airflow 53 is blown and irradiated onto the tumor area 60, either directly onto the tumor tissue 62 or indirectly onto the tumor tissue 62 via the epidermis 61.

In a method for treating a tumor using this immune cell induction device 1 for tumor tissue, the plasma airflow 53 is irradiated to the tumor area 60 for 1 second to 1 hour, preferably 1 to 20 minutes, and more preferably 10 to 15 minutes. The plasma airflow 53 may be sprayed evenly onto the tumor area 60 of a tumor-affected patient or tumor-affected animal in vivo, or may be concentrated in one location.

The method of treating a tumor using this immune cell induction device 1 for tumor tissue involves administering a single shot or multiple shots once to several times a day, preferably 1 to 3 times, either once or every day, or at intervals of 1 to 7 days or 1 to 4 weeks, continuously for 1 day to about 1 month until the tumor tissue disappears or decreases, or the tumor condition is tolerated.

Such tumor tissue is preferably a primary tumor site, but may also be tumor cells of the primary tumor site, or the primary tumor site and lymph nodes to be dissected. It may be a sentinel lymph node, which is a local lymph node that first receives lymph flow from the primary tumor site and where lymphatic micrometastasis may first occur, or a lymph node such as an afferent lymphatic vessel or an axillary lymph node closest to the sentinel lymph node that acts on micrometastatic tumor cells (microtumor cells), a surface lymph node, a blood vessel, or a tissue to which the tumor has metastasized.

According to the method for treating a tumor using the device 1 for inducing immune cells into tumor tissue, it is possible to induce immune cells into the tumor tissue. The immune cells include, for example, B lymphocytes (B cells), macrophages, neutrophils, T cells, NK cells, dendritic cells, and/or plasma cells.
Specifically, B lymphocytes (B cells) such as B lymphocytes, which are precursor cells of antibody-producing cells and cooperate with helper T cells to produce antibodies, and memory B cells that are stimulated by helper T cells and store information about antigens;
Macrophages are cytoplasm-rich, easily attached, and move by extending pseudopodia; they convey information about antigens (tumors) to helper T cells;
Neutrophils are a type of white blood cell that have a strong phagocytic effect, extend their pseudopods to ingest tumor cells, and kill them with the action of enzymes.
T cells such as helper T cells, which produce cytokines to activate B lymphocytes and help produce antibodies together with B lymphocytes, killer T cells, which kill and eliminate tumor cells under the control of helper T cells, and regulatory T cells, which suppress the immune system from acting against the self and prevent immune abnormalities;
NK cells (natural killer cells), which damage not only tumor cells but also cells that they recognize as abnormal;
Dendritic cells take in antigens from tumor cells and other sources, transmitting information about the antigen to T cells and initiating an immune response;
The terminally differentiated form of bone marrow-derived cells is the antibody-secreting plasma cell;
Examples include:

The plasma airflow 53 is ejected from the plasma airflow nozzle 15 and irradiated onto the epidermis 61 or tumor region 60, which is tumor tissue 62. Because the temperature of the epidermis 61 or tumor tissue 62 during plasma irradiation is at most 39-47°C, the tumor region 60 does not become inflamed and immune cells are not activated.

The details of the mechanism by which the tumor tissue 62 is induced to die by irradiating only a portion of the tumor tissue 62 without irradiating the entire tumor tissue 62, as a result of the increase and activation of immune cells induced by irradiating the tumor region 60 with the plasma airflow 53, are not entirely clear, but are presumed to be as follows.

Irradiation with the plasma airflow 53 results in the infiltration of neutrophils over a wide area of the tumor tissue 62, including around the area of tissue degeneration. Infiltration of lymphocytes and macrophages into the tumor nest is also observed. This shows that irradiation with the plasma airflow 53 can cause neutrophils to migrate over a wide area of the tumor tissue 62 in the subcutaneous tissue, and can also increase the infiltration of lymphocytes and macrophages into the tumor nest. This increases immune cells, which induces necrosis of tumor cells in the tumor tissue, and apoptosis can be used to treat, prevent the progression of, and prevent tumors.

Below, we will explain in detail an example in which the present invention is applied to a device for inducing immune cells into tumor tissue, and a comparative example in which the present invention is not applied.

Example 1
Human bladder cancer cell line 253J-BV; 4 x ¹⁰⁶ cells/100 μl was ectopic transplanted subcutaneously on the back of 5-week-old male nude mice (purchased from Japan SLC Co., Ltd.), and the tumor-bearing nude mice were prepared at 7-8 weeks of age. The tumor-bearing nude mice were irradiated for 15 minutes with a plasma airflow generated by a 10 kV sine wave with a frequency of 33 kHz for 1 L/min of helium gas using an immune cell induction device to which the present invention is applied. After that, the fluorescence intensity was measured using an IVIS spectrum by the luminol reaction emitting light with reactive oxygen species (ROS), and the antitumor effect was examined, and the temperature distribution of the epidermis was also examined using a FLIR T530 infrared thermography camera. The results of the luminol reaction emitting light with reactive oxygen species (ROS) are shown in the graph of FIG. 6(a) and the photograph of the same figure (b-2). In FIG. 7, the upper graph shows the change over time in the maximum temperature distribution, and the lower photograph shows the results of the temperature distribution of the epidermis.

(Comparative Example 1)
Helium was irradiated in the same manner as in Example 1, except that a non-plasma treated helium airflow was used without applying voltage instead of the plasma airflow generated by the immune cell induction device for tumor tissue. Then, the tissue was stained with luminol in the same manner. The results are shown in the graph of FIG. 6(a) and the photograph of FIG. 6(b-1).

(Staining comparison results between Example 1 and Comparative Example 1)
As is clear from Figure 6, the amount of active oxygen species increased significantly by about four times as much when the tumor tissue was irradiated with a plasma airflow generated by the immune cell induction device as in Example 1, compared to the case of irradiating the tumor tissue with a helium airflow as in Comparative Example 1. This shows that the active oxygen generated at the irradiated site by direct irradiation of the tumor tissue with low-temperature atmospheric pressure plasma using the immune cell induction device activates immune cells, which are then induced from the periphery of the tumor nest into the tumor nest.

Example 2
A human bladder cancer cell line, 253J-BV; 4 x ¹⁰⁶ cells/100 μl, was transplanted subcutaneously on the back of a 5-week-old male nude mouse (purchased from Japan SLC Co., Ltd.) to prepare a tumor-bearing model mouse. In the tumor area of the prepared nude mouse, a plasma airflow was directly irradiated from a distance of about 1.5 cm for 15 minutes to the tumor tissue through the epidermis, the plasma airflow being generated under the conditions of a 10 kV sine wave with a frequency of 33 kHz for 1 L/min helium gas using an immune cell induction device for tumor tissue to which the present invention is applied. The tumor tissue was stained with luminol in the same manner as in Example 1. In addition, a section was cut out from the tumor area, and HE staining (hematoxylin-eosin staining) and TUNEL staining (TdT-mediated UTPnickendlabeling staining) for staining apoptotic tissues and cells were performed. The results of luminol staining are shown in (a), the results of HE staining are shown in (b-1), and the results of TUNEL staining are shown in (b-2).

(Staining Results of Example 2)
As is clear from Figure 8, the luminol fluorescence measurement results showed the presence of reactive oxygen species, as shown in Figure 8(a). In addition, as shown in Figure 8(b-1), degenerated epithelium was formed between normal epithelium and normal epithelium, but irradiation with plasma airflow caused some bladder cancer cells to necrotize and undergo apoptosis, as shown in the dark area in Figure 8(b-2). As shown in the black frame in Figure 8(c), there was an increase in immune cells and exudate on the surface of the plasma-irradiated area. This indicates that B lymphocytes, macrophages, and neutrophils had accumulated in the tumor tissue, and immune cells had increased. This indicates that immune cells are activated by apoptosis caused by plasma irradiation and accumulate at the site.

Example 3
In the same manner as in Example 2, a plasma airflow was generated at a frequency of 33 kHz and a 10 kV sine wave for 1 L/min of helium gas by an immune cell induction device for tumor tissue according to the present invention, and was directly irradiated from a distance of about 1.5 cm to the tumor tissue in the tumor region of a nude mouse through the epidermis for 15 minutes. For the subcutaneous tissue and tumor tissue, immune cells such as B lymphocytes and neutrophils were stained with HE and counted under a microscope, and macrophages were stained with CD68 (KP-1) and counted.

(Comparative Example 2)
B lymphocytes, macrophages, and neutrophils were counted in the same manner as in Example 3, except that the epidermis at the tumor site was cauterized with an electric scalpel instead of irradiating the tumor tissue with a plasma airflow using an immune cell induction device.

(Comparative results of pathological analysis of immune cells between Example 3 and Comparative Example 2)
The numbers of B lymphocytes, macrophages, and neutrophils per unit area in the subcutaneous tissue and tumor tissue were compared for the plasma airflow irradiation treatment of Example 3 and the electric scalpel treatment of Comparative Example 2. The results are shown in FIG.

As is clear from Figure 9(a), in the subcutaneous tissue, electric scalpel treatment mainly increased macrophages, with only slight increases in B lymphocytes and neutrophils, whereas plasma airflow treatment mainly increased neutrophils, with only slight increases in B lymphocytes. On the other hand, in tumor cells, electric scalpel treatment showed little infiltration of B lymphocytes and macrophages, whereas plasma airflow treatment showed high infiltration of B lymphocytes and macrophages.

These results suggest that, unlike the group treated with an electric scalpel, the plasma therapy group induced lymphocytes and macrophages into the tumor nest, which was located slightly deeper than the irradiation site, thereby causing necrosis of the tumor tissue.

According to the device for inducing immune cells into tumor tissue and the method for using the same,
By injecting atmospheric pressure plasma into tumor tissue, immune cells can be induced and the local immune system can be activated, causing tumor tissue to necrose and undergo apoptosis, which can be used to treat, prevent the progression of, or prevent tumors.

1 is a device for inducing immune cells into tumor tissue, 10 is a low-temperature atmospheric pressure plasma generator, 11 is a gas flow path, 12 is an electrode, 12a is an upstream ground electrode, 12b is a middle electrode, 12c is a downstream ground electrode, 13 is a tubular dielectric, 14 is an outer cylindrical case, 15 is a plasma airflow outlet, 16 is a nozzle, 16a is a supporter, 17a is an endoscope, 17b is a catheter, 18 is a probe, 19 is a housing case, 20 is a gas supply device, 21 is a gas pipe, 22a is an opening/closing valve, 22b is a pressure regulator, 22c is a gas flow regulator, 23 is a gas cylinder, 30 is a voltage application device, 31 is a power source, 40 is a plasma generation control device, 51 is a carrier gas, 52 is plasma, 53 is a plasma airflow, 60 is a tumor area, 61 is the epidermis, and 62 is tumor tissue.

Claims (9)

An immune cell induction device for tumor tissue, comprising: a gas flow path for conducting gas within a tubular dielectric such as a glass tube or quartz tube; a tubular inner electrode surrounding the outside of the tubular dielectric, an upstream ground electrode arranged 70 mm upstream of the inner electrode, and a downstream ground electrode arranged 40 mm downstream of the inner electrode; a power source applied to generate plasma at room temperature under atmospheric pressure while the gas is conducted between the electrodes; and a single or multiple plasma airflow nozzles for spraying the plasma airflow generated by the plasma directly and/or indirectly into tumor tissue at the tip of the gas flow path, thereby inducing immune cells into the tumor tissue with the plasma. The device for inducing immune cells into tumor tissue according to claim 1, characterized in that the plasma airflow nozzle has a diameter of 200 to 700 μm, thereby inducing the immune cells and activating local immunity. 3. The device for inducing immune cells into tumor tissue according to claim 1, wherein the flow rate of the gas is set to 0.5 to 1.5 L/min, thereby preventing the tumor tissue from drying out. 4. The device for inducing immune cells into tumor tissue according to claim 1 , wherein the gas is helium gas, argon gas, nitrogen gas, oxygen gas, or a mixture of any of these gases. The device for inducing immune cells into tumor tissue described in any one of claims 1 to 4 , characterized in that the tip is tapered and/or the plasma airflow outlet has a diameter smaller than the diameter of the tubular dielectric. 6. The device for inducing immune cells into tumor tissue according to claim 1 , wherein the plasma airflow nozzle is provided at the tip of an endoscope, a catheter, or a probe. An apparatus for inducing immune cells into tumor tissue as described in any one of claims 1 to 6 , characterized in that the plasma airflow is sprayed in one direction or in multiple directions toward the tumor tissue in one shot or multiple shots . The device for inducing immune cells into tumor tissue described in claim 1, characterized in that the tumor tissue is a primary tumor lesion, tumor cells, microtumor cells, superficial lymph nodes, sentinel lymph nodes, lymph nodes, blood vessels, disseminated tumor tissue, and/or metastatic tissue in vivo . The device for inducing immune cells into tumor tissue according to claim 1, characterized in that the plasma stream is sprayed locally in order to increase B lymphocytes, macrophages, neutrophils, T cells, NK cells, dendritic cells, and / or plasma cells in the tumor tissue.

Images