System for treating cancer

Abstract

A system for treating cancer is disclosed. According to an embodiment of the present invention, the system comprises: an electric field applying device for applying an electric field for tumor treatment; the delivery system is used for delivering a drug, and the drug comprises at least one of an immune checkpoint inhibitor and a chemical drug; the immune checkpoint inhibitor is selected from one of a duvaleriumab, a palbolizumab, an atelizumab, a Nasuliumab and a tereprenil monoclonal antibody; the chemical drug is selected from at least one of gemcitabine, cis-platinum, GEMOX and lenvatinib. According to the system, proliferation of the biliary tract cancer cells can be inhibited, migration of the biliary tract cancer cells can be inhibited, mitosis of the biliary tract cancer cells can be interfered, and immunogenic death of the biliary tract cancer cells can be promoted.

Description

Technical Field

[0001] This application relates to the field of combination therapy, particularly to systems for treating cancer, and further to the use of an electric field application device in the preparation of a cancer treatment system, the use of a chemical drug or immune checkpoint inhibitor in the preparation of a drug, methods for treating cancer cells, methods for drug screening, methods for interfering with cancer cell mitosis, and methods for inhibiting cancer cell proliferation and promoting immunogenic cell death. Background Technology

[0002] Biliary tract cancer (BTC) is a group of malignant tumors originating from the epithelium of the gallbladder and bile ducts, exhibiting high heterogeneity. Surgery is the preferred treatment for BTC tumors, but many patients are diagnosed at an advanced stage, thus losing the opportunity for surgery. For patients with inoperable BTC, chemotherapy and radiotherapy are commonly used treatments. However, these treatments have limited efficacy and may be accompanied by significant side effects. Targeted therapy is a drug therapy that targets specific molecular markers of tumor cells, reducing damage to normal cells. However, the application of targeted therapy in BTC is still in its early stages. Immunotherapy attacks tumor cells by activating or enhancing the patient's own immune system. Immune checkpoint inhibitors, such as durvalumab, are a type of immunotherapy that blocks the interaction between PD-L1 and PD-1, thus preventing immune escape by tumor cells. Tumor therapeutic electric fields (TTFields) are a novel non-invasive treatment that interferes with tumor cell mitosis by applying a specific alternating electric field to the tumor region, leading to cell death.

[0003] Due to the complexity and heterogeneity of biliary tract cancer, single treatment methods often have limited effectiveness. Therefore, there is an urgent need to combine TTFields with other treatments such as chemotherapy, targeted therapy, or immunotherapy to achieve better therapeutic outcomes. Summary of the Invention

[0004] The present invention aims to at least partially address at least one of the technical problems existing in the prior art. To this end, the present invention provides a system for treating cancer.

[0005] This invention is based on the following discoveries of the inventors:

[0006] Patients with biliary tract cancer are often diagnosed at an advanced stage, making surgery an option. Chemotherapy and radiotherapy have limited efficacy against biliary tract tumors and significant side effects. To overcome this problem, the inventors used tumor therapeutic electric fields (TTFields) in combination with durvalumab and gemcitabine / cisplatin (GemCis) to treat biliary tract cancer, thereby inhibiting the proliferation and migration of biliary tract cancer cells, interfering with mitosis of biliary tract cancer cells, and promoting immunogenic death of biliary tract cancer cells.

[0007] In a first aspect, the present invention provides a system for treating cancer. According to an embodiment of the invention, the system includes: an electric field application device for applying an electric field for tumor treatment; and a delivery system for delivering a drug, the drug comprising at least one of an immune checkpoint inhibitor and a chemical drug; the immune checkpoint inhibitor being selected from durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; and the chemical drug being selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib. The system according to an embodiment of the invention is capable of inhibiting the proliferation of biliary tract cancer cells, inhibiting the migration of biliary tract cancer cells, interfering with the mitosis of biliary tract cancer cells, and promoting immunogenic death of biliary tract cancer cells.

[0008] In a second aspect, the invention proposes the use of an electric field application device in the preparation of a cancer treatment system in which cancer patients undergo drug treatment before, after, or simultaneously.

[0009] In a third aspect, the invention proposes the use of a chemical drug or an immune checkpoint inhibitor in the preparation of a medicament for treating a patient with cancer, the patient being treated with an electric field applied by an electric field application device before, after, or simultaneously with the treatment.

[0010] In a fourth aspect, the present invention provides a method for treating cancer cells. According to embodiments of the invention, the method includes: treating cancer cells under conditions where an electric field is applied by an electric field application device; pre-treatment, post-treatment, or simultaneous administration of a drug to the cancer cells; wherein the drug comprises at least one of an immune checkpoint inhibitor and a chemical drug; the immune checkpoint inhibitor is selected from durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; the chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib. The method according to embodiments of the invention can inhibit the proliferation of biliary tract cancer cells, inhibit the migration of biliary tract cancer cells, interfere with the mitosis of biliary tract cancer cells, and promote immunogenic death of biliary tract cancer cells.

[0011] In a fifth aspect, the present invention provides a method for drug screening. According to an embodiment of the invention, the method includes: subjecting cancer cells to an electric field applied by an electric field application device; contacting the cancer cells treated with the electric field with a candidate drug; and determining, based on the state of the cancer cells before and after the contact, whether the candidate drug is a drug suitable for treating cancer. The method according to an embodiment of the invention is capable of screening drugs for treating cancer.

[0012] In a sixth aspect, the present invention provides a method for interfering with the mitosis of cancer cells. According to an embodiment of the invention, the method includes: treating cancer cells under conditions where an electric field is applied by an electric field applying device; wherein the frequency of the electric field applied by the electric field applying device is 100–700 kHz, preferably 100–250 kHz, more preferably 140–160 kHz; and / or, the field strength of the electric field applied by the electric field applying device is 1–3 V / cm, preferably 1.6–2.5 V / cm, more preferably 2–2.2 V / cm. The method according to an embodiment of the invention is capable of interfering with the mitosis of biliary tract cancer cells.

[0013] In a seventh aspect, the present invention provides a method for inhibiting cancer cell proliferation and promoting immunogenic cell death. According to an embodiment of the present invention, the method includes: treating cancer cells under conditions where an electric field is applied by an electric field applying device; wherein the frequency of the electric field applied by the electric field applying device is 100–700 kHz, preferably 100–250 kHz, more preferably 140–160 kHz; and / or, the field strength of the electric field applied by the electric field applying device is 1–3 V / cm, preferably 1.6–2.5 V / cm, more preferably 2–2.2 V / cm. The method according to the embodiment of the present invention can inhibit cancer cell proliferation and promote immunogenic cell death.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0016] Figure 1 This is a three-dimensional assembly diagram of a TTF cell experimental apparatus according to one embodiment of this application.

[0017] Figure 2 for Figure 1 A partial three-dimensional exploded view of the TTF cell experimental setup shown.

[0018] Figure 3This is a cytotoxicity analysis diagram of HCCC-9810 cells obtained by applying TTFields at different frequencies for 96 hours in Example 1 of this application.

[0019] Figure 4 This is a graph showing the cytotoxicity analysis of RBE cells obtained by applying TTFields at different frequencies for 96 hours in Example 1 of this application.

[0020] Figure 5 This is a cytotoxicity analysis diagram of HCCC-9810 cells obtained by applying different electric field strengths to TTFields for 96 hours in Example 1 of this application.

[0021] Figure 6 This is a graph showing the cytotoxicity analysis of RBE cells obtained by applying different electric field strengths to RBE cells for 96 hours using TTFields in Example 1 of this application.

[0022] Figure 7 This is a graph showing the colony-forming ability of HCCC-9810 cells after 96 hours of TTFields application and without TTFields application, as shown in Example 1 of this application.

[0023] Figure 8 Example 1 of this application shows a statistical analysis of the colony-forming ability of HCCC-9810 cells after 96 hours of TTFields application and without TTFields application.

[0024] Figure 9 This is a diagram showing the colony-forming capacity of RBE cells after 96 hours of TTFields application and without TTFields application, as shown in Example 1 of this application.

[0025] Figure 10 Example 1 of this application shows a statistical analysis graph of the colony-forming ability of RBE cells after 96 hours of TTFields application and without TTFields application.

[0026] Figure 11 This is a graph showing the migration ability of HCCC-9810 cells with and without TTFields applied, as illustrated in Example 1 of this application.

[0027] Figure 12 This is a statistical analysis graph showing the migration ability of HCCC-9810 cells with and without TTFields applied in Example 1 of this application.

[0028] Figure 13This is a graph showing the migration ability of RBE cells after 72 hours of TTFields application and without TTFields application, as shown in Example 1 of this application.

[0029] Figure 14 This is a statistical analysis graph showing the migration ability of RBE cells after 72 hours of TTFields application and without TTFields application, as presented in Example 1 of this application.

[0030] Figure 15 The image shows mitosis in HCCC-9810 cells after 96 hours of TTFields application and without TTFields application, as shown in Example 1 of this application.

[0031] Figure 16 The image shows mitosis of RBE cells with and without TTFields applied for 96 hours, as shown in Example 1 of this application.

[0032] Figure 17 This is an analytical graph showing the LDH release capacity of HCCC-9810 cells after 96 hours of TTFields application and without TTFields application, as presented in Example 1 of this application.

[0033] Figure 18 This is an analytical graph showing the LDH release capacity of RBE cells after 96 hours of TTFields application and without TTFields application, as described in Example 1 of this application.

[0034] Figure 19 This is a graph showing the expression of calreticulin on the surface of HCCC-9810 cells after 96 hours of TTFields application and without TTFields application, as shown in Example 1 of this application.

[0035] Figure 20 This is a graph showing the expression of calreticulin on the surface of RBE cells after 96 hours of TTFields application and without TTFields application, as shown in Example 1 of this application.

[0036] Figure 21 This is an analysis graph showing the ATP content secreted by HCCC-9810 cells after 96 hours of TTFields application and without TTFields application, as presented in Example 1 of this application.

[0037] Figure 22 This is an analysis graph showing the ATP content secreted by RBE cells after 96 hours of TTFields application and without TTFields application, as presented in Example 1 of this application.

[0038] Figure 23This is an analysis graph showing the HMGB1 content secreted by HCCC-9810 cells after 96 hours of TTFields application and without TTFields application, as presented in Example 1 of this application.

[0039] Figure 24 This is a graph showing the analysis of HMGB1 content secreted by RBE cells after 96 hours of TTFields application and without TTFields application in Example 1 of this application.

[0040] Figure label:

[0041] Electric field application device 100; body 1; circuit board 2; petri dish 3; electrode sheet 4; bottom plate 11; top plate 12; support plate 13. Detailed Implementation

[0042] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0043] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0044] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0045] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0047] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0048] In this invention, the term "treatment" refers to the use of drugs to achieve desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of drugs or transgenic immune cells to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administration of drugs containing cells with chimeric antigen receptors as described herein to an individual in need.

[0049] In this invention, the term "mitosis" refers to a mode of cell division in an organism, a fundamental process for the growth, development, and tissue repair of multicellular organisms. Mitosis causes a single cell to divide into two daughter cells with identical genetic information. This process occurs continuously throughout the life cycle of an organism to maintain normal tissue function and the overall health of the organism. Mitosis can be divided into the following stages: Prophase: Chromosomes begin to condense, becoming more compact and visible. Each chromosome consists of two identical sister chromatids connected by centromeres. Simultaneously, the nuclear membrane and nucleolus disappear, and the spindle apparatus begins to form. Metaphase: Chromosomes align in the central plane of the cell, forming a structure called the centroplate. Spindle fibers from the spindle apparatus connect to the centromeres of the chromosomes. Anaphase: Sister chromatids are pulled towards the cell poles. In this stage, the two chromatids of each chromosome separate and are pulled towards opposite cell poles. Telophase: Chromosomes reach the cell poles and begin to decondense. The nuclear membrane and nucleolus reform, forming two new nuclei. Cytokinesis: The cytoplasm divides, forming two independent cells. In animal cells, this is usually accomplished by the formation of a contractile ring, while in plant cells, it is the formation of a cell plate. Mitosis is crucial for maintaining genetic stability because it ensures the accurate distribution of genetic material during cell division. However, any abnormality in the mitotic process can lead to genetic errors, such as variations in chromosome number, which are associated with the development of certain genetic diseases and cancers.

[0050] This invention proposes a system for treating cancer, the use of an electric field application device in the preparation of a cancer treatment system, the use of a chemical drug or immune checkpoint inhibitor in the preparation of a drug, a method for treating cancer cells, a method for drug screening, a method for interfering with cancer cell mitosis, and a method for inhibiting cancer cell proliferation and promoting immunogenic cell death, which will be described in detail below.

[0051] Systems for treating cancer

[0052] In a first aspect, the present invention provides a system for treating cancer. According to an embodiment of the invention, the system includes: an electric field application device for applying an electric field for tumor treatment; and a delivery system for delivering a drug, the drug comprising at least one of an immune checkpoint inhibitor and a chemical drug; the immune checkpoint inhibitor being selected from durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; and the chemical drug being selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib. The system according to an embodiment of the invention is capable of inhibiting the proliferation of biliary tract cancer cells, inhibiting the migration of biliary tract cancer cells, interfering with the mitosis of biliary tract cancer cells, and promoting immunogenic death of biliary tract cancer cells.

[0053] According to embodiments of the present invention, the method includes applying an alternating electric field of a certain frequency and field strength to a target site in the bile duct of a subject for a period of time via an electric field application device; and administering a recommended dose of durvalumab and gemcitabine / cisplatin to the target site in the bile duct of a subject in need. Specifically, the alternating electric field may be applied before, after, or simultaneously with the administration of durvalumab and / or gemcitabine / cisplatin. Alternatively, durvalumab and / or gemcitabine / cisplatin may be administered before, after, or simultaneously with the application of the alternating electric field. Applying the alternating electric field before the administration of durvalumab and / or gemcitabine / cisplatin may include several seconds, minutes, hours, or days prior to the administration of durvalumab and / or gemcitabine / cisplatin. Applying an alternating electric field after administration of durvalumab and / or gemcitabine / cisplatin can occur within seconds, minutes, hours, or days following administration. Applying an alternating electric field concurrently with administration of durvalumab and / or gemcitabine / cisplatin can occur within seconds or minutes before or after administration. Applying an alternating electric field simultaneously with durvalumab and / or gemcitabine / cisplatin can also include applying an alternating electric field while simultaneously administering durvalumab and / or gemcitabine / cisplatin.

[0054] Preferably, in the initial treatment phase, one cycle consists of 3 weeks (21 days). Durvalumab 1500 mg Q3W (once every 3 weeks) will be administered intravenously on day 1 of each cycle for a maximum of 8 cycles. Concurrently, patients will also receive cisplatin 25 mg / m² on days 1 and 8 of each cycle. 2 and gemcitabine 1000mg / m 2 Administer intravenously every 3 weeks for up to 8 cycles.

[0055] For subsequent treatment phases, after more than 8 cycles, each cycle will be changed to 4 weeks (28 days). Patients will receive durvalumab 1500 mg Q4W (once every 4 weeks) monotherapy on day 1 of each cycle until disease progression (PD) is confirmed or intolerable toxicity occurs.

[0056] The alternating electric field will be used for continuous treatment starting on day 1 of cycle 1 (C1D1) of the entire treatment course, with a daily treatment time of ≥18 hours, throughout the entire course of initial and subsequent treatments, until disease progression (PD) is confirmed or intolerable toxicity occurs. The frequency of the alternating electric field is 100–700 kHz, and the field strength is 1–3 V / cm.

[0057] According to an embodiment of the present invention, the electric field application device is adapted to perform electrotherapy on cancer cells.

[0058] According to a specific embodiment of the present invention, see Figure 1 and Figure 2 The electric field applying device 100 includes a body 1, a circuit board 2, multiple petri dishes 3, and multiple pairs of electrode plates 4. The body 1 includes a base plate 11, a top plate 12, and multiple support plates 13 supported between the base plate 11 and the top plate 12. The circuit board 2 is laid flat and fixed to the top surface of the top plate 12. The petri dishes 3 are placed horizontally on the base plate 11, located between the base plate 11 and the top plate 12. The electrode plates 4 are disposed between the petri dishes 3 and the top plate 12, with the bottom of the electrode plates 4 inserted into the petri dishes 3 and the top of the electrode plates 4 extending upwards through the top plate 12 and connecting to the circuit board 2. The petri dishes 3 contain culture media, which can be the same or different types, selected according to experimental needs. An electric field generator (not shown) of a tumor electric field therapy device (not shown) is connected to a circuit board 2. The alternating electrical signal generated by the electric field generator (not shown) is applied to tumor cells in the culture medium through the circuit board 2 and electrode plates 4 to create a tumor therapeutic electric field. The effects of the frequency, field strength, direction, and exposure time of the tumor therapeutic electric field on tumor cell growth are studied. The electric field application device can refer to the TTF cell experimental device in CN218561476U, which is incorporated herein by reference in its entirety.

[0059] According to an embodiment of the present invention, in order to ensure cell survival, the electrode 4 is equipped with a thermistor (not shown) that can record the temperature data of the culture medium in real time. During the experiment, the specific incubator ambient temperature is set to 33-34°C according to the voltage of the electric field therapy device (not shown), thereby controlling the temperature in the culture medium within the range of 36.5-37.5°C.

[0060] According to embodiments of the present invention, the cancer is selected from at least one of bile duct cancer, non-small cell lung cancer, glioma, ovarian cancer, gastric cancer, pleural mesothelioma, pancreatic cancer, and liver cancer.

[0061] According to an embodiment of the present invention, the frequency of the electric field applied by the electric field applying device is in the mid-frequency range, specifically 100–700 kHz, preferably 100–250 kHz, and more preferably 140–160 kHz. According to an embodiment of the present invention, the frequency of the electric field applied by the electric field applying device can be 100 kHz, 150 kHz, 200 kHz, 300 kHz, 400 kHz, 500 kHz, 600 kHz, 650 kHz, 700 kHz, or a range between these values ​​(100–650 kHz, 100–600 kHz, and 100–500 kHz). A suitable frequency can effectively interfere with the mitosis of biliary tract cancer cells, inhibit their proliferation, inhibit their migration, and promote immunogenic death of biliary tract cancer cells.

[0062] According to embodiments of the present invention, the electric field strength applied by the electric field applying device is 1-3 V / cm, preferably 1.6-2.5 V / cm, and more preferably 2-2.2 V / cm. According to embodiments of the present invention, the electric field strength applied by the electric field applying device can be 1 V / cm, 1.1 V / cm, 1.2 V / cm, 1.3 V / cm, 1.4 V / cm, 1.5 V / cm, 1.6 V / cm, 1.7 V / cm, 1.8 V / cm, 1.9 V / cm, 2.0 V / cm, 2.1 V / cm, 2.2 V / cm, 2.3 V / cm, 2.4 V / cm, 2.5 V / cm, 2.6 V / cm, 2.7 V / cm, 2.8 V / cm, 2.9 V / cm, 3 V / cm, or a range between these values, specifically 1-2.9 V / cm, 1-2.8 V / cm, and 1-2.7 V / cm. Moderate field strength can effectively interfere with the mitosis of bile duct cancer cells, inhibit their proliferation, suppress their migration, and promote their immunogenic death.

[0063] According to an embodiment of the present invention, the electric field applying device applies an electric field for a period of not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours, for example, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, and 96 hours.

[0064] According to embodiments of the present invention, TTFields exhibits field-strength-dependent cytotoxicity against biliary tract cancers (HCCC-9810 cells (human cholangiocarcinoma cells) and RBE cells (human hepatobiliary carcinoma cells)). Treatment with an alternating electric field at a frequency of 150 kHz and a field strength of 2.1 V / cm resulted in decreased colony formation and migration abilities in both HCCC-9810 and RBE cells, and increased abnormal mitosis in cancer cells. Furthermore, calreticulin (CRT), a biomarker of immunogenic cell death (ICD), significantly accumulated on the cell membrane surface of both HCCC-9810 and RBE cells, and the release of high-mobility group box 1 (HMGB1) and adenosine triphosphate (ATP) in the cell culture medium was increased.

[0065] Intracellular death (ICD) is a specific form of cell death that can activate a specific immune response in the host against its contained antigens, thus playing an important role in tumor immunotherapy. Cancer cells undergoing ICD express or release damage-associated molecular patterns (DAMPs) in their dying state, promoting the maturation of dendritic cells (DCs) and the infiltration of cytotoxic T lymphocytes (CTLs). This process can reverse the tumor immunosuppressive microenvironment and enhance the sensitivity to immunotherapy. DAMPs mainly include CRTs exposed on the cell surface, HMGB1 secreted by tumor cells, ATP molecules released by cells, and heat shock proteins (HSP70, HSP90), among others.

[0066] According to embodiments of the present invention, TTFields inhibits the proliferation of biliary tract cancer cells and induces immunogenic cell death.

[0067] Application of electric field application devices in the preparation of cancer treatment systems

[0068] In a second aspect, the invention proposes the use of an electric field application device in the preparation of a cancer treatment system in which cancer patients undergo drug treatment before, after, or simultaneously.

[0069] According to embodiments of the present invention, the cancer is selected from at least one of bile duct cancer, non-small cell lung cancer, glioma, ovarian cancer, gastric cancer, pleural mesothelioma, pancreatic cancer, and liver cancer.

[0070] According to embodiments of the present invention, the drug comprises at least one of an immune checkpoint inhibitor and a chemical drug;

[0071] The immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; the chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

[0072] Uses of chemical drugs or immune checkpoint inhibitors in drug preparation

[0073] In a third aspect, the invention proposes the use of a chemical drug or an immune checkpoint inhibitor in the preparation of a medicament for treating cancer, wherein the patient is treated with an electric field applied by an electric field application device before, after, or simultaneously with the treatment.

[0074] According to embodiments of the present invention, the cancer is selected from at least one of bile duct cancer, non-small cell lung cancer, glioma, ovarian cancer, gastric cancer, pleural mesothelioma, pancreatic cancer, and liver cancer.

[0075] According to an embodiment of the present invention, the immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; the chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

[0076] According to an embodiment of the present invention, the frequency of the electric field applied by the electric field applying device is 100-700 kHz, preferably 100-250 kHz, and more preferably 140-160 kHz.

[0077] According to an embodiment of the present invention, the electric field strength applied by the electric field applying device is 1-3V / cm, preferably 1.6-2.5V / cm, and more preferably 2-2.2V / cm.

[0078] According to an embodiment of the present invention, the electric field treatment time applied by the electric field application device is not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

[0079] A method for treating cancer cells

[0080] In a fourth aspect, the present invention provides a method for treating cancer cells. According to embodiments of the invention, the method includes: treating cancer cells under an electric field applied by an electric field application device; pre-treatment, post-treatment, or simultaneous administration of a drug to the cancer cells; wherein the drug comprises at least one of an immune checkpoint inhibitor and a chemical drug; the immune checkpoint inhibitor is selected from durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; the chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib. The method according to embodiments of the invention can inhibit the proliferation of biliary tract cancer cells, inhibit the migration of biliary tract cancer cells, interfere with the mitosis of biliary tract cancer cells, and promote immunogenic death of biliary tract cancer cells.

[0081] According to an embodiment of the present invention, the frequency of the electric field applied by the electric field applying device is 100-700 kHz, preferably 100-250 kHz, and more preferably 140-160 kHz.

[0082] According to an embodiment of the present invention, the electric field strength applied by the electric field applying device is 1-3V / cm, preferably 1.6-2.5V / cm, and more preferably 2-2.2V / cm.

[0083] According to an embodiment of the present invention, the electric field treatment time applied by the electric field application device is not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

[0084] According to embodiments of the present invention, the cancer cells are selected from at least one of biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

[0085] A method for drug screening

[0086] In a fifth aspect, the present invention provides a method for drug screening. According to an embodiment of the invention, the method includes: subjecting cancer cells to an electric field applied by an electric field application device; contacting the cancer cells treated with the electric field with a candidate drug; and determining, based on the state of the cancer cells before and after the contact, whether the candidate drug is a drug suitable for treating cancer. The method according to an embodiment of the invention is capable of screening drugs for treating cancer.

[0087] According to an embodiment of the present invention, the frequency of the electric field treatment is 100-700 kHz, preferably 100-250 kHz, and more preferably 140-160 kHz.

[0088] According to an embodiment of the present invention, the electric field strength of the electric field treatment is 1-3V / cm, preferably 1.6-2.5V / cm, and more preferably 2-2.2V / cm.

[0089] According to an embodiment of the present invention, the electric field treatment is applied for a period of not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

[0090] According to embodiments of the present invention, the cancer cells are selected from at least one of biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

[0091] A method to interfere with cancer cell mitosis

[0092] In a sixth aspect, the present invention provides a method for interfering with the mitosis of cancer cells. According to an embodiment of the invention, the method includes: treating cancer cells under an electric field applied by an electric field applying device; wherein the frequency of the electric field applied by the electric field applying device is 100–700 kHz, preferably 100–250 kHz, more preferably 140–160 kHz; and / or, the field strength of the electric field applied by the electric field applying device is 1–3 V / cm, preferably 1.6–2.5 V / cm, more preferably 2–2.2 V / cm. The method according to an embodiment of the invention can interfere with the mitosis of biliary tract cancer cells.

[0093] According to an embodiment of the present invention, the electric field applying device applies the electric field for a period of not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

[0094] According to embodiments of the present invention, the treatment further includes pre-treatment, post-treatment, or simultaneous administration of the cancer cell drug; wherein the drug comprises at least one of an immune checkpoint inhibitor and a chemical drug; the immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; and the chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

[0095] According to embodiments of the present invention, the cancer cells are selected from at least one of biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

[0096] A method to inhibit cancer cell proliferation and promote immunogenic cell death

[0097] In a seventh aspect, the present invention provides a method for inhibiting cancer cell proliferation and promoting immunogenic cell death. According to an embodiment of the invention, the method includes: treating cancer cells under an electric field applied by an electric field applying device; wherein the frequency of the electric field applied by the electric field applying device is 100–700 kHz, preferably 100–250 kHz, more preferably 140–160 kHz; and / or, the field strength of the electric field applied by the electric field applying device is 1–3 V / cm, preferably 1.6–2.5 V / cm, more preferably 2–2.2 V / cm. The method according to the embodiment of the invention can inhibit cancer cell proliferation and promote immunogenic cell death. According to an embodiment of the invention, the electric field applied by the electric field applying device is applied for a period of not less than 24 hours, preferably not less than 48 hours, more preferably 72–96 hours.

[0098] According to embodiments of the present invention, the treatment further includes pre-treatment, post-treatment, or simultaneous administration of the cancer cell drug; wherein the drug comprises at least one of an immune checkpoint inhibitor and a chemical drug; the immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; and the chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

[0099] According to embodiments of the present invention, the cancer cells are selected from at least one of biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

[0100] A method for treating bile duct cancer

[0101] In an eighth aspect, the present invention provides a method for treating biliary tract cancer. According to an embodiment of the invention, the method includes: treating cancer cells under an electric field applied by an electric field applying device; wherein the frequency of the electric field applied by the electric field applying device is 100–700 kHz, preferably 100–250 kHz, more preferably 140–160 kHz; and / or, the field strength of the electric field applied by the electric field applying device is 1–3 V / cm, preferably 1.6–2.5 V / cm, more preferably 2–2.2 V / cm. The method according to the embodiment of the invention can inhibit cancer cell proliferation and promote immunogenic cell death. According to an embodiment of the invention, the electric field applied by the electric field applying device is applied for a period of not less than 24 hours, preferably not less than 48 hours, more preferably 72–96 hours.

[0102] According to embodiments of the present invention, the treatment further includes pre-treatment, post-treatment, or simultaneous administration of the cancer cell drug; wherein the drug comprises at least one of an immune checkpoint inhibitor and a chemical drug; the immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; and the chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

[0103] According to embodiments of the present invention, the cancer cells are selected from at least one of biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

[0104] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0105] Example 1: TTFields inhibits the proliferation of cholangiocarcinoma cells (HCCC-9810 cells (human cholangiocarcinoma cells) and RBE cells (human hepatobiliary cancer cells)) and promotes their immunogenic cell death.

[0106] 1. The experimental procedure in this embodiment is as follows:

[0107] (1) Cell Culture

[0108] HCCC-9810 and RBE cells were purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection. The growth medium consisted of 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 90% RPMI-1640 (Roswell Park Memorial Institute-1640) medium. All cells were incubated in a humidified cell culture incubator at 37°C with 95% air and 5% CO2.

[0109] (2) In vitro application of TTFields

[0110] HCCC-9810 cells and RBE cells were digested with trypsin containing 0.25% EDTA and seeded onto 20mm circular spreaders in 12-well plates. HCCC-9810 cells were seeded onto 12 separate 20mm circular spreaders in one well of each 12-well plate, and RBE cells were seeded onto 12 separate 20mm circular spreaders in one well of each 12-well plate. After 24 hours of culture, spreaders with good cell condition for each cell type were selected and placed into 12 separate culture dishes, with 15mL of culture medium added to each dish. Each culture dish contained one cell spreader. In the control group (no TTFields treatment), six culture dishes containing HCCC-9810 cells and six culture dishes containing RBE cells were directly placed in a 37°C, 5% CO2 incubator for 96 hours under saturated humidity. The TTFields group involved placing six culture dishes containing HCCC-9810 cells and six culture dishes containing RBE cells in an electric field application device. An alternating electric field of 100-300 kHz was applied to the cell samples in both culture dishes using the device, with the voltage adjusted to maintain the field strength at 1-2.5 V / cm. The device was then placed in a 5% CO2 constant-temperature cell culture incubator at 29-34℃ (the temperature within the culture medium was adjusted according to the electric field strength, controlling it within the range of 36.5-37.5℃), and incubated for 96 hours under saturated humidity.

[0111] (3) Cell counting

[0112] After 96 hours of treatment with TTFields cells, the scaffolds were removed from each group. HCCC-9810 cells and RBE cells were digested with trypsin containing 0.25% EDTA and prepared into single-cell suspensions. The number of cells in each group was measured using a handheld cell counter Scepter 3.0.

[0113] (4) Cell clone formation

[0114] HCCC-9810 cells and RBE cells (control and TTFields treated for 96 hours) were collected and reseeded in 6-well plates (3.0 × 10⁶ cells / well). 3 Cells per well (3 replicates per well). Culture until the vast majority of individual clones have more than 50 cells, changing the medium and observing cell status every 3 days. After cloning, fix with 4% paraformaldehyde for 30 minutes, wash with phosphate-buffered saline (PBS), and stain each well with 1% crystal violet for 10 minutes. Wash cells several times with PBS, photograph, and count. Cloning rate = (number of clones / number of seeded cells) × 100%.

[0115] (5) Cell migration experiment

[0116] HCCC-9810 cells and RBE cells were plated according to their growth status, and after overnight incubation, the cells reached a confluence of 70-80%. Wounds were created using a 200 μL pipette tip, and the cells were washed away with PBS. Serum-free culture medium was then added, and after 0 hours, the cells were photographed and placed in an incubator for TTFields treatment. When significant migration was observed in the control group, cell slides from each group were removed for observation and photographing. After 24 hours of TTFields treatment, significant migration was observed in the HCCC-9810 cell control group; after 72 hours of TTFields treatment, significant migration was observed in the RBE cell control group. ImageJ (image processing software) was used to measure the scratch area and calculate the cell migration rate.

[0117] (6) Immunofluorescence staining

[0118] After 96 hours of TTFields treatment, cell slides from each group were removed and transferred to 12-well plates. Cells were fixed using 4% tissue cell fixative in a fume hood at room temperature. After washing with PBS, cells were blocked in blocking buffer for 60 minutes. After blocking, the blocking buffer was removed, and diluted primary antibody (tubulin, 1:500) was added and incubated overnight at 4°C. The next day, cells were incubated at room temperature in the dark. Goat anti-rabbit IgG (H&L) secondary antibody (antibody against the primary antibody, i.e., the main antibody) was incubated for 60 minutes. After washing, it was incubated with DAPI (4',6-diamino-2-phenylpyridine, a fluorescent dye that binds strongly to DNA) in the dark, followed by photography using a fluorescence microscope.

[0119] (7) Lactate dehydrogenase (LDH) assay

[0120] HCCC-9810 cells and RBE cells (1.0 × 10⁻⁶) treated with TTFields for 96 hours were compared. 4Cells (1 cell / well) are evenly seeded in a 96-well cell culture plate. Fresh culture medium is added (using low-serum medium containing 1% serum or appropriate serum-free medium). The wells are divided into the following groups: cell-free culture medium wells (background blank control wells), control cell wells (sample control wells) without TTFields treatment, cell wells for subsequent lysis without TTFields treatment (sample maximum enzyme activity control wells), and TTFields group cell wells (TTFields group treated samples). One hour before the scheduled detection time, the cell culture plate is removed from the incubator. Lactate dehydrogenase (LDH) release reagent provided in the kit is added to the "sample maximum enzyme activity control wells" at 10% of the original culture medium volume. After one hour, the plate is centrifuged (400g for 5 minutes). 120 μL of the supernatant from each well is transferred to the corresponding well in a new 96-well plate, and the sample is immediately measured. Add 60 μL of LDH detection working solution to each well, mix well, and incubate in the dark at room temperature (approximately 25°C) for 30 minutes. Measure the absorbance at 490 nm. LDH release rate (%) = (Absorbance of treated sample - Absorbance of sample control well) / (Absorbance of maximum enzyme activity in cells - Absorbance of sample control well) × 100.

[0121] (8) Determination of calreticulin expression

[0122] HCCC-9810 cells and RBE cells were collected from control and TTFields treatments for 96 hours. Cells were washed with PBS containing 1% bovine serum albumin (BSA), the supernatant was discarded, and cell particles were briefly vortexed to separate the cells. The cells were then resuspended at 1×10⁻⁶ cells / mL. 7 The concentration was set at / mL. 100 μL of cell suspension was aliquoted into individual test tubes for staining. The tubes were incubated at 4°C for 1 hour with primary antibody (CRT, 1 μg / mL) and rabbit IgG isotype control antibody. Secondary antibody was prepared using Alexa... Goat anti-rabbit IgG (H&L) was incubated at 4°C for 30 minutes. Cells were washed twice with PBS containing 1% BSA on ice, and then resuspended in 500 μL of PBS containing 1% BSA. Labeled cells were then detected using a BDAccuri C6 flow cytometer. Data were analyzed using FlowJo flow cytometry software.

[0123] (9) ATP assay

[0124] Cell culture media of HCCC-9810 and RBE cells treated with TTFields for 96 hours were collected, centrifuged at 4°C (2000g for 5 minutes), and the supernatant was collected for detection using an ATP assay kit. In short, 100 μL of working solution was added to a black 96-well plate, incubated at room temperature for 5 minutes, then 100 μL of the sample to be tested was added, and the sample was analyzed using a Tecan assay kit. Microplate reader measures chemiluminescence.

[0125] (10) HMGB1 determination

[0126] Cell culture media of HCCC-9810 and RBE cells treated with TTFields for 96 hours were collected. The supernatant was collected by centrifugation at 4°C (3000 rpm for 20 minutes) and analyzed using an enzyme-linked immunosorbent assay (ELISA) kit for HMGB1 (High Mobility Group Box-1 protein). 50 μL of the sample was added to each well of the ELISA-coated plate and incubated at 37°C for 30 minutes. After washing, 50 μL of enzyme-labeled reagent was added and the plate was incubated at 37°C for 30 minutes. After another wash, 50 μL of chromogenic buffer was added and the plate was incubated at 37°C in the dark for 30 minutes. Finally, stop solution was added, and the plate was analyzed using Tecan. The ELISA reader measures the OD value at 450 nm.

[0127] (11) Statistical processing

[0128] Statistical analysis and graphing were performed using GraphPad Prism 9.0 software. Each experiment was repeated at least three times, and data are expressed as mean ± standard deviation. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant and expressed as *P < 0.05; **P < 0.01; and ***P < 0.001.

[0129] 2. The experimental results of this embodiment are as follows:

[0130] (1) Optimal frequency selection for TTFields

[0131] Three electric fields with frequencies of 100 kHz, 150 kHz, and 200 kHz and an electric field strength of 1.7 V / cm were applied to HCCC-9810 cells and RBE cells for 96 hours to study the response of cholangiocarcinoma cells to different frequencies. Figure 3 and Figure 4 Frequency testing results showed that TTFields exhibited the greatest inhibitory effect on the growth of HCCC-9810 and RBE cells at 150 kHz. TTFields were then used at the optimal frequency of 150 kHz in subsequent experiments.

[0132] (2) Relationship between TTFields electric field strength and cytotoxicity

[0133] After applying TTFields at a frequency of 150 kHz to HCCC-9810 cells and RBE cells for 96 hours, with electric field strengths of 1.3 V / cm, 1.7 V / cm, and 2.1 V / cm, respectively, the cytotoxic effect of TTFields was evaluated by analyzing the relationship between the electric field strength of TTFields and cell count. Figure 5 and Figure 6 Cell counting results showed that the number of both HCCC-9810 cells and RBE cells decreased with increasing electric field strength. This indicates that the cytotoxic effect of TTFields depends on the magnitude of the electric field strength.

[0134] (3) TTFields inhibits the proliferation of bile duct cancer cells.

[0135] Cholangiocarcinoma cells from both control and control groups were collected and treated with TTFields at 150 kHz and 2.1 V / cm for 96 hours. Cell colony formation was observed to assess cell proliferation capacity. (See reference...) Figure 7-10 As shown, the colony-forming ability of surviving cells in the TTFields group was significantly reduced. Figure 7 The colony-forming ability of HCCC-9810 cells was shown to be significantly reduced after 96 hours of treatment with TTFields at 150 kHz and 2.1 V / cm. Figure 8 The study showed that after 96 hours of treatment with TTFields at 150 kHz and 2.1 V / cm, the colony formation rate of HCCC-9810 cells was 32.6% of that of the control group. Figure 9 The clonogenic ability of RBE cells was shown to be significantly reduced after 96 hours of treatment with TTFields at 150 kHz and 2.1 V / cm. Figure 10 The results showed that after 96 hours of treatment with TTFields at 150 kHz and 2.1 V / cm, the colony formation rate of RBE cells was 50.4% of that of the control group. This result indicates that TTFields inhibits the proliferation of cholangiocarcinoma cells.

[0136] (4) TTFields inhibits the migration of bile duct cancer cells.

[0137] To test the effect of TTFields treatment on the migration of cholangiocarcinoma cells, a wound healing experiment was conducted in this embodiment. Figure 11 and Figure 13 The study showed a reduction in wound area in HCCC-9810 and RBE cells after treatment with TTFields at 150 kHz and 2.1 V / cm. Figure 12 Quantitative data showed that after 24 hours of treatment with TTFields at 150 kHz and 2.1 V / cm, the relative migration rate of HCCC-9810 cells was 53.4% ​​compared to the control group. Figure 14 Quantitative data showed that after 72 hours of TTField treatment, the relative migration rate of RBE cells was 27.5% compared to the control group. These results indicate that TTFields inhibit the migration of cholangiocarcinoma cells.

[0138] (5) TTFields interfere with the mitosis of bile duct cancer cells.

[0139] Since an electric field with a strength of 2.1 V / cm has a good inhibitory effect, it will result in a small number of cells and make them difficult to observe. Therefore, after applying TTFields at 150 kHz and 1.7 V / cm for 96 hours to HCCC-8910 cells and RBE cells, we performed immunofluorescence staining of microtubules to observe the changes in spindle morphology of dividing cells. Figure 15 and Figure 16 The results showed that HCCC-9810 cells and RBE cells treated with TTFields at 150 kHz and 1.7 V / cm exhibited abnormal mitosis, including disordered spindle fiber arrangement and multipolar mitosis. This indicates that TTFields treatment interferes with the normal progression of mitosis in bile duct cancer cells.

[0140] (6) TTFields induce immunogenic death in cholangiocarcinoma cells.

[0141] Cells release LDH into the extracellular space when they are damaged or die. Figure 17 and Figure 18 The study revealed that after 96 hours of treatment with TTFields at 150 kHz and 2.1 V / cm, the LDH levels released by HCCC-9810 cells and RBE cells increased by 3.3-fold and 2.3-fold, respectively, further demonstrating that TTFields cause damage or death in BTC cells. To determine whether TTFields application promotes ICD in BTC cells, several identified ICD biochemical markers were detected in HCCC-9810 and RBE cells after 96 hours of treatment with TTFields at 150 kHz and 2.1 V / cm, including exposure of CRT on the cell membrane, and extracellular secretion of ATP and HMGB1. Figure 19 and Figure 20 Flow cytometry revealed an increase in CRT expression on the surface of HCCC-9810 cells and RBE cells in the TTFields treatment group. Figure 21 and Figure 22 The study revealed that ATP levels increased in the supernatant of HCCC-9810 and RBE cells after 96 hours of TTFields treatment at 150 kHz and 2.1 V / cm. Furthermore, Figure 23 and Figure 24The study revealed increased extracellular HMGB1 levels in HCCC-9810 and RBE cells treated with TTFields at 150 kHz and 2.1 V / cm. These results indicate that TTFields can induce ICD in BTC cell lines, specifically, TTFields-induced immunogenic cell death in cholangiocarcinoma cells.

[0142] Example 2: TTFields combined with durvalumab and gemcitabine / cisplatin inhibited the proliferation of cholangiocarcinoma cells (HCCC-9810 cells (human cholangiocarcinoma cells) and RBE cells (human hepatobiliary cancer cells)) and promoted their immunogenic cell death.

[0143] The experimental procedure in this embodiment is basically the same as that in Example 1. The difference is that an alternating electric field of 100-300 kHz is applied to the cell samples in the two culture dishes using an electric field application device. After adjusting the voltage to make the electric field strength 1-2.5 V / cm, the cell samples in the two culture dishes are treated with durvalumab and gemcitabine / cisplatin. The other procedures are the same as in Example 1.

[0144] The results showed that TTFields in combination with durvalumab and gemcitabine / cisplatin inhibited the proliferation and migration of cholangiocarcinoma cells, interfered with the mitosis of cholangiocarcinoma cells, and induced immunogenic death of cholangiocarcinoma cells.

[0145] Example 3: TTFields combined with pembrolizumab and gemcitabine / cisplatin (GemCis) inhibited the proliferation of cholangiocarcinoma cells (HCCC-9810 cells (human cholangiocarcinoma cells) and RBE cells (human hepatobiliary cancer cells)) and promoted their immunogenic cell death.

[0146] The experimental procedure in this embodiment is the same as in Example 2. The difference is that an alternating electric field of 100-300 kHz is applied to the cell samples in the two culture dishes using an electric field application device. After adjusting the voltage to make the electric field strength 1-2.5 V / cm, the cell samples in the two culture dishes are treated with pembrolizumab and gemcitabine / cisplatin (GemCis). The other procedures are the same as in Example 1.

[0147] The results showed that TTFields combined with pembrolizumab and gemcitabine / cisplatin (GemCis) inhibited the proliferation and migration of cholangiocarcinoma cells, interfered with the mitosis of cholangiocarcinoma cells, and induced immunogenic death of cholangiocarcinoma cells.

[0148] Example 4: TTFields combined with nivolumab and gemcitabine / cisplatin (GemCis) inhibited the proliferation of cholangiocarcinoma cells (HCCC-9810 cells (human cholangiocarcinoma cells) and RBE cells (human hepatobiliary cancer cells)) and promoted their immunogenic cell death.

[0149] The experimental procedure in this embodiment is the same as in Example 2. The difference is that an alternating electric field of 100-300 kHz is applied to the cell samples in the two culture dishes using an electric field application device. After adjusting the voltage to make the electric field strength 1-2.5 V / cm, the cell samples in the two culture dishes are treated with nivolumab and gemcitabine / cisplatin (GemCis). The other procedures are the same as in Example 1.

[0150] The results showed that TTFields in combination with nivolumab and gemcitabine / cisplatin (GemCis) inhibited the proliferation and migration of cholangiocarcinoma cells, interfered with the mitosis of cholangiocarcinoma cells, and induced immunogenic death of cholangiocarcinoma cells.

[0151] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0152] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A system for treating cancer, characterized in that, include: An electric field application device for applying an electric field for tumor treatment; and a delivery system for delivering a drug, the drug comprising at least one of an immune checkpoint inhibitor and a chemical drug; The immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; The chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

2. The system according to claim 1, characterized in that, The cancer is selected from at least one of bile duct cancer, non-small cell lung cancer, glioma, ovarian cancer, gastric cancer, pleural mesothelioma, pancreatic cancer, and liver cancer.

3. The system according to claim 1, characterized in that, The frequency of the electric field applied by the electric field applying device is 100-700 kHz, preferably 100-250 kHz, and more preferably 140-160 kHz; Optionally, the electric field strength applied by the electric field applying device is 1 to 3 V / cm, preferably 1.6 to 2.5 V / cm, and more preferably 2 to 2.2 V / cm; Optionally, the electric field applying device applies the electric field for a period of not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

4. The use of an electric field application device in the preparation of a cancer treatment system, characterized in that, Cancer patients undergo drug treatment before, during, or after treatment.

5. The use according to claim 4, characterized in that, The cancer is selected from at least one of bile duct cancer, non-small cell lung cancer, glioma, ovarian cancer, gastric cancer, pleural mesothelioma, pancreatic cancer, and liver cancer.

6. The use according to claim 4, characterized in that, The drug includes at least one of immune checkpoint inhibitors and chemical drugs; The immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; The chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

7. The use of chemical drugs or immune checkpoint inhibitors in the preparation of drugs, characterized in that, The drug is used to treat a patient's cancer, and the patient receives treatment with an electric field applied by an electric field application device before, during, or after treatment.

8. The use according to claim 7, characterized in that, The cancer is selected from at least one of bile duct cancer, non-small cell lung cancer, glioma, ovarian cancer, gastric cancer, pleural mesothelioma, pancreatic cancer, and liver cancer.

9. The use according to claim 7, characterized in that, The immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; The chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib; Optionally, the frequency of the electric field applied by the electric field applying device is 100-700 kHz, preferably 100-250 kHz, and more preferably 140-160 kHz; Optionally, the electric field strength applied by the electric field applying device is 1 to 3 V / cm, preferably 1.6 to 2.5 V / cm, and more preferably 2 to 2.2 V / cm; Optionally, the electric field treatment applied by the electric field application device is for a period of not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

10. A method for treating cancer cells, characterized in that, include: Treating cancer cells under conditions where an electric field is applied by an electric field application device; Pre-treatment, prognostic treatment, or simultaneous administration of drugs to the cancer cells; The drug includes at least one of immune checkpoint inhibitors and chemical drugs; The immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; The chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

11. The method according to claim 10, characterized in that, The frequency of the electric field applied by the electric field applying device is 100-700 kHz, preferably 100-250 kHz, and more preferably 140-160 kHz; Optionally, the electric field strength applied by the electric field applying device is 1 to 3 V / cm, preferably 1.6 to 2.5 V / cm, and more preferably 2 to 2.2 V / cm; Optionally, the electric field applying device applies the electric field for a period of not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

12. The method according to claim 10, characterized in that, The cancer cells are selected from at least one of the following: biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

13. A method for drug screening, characterized in that, include: Cancer cells are subjected to an electric field applied by an electric field application device; Cancer cells treated with the electric field come into contact with the candidate drug; Based on the state of the cancer cells before and after the contact, it is determined whether the candidate drug is a drug that can be used to treat cancer.

14. The method according to claim 13, characterized in that, The frequency of the electric field is 100–700 kHz, preferably 100–250 kHz, and more preferably 140–160 kHz; Optionally, the electric field strength of the electric field treatment is 1 to 3 V / cm, preferably 1.6 to 2.5 V / cm, and more preferably 2 to 2.2 V / cm; Optionally, the electric field treatment is applied for a period of not less than 248 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

15. The method according to claim 13, characterized in that, The cancer cells are selected from at least one of the following: biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

16. A method for interfering with the mitosis of cancer cells, characterized in that, include: Treating cancer cells under conditions where an electric field is applied by an electric field application device; The frequency of the electric field applied by the electric field applying device is 100-700 kHz, preferably 100-250 kHz, and more preferably 140-160 kHz. And / or, the electric field strength applied by the electric field applying device is 1 to 3 V / cm, preferably 1.6 to 2.5 V / cm, more preferably 2 to 2.2 V / cm.

17. The method according to claim 16, characterized in that, The electric field applying device applies the electric field for a period of not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

18. The method according to claim 16 or 17, characterized in that, Further, this includes pre-treatment, prognostic treatment, or simultaneous administration of drugs to the cancer cells; The drug includes at least one of immune checkpoint inhibitors and chemical drugs; The immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; The chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

19. The method according to claim 16, characterized in that, The cancer cells are selected from at least one of the following: biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

20. A method for inhibiting cancer cell proliferation and promoting immunogenic cell death, characterized in that, include: Treating cancer cells under conditions where an electric field is applied by an electric field application device; The frequency of the electric field applied by the electric field applying device is 100-700 kHz, preferably 100-250 kHz, and more preferably 140-160 kHz. And / or, the electric field strength applied by the electric field applying device is 1 to 3 V / cm, preferably 1.6 to 2.5 V / cm, more preferably 2 to 2.2 V / cm.

21. The method according to claim 20, characterized in that, The electric field applying device applies the electric field for a period of not less than 24 hours, preferably not less than 48 hours, and more preferably 72 to 96 hours.

22. The method according to claim 20 or 21, characterized in that, This further includes pre-treatment, prognostic treatment, or simultaneous administration of drug therapy to the cancer cells; The drug includes at least one of immune checkpoint inhibitors and chemical drugs; The immune checkpoint inhibitor is selected from one of durvalumab, pembrolizumab, atezolizumab, nivolumab, and toripalimab; The chemical drug is selected from at least one of gemcitabine, cisplatin, GEMOX, and lenvatinib.

23. The method according to claim 20, characterized in that, The cancer cells are selected from at least one of the following: biliary tract cancer cells, non-small cell lung cancer cells, glioma cells, ovarian cancer cells, gastric cancer cells, pleural mesothelioma cells, pancreatic cancer cells, and liver cancer cells.

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