Treatment of tumors with atmospheric pressure cold plasma (APFP)

Abstract

Cold plasma at atmospheric pressure for use in the treatment of an immunologically cold tumor in a patient requiring such treatment.

Description

Title of the invention: Treatment of tumors by atmospheric pressure cold plasma (APP) technical field

[0001] The invention relates to the treatment of tumors, in particular immunologically cold tumors, by atmospheric pressure cold plasma (APHP), in particular cholangiocarcinoma tumors, a cancer of the bile ducts. Previous technique

[0002] Based on their immunological characteristics, tumors can be classified as immunologically "cold" or immunologically "hot." The terms "hot" and "cold" are used to describe the presence or absence of functional immune cells in tumors, which has implications for the effectiveness of immunotherapy in cancer treatment.

[0003] Immunologically cold tumors (or “cold tumors”) have few tumor-infiltrating lymphocytes (TILs) and a low mutational burden. The immune system does not actively engage with these tumors, which may make them more resistant to immunotherapies that rely on a pre-existing immune response. Immunologically cold tumors can be classified as “immuno-deserted” tumors, i.e., those with very few immune cells, and “immune-excluded” tumors, where immune cells are present in the surrounding tissues but are unable to penetrate the tumor itself.

[0004] Immunologically hot tumors (or “hot tumors”) exhibit high levels of tumor-infiltrating lymphocytes, such as cytotoxic T cells, indicating an ongoing immune response against the tumor. Hot tumors also often exhibit high mutational burdens, resulting in the production of new antigens that can be recognized by the immune system. These tumors generally respond better to immunotherapies such as checkpoint inhibitors, which enhance the body's existing immune response against cancer cells.

[0005] Cholangiocarcinoma (CCA), or bile duct cancer, does not show immune infiltrate in 50% of cases. Approximately 50% of cholangiocarcinoma cases are cold tumors. The disease is classified according to its location: intrahepatic cholangiocarcinoma (CCAi), which develops in the liver, and extrahepatic cholangiocarcinoma (CCAe), which develops in the bile ducts at the hilum (liver-bile duct interface), or in the bile ducts. distal (away from the liver). CCA is typically a slow-growing cancer that causes no symptoms in its early stages. However, as the cancer progresses, patients may experience various symptoms, including jaundice (yellowing of the skin and eyes), abdominal pain, and weight loss. Once diagnosed, three lines of conventional treatment can be considered: surgery (a curative approach aimed at removing the tumor), chemotherapy, and targeted therapies. However, the latter are systemic treatments, also known as palliative treatments, and therefore not very effective.

[0006] One of the main characteristics of CCA is its high level of cancer-associated fibroblasts (CAFs). These cells are activated in the tumor microenvironment and can secrete large amounts of collagen and other extracellular matrix components, resulting in the formation of fibrotic and rigid tissue. This rigidity can, in turn, exert mechanical forces that promote the proliferation, invasion, and survival of cancer cells, potentially contributing to the aggressiveness of the cancer. In addition, the dense stromal environment can act as a barrier to the delivery of chemotherapy drugs, thus reducing the effectiveness of the treatment. It can also hinder the infiltration of immune cells, thereby protecting cancer cells from the immune system. All these characteristics are consistent with an "immunologically cold tumor."A tumor's resistance to cancer treatment may be due to the tumor's immunologically unresponsive nature.

[0007] Methods for treating cancer cells or cancer using cold plasma at atmospheric pressure have been described (EP 3 222 121 Bl, US 2017 / 0183632 Al, WO 2023 / 150338 Al)

[0008] To date there are few effective anticancer treatments against immunologically cold cancers.

[0009] To date, there are also few effective anticancer treatments against cholangiocarcinoma.

[0010] There is a need for new cancer treatments.

[0011] There is a need for new cancer treatments with little or fewer side effects than conventional systemic or local therapies, such as chemotherapy and radiotherapy, respectively.

[0012] There is a need for new anticancer treatments that can be applied locally to different cancers, in particular immunologically cold cancers.

[0013] There is a need for new treatments that modulate the tumor microenvironment (TME).

[0014] There is a need for new treatments to reduce the rigidity of the extracellular matrix of the MET.

[0015] There is a need for new treatments that restructure the tumor environment and thus increase the penetration of anti-tumor immune cells and treatments (antibodies for example).

[0016] There is a need for new anticancer treatments for cancers where MET rigidity and antitumor immunosurveillance are compromised and contribute to treatment resistance and low treatment efficacy.

[0017] There is a need for new anticancer treatments against so-called "cold" tumors.

[0018] There is a need for new anticancer treatments against cholangiocarcinomas.

[0019] There is a need for new treatments to reduce tumor rigidity in order to restore antitumor immunosurveillance and improve the effectiveness of anticancer therapies.

[0020] There is a need for new treatments for solid tumors exhibiting high rigidity and resistance to conventional therapies, such as chemotherapy, targeted therapy and immunotherapy.

[0021] There is a need for new treatments that can "transform" cold tumors into hot tumors in order to increase the effectiveness of the patient's immune system and / or immunotherapies.

[0022] There is a need for new treatments for chemoresistant tumors.

[0023] There is a need for new treatments for tumors resistant to immunotherapies or anticancer vaccines.

[0024] There is a need for new anticancer treatments capable of inducing immunogenic cell death.

[0025] There is a need for new anticancer treatments for "cold" tumors that can be used in combination with other anticancer therapies, such as immunotherapy.

[0026] There is a need for new treatments to restore the antitumor action of immunotherapies against "cold" tumors.

[0027] There is a need for new anticancer treatments for cholangiocarcinoma.

[0028] The present invention aims to satisfy all or part of these needs. Summary of the invention

[0029] According to one of its first objects, the present invention relates to a cold atmospheric pressure plasma for its use in the treatment of an immunologically cold tumor in a patient in need of such treatment.

[0030] According to one embodiment, the cold atmospheric pressure plasma is generated from a noble gas.

[0031] According to one embodiment, a noble gas can be chosen from helium, neon, argon, krypton, xenon and a mixture thereof.

[0032] According to one embodiment, a noble gas can be chosen from helium, neon, argon, krypton, and xenon

[0033] According to one embodiment, a noble gas can be helium, neon, or argon.

[0034] According to one embodiment, a noble gas can be helium.

[0035] According to one embodiment, the cold atmospheric plasma can produce at less one reactive species chosen from the group consisting of: reactive oxygen species (ROS), reactive nitrogen species (RNS), and combinations thereof.

[0036] According to one embodiment, the reactive oxygen species (ROS) can be chosen from the group consisting of: atomic oxygen (O), radical oxygen (O#), ozone (O3), hydroxyl radical (HO#), superoxide anion (O2#), hyperoxide radical (HOO#), hydroperoxyl (HO2), hydrogen peroxide (H2O2), nitric oxide (NO#), hydronium ion (H3O+), and combinations thereof.

[0037] According to one embodiment, the reactive nitrogen species (RNS) can be chosen from the group consisting of: nitric oxide (NO#), nitrous oxide (N2O), nitrogen dioxide (NO2), peroxynitrite (ONOO), nitrogen trioxide (N2O3), nitrogen tetroxide (N2O4), nitrite ion (NO2), nitrate ion (NO3), and combinations thereof.

[0038] According to one embodiment, the cold atmospheric pressure plasma can be generated by applying electrical pulses to a noble gas, the duration of each pulse being between 1 ns and 100 ms.

[0039] According to one embodiment, the time interval between two consecutive pulses may be fixed or variable. According to one embodiment, the time interval between two consecutive pulses is fixed.

[0040] According to one embodiment, the time separating two consecutive pulses can vary from about 1 ns to about 20 ns, or even from about 2 ns to about 15 ns, or even from about 5 ns to about 10 ns.

[0041] According to one embodiment, the atmospheric pressure cold plasma can be generated from a noble gas subjected to a voltage varying from approximately 100 V to approximately 100 kV, or approximately 1 kV to approximately 50 kV, or approximately 5 kV to approximately 30 kV, or approximately 10 kV.

[0042] According to one embodiment, the atmospheric pressure cold plasma can be generated from a noble gas subjected to an electrical signal whose frequency varies from about 0.1 Hz to about 10 MHz, or from about 1 Hz to about 100 kHz or from about 100 Hz to about 30 kHz or be about 10 kHz.

[0043] According to one embodiment, atmospheric pressure cold plasma can be applied to a portion of the patient's body surface at least at a surface power of about 0.1 pW / cm2 to about 100 mW / cm2 or about 1 pW / cm2 to about 10 mW / cm2, about 10 pW / cm2 to about 1 pW / cm2, or about 20 to about 500 pW / cm2.

[0044] According to one embodiment, atmospheric pressure cold plasma can be applied to a portion of the patient's body surface for approximately at least 1 second and commonly over a time interval ranging from approximately 10 seconds to approximately 30 minutes, and preferably over a time interval ranging from approximately 1 minute to approximately 5 minutes.

[0045] According to one embodiment, the atmospheric pressure cold plasma can be generated from a noble gas subjected to an electrical signal whose voltage varies from about 100 V to about 100 kV, or about 30 kV, and at a frequency varying from about 0.1 Hz to about 10 MHz, for at least 1 second.

[0046] According to one embodiment, the immunologically cold tumor can be chosen from among altered immunodeficient tumors, altered immune excluded tumors, cold immune tumors, tumors deserted by the immune system, tumors excluded from the immune system, and tumors with low immune infiltration.

[0047] According to one embodiment, the immunologically cold tumor may be a tumor of a cancer selected from liver cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, bladder cancer, kidney cancer, lung cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, Merkel cell carcinoma, and head and neck cancer.

[0048] According to one embodiment, bile duct cancer can be a cholangiocarcinoma.

[0049] According to one embodiment, the immunologically cold tumor may be a cholangiocarcinoma tumor and the atmospheric pressure cold plasma applied to a portion of the patient's bile duct surface may be generated from of a noble gas subjected to an electrical signal whose voltage varies from about 100 V to about 100 kV, or about 30 kV, and at a frequency varying from about 0.1 Hz to about 10 MHz, for at least 1 second.

[0050] According to one embodiment, cold atmospheric pressure plasma can be applied simultaneously or sequentially with the administration of an additional anticancer agent.

[0051] According to one embodiment, the additional anticancer agent can be chosen from electrotherapy, chemotherapy, radiotherapy, a therapeutic cancer vaccine, anticancer immunotherapy, and an oncolytic virus.

[0052] According to one embodiment, the additional anticancer agent is chosen from chemotherapy, immunotherapy, or targeted therapy based on at least one mutation identified in the tumor.

[0053] According to one embodiment, chemotherapy can be GEMCIS.

[0054] As illustrated by the examples given below, the inventors observed that the use of cold plasma on a preclinical cancer model, particularly a cold cancer model, made it possible to modulate the tumor microenvironment, notably by reducing the rigidity of the tissues surrounding the tumor and inducing immunogenic cell death. Immunogenic cell death (ICD) is a type of cell death that triggers an immune response. In the context of cancer, ICD actively signals the immune system to identify and eliminate cancer cells. This phenomenon can be particularly important in the fight against cancer cells.

[0055] Advantageously, such treatment makes it possible to improve and / or restore the sensitivity of tumors to the patient's immune system.

[0056] Advantageously, such treatment makes it possible to improve and / or restore the sensitivity of cold tumors to the patient's immune system.

[0057] Advantageously, such treatment makes it possible to improve and / or restore the sensitivity of cholangiocarcinomas to the patient's immune system.

[0058] Advantageously, such treatment makes it possible to improve and / or restore the effectiveness of antitumor drugs, in particular immune checkpoint inhibitors (i.e. immunotherapies) with respect to cold tumors.

[0059] Advantageously, such treatment makes it possible to improve and / or restore the therapeutic effect of anticancer treatments, particularly in cases where conventional anticancer therapies have failed.

[0060] Even more advantageously, such treatment makes it possible to improve and / or restore sensitivity to anticancer treatments in chemoresistant cancers.

[0061] Advantageously, modulation of the tumor microenvironment using cold plasma can be particularly effective in treating solid tumors exhibiting high rigidity and resistance to conventional therapies, such as chemotherapy and immunotherapy.

[0062] Advantageously, cold plasma therapy, due to its ability to target the tumor microenvironment, may have fewer side effects than conventional therapies which can lead to systemic toxicity. Brief description of the drawings

[0063] [Fig-1]: (a) Electrical diagram showing the electrical power deposited on a (a) Target mimicking the electrical response of a mouse model, as a function of frequency and applied voltage; (b) Time profile of tissue temperatures measured during a 10-minute cold plasma treatment; (c) Principal emitting species of the plasma phase measured by optical emission spectroscopy; (d) Long-lived and short-lived chemical species detected by mass spectrometry in the plasma phase considering three flow rate values: 0.2 L / min, 0.6 L / min, and 1.0 L / min. The relative intensity is calculated by subtracting the signal intensity when the plasma is off from the signal intensity when the plasma is on.

[0064] [Fig.2]: Comparison of the growth of cholangiocarcinoma (CCA) tumor volume carried by two groups of mice: 10 untreated mice with plasma (Control group) and 10 mice treated with cold plasma, with 6 treatments of 5 min each, spaced every 2-3 days (Plasma group).

[0065] [Fig. 3]: (a) Cell viability of hTERT-HSC cells (a human fibroblast cell line used as a cell model of cancer-associated fibroblasts) analyzed 24 hours after exposure to cold atmospheric plasma (CAP) using the crystal violet assay, (b) mRNA expression levels, (c) α-SMA protein expression levels (fibroblast activation markers) and γH2AX protein expression levels (a marker of DNA double-brown breaks) 24 hours after PFA exposure of hTERT-HSC cells, (d) Image of an hTERT-HSC migration assay taken 24 hours after PFA exposure showing that the treated cells lose their ability to recapture the scratch created before treatment, and therefore to migrate. Detailed description

[0066] Definitions

[0067] Unless otherwise specified, the scientific and technical terms used in this document have the meanings commonly accepted in the field to which it relates. Examples of methods, devices, and materials are described in this document, but similar methods, devices, and materials or Equivalent terms may also be used in the implementation of this description. In case of conflict, this description shall prevail.

[0068] Unless the context otherwise indicates, singular terms include the plural and plural terms include the singular. The terms "a", "one or more" and "at least one" may be used interchangeably in the description.

[0069] Certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for the sake of brevity, described in the context of a single embodiment, may also be implemented separately or in any suitable sub-combination.

[0070] Numeric ranges include all numbers defining the range. Each maximum numeric limit given throughout the description includes any lower numeric limit, as if those lower numeric limits were expressly stated. Each minimum numeric limit given throughout the description includes any upper numeric limit, as if those higher numeric limits were expressly stated herein. Each numeric range given throughout the description includes any narrower numeric range that lies within such a wide numeric range, as if all such narrower numeric ranges were expressly stated herein.

[0071] In the description, embodiments described herein with the terms "having" or "comprising" include embodiments described with the terms "comprising only," "consisting of," and / or "consisting essentially of." The expression "consisting of" implies the inclusion of the stated elements to the exclusion of any other element. The expression "consisting essentially of" implies the inclusion of the stated elements, and possibly other elements when the other elements do not significantly affect the fundamental characteristic(s) of the disclosure.

[0072] Furthermore, the expression "and / or" should be considered as a specific disclosure of each of the two features with or without the other. Thus, the expression "and / or" used in an expression such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone) and "B" (alone).

[0073] The terms "about" or "approximately" mean an acceptable measurement error for a particular value of a parameter determined by the usual measurement methods in the field and which will depend in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" may mean within a range of two or fewer than two deviations types, according to the practice of the art. Alternatively, "about" can mean a numerical range or deviation of up to 20%, e.g. up to 10%, e.g. up to 5%, and even up to 1% of a given value.

[0074] Atmospheric pressure cold plasma (or atmospheric cold plasma). An atmospheric pressure cold plasma is a plasma generated at atmospheric pressure such that it operates out of thermal equilibrium and therefore out of thermodynamic equilibrium. This means that the elementary species that compose it (electrons, ions, and neutral species) each have their own temperature. Typically, electrons are very few in number and have temperatures on the order of 10,000 °C or even higher. Ions, on the other hand, have temperatures on the order of 1,000 °C. Finally, the neutral species—by far the most numerous—have a temperature lower than that of the ions, typically between 20 °C and 300 °C. For the medical applications targeted by the present invention, it is understood that the temperature of the neutral species must remain low, typically less than or equal to 50 °C, preferably less than or equal to 45 °C, and even better less than or equal to 40 °C.Thus, in this application, "atmospheric pressure cold plasma" means a plasma generated at atmospheric pressure with a temperature of 50°C or less. Atmospheric pressure cold plasma is commonly produced by applying an electrical discharge to a gaseous medium. This discharge is delivered by a high-voltage power supply, which causes partial ionization of the gas. The gaseous medium can be air, but it is common to use a noble gas, which, due to its chemical inertness, allows for the generation of a stable and easily controllable cold plasma. The gaseous medium may contain, in particular, helium, argon, neon, or a combination of these gases; preferably, the gaseous medium contains helium and / or argon and / or neon. Upon contact with ambient air, the atmospheric pressure cold plasma can then produce reactive oxygen and nitrogen species.

[0075] Treatment. The terms “treat” or “treatment” mean that the patient’s pathology is stabilized, reduced or at least partially improved and that some relief, attenuation or reduction of at least one clinical symptom of the pathology is obtained.

[0076] Exposure. In the context of the description, when used with respect to atmospheric pressure cold plasma, the terms "expose", "apply", or "administer" are interchangeable and refer to the exposure of, or the application to, a portion of the body surface of a patient in need of, atmospheric pressure cold plasma as described herein.

[0077] Immunologically cold tumor. The expressions "immunologically cold tumor" and "cold tumor" are used interchangeably and refer to a tumor characterized by low infiltration of immune cells, particularly T lymphocytes, or by a tumor microenvironment (TME) that inhibits or prevents immune activity against the tumor. In particular, as used here, the expression "immunologically cold tumor" tends to cover immunosuppressed altered tumors, immunoexcluded altered tumors, and cold tumors as they can be identified using an Immunoscore (Bruni, D. et al. (2020). Nature Rev Cancer 20: 662-680.; Galon, J. et al. (2006). Science. 313: 1960-1964; Lanzi A. et al. (2020). Oncolmmunology, 9: 1-3; Galon J, and Bruni D. Nat Rev Drug Discov. 2019;18(3): 197-218; Angell, HK et al. (2020). Clinical Cancer Research 26: 332-339). The term "cold tumor" also encompasses tumors defined as those deserted by the immune system, tumors excluded from the immune system, and tumors with low immune infiltration. Conversely, an immunologically hot tumor is a tumor characterized by infiltration (presence) of immune cells, particularly T lymphocytes, or by a tumor microenvironment (TME) that does not prevent immune activity against the tumor.

[0078] Patient. Subject. Individual. For the purposes of this description, these terms are used interchangeably and refer to a mammal, such as a rodent, feline, canid, or primate. In particular, a patient is a human being. A "patient in need" is a patient known or presumed to have a tumor.

[0079] Patient body surface. For the purposes of this description, "patient body surface" means a surface of a part of the patient's body in contact with the outside or with another part of the patient's body. Such a surface may be in contact with the external environment or a medical device. This term also includes the surfaces of internal cavities of a patient's body that are accessible naturally, endoscopically, or surgically. Examples of a patient body surface include the skin, the surface of an eyeball, the mucous membrane of the stomach, intestine, or bladder, and the surface of the bile ducts (including the gallbladder) or endothelium.

[0080] Portion of the patient's body surface. For the purposes of this description, "portion of the patient's body surface" means all or part of that surface.

[0081] Atmospheric pressure cold plasma

[0082] The cold plasma at atmospheric pressure is preferably a plasma generated from a noble gas.

[0083] According to one embodiment, a noble gas can be chosen from helium, neon, argon, krypton, xenon and a mixture thereof.

[0084] According to one embodiment, a noble gas can be chosen from helium, neon, argon, krypton, and xenon

[0085] According to one embodiment, a noble gas can be helium, neon, or argon.

[0086] According to one embodiment, a noble gas can be helium.

[0087] According to one embodiment, the cold atmospheric plasma can produce at less one reactive species chosen from the group consisting of: reactive oxygen species (ROS), reactive nitrogen species (RNS), and combinations thereof.

[0088] According to one embodiment, reactive oxygen species (ROS) can be chosen from the group consisting of: atomic oxygen (O), radical oxygen (O#), ozone (O3), hydroxyl radical (HO#), superoxide anion (O2#), hyperoxide radical (HOO#), hydroperoxyl (HO2), hydrogen peroxide (H2O2), nitric oxide (NO#), hydronium ion (H3O+), and combinations thereof.

[0089] According to one embodiment, reactive oxygen species (ROS) can be chosen from the group consisting of: atomic oxygen (O), radical oxygen (O#), ozone (O3), hydroperoxyl (HO2), hydrogen peroxide (H2O2), hydronium ion (H3O+), and combinations thereof.

[0090] According to one embodiment, the reactive nitrogen species (RNS) can be chosen from the group consisting of: nitric oxide (NO#), nitrous oxide (N2O), nitrogen dioxide (NO2), peroxynitrite (ONOO), nitrogen trioxide (N2O3), nitrogen tetroxide (N2O4), nitrite ion (NO2), nitrate ion (NO3), and combinations thereof.

[0091] According to one embodiment, the reactive nitrogen species (RNS) can be chosen from the group consisting of: nitric oxide (NO#), nitrous oxide (N2O), nitrogen dioxide (NO2), and combinations thereof.

[0092] According to one embodiment, the reactive species produced by a cold atmospheric plasma can be chosen from the group consisting of: atomic oxygen (O), radical oxygen (O#), ozone (O3), hydroxyl radical (HO#), hydrogen peroxide (H2O2), hydroperoxyl (HO2), nitric oxide (NO#), nitrogen dioxide (NO2), nitrous oxide (N2O), and combinations thereof.

[0093] According to one embodiment, a cold atmospheric plasma can produce a combination of reactive species consisting of: atomic oxygen (O), radical oxygen (O#), ozone (O3), hydroxyl radical (HO#), hydrogen peroxide (H2O2), hydroperoxyl (HO2), nitric oxide (NO#), nitrogen dioxide (NO2), and nitrous oxide (N2O).

[0094] Cold plasma at atmospheric pressure can be generated from at least one noble gas, for example helium, subjected to electrical pulses of, in particular, a few microseconds or a few milliseconds, preferably a few microseconds, also called "nano-pulses". The duration of each pulse can be between 1 ns and 100 ms.

[0095] According to one embodiment, the time interval between two consecutive pulses may be fixed or variable. According to one embodiment, the time interval between two consecutive pulses is fixed.

[0096] According to one embodiment, the time separating two consecutive pulses can vary from about 1 ns to about 20 ns, or even from about 2 ns to about 15 ns, or even from about 5 ns to about 10 ns.

[0097] Alternatively, the atmospheric pressure cold plasma can be generated from at least one noble gas, for example helium, subjected to a sinusoidal voltage.

[0098] According to one embodiment, the atmospheric pressure cold plasma can be generated from a noble gas subjected to a voltage varying from about 100 V to about 100 kV, or from about 1 kV to about 50 kV, or from about 5 kV to about 30 kV or be about 10 kV.

[0099] In general, atmospheric pressure cold plasma can be generated from at least one noble gas, in particular containing helium, subjected to a voltage varying from about 100 V to about 30 kV, and more commonly varying from about 1 kV to about 10 kV.

[0100] The atmospheric pressure cold plasma is generated by means of a conventional plasma generation device. A conventional plasma generation device may be a device such as that described in patent application WO 2022 / 229515.

[0101] A cold plasma generation device at atmospheric pressure conventionally comprises a plasma generation chamber having a noble gas inlet, the plasma generation chamber being subjected to an electric field via one or more electrodes. The electrodes are configured to emit an electric field which, when its value exceeds the disruption potential of the gas contained between the electrodes, causes partial ionization of the gas confined in the plasma generation chamber.

[0102] The electrodes can be configured to generate an electrical signal at a frequency of approximately 0.1 Hz to approximately 10 MHz, or approximately 1 Hz to 100 kHz, or approximately 100 Hz to 30 kHz, or possibly approximately 10 kHz

[0103] The noble gas flow rate at the inlet of the plasma generation chamber of the plasma generation device can vary from about 1 pL / min to about 10 L / min, better from about 1 mL / min to about 5 L / min, better from about 50 mL / min to about 2 L / min, even better from about 500 mL / min to about 1 L / min.

[0104] Such devices make it possible to generate a cold plasma at atmospheric pressure whose jet distribution is spatially homogeneous allowing application on a surface between 0.1 cm2 and 100 cm2.

[0105] Upon contact with air or the external environment, at the outlet of the plasma generation device, the plasma can advantageously react with oxygen and / or nitrogen and / or water vapor contained in the air, in order to generate reactive species with added therapeutic value.

[0106] The temperature of the cold plasma at atmospheric pressure, at the outlet of the plasma generation device, is preferably less than or equal to 50°C, better less than or equal to 45°C, even better less than or equal to 40°C. At the outlet of the plasma generation device, the temperature of the cold plasma at atmospheric pressure is preferably greater than or equal to 10°C, better greater than or equal to 20°C.

[0107] The plasma generation device is not necessarily in direct contact with the surface to be treated. In particular, a distance of between 0.1 mm and 100 mm can separate the plasma generation device from the surface to be treated.

[0108] Tumors

[0109] According to one embodiment, a tumor considered in this description may be a tumor including a carcinoma, a sarcoma, or a cancer of the central nervous system.

[0110] According to one embodiment, a tumor considered in this description may be a tumor among head and neck cancer, thoracic cancer, digestive cancer, genitourinary cancer, gynecological cancer, skin cancer.

[0111] According to one embodiment, a tumor can be selected from liver cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, gastric cancer, prostate cancer, kidney cancer, lung cancer, cervical cancer, endometrial cancer, vulvar cancer, Merkel cell carcinoma (skin cancer), and head and neck cancer.

[0112] The tumors considered in the present description may in particular be immunologically cold tumors.

[0113] Immunologically cold tumors are tumors that are weakly infiltrated by inflammatory immune cells, particularly T cells and especially CD8+ T cells. Immune infiltration of tumors, and in particular CD8+ T cell infiltration, has been shown to correlate with longer disease-free survival and / or overall survival in cancers with different histological characteristics and anatomical locations.

[0114] The density of CD8+ T cells in the tumor or its periphery may be a way to identify patients with cold tumors. Recently, an immune test called "Immunoscore" was developed to quantify in situ the infiltration of CD3+ CD8+ T cells into tumors of cancer patients (Bruni, D. et al. (2020). Nature Rev Cancer 20: 662-680.; Galon, J. et al. (2006). Science. 313: 1960-1964.; Lanzi A. et al. (2020). Oncolmmunology, 9: 1-3.).

[0115] The Immunoscore is a scoring system based on immunohistochemistry and digital pathology that assesses CD3+ and CD8+ T cell densities in the tumor and its invasive margin. In short, two adjacent slides of formalin-fixed, paraffin-embedded tumor blocks are stained with anti-CD3 and anti-CD8 antibodies in an automated staining system. The slides are then scanned, and the digital images are used to quantify the densities of the cells of interest using digital pathology software. The densities are then translated into an Immunoscore, ranging from a low Immunoscore (10) to a high Immunoscore (14).

[0116] Galon and Bruni proposed that the immunoscore be aligned with the concept of "hot and cold tumors" (Galon J, and Bruni D. Nat Rev Drug Discov. 2019; 18(3): 197-218.; Angell, HK et al. (2020). Clinical Cancer Research 26: 332-339.). With this approach, tumors were classified into four categories based on T-cell infiltration: warm immune tumors, altered immunosuppressed tumors, altered immune excluded tumors, and cold tumors.

[0117] The characteristics of these four types of tumors can be defined as follows:

[0118] 1. Hot immune tumors (also referred to herein as hot tumors).

[0119] High degree of T cell and cytotoxic T cell infiltration (high immunoscore).

[0120] Activation of checkpoints (programmed cell death protein 1 (PD-1), cytotoxic T-cell associated antigen 4 (CTLA-4), T-cell mucin immunoglobulin receptor 3 (TIM-3) and lymphocyte activation gene 3 (LAG-3)) or other altered T-cell functions (e.g., extracellular potassium-induced T-cell suppression).

[0121] 2. Altered immunocompromised tumors

[0122] Weak, but not absent, infiltration of T lymphocytes and cytotoxic T lymphocytes (Intermediate Immunoscore)

[0123] Presence of soluble inhibitory mediators (TGFγ, interleukin-10 (IL-10) and vascular endothelial growth factor (VEGF))

[0124] Presence of immunosuppressive cells (myeloid-derived suppressor cells and regulatory T cells)

[0125] Presence of T cell checkpoints (PD-1, CTLA-4, TIM-3 and LAG-3)

[0126] 3. Immune tumors excluded by alteration

[0127] No T-cell infiltration within the tumor bed

[0128] Accumulation of T cells at the tumor margins (invasive margin) (Intermediate Immunoscore)

[0129] Activation of oncogenic pathways

[0130] Epigenetic regulation and reprogramming of the tumor microenvironment

[0131] Aberrant tumor vascularization and / or stroma

[0132] Hypoxia

[0133] 4. Cold immune tumors

[0134] Absence of T cells inside the tumor and at the tumor edges (low Immunoscore)

[0135] Failure of T cell priming (low tumor mutational burden, poor antigen presentation and intrinsic insensitivity to T cell clearance).

[0136] A patient is considered to have a cold tumor if they have a tumor corresponding to the definition of an altered immunosuppressed tumor, an altered immune-excluded tumor, or an immune-cold tumor as defined above. This is equivalent to cancer patients with immunoscores I, II, or III, but not IV (the latter being tumors inflamed by T lymphocytes). The present invention applies to patients with immunoscores I, II, and III, but not IV.

[0137] Other tests and technologies besides immunoscore or T-cell density can help identify cold tumors.

[0138] In some cases, a cold tumor presents anergic (or anergic) lymphocytes. This means that the lymphocytes do not respond to the antigen. The methods for determining anergic lymphocytes are well known in art.

[0139] In other cases, a cold tumor exhibits a low percentage of CD3-positive cells. For example, the cold tumor may have less than 10% CD3-positive cells (as a percentage of the total tumor cells), i.e., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, or no CD3-positive cells. Methods for measuring the percentage of CD3-positive cells are known in Art.

[0140] The expression "cold tumor" also encompasses all tumors deserted by the immune system, tumors excluded from the immune system and tumors with low immune infiltration.

[0141] "Immune system deserted tumors" refers to tumors for which there is a total absence of immune response in the tumor due to a lack of tumor-infiltrating T cells.

[0142] "Immune system excluded tumors" refer to tumors for which reactive T cells are generated but are unable to penetrate the tumor to organize a response against it; T cells may be present at the periphery of the tumor.

[0143] “Low immune infiltration tumors” refers to tumors in which the level of penetration of immune cells (T lymphocytes) into the tumor microenvironment is reduced.

[0144] The term "cancer including a cold tumor" refers to both cold and non-cold tumors. For example, this may occur if the primary cancer (which may be a cold tumor) has metastasized and formed secondary tumors that are not cold tumors. A patient covered by this description may have multiple tumors, only one of which is a cold tumor.

[0145] By "cold tumor(s)", reference is made to cancers in which all tumors (primary and secondary) are cold.

[0146] According to one embodiment, an immunologically cold tumor can be selected from a sarcoma or a carcinoma.

[0147] According to one embodiment, an immunologically cold or non-immunological tumor may be a tumor of a cancer selected from liver cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, gastric cancer, prostate cancer, kidney cancer, lung cancer, cervical cancer, endometrial cancer, Merkel cell carcinoma, and head and neck cancer.

[0148] According to one embodiment, an immunologically cold tumor may be a tumor of a cancer selected from liver cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, bladder cancer, kidney cancer, lung cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, Merkel cell carcinoma, and head and neck cancer.

[0149] According to one embodiment, a bile duct cancer can be a cancer comprising immunologically cold or hot tumors.

[0150] According to one embodiment, a bile duct cancer can be a cancer comprising immunologically cold and hot tumors.

[0151] According to one embodiment, a cancer of the bile ducts can be a cancer consisting of immunologically cold tumors.

[0152] According to one embodiment, a cancer of the bile ducts may be a cholangiocarcinoma.

[0153] Cholangiocarcinoma is a malignant tumor that develops from the epithelial cells of the bile ducts, which are the channels that carry bile from the liver to the gallbladder and then to the small intestine. A cholangiocarcinoma can develop in any part of the bile ducts. It can be seen in the liver (intrahepatic cholangiocarcinoma) or at the point where the bile ducts leave the liver (extrahepatic cholangiocarcinoma, including perihilar and distal cholangiocarcinomas, and gallbladder cancers).

[0154] According to one embodiment, a cholangiocarcinoma can be selected from an intrahepatic cholangiocarcinoma and an extrahepatic cholangiocarcinoma.

[0155] Symptoms of cholangiocarcinoma may include abdominal pain, fever, jaundice, itching, weight loss, and fatigue. Diagnosis of cholangiocarcinoma may be made by a combination of tests, including abdominal ultrasound, computed tomography (CT) scan, or magnetic resonance imaging (MRI).

[0156] According to one embodiment, an immunologically cold tumor can be a cancer selected from among a cancer of the bile ducts.

[0157] According to one embodiment, an immunologically cold tumor can be a cholangiocarcinoma tumor.

[0158] Combination of treatments

[0159] According to one embodiment, a cold atmospheric pressure plasma as described for cancer treatment can be used in combination with at least one other anticancer therapeutic agent.

[0160] The expressions "in combination with" or "combined with" mean that atmospheric pressure cold plasma is applied before, after, or concurrently with the administration of the additional anticancer agent. In some cases, the expression "in combination with" includes the simultaneous or sequential application and administration of atmospheric pressure cold plasma and the additional anticancer agent.

[0161] According to one embodiment, a cold atmospheric pressure plasma as described may be applied simultaneously or sequentially with the administration of at least one additional anticancer therapeutic agent.

[0162] In some embodiments, atmospheric pressure cold plasma and the additional anticancer agent are applied and administered sequentially to a patient in need. For example, atmospheric pressure cold plasma and the additional anticancer agent are applied and administered on the first day and the following day of the treatment period, respectively, or on the same day, one at a first time and the other at a subsequent time. For example, atmospheric pressure cold plasma is applied on morning of one day, and the additional anticancer agent is administered in the afternoon of the same day, or vice versa.

[0163] In some embodiments, the atmospheric pressure cold plasma and the additional anticancer agent are, respectively, applied and administered simultaneously to a subject in need. For example, the atmospheric pressure cold plasma and the additional anticancer agent are, respectively, applied and administered on the same day, at approximately the same time.

[0164] According to one embodiment, a cold atmospheric pressure plasma for its use as described herein can be applied simultaneously or sequentially to the administration of an additional anticancer agent.

[0165] According to one embodiment, an additional anticancer therapeutic agent is administered following the application of atmospheric pressure cold plasma. This administration may be performed immediately after the application of atmospheric pressure cold plasma or within 5 hours following this application.

[0166] According to one embodiment, an additional anticancer therapeutic agent is administered before the application of atmospheric pressure cold plasma. This administration may be performed immediately before the application of atmospheric pressure cold plasma or within 5 hours preceding this application.

[0167] An additional anticancer therapeutic agent is necessarily distinct from atmospheric pressure cold plasma therapy as described herein.

[0168] An additional cold anticancer agent at atmospheric pressure may be selected from electrotherapy, for example pulsed electric field therapy, immunogenic chemotherapy, radiotherapy, a therapeutic cancer vaccine, anticancer immunotherapy, and an oncolytic virus.

[0169] An anticancer immunotherapy may be chosen from immune checkpoint inhibitors.

[0170] Immunogenic chemotherapy induces the death of cancer cells in a way that triggers an immune response, leading to the recruitment of immune cells to the tumor. Examples include 5-FU, gemcitabine, capecitabine, and oxaliplatin.

[0171] Radiotherapy, like chemotherapy, can induce immunogenic cell death of tumor cells leading to an anti-tumor immune response, such as SIRT (selective internal radiotherapy also called radioembolization) which uses Yttrium-90, Holmium-166 or Rhenium-186 or 188.

[0172] Cancer vaccines stimulate an immune response against specific tumor antigens, which can attract immune cells to the tumor. Targets include CTLA-4, PD-1 (DNA vaccine), and Wilms' tumor gene antigen (WT1) and mucin 1 (MUC1) (RNA vaccine). Other tumor antigens tested are: cell division associated 1 (CDCA1), cadherin 3 (CDH3) and kinesin family member 20A (KIF20A).

[0173] Immune checkpoint inhibitors include anti-PD-1, anti-PD-L1, and anti-CTLA-4 therapies, which can suppress T cell feedback controls, thereby enhancing their activity and ability to infiltrate tumors.

[0174] According to one embodiment, an immune checkpoint inhibitor may be an antibody that inhibits programmed cell death transmembrane protein 1 (PDCD1 or PD-1 or CD279), the PD-L1 ligand (B7H1 or CD274) and / or cytotoxic T lymphocyte-associated molecule 4 (CTLA-4).

[0175] Such an antibody may be a monoclonal antibody.

[0176] According to one embodiment, a PD-1 blocking antibody may be selected from pembrolizumab, nivolumab, spartalizumab, tislelizumab and cemiplimab.

[0177] According to one embodiment, a PD-L1 blocking antibody may be selected from atezolizumab, avelumab and durvalumab.

[0178] According to one embodiment, a CTLA-4 blocking antibody may be ipilimumab.

[0179] Oncolytic viruses can selectively infect and kill cancer cells, causing the release of tumor antigens and triggering an immune response.

[0180] The term “oncolytic” is intended to designate the ability of a virus to replicate selectively in dividing cells (e.g., a proliferative cell such as a cancer cell) with the aim of slowing the growth and / or lysing said dividing cell, whether in vitro or in vivo, while showing no replication or minimal replication in non-dividing cells (e.g., normal or healthy cells).

[0181] Examples of oncolytic viruses suitable for the invention include adenoviruses, protoparvoviruses, vaccinia viruses, reoviruses; herpes simplex virus (HSV); vesicular stomatitis virus (VSV); Newcastle disease virus (NDV); measles virus; picomaviruses; and Maraba virus. These viruses are known to those competent in medicine and virology.

[0182] Uses and methods

[0183] According to one of its objects, the invention relates to a cold plasma at atmospheric pressure for its use in the treatment of a tumor in a patient in need of such treatment.

[0184] According to one embodiment, the tumor may be an immunologically cold tumor.

[0185] According to one embodiment, the tumor may be an immunologically warm tumor.

[0186] According to one of its objects, the invention relates to a method of treating a tumor, in particular immunologically cold, in a patient in need of such treatment, the method comprising at least one step consisting of applying cold plasma at atmospheric pressure to a portion of the patient's body surface.

[0187] The step of applying cold plasma at atmospheric pressure to a portion of the patient's body surface allows the treatment of a tumor, in particular an immunologically cold one.

[0188] According to one embodiment, use or method as described herein, allow the treatment of altered immunocompromised tumors.

[0189] According to one embodiment, use or method as described herein, allow the treatment of altered immune excluded tumors.

[0190] According to one embodiment, use or method as described herein, allow the treatment of cold immune tumors.

[0191] According to one embodiment, use or method as described herein, allow the treatment of tumors deserted by the immune system.

[0192] According to one embodiment, use or method as described herein, allow the treatment of tumors excluded from the immune system.

[0193] According to one embodiment, use or method as described herein, allow the treatment of tumors with low immune infiltration.

[0194] According to one embodiment, atmospheric pressure cold plasma can be applied to a portion of the patient's body surface for a duration ranging from about 1 s to about 120 min., or from about 10 s to about 45 min., or from about 1 min to about 10 min., or from about 1 to about 5 min., or be about 5 min.

[0195] According to one embodiment, the atmospheric pressure cold plasma can be applied to a portion of the patient's body surface at least at a frequency ranging from 0.1 Hz to about 10 MHz, or from about 1 Hz to 100 kHz or from about 100 Hz to 30 kHz or be about 10 kHz.

[0196] According to one embodiment, atmospheric pressure cold plasma can be applied to a portion of the patient's body surface at least at a generator voltage ranging from 100 V to 100 kV, or from about 1 kV to 50 kV, or from about 5 kV to 30 kV or from about 10 kV.

[0197] According to one embodiment, atmospheric pressure cold plasma can be applied to a portion of the patient's body surface at least at a surface power of 0.1 pW / cm2 to 100 mW / cm2 or from 1 pW / cm2 to 10 mW / cm2, from 10 pW / cm2 to 1 pW / cm2, or from 20 to 500 pW / cm2.

[0198] According to one embodiment, atmospheric pressure cold plasma can be applied to a portion of the patient's body surface for a minimum of 1 treatment, typically between 2 and 10 treatments. The interval between two consecutive treatments is between approximately 1 hour and approximately 1 week, or between approximately 10 hours and 3 days, or between approximately 1 and 2 days.

[0199] According to one embodiment, the atmospheric pressure cold plasma can be generated from at least one noble gas, in particular helium, subjected to an electric field via at least one electrode delivering an electrical signal at a generator voltage varying between 100 V and 100 kV, or about 1 kV and 50 kV, or about 5 kV and 30 kV or about 10 kV, and at a frequency of 0.1 Hz to about 10 MHz, or about 1 Hz to 100 kHz or about 100 Hz to 30 kHz or about 10 kHz, the noble gas flowing at a rate of between 1 pL / min and 10 L / min, better between 1 mL / min and 5 L / min, better between 50 mL / min and 2 L / min, even better between 500 mL / min and 1 L / min.

[0200] According to one embodiment, atmospheric pressure cold plasma can be applied to a portion of the patient's body surface for at least 1 second and commonly over a time interval of between 10 seconds and 30 minutes, and preferably over a time interval of between 1 minute and 5 minutes.

[0201] According to one embodiment, the cancer is a cholangiocarcinoma and cold plasma at atmospheric pressure can be applied to a portion of the surface of the bile ducts of the patient for at least 1 second and preferably over a time interval of between 10 seconds and 10 minutes. Examples

[0202] The examples described below are given for the purpose of illustrating the claimed invention and shall not be construed as limiting it.

[0203] Example 1: Materials and Methods cold plasma device

[0204] The cold plasma device generates a non-thermal plasma at atmospheric pressure with adjustable parameters such as power, frequency, and treatment duration. The device ensures uniform plasma treatment of the target area, and the results presented below indicate that no risk of thermal / electrical damage to surrounding tissues was observed.

[0205] The device for generating the cold plasma has a plasma generation chamber, designed to contain helium and allow plasma formation. It is made of heat- and ionization-resistant materials to ensure safety and durability. Typically, these are dielectric materials such as quartz or alumina. The device also includes a A helium supply system injects this gas into the plasma generation chamber. This system is equipped with flow regulators to precisely control the amount of helium injected. Two concentric rings serve as electrodes. These rings are strategically positioned outside the chamber to create a uniform and efficient electric field, preventing any risk of arcing and thus ensuring consistent cold plasma generation. One of these electrodes must be connected to a high-voltage power supply, preferably a high-voltage pulse supply. The pulse voltage is adjustable, allowing for fine control of the pulse intensity and frequency, which is crucial for modulating the characteristics of the generated plasma. Thanks to the precision of the pulse voltage supply and the optimized design of the dual-ring electrodes, the device allows for advanced control of plasma temperature and density.This feature is essential for specific applications where plasma accuracy is crucial. Integrated safety systems, such as automatic circuit breakers and overvoltage detectors, can be used to ensure operator safety and device reliability. Determination of reactive species

[0206] The present plasma jet device is designed to generate a variety of reactive species, the composition and characteristics of which can be accurately analyzed and quantified using Optical Emission Spectroscopy (OES) and Mass Spectrometry (MS).

[0207] Optical Emission Spectroscopy is used to detect and characterize radiative species, primarily ions, atoms, and molecules in excited states, which emit light upon returning to a lower energy state. This method is particularly effective for the analysis of short-lived radiative species, such as certain free radicals and excited ions, which rapidly emit light after excitation. In particular, we were able to highlight the generation of OH and O radicals.

[0208] Mass spectrometry is used to identify and quantify ionic species and certain neutral molecules present in the plasma. This technique is suitable for the detection of non-radiative species, thus providing a complete analysis of the stable components of the plasma.

[0209] The combined use of OES and MS in this device allows for an exhaustive analysis of the species present in the plasma jet, covering both radiative and non-radiative aspects, as well as the short- and long-term characteristics of the generated species. Thermal safety

[0210] In order to ensure safe use in accordance with safety standards, the device is designed to maintain the treatment temperature below the critical threshold of 40 °C, regardless of the duration of the plasma treatment.

[0211] To ensure continuous compliance with this temperature constraint, thermal monitoring is implemented using infrared thermographic imaging. This monitoring system captures real-time thermal data from the plasma jet device and the processing environment, enabling precise and instantaneous temperature assessment.

[0212] The data captured by thermographic imaging are analyzed to identify any area or component of the device exceeding the temperature limit of 40 °C. If this temperature is exceeded, thermal regulation mechanisms are automatically activated to reduce the temperature to a safe level. These systems may include modulating plasma intensity, adjusting gas flow rates, and activating cooling systems.

[0213] Measurement of tumor stiffness by shear wave elastography

[0214] Shear wave elastography (SWE) measurements are performed with an Aixplorer ultrasound device (SuperSonic Imagine, Aix-en-Provence, France) using a 15 MHz surface probe dedicated to research (256 elements, 0.125 pm step size).

[0215] Mice were anesthetized with 2% isoflurane and their body temperature was maintained at a physiological level using a hot plate. B-mode and SWE images were acquired simultaneously. The B-mode image allowed for manual determination of the region of interest (ROI) corresponding to the tumor contours. The SWE image was acquired in penetration mode with a color scale ranging from 0 (transparent) to 40 kPa (red), arbitrarily chosen at the beginning of the study based on the expected stiffness values. The area, diameter, and a set of stiffness values ​​(mean, minimum, maximum, and standard deviation) were recorded for the ROI as defined above. The SWE images were also analyzed using internal MATLAB code to retrieve the stiffness map.

[0216] Mouse model of cholangiocarcinoma (ectopic subcutaneous)

[0217] Two models were used.

[0218] Xenograft: The injected cholangiocarcinoma cells are of a different species than those in mice. Human CCA cells were injected under the skin of the right or left flank of so-called immunodeficient mice (strain Rj:ATHYM-Foxnlnu / nu); their immune system is incomplete. See the following publication for a detailed description (Lekbaby et al., Experimental model of biliary tract cancers: subcutaneous xenograft of human cell Nus in immunodeficient nude mouse. Methods in Cell Biology, 2024).

[0219] Syngeneic: The injected cholangiocarcinoma cells are of the same species as the mouse. Murine cholangiocarcinoma cells (SB1) were injected under the skin of the right or left flank of immunocompetent mice (B6 / Rj-Tyr c / c); their immune system is complete. Three million cells were injected per mouse using a syringe (see the publication cited above in the "xenograft" section). Tumors appeared 3 to 4 weeks post-injection; they were measured using calipers 3 times per week.

[0220] In both cases, treatments with atmospheric pressure cold plasma began when the tumors measured between 50 and 100 mm3. Artificial bile duct models

[0221] Two models were used: an "ERCP trainer" and a post-mortem porcine anatomical model.

[0222] The ERCP trainer is a training simulator specifically designed for training in retrograde cholangiopancreatic endoscopy (ERCP), an advanced medical technique used to diagnose and treat diseases of the bile ducts and pancreas. This device realistically reproduces the relevant anatomy, thus providing healthcare professionals with a safe environment to practice and improve their skills in various ERCP procedures, such as bile duct cannulation, dye injection, stent placement, sphincterotomy, and lithotripsy.

[0223] The post-mortem porcine anatomical model. This model includes the esophagus, stomach, duodenum, biliary tree, liver, and gallbladder. It is placed in a dedicated plastic structure designed to resemble the human peritoneal cavity. This structure is known as EASIE (Erlangen active simulator for interventional endoscopy) and was used in its EASIE-RTM version, supplied by the company ENDOSIM. EASIE-RTM is recognized as an outstanding educational tool for interventional endoscopy.

[0224] Cell viability, DNA alterations, cell migration

[0225] Cell viability analyzed by the crystal violet method: Cells were seeded in 24-well plates the day before plasma treatment. Cell viability was assessed between 24 and 72 hours post-treatment on all cell lines studied, with the medium having been renewed beforehand. Cells were stained with crystal violet (gentian violet diluted in methanol) for 15 min and then rinsed with water. The crystal violet was then dissolved in SDS. The plates were incubated at 37 °C for 2 h, and then the absorbance was quantified with a spectrophotometer (TECAN®) at a wavelength of 595 nm.

[0226] Study of real-time fibroblast migration by scratch assay: Myofibroblastic cells (hTERT-HSC) were seeded confluently in a 24-well plate the day before treatment. Using a cone, a tear in the cell mat was made at the bottom of each well. After washing, the cells were directly treated with cold plasma. Cell migration was filmed in real time for 24 hours using an Olympus 1X83 microscope with a motorized stage and a temperature-controlled chamber. Images were taken every 30 minutes and then analyzed with ImageJ software.

[0227] DNA analysis by Western blot: 24 h following cold plasma treatment, cells were lysed with RIPA (Radioimmunoprecipitation Assay, Merck) solution on ice for 15 min with shaking. After centrifugation (15 min at 4 °C), the supernatants were collected and the proteins were quantified using a BCA kit (Pierce™ BCA Protein Assay Kit, ThermoFisher 23225), which operates based on bicinchoninic acid. The protein concentration was then determined by measuring the absorbance at 570 nm (TECAN®) using a BSA calibration curve. The proteins were then diluted to a concentration of 2 pg / pL in the loading buffer, which is Laemmli blue, in the presence of 2-mercaptoethanol, which allows the proteins to be denatured for 5 min at 98 °C.Protein migration was performed on a 12% concentrated acrylamide gel in a migration solution (Tris-Glycine-SDS diluted in distilled water) for approximately 1 hour at 150 V. The proteins were then transferred to a nitrocellulose membrane for approximately 1 hour 20 minutes at 50 V (using a transfer solution of diluted Tris-Glycine). The membranes were then saturated with 5% BSA diluted in 0.1% TBS Tween (TBS-T) for 1 hour with shaking at room temperature, and then cut with a scalpel to the desired protein size. These were incubated with the primary antibody (Table 1) overnight at 4°C with shaking, and then washed with TBS-T. The membranes were then exposed to the secondary antibody (coupled to peroxidase) for 1 h at room temperature under agitation.Protein visualization was performed using the ECL Prime kit (Life Sciences) by chemiluminescence (Imager iBright, Thermo Fisher). A stripping buffer (GeBa) was applied to the membranes for 15 min to visualize proteins with similar molecular weights. The γ-H2AX antibody was used as a marker for DNA breaks.

[0228] Table 1: Primary Antibodies Protein Molecular Weight (kDa) Reference Company Dilution Source yH2AX 15 #9718 Cell Signaling 1 / 1000 (WB) Rabbit GAPDH 36 sc-32233 Santa Cruz 1 / 1000 Mouse

[0229] Gbroblast phenotype analysis by RT-qPCR: Total mRNAs were extracted using the NucleoSpin RNA kit (Macherey-Nagel) 24 h after treatment with cold plasma. The RNA was eluted in 40 pL of RNAse-free water and then quantified using a Nanodrop (ThermoScientific). cDNAs were then synthesized using a kit (Invitrogen) and a thermocycler (Agilent Technologies). Quantitative PCR was performed on the LightCycler 96 (Roche) using cDNAs concentrated at 2.5 ng / pL, with SYBR Green as the fluorescent probe. The expression of genes of interest is analyzed by duplicate method 2 [(CT gene of interest-CT housekeeping gene)-CT control sample], in GApDH„6 gene of standardization.

[0230] Table 2: Sequences of primers used for RT-qPCR. Protein Gene Sense primer (5'- 3') Antisense primer (5'- 3') a-SMA ACTA2 SEQ ID NO: 1: GACAATGGCTC TGGGCTCTGTAA SEQ ID NO: 2: CTGTGCTTCGT CACCCACGTA COL1A1 COL1A 1 SEQ ID NO: 3: AGTTCGAGTAT GGCGG SEQ ID NO: 4: CAGTGACGCTG TAGGT COL4A1 COL4A 1 SEQ ID NO: 5: CCGCTGCGAAG GGTGG SEQ ID NO: 6: CCCTTTTGTCCT GGTGGTCCC GAPDH GAPD H SEQ ID NO: 7: AGCCACATCGC TCAGACAC SEQID NO: 8: GCCCAATACGA CCAAATCC Example 2: Results

[0231] Determination of the operating parameters of atmospheric pressure cold plasma

[0232] In order to determine the optimal parameters for treating tumors with cold plasma, a series of in vitro and in vivo experiments were conducted to identify the most effective power, frequency, and duration for modulating the tumor microenvironment (TME). These parameters are adjusted according to the type of tumor, its size, and its location.

[0233] A parametric study was conducted to investigate the electrical operating conditions of cold plasma treatment using artificial bile duct models that mimic the properties of real ducts.

[0234] During the study, a voltage of 2500 to 6000 V and a frequency of 1 to 20 kHz were applied (see Fig. 1a). The resulting electrical power deposited in the target is indicated by the color bar. The dotted area in the lower left corner indicates a combination of electrical parameters where no plasma can be generated, while the black area in the upper right corner indicates that the electrical discharge has turned into a thermal arc, which can be dangerous for the patient. The power values ​​in the region between these two areas are between 3 and 350 pW, which are safe and effective for use on human patients. Determination of reactive species

[0235] Reactive oxygen and nitrogen species in the plasma phase that induce antitumor effects and MET remodeling have been identified, as shown in Fig. 1b. The main species include radicals (such as OH and O), N2+ ions and excited species (He and N2*).

[0236] Reactive nitrogen species are the most emissive, followed by helium atoms. These species result from complex reaction mechanisms that induce oxidative stress, antitumor effects, and a reduction in tumor rigidity. Thermal safety

[0237] Thermal safety was verified on post-mortem porcine models using optical diagnostics.

[0238] Infrared imaging was used to monitor the temperature increase in the anatomical region where the tumor is located before, during and after exposure to cold plasma, as shown in Figs. 1 and 1d. The results indicate that plasma-induced warming remains quite low, with the temperature increase never exceeding 10 °C and easily being reduced to 5 °C, even after a 5-minute treatment.

[0239] Mouse model of subcutaneous ectopic cholangiocarcinoma tumor

[0240] The antitumor effect of cold plasma at atmospheric pressure was studied in an immunocompromised model of cholangiocarcinoma, a desmoplastic tumor of the biliary epithelium characterized primarily by a large fibrous stroma. This tumor is characterized by a high rigidity established by specific cell populations called cancer-associated fibroblasts (CAFs), which secrete a collagen-rich extracellular matrix responsible for the ineffectiveness of therapies against cholangiocarcinoma. This “barrier” prevents the action of chemotherapies, limits the infiltration of immune cells, and compromises antitumor surveillance.

[0241] In vivo results showed that repeated treatment of the subcutaneous tumor with cold plasma resulted in a significant decrease in tumor burden compared to the control group ([Fig. 2]). Furthermore, shear wave elastography observed that this reduction was associated with a slight decrease in tumor stiffness. Stiffness is represented by internal pressure expressed in kPa. While this value can reach 40 kPa in untreated tumors, it typically decreases to average values ​​close to 10 kPa in tumors treated with cold plasma.

[0242] Cancer-associated fibroblast cell models

[0243] In vitro experiments were carried out to expose human hepatic myofibroblastic cell lines (hTERT-HSC used as CAF models) to cold plasma at atmospheric pressure in order to study the phenotypic changes induced by such treatment.

[0244] We have demonstrated that plasma treatment leads to a significant decrease in cell viability (Fig. 3a), and that the remaining live cells underexpress certain myofibroblastic markers, such as smooth muscle actin α (α-SMA) protein (Fig. 3b). Exposure to cold plasma at atmospheric pressure resulted in DNA alterations, as shown by the increased phosphorylation of histone H2AX:gamma-H2AX (Fig. 3c). Furthermore, the remaining live cells lost their ability to migrate, as observed by real-time microscopy (Fig. 3d).

[0245] These results demonstrate the ability of a cold plasma to modulate the TEM.

[0246] Example 3: Conclusion

[0247] The atmospheric pressure cold plasma treatment of tumors described herein represents a novel approach to modulating the tumor microenvironment, which may enhance the efficacy of anticancer therapies and improve patient outcomes (in locally advanced or metastatic stages, 1-year, 3-year, and 5-year survival rates are 25%, 10%, and 7%, respectively). By reducing matrix rigidity and increasing the accessibility of antitumor immune cells and therapeutic agents to the tumor, cold plasma treatment has demonstrated its efficacy in treating cancers with a rigid microenvironment.

Claims

Demands

1. Atmospheric pressure cold plasma for use in the treatment of an immunologically cold tumor in a patient in need of such treatment.

2. Atmospheric pressure cold plasma for use according to claim 1, wherein the atmospheric pressure cold plasma is generated from a noble gas.

3. Atmospheric pressure cold plasma for use according to claim 2, the cold plasma producing at least one reactive species selected from the group consisting of: reactive oxygen species (ROS), reactive nitrogen species (RNS), and combinations thereof.

4. Atmospheric pressure cold plasma for use according to claim 2, wherein the atmospheric pressure cold plasma is generated by applying electrical pulses to said noble gas, the duration of each pulse being between 1 ns and 100 ms.

5. Atmospheric pressure cold plasma for use according to claim 2 or 3, wherein the atmospheric pressure cold plasma is generated from said noble gas subjected to a voltage ranging from 100 V to 100 kV, or from 1 kV to 50 kV, or from 5 kV to 30 kV or be 10 kV.

6. Atmospheric pressure cold plasma for use according to any one of claims 2 to 4, wherein the atmospheric pressure cold plasma is generated from said noble gas subjected to an electrical signal whose frequency varies from 0.1 Hz to 10 MHz, or from 1 Hz to 100 kHz or from 100 Hz to 30 kHz or be 10 kHz.

7. Atmospheric pressure cold plasma for use according to any one of claims 1 to 5, wherein the atmospheric pressure cold plasma is applied to a portion of the patient's body surface at least at a surface power of 0.1 pW / cm2 to 100 mW / cm2 or 1 pW / cm2 to 10 mW / cm2, 10 pW / cm2 to 1 pW / cm2, or 20 to 500 pW / cm2.

8. Atmospheric pressure cold plasma for use according to any one of claims 1 to 6, wherein the atmospheric pressure cold plasma is applied to a portion of the surface of the patient's body for at least 1 second and commonly over a time interval ranging from 10 seconds to 30 minutes, and preferably over a time interval ranging from 1 minute to 5 minutes.

9. Atmospheric pressure cold plasma for use according to any one of claims 1 to 7, wherein the immunologically cold tumor is selected from among altered immunodeficient tumors, altered immune excluded tumors, cold immune tumors, immune deserted tumors, immune excluded tumors, and tumors with low immune infiltration.

10. Atmospheric pressure cold plasma for use according to any one of claims 1 to 8, wherein the immunologically cold tumor is a cancer tumor selected from liver cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, bladder cancer, kidney cancer, lung cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, Merkel cell carcinoma, and head and neck cancer.

11. Atmospheric pressure cold plasma for use according to any one of claims 1 to 9, wherein the atmospheric pressure cold plasma is applied simultaneously or sequentially with the administration of an additional anticancer agent.

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