WO2024008745A1 - Methods and composition to identify and treat subjects resisting to chemotherapy treatment - Google Patents

Methods and composition to identify and treat subjects resisting to chemotherapy treatment Download PDF

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Publication number
WO2024008745A1
WO2024008745A1 PCT/EP2023/068444 EP2023068444W WO2024008745A1 WO 2024008745 A1 WO2024008745 A1 WO 2024008745A1 EP 2023068444 W EP2023068444 W EP 2023068444W WO 2024008745 A1 WO2024008745 A1 WO 2024008745A1
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Prior art keywords
cancer
nets
inhibitor
subject
chemotherapy
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PCT/EP2023/068444
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French (fr)
Inventor
Cédric GAGGIOLI
Jean ALBRENGUES
Alexandra MOUSSET
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Institut National de la Santé et de la Recherche Médicale
Centre National De La Recherche Scientifique
Universite Cote D'azur
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Publication of WO2024008745A1 publication Critical patent/WO2024008745A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention is in the field of oncology, more particularly, the invention relates to methods and composition to identify and treat subjects resisting to chemotherapy treatment.
  • TME tumor microenvironment
  • ECM extracellular cellular matrix
  • inflammatory cells such as monocytes and macrophages
  • monocytes and macrophages provide cues for the behavior of cancer cells during cancer progression [5] and have emerged as potent regulators of the therapeutic response in primary cancer [6-11].
  • chemotherapy has itself been linked to inflammation, which is considered a common, persistent, and potentially debilitating complication of chemotherapy [12-14], Altogether, while inflammation has been linked to cancer progression, it is still unclear whether and how chemotherapy-induced inflammation is responsible for chemoresistance, and if so, by which mechanisms.
  • the invention relates to a method for identifying a subject suffering from a cancer is at risk of having or developing a resistance to chemotherapy treatment comprising following steps: i) determining in a biological sample obtained from said subject the level of neutrophil extracellular traps (NETs); ii) comparing the level and/or the number determined in step i) with a predetermined reference value and iii) concluding that the subject has or is at risk of having or developing a resistance to chemotherapy treatment when the level and/or the number of NETs is higher than the predetermined reference value or concluding that the subject has not or is not at risk of having or not develop a resistance to the chemotherapy treatment when the level and/or the number of NETs is lower than the predetermined reference value.
  • the invention is defined by claims.
  • NET Neutrophil Extracellular Trap
  • the NET -mediated TGF0 activation caused cancer cell to undergo epithelial to mesenchymal transition and correlated with chemoresistance.
  • pharmacologically targeting of IL- I ⁇ , NETs, integrin-av01 , MMP9, and TGF0 all dramatically improved chemotherapy response in our mice model.
  • Their work establishes a novel paradigm for how NETs regulate activities of neighboring cells by trapping and activating cytokines.
  • chemotherapy resistance in the metastatic setting can be reduced or prevented by targeting the previously unrecognized IL- I ⁇ -NET-TGF0 axis.
  • the invention relates to a method for identifying whether a subject suffering from a cancer is at risk of having or developing a resistance to chemotherapy treatment comprising following steps: i) determining in a biological sample obtained from said subject the level of neutrophil extracellular traps (NETs), ii) ii) comparing the level and/or the number determined in step i) with a predetermined reference value and iii) iii) concluding that the subject has or is at risk of having or developing a resistance to chemotherapy treatment when the level and/or the number of NETs is higher than the predetermined reference value or concluding that the subject has not or is not at risk of having or not develop a resistance to the chemotherapy treatment when the level and/or the number of NETs is lower than the predetermined reference value.
  • NETs neutrophil extracellular traps
  • the invention relates to a method for monitoring the response of a subject suffering from a cancer to chemotherapy treatment comprising following steps: i) determining in a sample obtained from said subject the level of neutrophil extracellular traps (NETs); ii) comparing the level and/or the number determined in step i) with a reference value and iii) concluding that the subject will not respond to chemotherapy treatment when the level and/or the number of NETs is higher than the reference value or concluding that the subject will respond to chemotherapy treatment when the level and/or the number of NETs is lower than the reference value.
  • NETs neutrophil extracellular traps
  • the terms "will achieve a response” or “respond” refer to the response to a treatment of the subject suffering from a cancer. Typically such treatment induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a cancer.
  • the term “respond” refers to the ability of a chemotherapeutic agent to an improvement of the pathological symptoms, thus, the subject presents a clinical improvement compared to the subject who does not receive the treatment.
  • the said subject is considered as a "responder” to the treatment.
  • the term “not respond” refers to a subject who does not present any clinical improvement to the treatment with an immune checkpoint inhibitor treatment. This subject is considered as a "non-responder" to the treatment.
  • the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human.
  • the subject has or is susceptible to have breast cancer primary tumor.
  • the subject has or is susceptible to have breast cancer with metastasis.
  • the subject has or is susceptible to have breast cancer with lung metastasis.
  • the subject has or is susceptible to have breast cancer with brain metastasis.
  • the subject has or is susceptible to have breast cancer with liver metastasis. In a further embodiment, the subject has or is susceptible to have breast cancer with intestine metastasis.
  • the subject has or is susceptible to have breast cancer with melanoma metastasis.
  • cancer refers to a malignant growth or tumour resulting from an uncontrolled division of cells.
  • cancer includes primary tumors and metastatic tumors.
  • the cancer is solid or liquid cancer.
  • the cancer is selected from the group consisting of but not limited to: bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g.
  • bile duct cancer e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer
  • bladder cancer e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcom
  • breast cancer e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia
  • Castleman disease e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia
  • cervical cancer colorectal cancer
  • endometrial cancer e.g.
  • lung cancer e g. small cell lung cancer, non-small cell lung cancer
  • mesothelioma plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g.
  • the cancer is breast cancer.
  • the cancer is lung cancer.
  • the cancer is the cancer is breast cancer with lung metastasis. In a further embodiment, the cancer is the cancer is breast cancer with brain metastasis. In a further embodiment, the subject has or is susceptible to have breast cancer with liver metastasis.
  • the subject has or is susceptible to have breast cancer with intestine metastasis.
  • the subject has or is susceptible to have breast cancer with melanoma metastasis.
  • the subject has or is susceptible to have breast cancer with lung metastasis, brain metastasis, liver metastasis and/or melanoma metastasis.
  • biological sample refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a tissue biopsy.
  • the biological sample is a blood sample. In another embodiment, the biological sample is a plasma sample.
  • the biological sample is a blood sample, more particularly, peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma.
  • PBMC can be extracted from whole blood using a hypotonic lysis, which will preferentially lyse red blood cells. Such procedures are known to the experts in the art.
  • the biological sample is tumor tissue sample.
  • tumor tissue sample has its general meaning in the art and encompasses pieces or slices of tissue that have been removed including following a surgical tumor resection.
  • the tumor tissue sample can be subjected to a variety of well-known post- collection preparative and storage techniques (e g., fixation, storage, freezing, etc.) prior to determining the cell densities.
  • the tumor tissue sample is fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a block which is readily cut.
  • Thin slices of material can be then prepared using a microtome, placed on a glass slide and submitted e.g.
  • the term “resistance” refers to the proliferation of cancer cells which cannot be prevented or inhibited by means of a chemotherapeutic agent or a combination of chemotherapeutic agents usually used to treat cancer.
  • the chemotherapeutic resistance can be intrinsically resistant prior to chemotherapy, or resistance may be acquired during treatment of cancer that is initially sensitive to chemotherapy.
  • chemotherapeutic treatment refers to use of a chemotherapeutic agent to reduce and/or inhibit tumor growth.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); crypto
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrol
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and phannaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.
  • the chemotherapeutic agent is cisplatin or Adriamycin/Cyclophosphamide
  • NET Neurotrophil Extracellular Traps
  • Neutrophils are the immune system's first line of defense against infection and have conventionally been thought to kill invading pathogens through two strategies: engulfment of microbes and secretion of anti-microbials.
  • NETs allow neutrophils to kill extracellular pathogens while minimizing damage to the host cells.
  • NET extracellular DNA fibril matrix
  • PMA phorbol myristate acetate
  • IL-8 Interleukin 8
  • LPS lipopolysaccharide
  • neutrophils Upon in vitro activation with the exogenous pharmacological agent phorbol myristate acetate (PMA), Interleukin 8 (IL-8) or lipopolysaccharide (LPS), neutrophils release granule proteins and chromatin to form an extracellular DNA fibril matrix known as NET through an active process.
  • NETs are characterized by different protein markers.
  • NET protein markers denotes protein in the neutrophil extracellular traps like MPO (myeloperoxidase) and NE (neutrophil elastase).
  • NET protein markers can be the myeloperoxidase/DNA complex, the elastase/DNA complex, myeloperoxidase, elastase, citrullinated histones, proteinase 3, cathepsin, lactoferrin, or gelatinase.
  • Indirect associated NET protein markers like anti-phospholipid (anti-cardiolipin (aCL) and, anti-phosphatidylserine) can also be used and are considered as NET protein markers.
  • the expression level of histone H2B and myeloperoxidase are used to assess NET formation.
  • the terms “expression level”, “level” and “concentration” “quantity” can be used in a equivalent manner.
  • Methods to determine the expression levels of NETs can be performed by any method known in the art, including without limitation: immunostaining, immunohistochemistry, immunofluorescence ELISA, flow cytometry, chromatography, direct sequencing or Q-PCR.
  • the level of NETs is determined by immunostaining, immunohistochemistry, immunofluorescence ELISA or flow cytometry.
  • protein or antibody concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
  • CE-MS capillary electrophoresis-mass spectroscopy technique
  • ELISA ELISA
  • Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the presence of the protein or antibody can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays.
  • immunoassays such as competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis-mass spectroscopy technique (CE-MS). etc.
  • the reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e.
  • polystyrene latex e.g., beads or microtiter plates
  • polyvinylidine fluoride e.g., diazotized paper
  • nylon membranes e.g., nylon membranes
  • activated beads e.g., magnetically responsive beads, and the like.
  • an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
  • the predetermined corresponding reference value can be relative to a number or value derived from population studies, including without limitation, subjects of the same or similar age range, subjects in the same or similar ethnic group, subjects at risk of having resistance to chemotherapeutic treatment, and subject without cancer (healthy subject).
  • Such predetermined corresponding reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of the disease.
  • the term “predetermined reference value” refers to a threshold value or a cut-off value.
  • a “threshold value”, “reference value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the concentration of the markers of the invention (e g. NETs) in properly banked historical subject samples may be used in establishing the predetermined corresponding reference value.
  • the predetermined corresponding reference value is the median measured in the population of the subjects for the marker of in the invention (NETs for example).
  • the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • concentration of the marker of the invention NETs for example
  • NETs concentration of the marker of the invention
  • algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification.
  • the full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator the reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5.
  • AUC>0.5 the diagnostic result gets better and better as AUC approaches 1.
  • AUC is between 0.5 and 0.7, the accuracy is low.
  • AUC is between 0.7 and 0.9, the accuracy is moderate.
  • AUC is higher than 0.9, the accuracy is quite high.
  • This algorithmic method is preferably done with a computer.
  • Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
  • the predetermined corresponding reference value is typically determined by carrying out a method comprising the steps of: a) providing a collection of samples from subjects; b) providing, for each sample provided at step a), information relating to the actual clinical profile of the subject (healthy or suffering from a cancer); c) providing a serial of arbitrary quantification values; d) determining the concentration of the marker of the invention (NETs for example) for each sample contained in the collection provided at step a); e) classifying said blood samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples are obtained for the said specific quantification value, wherein the samples of each group are separately
  • the predetermined corresponding reference value thus allows discrimination between healthy subject and subjects suffering from an inflammatory disese.
  • high statistical significance values e.g. low P values
  • a range of values is provided instead of using a definite predetermined corresponding reference value. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g.
  • a range of quantification values includes a "cut-off value as described above.
  • the diagnosis can be determined by comparing the co centration of the marker of the invention (NETs for example) with the range of values which are identified.
  • a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found).
  • the invention relates to a method for treating a cancer resistant to a chemotherapeutic agent in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of an inhibitor of NETs.
  • the method according to the invention wherein such inhibitor reduces NETs formation by reducing the neutrophil recruitment.
  • treating refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • the term “inhibitor of NETs” refers to a natural or synthetic compound that has a biological effect to inhibit the formation of NETs typically, inhibits the activity or the expression of NETs.
  • the term “inhibit” means to prevent something from happening, to delay occurrence of something happening, and/or to reduce the extent or likelihood of something happening.
  • the terms “inhibiting metastasis”, “inhibiting metastases” and “inhibiting the formation of metastases”, which are used herein interchangeably, are intended to encompass preventing, delaying, and/or reducing the likelihood of occurrence of metastases as well as reducing the number, growth rate, size, etc... of metastases. In the context of the invention, such inhibitor inhibits metastasis more particularly, inhibits lung metastasis and reduces neutrophil recruitment to the lungs.
  • the inhibitor of NETs is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.
  • the inhibitor of NETs is PAD4 inhibitor, CXCR2 inhibitor, IL-1 ⁇ inhibitor, TGF ⁇ inhibitor, MMP9 inhibitor or ITG ⁇ v ⁇ 1 inhibitor.
  • the inhibitor of NETs is PAD4 inhibitor, CXCR2 inhibitor, IL-1 ⁇ inhibitor, TGF ⁇ inhibitor, MMP9 inhibitor and ITG ⁇ v ⁇ 1 inhibitor.
  • the inhibitor of NETs is DNase I.
  • DNase I has the following CAS number in the art: 9003-98-9.
  • the inhibitor of NETs is PAD4 inhibitor.
  • PAD Protein Arginine Deiminase
  • PAD belongs to the family of enzymes that catalyzes the hydrolysis of peptidyl-arginine to form peptidyl- citrulline on histones, fibrinogen, and other biologically relevant proteins.
  • the inhibitor of NETs is PAD4 inhibitor.
  • the inhibitor of NETs is GSK484.
  • GSK484 has the following structure and CAS number in the art : 1652591-81-5
  • the inhibitor of NETs is a compound of Formula (II) and its derivatives as described in WO2021/222353.
  • X 1 and X 2 are C or N;
  • X 3 is N-R 3 or C-R 3 ; provided that two of X 1 , X 2 , and X 3 are C; where each dashed line represents an optional bond to complete valency requirements of each X 1 , X 2 and X 3 ;
  • X 4 is N or C-R 2 ;
  • X 5 is N or CR 6 ;
  • X 7 is N or CR 7 ;
  • R 1 is hydrogen, halo, -CN, -OR 12 , -N(R 12 )2, -SR 12 , -C1 -8 alkyl optionally substituted with 1 to 3 Z 1 , C 3-6 cycloalkyl optionally substituted with 1 to 3 Z 1 , or 4-6 membered heterocyclyl optionally substituted with 1 to 3 Z 1 ;
  • R 2 is hydrogen, halo, -CN, -OR 12 , -N(R 12 )2, -SR 12 , -Cl -8 alkyl optionally substituted with 1 to 3 Z 2 , C 3-6 cycloalkyl optionally substituted with 1 to 3 Z 2 , or 4-6 membered heterocyclyl optionally substituted with 1 to 3 Z 2 ;
  • L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 are each independently:
  • Ci-10 alkylene optionally substituted with 1 to 3 Z 8 ;
  • R 10 is hydrogen, - C 1-8 alkyl optionally substituted with 1 to 3 Z 10 , or C3-6 cycloalkyl optionally substituted with 1 to 3 Z 10 ;
  • R 11 is hydrogen, -C1-8 alkyl optionally substituted with 1 to 4 Z 11 , -C3-8 cycloalkyl optionally substituted with 1 to 4 Z 11 , or 4-12-membered heterocyclyl optionally substituted with 1 to 4 Z 11 ; or
  • R 10 and R 11 are taken together with nitrogen to which they are attached to form a 4-12- membered heterocyclyl optionally substituted with 1 to 4 Z 11 ; each R 12 and R 13 are independently hydrogen, C 1-8 alkyl optionally substituted with 1 to 3 Z 1b , C2-8 alkenyl optionally substituted with 1 to 3 Z 1b , C2-8 alkynyl optionally substituted with 1 to 3 Z 1b , C3-10 cycloalkyl optionally substituted with 1 to 3 Z 1b , 4-10 membered heterocyclyl optionally substituted with 1 to 3 Z 1b , C 6-10 aryl optionally substituted with 1 to 3 Z 1b , or 5-10 membered heteroaryl optionally substituted with 1 to 3 Z 1b ; each Z 1 , Z 2 , Z 3 , Z 6 , Z 7 , and Z 8 is independently oxo, halo, -NO2, -N3, -CN, C1-8 alkyl optionally substituted by
  • each Z 2 , Z 3 , Z 6 , Z 7 , and Z 8 is independently optionally substituted with 1 to 3 Z 1a ; each Z la is independently oxo, halo, -NO 2 , -N 3 , -CN, C1-8 alkyl optionally substituted by 1 to 3 Z 1b , C2-8 alkenyl optionally substituted by 1 to 3 Z 1b , C2-8 alkynyl optionally substituted by 1 to 3 Z 1b , C3-8 cycloalkyl optionally substituted by 1 to 3 Z 1b , 6-10 membered aryl optionally substituted by 1 to 3 Z 1b , 4-10 membered heterocyclyl optionally substituted by 1 to 3 Z 1b , 5- 10 membered heteroaryl optionally substituted with 1 to 3 Z 1b ,
  • the NETs inhibitor according to the invention wherein the inhibitor is CXCR2 inhibitor.
  • CXCR2 refers to CXC Chemokine Receptor 2, also known as CD128, IL8RB and IL8 receptor type B, whose gene is encoded on human chromosome 2q35.
  • CXCR2 belongs the G-protein-coupled receptor family. This protein is a receptor for interleukin 8 (IL8). It binds to IL8 with high affinity, and transduces the signal through a G- protein activated second messenger system.
  • IL8 interleukin 8
  • the CXCR2 inhibitor is selected in following group consisting of but not limited to: AZD5069, SB 265610, SB225002, or Navarixin (SCH- 527123).
  • the NETs inhibitor according to the invention wherein the inhibitor is IL-1 ⁇ inhibitor.
  • IL-1 ⁇ also known as leukocytic pyrogen, leukocytic endogenous mediator, mononuclear cell factor, lymphocyte activating factor and other names, is a cytokine protein that in humans is encoded by the IL IB gene.
  • the IL-1 ⁇ inhibitor is an antibody. In a particular embodiment, the IL-1 ⁇ inhibitor is neutralizing monoclonal anti-IL-Ib antibody.
  • the IL-1 ⁇ inhibitor is an antibody.
  • the IL-1 ⁇ inhibitor is canakinumab (trade name Haris, developed by Novartis).
  • the NETs inhibitor according to the invention wherein the inhibitor is TGF ⁇ inhibitor.
  • TGF ⁇ refers to Transforming Growth Factor or Tumor Growth Factor is a cytokine that controls many key cellular functions including proliferation, differentiation, survival, migration and epithelial mesenchymal transition. It is a member of a superfamily of 38 cytokines that include TGFp, bone morphogenetic proteins (BMP), growth differentiation factors, inhibins, and activins.
  • BMP bone morphogenetic proteins
  • the TGF ⁇ inhibitor is selected in following group consisting of but not limited to: Galunisertib (LY21557299, Eli Lilly & Co.), LY3200882 (Eli Lilly), LY573636 (Tasisulam, Eli Lilly), LY2109761 (Eli Lilly), LY364937 (Eli Lilly), Ki26894 (Kirin Brewery Company), LY580276 (Eli Lilly), SB-431542 and SB-505124 (GlaxoSmithKline), SD-093 and SD-208, IN-1130 (In2Gen), SRK181-mIgGl (Scholar Rock), Fresolimumab (GC1008, Genzyme), LY3022859 (Eli Lilly), LY580276 (Eli Lilly), 264RAD (AstraZeneca), ID 11 (Genzyme Corp., Sanofi), 2G7 (Genentech).
  • Galunisertib LY21557299, Eli Lilly
  • the TGF ⁇ inhibitor is SB-431542.
  • the NETs inhibitor according to the invention wherein the inhibitor is MMP9 inhibitor.
  • MMP9 also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB)
  • GELB gelatinase B
  • Matrix MetalloProteinase 9 is a matrixin, a class of enzymes that belong to the zinc-metalloproteinases family involved in the degradation of the extracellular matrix.
  • the MMP9 inhibitor is SB-3 CT.
  • the NETs inhibitor according to the invention wherein the inhibitor is ITG ⁇ v ⁇ 1 inhibitor.
  • IGF ⁇ v ⁇ 1 also known as CD29 refers to Integrin beta-1 is a cell surface receptor that in humans is encoded by the ITGB1 gene.
  • the inhibitor of ITG ⁇ v ⁇ 1 is disclosed in Reed et al 2015 (Sci Transl Med. 2015 May 20; 7(288): 288ra79. doi:10.1126/scitranslmed.aaa5094).
  • the ITG ⁇ v ⁇ 1 inhibitor is avpi integrin-IN-1 TFA.
  • the invention relates to i) a chemotherapeutic agent and (ii) a NETs inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
  • the invention relates to i) a chemotherapeutic agent and (ii) DNase I, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
  • the invention relates to i) a chemotherapeutic agent and (ii) PAD4 inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
  • the invention relates to i) a chemotherapeutic agent and (ii) CXCR2 inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
  • the invention relates to i) a chemotherapeutic agent and (ii) IL- I ⁇ inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
  • the invention relates to i) a chemotherapeutic agent and (ii) TGF inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
  • the invention relates to i) a chemotherapeutic agent and (ii) MMP9 inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
  • the invention relates to i) a chemotherapeutic agent and (ii) ITG ⁇ v ⁇ 1 inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
  • the combined preparation according to the invention wherein the cancer is breast cancer.
  • the combined preparation according to the invention wherein the cancer is lung cancer.
  • the combined preparation according to the invention wherein the cancer is breast cancer with lung metastasis.
  • the cancer is the cancer is breast cancer with brain metastasis.
  • the subject has or is susceptible to have breast cancer with liver metastasis.
  • the subject has or is susceptible to have breast cancer with intestine metastasis.
  • the subject has or is susceptible to have breast cancer with melanoma metastasis.
  • the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication.
  • the combined therapy may be dual therapy or bi-therapy.
  • inhibitors When several inhibitors are used, a mixture of inhibitors is obtained.
  • multi- therapy for example, bi-, tri- or quadritherapy
  • at least on other inhibitor can accompany the
  • cancer resistant refers to a cancer which does not respond to a treatment.
  • the cancer may be resistant at the beginning of treatment, or it may become resistant during treatment.
  • the resistance to drug leads to rapid progression of metastatic of the cancer.
  • the resistance of cancer for the medication is caused by mutations in the gene which are involved in the proliferation, divisions, or differentiation of cells.
  • the cancer is resistant to chemotherapeutic treatment.
  • chemotherapy or “chemotherapeutic treatment” refers to use of chemotherapeutic agents to treat a subject.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,
  • the chemotherapeutic agents is cisplatin or Adriamycin/Cyclophosphamide.
  • the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human.
  • the subject is identified as resistant to chemotherapy treatment according to the method as described above.
  • the subject has or is susceptible to have breast cancer primary tumor.
  • the subject has or is susceptible to have breast cancer with lung metastasis.
  • the cancer is lung cancer.
  • the cancer is the cancer is breast cancer with brain metastasis.
  • the subject has or is susceptible to have breast cancer with liver metastasis.
  • the subject has or is susceptible to have breast cancer with intestine metastasis.
  • the subject has or is susceptible to have breast cancer with melanoma metastasis.
  • the subject has or is susceptible to have breast cancer with lung metastasis, brain metastasis, liver metastasis and/or melanoma metastasis.
  • administering refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor ofNETs) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a disease, or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • a “therapeutically effective amount” is meant a sufficient amount of inhibitor of NETs for use in a method for the treatment of cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • administration simultaneously refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time.
  • administration separately refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes.
  • administration sequentially refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
  • the NETs inhibitor can be used alone as a single inhibitor or in combination with chemotherapeutic treatment. When several inhibitors are used, a mixture of inhibitors is obtained. In the case of multi-therapy (for example, bi-, tri- or quadritherapy), at least on other inhibitor can accompany the NETs inhibitor.
  • the invention in another aspect, relates to a pharmaceutical composition comprising a NETs inhibitor for use in the treatment of resistant cancer to chemotherapeutic treatment.
  • the pharmaceutical composition according to the invention wherein, the cancer resistant is breast cancer.
  • the pharmaceutical composition according to the invention wherein, the cancer resistant is breast cancer with lung metastasis.
  • the cancer is lung cancer.
  • the cancer is the cancer is breast cancer with brain metastasis.
  • the cancer is breast cancer with liver metastasis.
  • the cancer is breast cancer with intestine metastasis.
  • the cancer is breast cancer with melanoma metastasis.
  • the NETs inhibitors as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as a carboxylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol, glycerol, glycerol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, arate, arate, arate, arate, arate, arate, arate, arate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dimethacrylate, ethylene glycol dim
  • compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile inj ectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by fdtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • FIGURES are a diagrammatic representation of FIGURES.
  • PMN_CM CM from unstimulated neutrophils
  • PMN_C5a CM CM from neutrophils treated with C5a
  • NET CM CM from neutrophils induced to form NETs with PMA.
  • FIG. 1 Chemotherapy triggers the secretion of IL-1 ⁇ by cancer cells, which drives NET formation.
  • A, B Quantification of images from the lungs of mice treated as indicated at day 21 and stained for myeloperoxidase (neutrophils, A) and citrullinated histone H3 (NETs, B).
  • FIG. 3 Targeting NETs ameliorates chemotherapy efficacy against breast cancer lung metastasis.
  • FIG. 4 Targeting NETs ameliorates chemotherapy efficacy against breast cancer liver metastasis.
  • A Schematic showing experimental design.
  • mice Six to eight-week-old female BALB/c mice were purchased from Charles River Laboratories. All procedures were conducted in accordance with French laws and European recommendations following a protocol approved by the French Ministry of Education and Research and by the local ethical committee for animal experimentation (CIEPAL Azur).
  • DNase (300 U/mouse) treatment was used to digest the DNA strands of NETs.
  • Antibodies were injected intraperitoneally 3 times a week using anti-Ly6G (200 pg/mouse) and its IgG control (200 pg/mouse); anti-IL-1 ⁇ (200pg/mouse) and its IgG control (200pg/mouse); anti-TGF ⁇ (200pg/mouse) and its IgG control (200 pg/mouse).
  • anti-Ly6G 200 pg/mouse
  • anti-IL-1 ⁇ 200pg/mouse
  • anti-TGF ⁇ 200pg/mouse
  • mice were sacrificed for analysis.
  • Neutrophils were harvested from six to eight- week-old female BALB/c mice.
  • the bone marrow of the femurs and tibias was isolated in sterile Hank’s buffered salt solution (HBSS IX) without Ca2+/Mg2+.
  • Bone marrow cells were flushed in HBSS and after centrifugation (400g, 5 min, 4°C), the cells were resuspended for 3 min on ice in an ammonium-chloride- potassium (ACK) buffer, and centrifuged (400g, 5 min, 4°C) before being washed twice with HBSS IX (400g centrifugation was used for the washing steps, 5 min, 4°C).
  • Neutrophils were then separated from mononuclear cells by plating 2 mL of the cell suspension onto a Percoll gradient consisting of 3 mL of 81% Percoll under 3 mL of 62% Percoll, followed by centrifugation at 700g for 20 min at 4°C. The middle layer containing the neutrophils was washed twice in HBSS IX and cells were resuspended in serum-free DMEM.
  • CM conditioned media
  • PMN CM unstimulated neutrophils
  • NET CM NET-containing conditioned media
  • CM degranulated neutrophils
  • Luciferase-expressing cancer cells (2000) were plated in 96-well culture plates and incubated in complete media at 37°C overnight. For experiments with neutrophil CM, media were replaced the next day with 100 pL of neutrophil CM with the indicated treatment. For co- culture experiment, the next day, media were replaced, and neutrophils (10 000 per well) were plated with cancer cells in a serum-free medium with the indicated treatment. After 2 days, 100 pL of medium containing 5 pg/mL of luciferin were added and the number of cancer cells was measured by bioluminescence imaging (BLI) using a plate reader.
  • BLI bioluminescence imaging
  • Neutrophils (100,000) were seeded onto 96-well plates and incubated in serum free DMEM or CM (with vehicle or cytokines and/or inhibitors as indicated) for 4 hours.
  • Sytox green 50 nM was then added to the plate and after 5 minutes, fluorescence intensity was measured at excitation/emission wavelengths of 488/520 nm using a plate reader.
  • CM generated from cells treated with cisplatin was referred as 410.4 Cis CM while the CM generated from cells treated with AC was referred as 410.4 AC CM.
  • Chemotaxis was assayed using a boyden chamber with a 0.3 pm pore size cell culture insert.
  • the upper and the lower wells were separated by a light-tight polyethylene terephthalate (PET) membrane.
  • PET polyethylene terephthalate
  • Serum-free DMEM or CM containing the indicated neutralizing antibodies and inhibitor were applied to the lower wells, and the cells (50,000) were seeded in each of the upper wells and incubated overnight at 37°C with 5% CO2.
  • the membranes were fixed in 4% PFA for 15 min, washed twice with PBS and stained for 10 min with Sytox green (5pM).
  • the membranes were then mounted between glass slides and cover slips using mounting media. The number of migrating cells was then manually counted using confocal microscopy and a 20x objective. Flow cytometry analysis
  • mice After treatment, the mice were sacrificed, and the lungs were removed. Lungs were then mechanically dissociated and digested at 37°C for 30 min with 2% FCS RPMI containing Dispase (2.5 U/mL), Collagenase D (0.1 mg/mL), DNase I (25 U/mL) and Liberase DL (0.2 mg/mL). Suspensions were then passed through a 70pm nylon cell strainer with RPMI and centrifuged at 600g for 1 min at 4°C.
  • FCS RPMI containing Dispase 2.5 U/mL
  • Collagenase D 0.1 mg/mL
  • DNase I 25 U/mL
  • Liberase DL 0.2 mg/mL
  • FACS Fluorescence Activated Cell Sorter
  • CD8 Lymphocytes were detected as CD8+ cells
  • CD4 Lymphocytes were detected as CD4+ cells
  • B cells were detected as CD19+ cells
  • NK cells were detected as NKp46+ cells.
  • Dendritic cells were detected as CDl lc+ and CDl lb- cells.
  • monocytes were detected as Ly6C+ cells
  • neutrophils as Ly6G+ cells
  • macrophages as F4/80+ cells.
  • the neutralizing/blocking antibodies used in vitro were: anti-integrin pi antibody clone Ha2/5, anti-integrin ⁇ V, anti-mouse CXCL5, CXCL1 and IL-1 ⁇ and were used at 10 pg/mL
  • NET -ELISA an anti -neutrophil elastase antibody and an anti-DNA peroxidase conjugated antibody were used.
  • antibodies against CD4 (1 :50), CD8a (1:100), TCR P (1:50), CD19 (1:50), CD45 (1:50), NKp46 (1 :20), F4/80 (1 :20), CDl lb (1 :50), CDl lc (1 :50), Ly6C (1:50), Ly6G (1:50), and CD107a (1 :20) were used.
  • sections were incubated overnight at 4°C with anti-myeloperoxidase (1: 100) and anti-citrullinated histone H3 antibodies (1:250) in 0.5X blocking buffer. After three washes with PBS, the sections were incubated with the suitable fluorochrome-conjugated secondary antibodies (1:150) in 0.5X blocking buffer for 45 min in the dark at room temperature. After two washes with PBS and one with water, sections were counterstained with DAPI and rinsed in water, and the slides were mounted onto coverslips using mounting media).
  • Paraffin-embedded tissue sections were first deparaffinized and rehydrated. The slides were then incubated with hematoxylin (15 min), an ammonia solution (0.08 % in water), and eosin (30 sec) and washed with tap water between each step. After dehydration, the slides were mounted onto cover slips using mounting media. Metastasis areas and foci were then quantified with Imaged software.
  • Plasma samples were collected from cardiac blood using a syringe with a 25 G needle and placed into EDTA tubes. Whole blood was centrifuged at 4°C at 1300g for 10 min, and the top plasma layer was collected.
  • 96-well Enzyme Immuno As say /Radio ImmunoAssay (EIA/RIA) plates were coated overnight at 4°C with an anti-elastase antibody (1:250) in 15 mM of Na2CO3, 35 mM of NaHCO3, at pH 9.6. The next day, the wells were washed three times with PBS, blocked in 5% BSA for two hours at room temperature, and washed three times with PBS. Then, 50 mL of plasma samples were added to the wells, incubated for two hours at room temperature on a shaker, and plates were washed three times with wash buffer (1% BSA, 0.05% Tween 20 in PBS).
  • wash buffer 1% BSA, 0.05% Tween 20 in PBS.
  • an anti-DNA-peroxidase conjugated antibody (1:50) in 1% BSA in PBS was added to the wells for 2 hours at room temperature, and the wells were washed five times with wash buffer before the addition of 2,2 Z -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid).
  • Optical density was read 40 min later at 405 nm using a plate reader.
  • An IL-1 ⁇ kit and a TGF-pi kit were used on CM as indicated and following the manufacturer’s instructions.
  • ELISA kits were used to evaluate the plasma concentration of creatinine and urea, following the manufacturer’s instructions.
  • ATP was quantified in CM using an ATP determination kit following the manufacturer’s instructions.
  • Phorbol 12-myristate 13-acetate (PMA) was used at 20 nM; recombinant mouse complement 5a was used at 100 ng/mL. Recombinant IL-1 ⁇ and TGF ⁇ -1 were used at 1 ng/mL.
  • Cisplatin was used at 10 pM and Adriamycin/Cyclophosphamide (referred as AC) were used at 0.1 pM and 1 pM respectively.
  • the PAD4 inhibitor GSK484 was used at 10 pM to inhibit NET formation, and 1.5 units/mL of DNase I was used to digest NET scaffolds.
  • the NRPL3 inhibitor MCC950 was used at 5 pM; AC-YVAD-CMK was used at 100 pM to inhibit caspase 1 and Bayl 1-7082 was used at 10 pM to inhibit NF-kB activity; Sivelestat, MMP9 inhibitor 1, Cathepsin G inhibitor 1, 4-Aminoebnzoic Acid hydrazide were used at 10 pM to inhibit NE, MMP9, Cathepsin G and MPO activity respectively, RGD peptide was used at 1 mM to counteract integrin pi-dependant adhesion; aVpi integrin-IN-1 TFA (HY-100445A, Medchem Express) was used at 100 pM to inhibit ITGavpi activity; anti -Integrin pi antibodies clone Ha2/5 and anti-integrin Dv were used at 10 pg/mL to inhibit integrin pi and av activity respectively; anti-mouse CXCL5, CXCL1 and IL-1 ⁇ were used at
  • Cells were fixed with 4% paraformaldehyde (PF A) for 20 min. After fixation, they were rinsed twice in PBS, incubated in 50 mM of NH4C1 for 10 min and permeabilized with 0.5% Triton X-100 for 5 min. Cells were next blocked in PBS containing 1% bovine serum albumin (BSA) for 60 min and incubated with anti-H2B (1:200), anti-myeloperoxidase (1:400) antibodies in blocking buffer overnight at 4°C.
  • BSA bovine serum albumin
  • Quantification of neutrophils and NETs from immunofluorescence images were analyzed using two-way ANOVA followed by Tukey’s procedure, and one-way ANOVA followed by Dunnett’s procedure, where the veh. group was used as the reference group and and using two-way ANOVA followed by Sidak’s procedure.
  • ATP quantification was analyzed using one-way ANOVA followed by Dunnett’s procedure, where the veh. group was used as the reference group.
  • ATP quantification was analyzed using two-way ANOVA followed by Tukey’s procedure.
  • mice were injected intravenously with 410.4 murine breast cancer cells and metastases were allowed to form for 7 days. Then, mice were treated with cisplatin or Adriamycin/Cyclophosphamide (AC) chemotherapies (data not shown). While chemotherapy caused no significant changes in the lungs of non-tumor bearing mice, we observed marked effects of chemotherapy of inflammatory cell infiltrate in the metastatic lungs: it triggered an increased neutrophil recruitment as determined by flow cytometry (data not shown) and confirmed by immunofluorescence (data not shown).
  • AC Adriamycin/Cyclophosphamide
  • CM conditioned media
  • 410.4 Cis CM conditioned media
  • Chemotherapy triggers NLRP3-mediated IL-1 ⁇ secretion in cancer cells, leading to NET formation and chemoresistance
  • IL-1 ⁇ blocking antibodies improved chemotherapy response in the co-culture assay in vitro (data not shown). In contrast, IL-1 ⁇ blocking antibodies did not increase the chemotherapy response when using NET CM, where the NETs had already been formed (data not shown). These data support a role for IL-1 ⁇ in NET formation but not in the downstream effects causing chemotherapy resistance. Also, in the context of lung metastasis, the IL-1 ⁇ blocking antibodies inhibited NET formation, reduced neutrophil recruitment ( Figures 2A-C), improved chemotherapy response ( Figures 2D and 2E) and improved kidney function following cisplatin treatment (data not shown).
  • IL-1 ⁇ is generally secreted after activation of inflammasomes, large protein complexes assembled when cells sense danger, e.g., components such as those from dead cells [38], The assembly of the inflammasome complex activates caspase- 1, which then cleaves pro-IL-1 ⁇ to generate mature IL-1 ⁇ [39],
  • NLRP3 NOD-like receptor family pyrin domain containing 3
  • NLRP3 inflammasome activation requires two signals: (i) a priming signal, which leads to Nuclear Factor-K-light-chain-enhancer of activated B cells (NF- KB)-mediated inflammasome and pro-IL- I ⁇ transcription; and (ii) a danger signal, which leads to inflammasome assembly and caspase-1 activation.
  • a priming signal which leads to Nuclear Factor-K-light-chain-enhancer of activated B cells (NF- KB)-mediated inflammasome and pro-IL- I ⁇ transcription
  • a danger signal which leads to inflammasome assembly and caspase-1 activation.
  • Adenosine triphosphate (ATP) binding to its purigenic receptor P2X 7 (P2RX7) is a well-known NLRP3 danger signal and cancer cell death induced by chemotherapy causes the release of ATP into the extracellular space [41].
  • chemotherapy treated 410.4 cancer cells released ATP (data not shown)
  • both NF-KB and P2RX7 inhibition blocked chemotherapy-induced IL- I ⁇ secretion (data not shown).
  • ATP released by dying chemotherapy-treated tumor cells triggers NLRP3 inflammasome activation in neighboring non-dying cancer cells.
  • CM from cisplatin-treated cancer cells triggered ASC assembly, and second, it was dependent on NF- ⁇ B, P2RX7, and NLRP3 activity.
  • caspase 1 inhibition did not block ASC assembly as caspase-1 only mediates the cleavage of pro-IL-1 ⁇ into IL-1 ⁇ following ASC assembly.
  • NLRP3 inhibition improved chemotherapy efficacy in the co-culture system (data not shown) but had no effect when NETs were already formed (data not shown). Additionally, NLRP3 inhibition blocked chemotherapy-induced NET formation in vivo and significantly improved both treatment efficacy (data not shown).
  • NETs activate TGF ⁇ signaling pathway in cancer cells, causing EMT and chemoresistance
  • EMT Epithelial-to-Mesenchymal Transition
  • TGFp Transforming Growth Factor P
  • TGF ⁇ R1 TGF ⁇ Receptor 1
  • NET-associated MMP9 activates latent TGF ⁇ which counteracts chemotherapy efficacy
  • TGF ⁇ played a role in NET-mediated chemoresistance.
  • TGF ⁇ is synthesized and secreted as a latent complex which is then processed to its active form [45], Using an ELISA test to detect both total- and active- TGF ⁇ , we found a large amount of the latent form of TGF ⁇ in the CM from 410.4 cancer cells, but not in the CM from neutrophils including after NET formation. The active form of TGF ⁇ was barely detectable in the CMs (data not shown). Chemotherapy did not change the levels of active and total TGF ⁇ present in the CM from cancer cells (data not shown).
  • NETs activated latent TGF ⁇ released by 410.4 cells (data not shown).
  • NETs did not modulate transcription of TGF ⁇ family members or receptors in 410.4 cells (data not shown) but targeting of NETs abrogated the activation of latent TGF ⁇ (data not shown).
  • Latent TGF ⁇ is secreted either as a Small Latent Complex (SLC), associated with Latency Associated Protein (LAP) or as a Large Latent Complex (LLC), associated with LAP and Latent TGF ⁇ Binding Protein (LBTP) [46], LBTP was not detected in cell lysate, ECM, or CM from 410.4 cancer cells, but the LAP-TGFp complex was detectable in all three isolates of the cancer cells (data not shown). In agreement with the ELISA results (data not shown), Western Blot revealed that incubation of cancer cell CM with NETs led to the degradation of LAP and the release of active TGFp and targeting NETs abrogated LAP degradation (data not shown).
  • SLC Small Latent Complex
  • LLC Large Latent Complex
  • LBTP Latent TGF ⁇ Binding Protein
  • CM from unstimulated and degranulated neutrophils had no effect (data not shown).
  • Activation of latent TGF ⁇ into active TGF ⁇ was also observed when culturing cancer cells with NET CM or when using a co-culture system and targeting NETs abrogated the activation of TGFp also under these conditions (data not shown).
  • NETs are characterized by the association of neutrophil proteases with the DNA scaffold [47, 48], MMP9 can proteolytically activate TGFp and is present within the NET-DNA scaffold (data not shown) [18, 49], Using inhibitors against some of the major NET-associated proteases - Neutrophil Elastase (NE), MMP9 and Cathepsin G - and MPO (which indirectly can activate TGF ⁇ activation [50]), we found that only MMP9 inhibition inhibited NET-mediated TGF ⁇ activation (data not shown) and chemoresistance in vitro (data not shown). MMP9 inhibition also sensitized cancer cells to cisplatin and AC chemotherapy in vivo (data not shown).
  • MMP9 inhibition additionally decreased the number of neutrophils and NETs in the lungs (data not shown) but did not block the ability of chemotherapy-treated cancer cells to promote NET formation in vitro (data not shown).
  • NET- associated MMP9 can activate latent TGF ⁇ , secreted e.g., by cancer cells, and that this activation induces EMT in cancer cells and correlates with chemoresistance.
  • NET-mediated proteolytic activation of TGF ⁇ was required for cancer cell resistance to treatment and digesting NETs with DNase I improved treatment efficacy in vivo and in vitro (data not shown). Yet, we previously showed that DNase I digestion of NETs does not reduce NET-MMP9 activity [18], Therefore, we tested whether the NET-DNA scaffold would contribute to TGF ⁇ activation through a different means.
  • Integrins are large transmembrane proteins, but parts of them are also found in NETs [51], Moreover, integrins, including integrin (ITG) ocvpi, have been shown to bind latent TGF ⁇ through the LAP-RGD domain [45, 46], We hypothesized that latent TGF ⁇ was trapped and then processed within the NET-DNA scaffold. We found that ITGavp 1 and MMP9 were both present within the NET-DNA scaffold by immunofluorescence (data not shown).
  • ITG ⁇ v ⁇ 1 improved chemotherapy response both in vitro (data not shown) and in vivo (data not shown) and reduced neutrophil recruitment and NET formation (data not shown).
  • ITG ⁇ v ⁇ 1 targeting did not block NET formation induced by chemotherapy-treated cancer cell CM in vitro (data not shown).
  • NETs act as a scaffold that traps cancer-cell derived latent TGFp, which can then be cleaved efficiently by NET-associated MMP9 and released as active TGF ⁇ to promote chemoresistance.
  • the inventors also show that targeting NETs ameliorates chemotherapy efficacy against breast cancer lung metastasis ( Figures 3A and 3B) and against breast cancer liver metastasis ( Figures 4A to 4C).

Abstract

The present invention relates to a method for treating a subject suffering from a cancer comprising a step of administering said subject with a therapeutically effective amount of an inhibitor of NETs. By in vitro experiments, inventors have demonstrated that inhibiting the formation of NETs with a PAD4 inhibitor or digesting the NET-DNA scaffold with DNase I during PMA- induced NET formation overcame the chemoresistance induced by the NET CM in vitro. They have also generated data in vivo and shown that targeting of NETs with either a PAD4 inhibitor or DNase I enhanced chemotherapy efficacy. PAD4 inhibition and DNase I treatment not only eliminated NETs in the metastatic lungs and in the plasma, but also reduced neutrophil recruitment to the lungs.

Description

METHODS AND COMPOSITION TO IDENTIFY AND TREAT SUBJECTS RESISTING TO CHEMOTHERAPY TREATMENT
FIELD OF THE INVENTION:
The present invention is in the field of oncology, more particularly, the invention relates to methods and composition to identify and treat subjects resisting to chemotherapy treatment.
BACKGROUND OF THE INVENTION:
Breast cancer accounts for the highest incidence and death rates in women worldwide, and metastasis is responsible for most breast cancer-related deaths [1], Most patients with metastases are treated with chemotherapy, but in the majority of cases, resistance to chemotherapy develops [2], Chemotherapy resistance is therefore a major challenge in treating metastatic cancers [3], While cancer cell intrinsic factors, such as its genetic and epigenetic traits, play a role in therapy resistance, it is now well established that the tumor microenvironment (TME) modulates response to therapy [4], Cancer extrinsic resistance is e.g., mediated by growth factors, cytokines and extracellular cellular matrix (ECM) proteins secreted by host cells. Among host cells, inflammatory cells, such as monocytes and macrophages, provide cues for the behavior of cancer cells during cancer progression [5] and have emerged as potent regulators of the therapeutic response in primary cancer [6-11], Interestingly, chemotherapy has itself been linked to inflammation, which is considered a common, persistent, and potentially debilitating complication of chemotherapy [12-14], Altogether, while inflammation has been linked to cancer progression, it is still unclear whether and how chemotherapy-induced inflammation is responsible for chemoresistance, and if so, by which mechanisms.
There is thus a need to understand chemoresistance of cancer and identify new biomarker and/or target to avoid chemoresistance.
SUMMARY OF THE INVENTION:
The invention relates to a method for identifying a subject suffering from a cancer is at risk of having or developing a resistance to chemotherapy treatment comprising following steps: i) determining in a biological sample obtained from said subject the level of neutrophil extracellular traps (NETs); ii) comparing the level and/or the number determined in step i) with a predetermined reference value and iii) concluding that the subject has or is at risk of having or developing a resistance to chemotherapy treatment when the level and/or the number of NETs is higher than the predetermined reference value or concluding that the subject has not or is not at risk of having or not develop a resistance to the chemotherapy treatment when the level and/or the number of NETs is lower than the predetermined reference value. In particular, the invention is defined by claims.
DETAILED DESCRIPTION OF THE INVENTION:
Inventors have shown that chemotherapy induced neutrophil recruitment and Neutrophil Extracellular Trap (NET) formation which reduced therapy response in a mouse model of breast cancer lung metastasis. They found that chemotherapy-treated cancer cells released adenosine triphosphate causing other cancer cells to secrete IL- Iβ, which in turn triggered neutrophils to form NETs. Two NET-associated proteins where required for NETs’ ability to induce chemoresistance: first, integrin-av01 in NETs trapped latent TGF0. Then, matrix metalloproteinase 9 cleaved and activated the trapped latent TGF0. The NET -mediated TGF0 activation caused cancer cell to undergo epithelial to mesenchymal transition and correlated with chemoresistance. Critically, pharmacologically targeting of IL- Iβ, NETs, integrin-av01 , MMP9, and TGF0 all dramatically improved chemotherapy response in our mice model. Their work establishes a novel paradigm for how NETs regulate activities of neighboring cells by trapping and activating cytokines. Additionally, their data suggest that chemotherapy resistance in the metastatic setting can be reduced or prevented by targeting the previously unrecognized IL- Iβ-NET-TGF0 axis.
Method to identify subjects resisting to chemotherapy treatment
Accordingly, in the first aspect, the invention relates to a method for identifying whether a subject suffering from a cancer is at risk of having or developing a resistance to chemotherapy treatment comprising following steps: i) determining in a biological sample obtained from said subject the level of neutrophil extracellular traps (NETs), ii) ii) comparing the level and/or the number determined in step i) with a predetermined reference value and iii) iii) concluding that the subject has or is at risk of having or developing a resistance to chemotherapy treatment when the level and/or the number of NETs is higher than the predetermined reference value or concluding that the subject has not or is not at risk of having or not develop a resistance to the chemotherapy treatment when the level and/or the number of NETs is lower than the predetermined reference value. In a particular embodiment, the invention relates to a method for monitoring the response of a subject suffering from a cancer to chemotherapy treatment comprising following steps: i) determining in a sample obtained from said subject the level of neutrophil extracellular traps (NETs); ii) comparing the level and/or the number determined in step i) with a reference value and iii) concluding that the subject will not respond to chemotherapy treatment when the level and/or the number of NETs is higher than the reference value or concluding that the subject will respond to chemotherapy treatment when the level and/or the number of NETs is lower than the reference value.
As used herein, the terms "will achieve a response" or "respond" refer to the response to a treatment of the subject suffering from a cancer. Typically such treatment induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a cancer.
In particular, in the context of the invention, the term "respond" refers to the ability of a chemotherapeutic agent to an improvement of the pathological symptoms, thus, the subject presents a clinical improvement compared to the subject who does not receive the treatment. The said subject is considered as a "responder" to the treatment. The term "not respond" refers to a subject who does not present any clinical improvement to the treatment with an immune checkpoint inhibitor treatment. This subject is considered as a "non-responder" to the treatment.
As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human.
In a particular embodiment, the subject has or is susceptible to have breast cancer primary tumor.
In a particular embodiment, the subject has or is susceptible to have breast cancer with metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with lung metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with brain metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with liver metastasis. In a further embodiment, the subject has or is susceptible to have breast cancer with intestine metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with melanoma metastasis.
As used herein, the term “cancer” refers to a malignant growth or tumour resulting from an uncontrolled division of cells. The term “cancer” includes primary tumors and metastatic tumors.
In a particular embodiment, the cancer is solid or liquid cancer.
Typically, the cancer is selected from the group consisting of but not limited to: bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, glioblastoma, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia), Castleman disease (e g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinroma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, nonHodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma). In a particular embodiment, the cancer is breast cancer.
In a further embodiment, the cancer is lung cancer.
In a further embodiment, the cancer is the cancer is breast cancer with lung metastasis. In a further embodiment, the cancer is the cancer is breast cancer with brain metastasis. In a further embodiment, the subject has or is susceptible to have breast cancer with liver metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with intestine metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with melanoma metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with lung metastasis, brain metastasis, liver metastasis and/or melanoma metastasis.
As used herein, the term “biological sample” refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a tissue biopsy.
In a particular embodiment, the biological sample is a blood sample. In another embodiment, the biological sample is a plasma sample.
In a particular embodiment, the biological sample is a blood sample, more particularly, peripheral blood mononuclear cells (PBMC). Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis, which will preferentially lyse red blood cells. Such procedures are known to the experts in the art.
In another embodiment, the biological sample is tumor tissue sample.
As used herein, the term “tumor tissue sample” has its general meaning in the art and encompasses pieces or slices of tissue that have been removed including following a surgical tumor resection. The tumor tissue sample can be subjected to a variety of well-known post- collection preparative and storage techniques (e g., fixation, storage, freezing, etc.) prior to determining the cell densities. Typically the tumor tissue sample is fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a block which is readily cut. Thin slices of material can be then prepared using a microtome, placed on a glass slide and submitted e.g. to immunohistochemistry (IHC) (using an IHC automate such as BenchMark® XT or Autostainer Dako, for obtaining stained slides). As used herein, the term “resistance” refers to the proliferation of cancer cells which cannot be prevented or inhibited by means of a chemotherapeutic agent or a combination of chemotherapeutic agents usually used to treat cancer. The chemotherapeutic resistance can be intrinsically resistant prior to chemotherapy, or resistance may be acquired during treatment of cancer that is initially sensitive to chemotherapy.
As used herein, the term "chemotherapeutic treatment" refers to use of a chemotherapeutic agent to reduce and/or inhibit tumor growth.
As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and phannaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.
In the context of the invention, the chemotherapeutic agent is cisplatin or Adriamycin/Cyclophosphamide
As used herein, the term “NET” for “Neutrophil Extracellular Traps” denotes networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind pathogens. Neutrophils are the immune system's first line of defense against infection and have conventionally been thought to kill invading pathogens through two strategies: engulfment of microbes and secretion of anti-microbials. In 2004, a third function was identified: formation of NETs. NETs allow neutrophils to kill extracellular pathogens while minimizing damage to the host cells. Upon in vitro activation with the exogenous pharmacological agent phorbol myristate acetate (PMA), Interleukin 8 (IL-8) or lipopolysaccharide (LPS), neutrophils release granule proteins and chromatin to form an extracellular DNA fibril matrix known as NET through an active process. NETs are characterized by different protein markers.
As used herein, the term “NET protein markers” denotes protein in the neutrophil extracellular traps like MPO (myeloperoxidase) and NE (neutrophil elastase). NET protein markers can be the myeloperoxidase/DNA complex, the elastase/DNA complex, myeloperoxidase, elastase, citrullinated histones, proteinase 3, cathepsin, lactoferrin, or gelatinase. Indirect associated NET protein markers like anti-phospholipid (anti-cardiolipin (aCL) and, anti-phosphatidylserine) can also be used and are considered as NET protein markers. In the context of the invention, the expression level of histone H2B and myeloperoxidase are used to assess NET formation.
According to the invention, the terms “expression level”, “level” and “concentration” “quantity” can be used in a equivalent manner.
Methods to determine the expression levels of NETs can be performed by any method known in the art, including without limitation: immunostaining, immunohistochemistry, immunofluorescence ELISA, flow cytometry, chromatography, direct sequencing or Q-PCR. In a particular embodiment, the level of NETs is determined by immunostaining, immunohistochemistry, immunofluorescence ELISA or flow cytometry.
Typically, protein or antibody concentration may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
The presence of the protein or antibody can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis-mass spectroscopy technique (CE-MS). etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
Typically, the predetermined corresponding reference value can be relative to a number or value derived from population studies, including without limitation, subjects of the same or similar age range, subjects in the same or similar ethnic group, subjects at risk of having resistance to chemotherapeutic treatment, and subject without cancer (healthy subject). Such predetermined corresponding reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of the disease.
As used herein, the term “predetermined reference value” refers to a threshold value or a cut-off value. A "threshold value", “reference value” or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of the concentration of the markers of the invention (e g. NETs) in properly banked historical subject samples may be used in establishing the predetermined corresponding reference value. In some embodiments, the predetermined corresponding reference value is the median measured in the population of the subjects for the marker of in the invention (NETs for example). In some embodiments, the threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the concentration of the marker of the invention (NETs for example) in a group of reference, one can use algorithmic analysis for the statistic treatment of the expression levels determined in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator the reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
In some embodiments, the predetermined corresponding reference value is typically determined by carrying out a method comprising the steps of: a) providing a collection of samples from subjects; b) providing, for each sample provided at step a), information relating to the actual clinical profile of the subject (healthy or suffering from a cancer); c) providing a serial of arbitrary quantification values; d) determining the concentration of the marker of the invention (NETs for example) for each sample contained in the collection provided at step a); e) classifying said blood samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples are obtained for the said specific quantification value, wherein the samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical profile of the subjects from which samples contained in the first and second groups defined at step f) derive; g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested; h) setting the said predetermined corresponding reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant) has been calculated at step g).
Thus in some embodiments, the predetermined corresponding reference value thus allows discrimination between healthy subject and subjects suffering from an inflammatory disese. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in one alternative embodiment of the invention, instead of using a definite predetermined corresponding reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off value as described above. For example, according to this specific embodiment of a "cut-off value, the diagnosis can be determined by comparing the co centration of the marker of the invention (NETs for example) with the range of values which are identified. In certain embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found). Method for treating a subject resistant to chemotherapeutic treatment
By in vitro experiments, inventors have demonstrated that inhibiting the formation of NETs with a PAD4 inhibitor or digesting the NET-DNA scaffold with DNase I during PMA- induced NET formation overcame the chemoresistance induced by the NET CM in vitro.
They have also generated data in vivo and shown that targeting of NETs with either a PAD4 inhibitor or DNase I enhanced chemotherapy efficacy. PAD4 inhibition and DNase I treatment not only eliminated NETs in the metastatic lungs and in the plasma, but also reduced neutrophil recruitment to the lungs.
Accordingly, in a second aspect, the invention relates to a method for treating a cancer resistant to a chemotherapeutic agent in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of an inhibitor of NETs.
In a particular embodiment, the method according to the invention, wherein such inhibitor reduces NETs formation by reducing the neutrophil recruitment.
As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
As used herein, the term “inhibitor of NETs” refers to a natural or synthetic compound that has a biological effect to inhibit the formation of NETs typically, inhibits the activity or the expression of NETs. As used herein, the term "inhibit" means to prevent something from happening, to delay occurrence of something happening, and/or to reduce the extent or likelihood of something happening. Thus, the terms "inhibiting metastasis", "inhibiting metastases" and "inhibiting the formation of metastases", which are used herein interchangeably, are intended to encompass preventing, delaying, and/or reducing the likelihood of occurrence of metastases as well as reducing the number, growth rate, size, etc... of metastases. In the context of the invention, such inhibitor inhibits metastasis more particularly, inhibits lung metastasis and reduces neutrophil recruitment to the lungs.
In a particular embodiment, the inhibitor of NETs is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.
In a particular embodiment, the inhibitor of NETs is PAD4 inhibitor, CXCR2 inhibitor, IL-1β inhibitor, TGFβ inhibitor, MMP9 inhibitor or ITGαvβ1 inhibitor.
In a particular embodiment, the inhibitor of NETs is PAD4 inhibitor, CXCR2 inhibitor, IL-1β inhibitor, TGFβ inhibitor, MMP9 inhibitor and ITGαvβ1 inhibitor.
In a particular embodiment, the inhibitor of NETs is DNase I. Typically such DNase I has the following CAS number in the art: 9003-98-9.
In another embodiment, the inhibitor of NETs is PAD4 inhibitor.
As used herein, the term “PAD” refers to Protein Arginine Deiminase. PAD belongs to the family of enzymes that catalyzes the hydrolysis of peptidyl-arginine to form peptidyl- citrulline on histones, fibrinogen, and other biologically relevant proteins.
In the context of the invention, the inhibitor of NETs is PAD4 inhibitor.
In a particular embodiment, the inhibitor of NETs is GSK484. GSK484 has the following structure and CAS number in the art : 1652591-81-5
Figure imgf000015_0001
In another embodiment, the inhibitor of NETs is a compound of Formula (II) and its derivatives as described in WO2021/222353.
Figure imgf000015_0002
Formula II wherein:
X1 and X2 are C or N;
X3 is N-R3 or C-R3; provided that two of X1, X2, and X3 are C; where each dashed line represents an optional bond to complete valency requirements of each X1, X2 and X3;
X4 is N or C-R2;
X5 is N or CR6;
X7 is N or CR7;
R1 is hydrogen, halo, -CN, -OR12, -N(R12)2, -SR12, -C1 -8 alkyl optionally substituted with 1 to 3 Z1, C3-6 cycloalkyl optionally substituted with 1 to 3 Z1, or 4-6 membered heterocyclyl optionally substituted with 1 to 3 Z1;
R2 is hydrogen, halo, -CN, -OR12, -N(R12)2, -SR12, -Cl -8 alkyl optionally substituted with 1 to 3 Z2, C3-6 cycloalkyl optionally substituted with 1 to 3 Z2, or 4-6 membered heterocyclyl optionally substituted with 1 to 3 Z2;
L1, L2, L3, L4, L5, and L6 are each independently:
Ci-10 alkylene, optionally substituted with 1 to 3 Z8;
C2-10 alkenylene, optionally substituted with 1 to 3 Z8; C2-10 alkynylene, optionally substituted with 1 to 3 Z8;
2-6 membered heteroalkylene, optionally substituted with 1 to 3 Z8;
C3-C10 cycloalkylene, optionally substituted with 1 to 3 Z8;
4-10 membered heterocyclene, optionally substituted with 1 to 3 Z8;
C6-10 arylene, optionally substituted with 1 to 3 Z8;
5-10 membered heteroarylene, optionally substituted with 1 to 3 Z8; or
-O-, -N(R8)-, -S-, -C(O)-, -C(O)O-, -C(O)N(R8)-, -SO-, -SO2-, -SO2N(R8)-,-N(R8)C(O)O-, - OC(O)O-, -N(R8)C(O)N(R8)-, -N(R8)S(O)2N(R8)-, -N(R8)C(N-CN)-, -S(O)(NR8)-, or - S(O)(NR8)N(R8)-; and ml, m2, m3, m4, m5, and m6 are each independently 0 or 1; provided that L1m1, L2m2, L3m3, L4m4, L5m5, and L6m6 taken together with the four consecutive atoms between which they are attached form an optionally substituted 11 to 20 membered macrocyclic ring; each R8 and R9 is independently hydrogen, C1-8 alkyl optionally substituted with 1 to 3 Z1b, C2-8 alkenyl optionally substituted with 1 to 3 Z1b, C2-8 alkynyl optionally substituted with 1 to 3 Z1b, C3-10 cycloalkyl optionally substituted with 1 to 3 Z1b, 4-10 membered heterocyclyl optionally substituted with 1 to 3 Z1b, C6-10 aryl optionally substituted with 1 to 3 Z1b, or 5-10 membered heteroaryl optionally substituted with 1 to 3 Z1b;
R10 is hydrogen, - C1-8 alkyl optionally substituted with 1 to 3 Z10, or C3-6 cycloalkyl optionally substituted with 1 to 3 Z10;
R11 is hydrogen, -C1-8 alkyl optionally substituted with 1 to 4 Z11, -C3-8 cycloalkyl optionally substituted with 1 to 4 Z11, or 4-12-membered heterocyclyl optionally substituted with 1 to 4 Z11; or
R10 and R11 are taken together with nitrogen to which they are attached to form a 4-12- membered heterocyclyl optionally substituted with 1 to 4 Z11; each R12 and R13 are independently hydrogen, C1-8 alkyl optionally substituted with 1 to 3 Z1b, C2-8 alkenyl optionally substituted with 1 to 3 Z1b, C2-8 alkynyl optionally substituted with 1 to 3 Z1b, C3-10 cycloalkyl optionally substituted with 1 to 3 Z1b, 4-10 membered heterocyclyl optionally substituted with 1 to 3 Z1b, C6-10 aryl optionally substituted with 1 to 3 Z1b, or 5-10 membered heteroaryl optionally substituted with 1 to 3 Z1b; each Z1, Z2, Z3, Z6, Z7, and Z8 is independently oxo, halo, -NO2, -N3, -CN, C1-8 alkyl optionally substituted by 1 to 3 Zla, C2-8 alkenyl optionally substituted by 1 to 3 Zla, C2-8 alkynyl optionally substituted by 1 to 3 Zla, C3-8 cycloalkyl optionally substituted by 1 to 3 Zla, 6-10 membered aryl optionally substituted by 1 to 3 Zla, 4-10 membered heterocyclyl optionally substituted by 1 to 3 Zla, 5-10 membered heteroaryl optionally substituted with 1 to 3 Zla, -OR9, -C(O)R9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)3 +, -N(R9)C(O)R9, -N(R9)C(O)OR9, - N(R9)C(O)N(R9)2, -N(R9)S(O)2(R9), -NR9S(O)2N(R9)2, -NR9S(O)2O(R9), -NS(O)(R9)2, - OC(O)R9, -OC(O)OR9, -OC(O)N(R9)2, -Si(R9)3, -SR9, -S(O)R9, -SF5, -S(O)(NR9)R9, - S(NR9)(NR9)R9, -S(O)(NR9)N(R9)2, -S(O)(NCN)R9, -S(O)2R9, -S(O)2N(R9)2, -
C(O)N(R9)S(O)2R9, or -S(O)2N(R9)C(O)R9, wherein each Z2, Z3, Z6, Z7, and Z8 is independently optionally substituted with 1 to 3 Z1a; each Zla is independently oxo, halo, -NO2, -N3, -CN, C1-8 alkyl optionally substituted by 1 to 3 Z1b, C2-8 alkenyl optionally substituted by 1 to 3 Z1b, C2-8 alkynyl optionally substituted by 1 to 3 Z1b, C3-8 cycloalkyl optionally substituted by 1 to 3 Z1b, 6-10 membered aryl optionally substituted by 1 to 3 Z1b, 4-10 membered heterocyclyl optionally substituted by 1 to 3 Z1b, 5- 10 membered heteroaryl optionally substituted with 1 to 3 Z1b, -OR13, -C(O)R13, -C(O)OR13, - C(O)N(R13)2, -N(R13)2, -N(R13)3+, -N(R13)C(O)R13, -N(R13)C(O)OR13, -N(R13)C(O)N(R13)2, -N(R13)S(O)2(R13), -NR13S(O)2N(R13)2, -NR13S(O)2O(R13), -NS(O)(R13)2, -OC(O)R13, - OC(O)OR13, -OC(O)N(R13)2, -Si(R13)3, -SR13, -S(O)R13, -SF5, -S(O)(NR13)R13, - S(NR13)(NR13)R13, -S(O)(NR13)N(R13)2, -S(O)(NCN)R13, -S(O)2R13, -S(O)2N(R13)2, - C(O)N(R13)S(O)2R13, or -S(O)2N(R13)C(O)R13; each Z10 and Z11 is independently selected from oxo, halo, -CN, C1-8 alkyl optionally substituted by 1 to 3 Z1b, C3-8 cycloalkyl optionally substituted by 1 to 3 Z1b, aryl optionally substituted by 1 to 3 Z1b, 4-10 membered heterocyclyl optionally substituted by 1 to 3 Z1b, 5-10 membered heteroaryl optionally substituted with 1 to 3 Z1b, -OR13, -C(O)R13, -C(O)OR13, -C(O)N(R13)2, - N(R13)2, -N(R13)3 +, -N(R13)C(O)R13, -N(R13)C(O)OR13, -N(R13)C(O)N(R13)2, -OC(O)R13, - OC(O)OR13, -OC(O)-N(R13)2, and -S-R13; and each Z1b is independently oxo, hydroxy, halo, -NO2, -N3, -CN, C1-9 alkyl, C2-6 alkenyl, C2. 6 alkynyl, C3-15 cycloalkyl, C1-8 haloalkyl, aryl, heteroaryl, heterocyclyl, -O(C1-9 alkyl), -O(C2- 6 alkenyl), -O(C2-6 alkynyl), -O(C3-15 cycloalkyl), -O(C1-8 haloalkyl), -O(aryl), - O(heteroaryl), -O(heterocyclyl), -OC(O) (C1 -9 alkyl), -OC(O)(C2-6 alkenyl), -OC(O)(C2-6 alkenyl), -OC(O)(C2-6 alkynyl), -OC(O)(C3-15 cycloalkyl), -OC(O)(C1-8 haloalkyl), OC(O)(aryl), -OC(O)(heteroaryl), -OC(O)(heterocyclyl), -NH2, -NH(C1-9 alkyl), -NH(C2-6 alkenyl), -NH(C2-6 alkynyl), -NH(C3-15 cycloalkyl), -NH(C1-8 haloalkyl), -NH(aryl), - NH(heteroaryl), -NH(heterocyclyl), -N(C1-9 alkyl)2, -N(C3-15 cycloalkyl)2, -N(C2-6 alkenyl)2, - N(C2-6 alkynyl)2, -N( C3-15 cycloalkyl)2, -N( C1-8 haloalkyl)2, -N(aryl)2, -N(heteroaryl)2, - N(heterocyclyl)2, -N(C1-9 alkyl)( C3-15 cycloalkyl), -N(C1-9 alkyl)(C2-6 alkenyl), -N(C1-9 alkyl)(C2-6 alkynyl), -N(C1-9 alkyl)( C1-8 haloalkyl), -N(C1-9 alkyl)(aryl), -N(Ci. 9 alkyl)(heteroaryl), -N(Cn 9 alkyl)(heterocyclyl), -C(O)(Ci-9 alkyl), -C(O)(C2-6 alkenyl), - C(O)(C2-6 alkynyl), -C(O)( C3-15 cycloalkyl), -C(O)(C1-8 haloalkyl), -C(O)(aryl), C(O)(heteroaryl), -C(O)(heterocyclyl), -C(O)O(C1-9 alkyl), -C(O)O(C2-6 alkenyl), - C(O)O(C2-6 alkynyl), -C(O)O(C3-15 cycloalkyl), -C(O)O(C1-8 haloalkyl), -C(O)O(aryl), - C(O)O(heteroaryl), -C(O)O(heterocyclyl), -C(O)NH2, -C(O)NH(C1-9 alkyl), -C(O)NH(C2-6 alkenyl), -C(O)NH(C2-6 alkynyl), -C(O)NH(C3-15 cycloalkyl), -C(O)NH(C1-8 haloalkyl), - C(O)NH(aryl), -C(O)NH(heteroaryl), -C(O)NH(heterocyclyl), -C(O)N(C1-9 alkyl)2, C(O)N(C3-15 cycloalkyl)2, -C(O)N(C2.6 alkenyl)2, -C(O)N(C2-6 alkynyl)2, -C(O)N(C1-8 haloalkyl)2, -C(O)N(aryl)2, -C(O)N(heteroaryl)2, -C(O)N(heterocyclyl)2, -NHC(O)(C1-9 alkyl), -NHC(O)(C2-6 alkenyl), -NHC(O)(C2-6 alkynyl), -NHC(O)(C3-15 cycloalkyl), - NHC(O)( C1-8 haloalkyl), -NHC(O)(aryl), -NHC(O)(heteroaryl), -NHC(O)(heterocyclyl), - NHC(O)O(C1-9 alkyl), -NHC(O)O(C2-6 alkenyl), -NHC(O)O(C2-6 alkynyl), -NHC(O)O(C3-15 cycloalkyl), -NHC(O)O(C1-8 haloalkyl), -NHC(O)O(aryl), -NHC(O)O(heteroaryl), NHC(O)O(heterocyclyl), -NHC(O)NH(C 1 -9 alkyl), -NHC(O)NH(C2.6 alkenyl), -
NHC(O)NH(C2-6 alkynyl), -NHC(O)NH( C3-15 cycloalkyl), -NHC(O)NH(C1-8 haloalkyl), -NHC(O)NH(aryl), -NHC(O)NH(heteroaryl), - NHC(O)NH(heterocyclyl), -SH, -S(C1-9 alkyl), -S(C2-6 alkenyl), -S(C2-6 alkynyl), -S(C3-15 cycloalkyl), -S( C1-8 haloalkyl), -S(aryl), -S(heteroaryl), -S(heterocyclyl), -NHS(O)(C1-9 alkyl), - N(C1-9 alkyl)(S(O)(C1-9 alkyl), -S(O)N(C1-9 alkyl)2, -S(O)(Ci-9 alkyl), -S(O)(NH)(C1-9 alkyl), -S(O)(C2-6 alkenyl), -S(O)(C2-6 alkynyl), -S(O)(C3-15 cycloalkyl), -S(O)(C1-8 haloalkyl), - S(O)(aryl), -S(O)(heteroaryl), -S(O)(heterocyclyl), -S(O)2(C1-9 alkyl), -S(O)2(C2-6 alkenyl), -S(O)2(C2-6 alkynyl), -S(O)2(C3-15 cycloalkyl), -S(O)2( C1-8 haloalkyl), -S(O)2(aryl), - S(O)2(heteroaryl), -S(O)2(heterocyclyl), -S(O)2NH(C1-9 alkyl), or -S(O)2N(C1-9 alkyl)2; wherein any alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl of Z1b is optionally substituted with one or more halo, C1-9 alkyl, C1-8 haloalkyl, -OH, -NH2, -NH(C1-9 alkyl), -NH(C3- 15 cycloalkyl), -NH(C1-8 haloalkyl), -NH(aryl), -NH(heteroaryl), -NH(heterocyclyl), -N(C1-9 alkyl)2, - N(C3-15 cycloalkyl )2, -NHC(O)(C3-15 cycloalkyl), -NHC(O)(C1-8 haloalkyl), - NHC(O)(aryl), -NHC(O)(heteroaryl), -NHC(O)(heterocyclyl), -NHC(O)O(C1-9 alkyl), - NHC(O)O(C2-6 alkynyl), -NHC(O)O(C3-15 cycloalkyl), -NHC(O)O(C1-8 haloalkyl), - NHC(O)O(aryl), -NHC(O)O(heteroaryl), -NHC(O)O(heterocyclyl), -NHC(O)NH(C1-9 alkyl), -S(O)(NH)(C1-9 alkyl), -S(O)2(C1-9 alkyl), -S(O)2(C3-15 cycloalkyl), -S(O)2(Ci-
8 haloalkyl), -S(O)2(aryl), -S(O)2(heteroaryl), -S(O)2(heterocyclyl), -S(O)2NH(C1-9 alkyl), - S(O)2N(C1-9 alkyl)2j -O(C3-15 cycloalkyl), -O(C1-8 haloalkyl), -O(aryl), -O(heteroaryl), - O(heterocyclyl), or -O(C1-9 alkyl).
In a particular embodiment, the NETs inhibitor according to the invention, wherein the inhibitor is CXCR2 inhibitor.
As used herein, the term “CXCR2” refers to CXC Chemokine Receptor 2, also known as CD128, IL8RB and IL8 receptor type B, whose gene is encoded on human chromosome 2q35. CXCR2 belongs the G-protein-coupled receptor family. This protein is a receptor for interleukin 8 (IL8). It binds to IL8 with high affinity, and transduces the signal through a G- protein activated second messenger system.
In a particular embodiment, the CXCR2 inhibitor is selected in following group consisting of but not limited to: AZD5069, SB 265610, SB225002, or Navarixin (SCH- 527123).
In a particular embodiment, the NETs inhibitor according to the invention, wherein the inhibitor is IL-1β inhibitor.
As used herein, the term “IL-1β ” also known as leukocytic pyrogen, leukocytic endogenous mediator, mononuclear cell factor, lymphocyte activating factor and other names, is a cytokine protein that in humans is encoded by the IL IB gene.
In a particular embodiment, the IL-1β inhibitor is an antibody. In a particular embodiment, the IL-1β inhibitor is neutralizing monoclonal anti-IL-Ib antibody.
In a particular embodiment, the IL-1β inhibitor is an antibody. In a particular embodiment, the IL-1β inhibitor is canakinumab (trade name Haris, developed by Novartis).
In a particular embodiment, the NETs inhibitor according to the invention, wherein the inhibitor is TGFβ inhibitor.
As used herein, the term “TGFβ ” refers to Transforming Growth Factor or Tumor Growth Factor is a cytokine that controls many key cellular functions including proliferation, differentiation, survival, migration and epithelial mesenchymal transition. It is a member of a superfamily of 38 cytokines that include TGFp, bone morphogenetic proteins (BMP), growth differentiation factors, inhibins, and activins.
In a particular embodiment, the TGFβ inhibitor is selected in following group consisting of but not limited to: Galunisertib (LY21557299, Eli Lilly & Co.), LY3200882 (Eli Lilly), LY573636 (Tasisulam, Eli Lilly), LY2109761 (Eli Lilly), LY364937 (Eli Lilly), Ki26894 (Kirin Brewery Company), LY580276 (Eli Lilly), SB-431542 and SB-505124 (GlaxoSmithKline), SD-093 and SD-208, IN-1130 (In2Gen), SRK181-mIgGl (Scholar Rock), Fresolimumab (GC1008, Genzyme), LY3022859 (Eli Lilly), LY580276 (Eli Lilly), 264RAD (AstraZeneca), ID 11 (Genzyme Corp., Sanofi), 2G7 (Genentech).
In a particular embodiment, the TGFβ inhibitor is SB-431542.
In a particular embodiment, the NETs inhibitor according to the invention, wherein the inhibitor is MMP9 inhibitor.
As used herein, the term “MMP9” also known as 92 kDa type IV collagenase, 92 kDa gelatinase or gelatinase B (GELB), refers to Matrix MetalloProteinase 9 is a matrixin, a class of enzymes that belong to the zinc-metalloproteinases family involved in the degradation of the extracellular matrix.
In a particular embodiment, the MMP9 inhibitor is SB-3 CT.
In a particular embodiment, the NETs inhibitor according to the invention, wherein the inhibitor is ITGαvβ1 inhibitor.
As used herein, the term “ITGαvβ1” also known as CD29 refers to Integrin beta-1 is a cell surface receptor that in humans is encoded by the ITGB1 gene.
In a particular embodiment, the inhibitor of ITGαvβ1 is disclosed in Reed et al 2015 (Sci Transl Med. 2015 May 20; 7(288): 288ra79. doi:10.1126/scitranslmed.aaa5094).
In a particular embodiment, the ITGαvβ1 inhibitor is avpi integrin-IN-1 TFA.
In a third aspect, the invention relates to i) a chemotherapeutic agent and (ii) a NETs inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
In a particular embodiment, the invention relates to i) a chemotherapeutic agent and (ii) DNase I, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
In a particular embodiment, the invention relates to i) a chemotherapeutic agent and (ii) PAD4 inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer. In a particular embodiment, the invention relates to i) a chemotherapeutic agent and (ii) CXCR2 inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
In a particular embodiment, the invention relates to i) a chemotherapeutic agent and (ii) IL- Iβ inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
In a particular embodiment, the invention relates to i) a chemotherapeutic agent and (ii) TGF inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
In a particular embodiment, the invention relates to i) a chemotherapeutic agent and (ii) MMP9 inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
In a particular embodiment, the invention relates to i) a chemotherapeutic agent and (ii) ITGαvβ1 inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
In a particular embodiment, the combined preparation according to the invention wherein the cancer is breast cancer.
In a particular embodiment, the combined preparation according to the invention wherein the cancer is lung cancer.
In a further embodiment, the combined preparation according to the invention wherein the cancer is breast cancer with lung metastasis.
In a further embodiment, the cancer is the cancer is breast cancer with brain metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with liver metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with intestine metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with melanoma metastasis.
As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.
When several inhibitors are used, a mixture of inhibitors is obtained. In the case of multi- therapy (for example, bi-, tri- or quadritherapy), at least on other inhibitor can accompany the
NET s inhibitor. As used herein, the term “cancer resistant” refers to a cancer which does not respond to a treatment. The cancer may be resistant at the beginning of treatment, or it may become resistant during treatment. The resistance to drug leads to rapid progression of metastatic of the cancer. The resistance of cancer for the medication is caused by mutations in the gene which are involved in the proliferation, divisions, or differentiation of cells. In the context of the invention, the cancer is resistant to chemotherapeutic treatment.
As used herein, the term “chemotherapy” or “chemotherapeutic treatment” refers to use of chemotherapeutic agents to treat a subject. As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth.
Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston), and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In the context of the invention, the chemotherapeutic agents is cisplatin or Adriamycin/Cyclophosphamide.
As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human.
In a particular embodiment, the subject is identified as resistant to chemotherapy treatment according to the method as described above. In a particular embodiment, the subject has or is susceptible to have breast cancer primary tumor. In a further embodiment, the subject has or is susceptible to have breast cancer with lung metastasis.
In a further embodiment, the cancer is lung cancer.
In a further embodiment, the cancer is the cancer is breast cancer with brain metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with liver metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with intestine metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with melanoma metastasis.
In a further embodiment, the subject has or is susceptible to have breast cancer with lung metastasis, brain metastasis, liver metastasis and/or melanoma metastasis.
As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor ofNETs) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.
By a "therapeutically effective amount" is meant a sufficient amount of inhibitor of NETs for use in a method for the treatment of cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
The NETs inhibitor can be used alone as a single inhibitor or in combination with chemotherapeutic treatment. When several inhibitors are used, a mixture of inhibitors is obtained. In the case of multi-therapy (for example, bi-, tri- or quadritherapy), at least on other inhibitor can accompany the NETs inhibitor.
In another aspect, the invention relates to a pharmaceutical composition comprising a NETs inhibitor for use in the treatment of resistant cancer to chemotherapeutic treatment.
In a particular embodiment the pharmaceutical composition according to the invention wherein, the cancer resistant is breast cancer.
In a particular embodiment the pharmaceutical composition according to the invention wherein, the cancer resistant is breast cancer with lung metastasis.
In a further embodiment, the cancer is lung cancer.
In a further embodiment, the cancer is the cancer is breast cancer with brain metastasis.
In a further embodiment, the cancer is breast cancer with liver metastasis.
In a further embodiment, the cancer is breast cancer with intestine metastasis.
In a further embodiment, the cancer is breast cancer with melanoma metastasis.
The NETs inhibitors as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi- solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile inj ectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1: NETs formed during chemotherapy counteract treatment efficacy (A, B) Mice injected intravenously with 410.4 cells were treated for 21 days as indicated, and the lung metastatic burden was quantified from hematoxylin and eosin staining (in (A) n=4 mice per group; in (B) n=12 mice for the Veh. group, n=8 mice for the Cis and AC groups, n=5 mice for the CXCR2 inh. group and n=4 for the CXCR2 inh. + Cis and CXCR2 inh. + AC groups; mean±SD). (C) Bioluminescence imaging (BLI) signal from luciferase- expressing 410.4 cells cultured alone or with neutrophils at day 2 after indicated treatments (n=3; mean±SD). (D) BLI signal from luciferase-expressing 410.4 cells at day 2 after indicated treatments. PAD4 inh. and DNase I were used during the neutrophil culture to block or digest NET formation, respectively (n=3; mean±SD). PMN_CM: CM from unstimulated neutrophils; PMN_C5a CM: CM from neutrophils treated with C5a; NET CM: CM from neutrophils induced to form NETs with PMA. (E, F) Mice injected intravenously with 410.4 cells were treated for 21 days as indicated, and the lung metastatic burden was quantified from hematoxylin and eosin staining (in (E) n=13 mice for the Veh. group, n=5 for the Cis and AC groups, n=4 for the PAD4 inh. Cis group and n=6 for the PAD4 inh. + Cis and PAD4 inh. + AC groups; in (F) n= 10 mice for the Veh. group, n=5 mice for the Cis, AC and DNase I + Cis groups, n=4 for the DNase I group and n=6 for the DNase I + AC group; mean±SD).
Figure 2. Chemotherapy triggers the secretion of IL-1β by cancer cells, which drives NET formation. (A, B) Quantification of images from the lungs of mice treated as indicated at day 21 and stained for myeloperoxidase (neutrophils, A) and citrullinated histone H3 (NETs, B). (C) ELISA quantification of NETs in the plasma of mice treated as indicated at day 21 (n=3 mice per group; mean±SD). (D, E) Mice injected intravenously with 410.4 cells were treated for 21 days as indicated, and the size (D) and the number of metastatic foci (E) in the lungs were quantified from hematoxylin and eosin staining (n=4 mice for the Veh., IL-1β Ab, IL-lp Ab + Cis and the IL-1β Ab + AC groups and n=6 for the Cis and AC groups; mean±SD).
Figure 3. Targeting NETs ameliorates chemotherapy efficacy against breast cancer lung metastasis. (A, B) Mice injected intravenously with PyMT (A) or TS/A (B) cells were treated for 21 days as indicated (treatment starting from day 7 to day 21, daily for the PAD4 inhibitor and Dnase I, twice weekly for AC chemotherapy), and the lung metastatic burden was quantified from H&E staining (n=4 mice for the Veh. group, n=5 mice for the AC group and n=6 mice for the other groups, n=3 mice for the Veh., PAD4 inh. and AC + PAD4 inh. groups and n=4 mice for the other groups).
Figure 4. Targeting NETs ameliorates chemotherapy efficacy against breast cancer liver metastasis. (A) Schematic showing experimental design. (B, C) Mice injected in the mammary fat pad with 410.4 cells were treated as indicated in (A), and the size (B) and the number of metastatic foci (C) in the liver were quantified from hematoxylin and eosin staining (n=4 mice per group mean±SD).
Figure 5. Resistant metastatic breast cancer patients exhibit high level of NETs after chemotherapy. (A, B and C) ELISA quantification of NETs in the plasma of patients treated as indicated.
EXAMPLE:
Material & Methods
Animals
Six to eight-week-old female BALB/c mice were purchased from Charles River Laboratories. All procedures were conducted in accordance with French laws and European recommendations following a protocol approved by the French Ministry of Education and Research and by the local ethical committee for animal experimentation (CIEPAL Azur).
Lung metastasis model and treatments
To generate lung metastasis, we intravenously injected 410.4 mouse cancer cells (500,000 per mouse) in 100 pL of PBS into eight-week-old female BALB/c mice. After 7 days, chemotherapy treatment was started twice a week with cisplatin (2.5 mg/kg) or Adriamycin/Cyclophosphamide (AC, Img/kg and 30 mg/kg, respectively). Inhibitors were injected intraperitoneally daily using compounds targeting PAD4 (20mg/kg), CXCR2 (Img/kg), NLRP3 (40 mg/kg), MMP9 (50 mg/kg), TGFβR1 (lOmg/kg), and Integrin aVpi (70 mg/kg). DNase (300 U/mouse) treatment was used to digest the DNA strands of NETs. Antibodies were injected intraperitoneally 3 times a week using anti-Ly6G (200 pg/mouse) and its IgG control (200 pg/mouse); anti-IL-1β (200pg/mouse) and its IgG control (200pg/mouse); anti-TGFβ (200pg/mouse) and its IgG control (200 pg/mouse). Three weeks after cancer cells injection, mice were sacrificed for analysis.
Primary tumor model and treatments
To generate mammary tumors, we injected 410.4 mouse cancer cells (500,000 per mouse) in 100 pL of Matrigel/PBS (1:1 ratio) into the mammary fat pad of eight-week-old female BALB/c mice. After 21 days, when the tumor reached approximately 35mm3, chemotherapy treatment was started twice a week with cisplatin (2.5mg/kg) or Adriamycin/cyclophosphamide (AC, Img/kg and 30 mg/kg, respectively). Inhibitors were injected intraperitoneally daily using PAD4 inhibitor (20mg/kg) and DNase (300 U/mouse). Two weeks after chemotherapy treatment, mice were sacrificed, the tumor removed and pictured on millimeter paper to quantify tumor size using ImageJ.
Cell culture
410.4 murine cancer cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) (FCII, Hyclone), 2 mM L-Glutamine, 100 units/mL penicillin, and 100 pg/mL streptomycin. To generate mCherry- and luciferase- expressing cells, a pGIPz vector containing cDNA for mCherry and luciferase was used. Lentiviral supernatants were collected from a 293 T packaging cell line transfected with the vector. 410.4 cells were infected with the viral supernatants overnight in the presence of 10 pg/mL polybrene and selected with 1 pg/ml puromycin. Cells were tested repeatedly for mycoplasma over the course of the study and were never positive. Neutrophils and lung cells were isolated freshly as described below.
Isolation of mouse neutrophils
Neutrophils were harvested from six to eight- week-old female BALB/c mice. The bone marrow of the femurs and tibias was isolated in sterile Hank’s buffered salt solution (HBSS IX) without Ca2+/Mg2+. Bone marrow cells were flushed in HBSS and after centrifugation (400g, 5 min, 4°C), the cells were resuspended for 3 min on ice in an ammonium-chloride- potassium (ACK) buffer, and centrifuged (400g, 5 min, 4°C) before being washed twice with HBSS IX (400g centrifugation was used for the washing steps, 5 min, 4°C). Neutrophils were then separated from mononuclear cells by plating 2 mL of the cell suspension onto a Percoll gradient consisting of 3 mL of 81% Percoll under 3 mL of 62% Percoll, followed by centrifugation at 700g for 20 min at 4°C. The middle layer containing the neutrophils was washed twice in HBSS IX and cells were resuspended in serum-free DMEM.
Activation of neutrophils and preparation of neutrophil-conditioned media
Primary isolated neutrophils were cultured in 24-well plates (250,000 per well) containing 500 pL of serum -free DMEM and were activated overnight with PMA to induce NETs or with recombinant complement 5a (C5a) to induce degranulation. The study used conditioned media (CM) from unstimulated neutrophils (PMN CM), NET-containing conditioned media (CM, induced with PMA and referred as NET CM) and CM from degranulated neutrophils (obtained using complement 5a, PMN C5a CM). GSK484 was added to inhibit PAD4 and therefore NET production and DNase I to digest NET-DNA. The next day, the neutrophil CM were collected and used to treat the 410.4 cancer cell culture. To assess NET formation, an immunofluorescence was performed using histone H2B and myeloperoxidase on neutrophils cultured overnight on poly-L-lysine-coated coverslips in serum-free DMEM or CM (with vehicle or cytokines and/or inhibitors as indicated).
Effect of chemotherapy on cancer cells in vitro
Luciferase-expressing cancer cells (2000) were plated in 96-well culture plates and incubated in complete media at 37°C overnight. For experiments with neutrophil CM, media were replaced the next day with 100 pL of neutrophil CM with the indicated treatment. For co- culture experiment, the next day, media were replaced, and neutrophils (10 000 per well) were plated with cancer cells in a serum-free medium with the indicated treatment. After 2 days, 100 pL of medium containing 5 pg/mL of luciferin were added and the number of cancer cells was measured by bioluminescence imaging (BLI) using a plate reader.
NETs quantification using sytox green
Neutrophils (100,000) were seeded onto 96-well plates and incubated in serum free DMEM or CM (with vehicle or cytokines and/or inhibitors as indicated) for 4 hours. Sytox green (50 nM) was then added to the plate and after 5 minutes, fluorescence intensity was measured at excitation/emission wavelengths of 488/520 nm using a plate reader.
Conditioned media preparation
Cancer cells were grown in complete medium, washed twice with PBS, and subsequently incubated at 37°C in a serum-free medium with cisplatin, AC. After 48 hours, CM were collected, centrifuged at 500g for 5 min to remove cell debris and the supernatant were stored at -80°C. The CM generated from cells treated with cisplatin was referred as 410.4 Cis CM while the CM generated from cells treated with AC was referred as 410.4 AC CM.
Cytokine array
A Proteome Profiler Mouse XL Cytokine Array test was performed following the manufacturer’s instructions.
Chemotaxis assay
Chemotaxis was assayed using a boyden chamber with a 0.3 pm pore size cell culture insert. The upper and the lower wells were separated by a light-tight polyethylene terephthalate (PET) membrane. Serum-free DMEM or CM containing the indicated neutralizing antibodies and inhibitor were applied to the lower wells, and the cells (50,000) were seeded in each of the upper wells and incubated overnight at 37°C with 5% CO2. The next day, the membranes were fixed in 4% PFA for 15 min, washed twice with PBS and stained for 10 min with Sytox green (5pM). The membranes were then mounted between glass slides and cover slips using mounting media. The number of migrating cells was then manually counted using confocal microscopy and a 20x objective. Flow cytometry analysis
After treatment, the mice were sacrificed, and the lungs were removed. Lungs were then mechanically dissociated and digested at 37°C for 30 min with 2% FCS RPMI containing Dispase (2.5 U/mL), Collagenase D (0.1 mg/mL), DNase I (25 U/mL) and Liberase DL (0.2 mg/mL). Suspensions were then passed through a 70pm nylon cell strainer with RPMI and centrifuged at 600g for 1 min at 4°C. The supernatant was removed, and the cells were incubated at 4°C for 15 min in ice-cold Fluorescence Activated Cell Sorter (FACS) buffer (2% FCS and 0.5 mM EDTA) with Fc Block diluted 1 :25). After one wash in FACS buffer, the cells were incubated with conjugated antibodies for 30 min at 4°C in the dark which allowed the detection of different immune cell populations. The cells were then washed twice in FACS buffer and resuspended in 450 pL of FACS buffer before being analyzed using a CytoFLEX operated by Cytexpert software. The different immune cell populations were detected among CD45 positive cells. Among TCRp cells, CD8 Lymphocytes were detected as CD8+ cells, and CD4 Lymphocytes were detected as CD4+ cells. B cells were detected as CD19+ cells. NK cells were detected as NKp46+ cells. Dendritic cells were detected as CDl lc+ and CDl lb- cells. Among CDl lb+ cells, monocytes were detected as Ly6C+ cells, neutrophils as Ly6G+ cells, and macrophages as F4/80+ cells.
Antibodies
For Western Blot analysis, antibodies against Latent Associated Protein, TGFp, LB TP, plKB, IKB and tubulin were used.
For immunofluorescence staining of in vitro cultures, antibodies against myeloperoxidase and Histone H2B were used to assess NET formation, and an antibody against ASC was used to assess inflammasome oligomerization. Antibodies against ITGpi, ITGDv, MMP9 and TGFβ were used to assess the presence of these proteins on NETs.
For immunofluorescence on tissue samples, antibodies against myeloperoxidase and citrullinated histone H3 were used to detect NETs.
The neutralizing/blocking antibodies used in vitro were: anti-integrin pi antibody clone Ha2/5, anti-integrin αV, anti-mouse CXCL5, CXCL1 and IL-1β and were used at 10 pg/mL
For NET -ELISA, an anti -neutrophil elastase antibody and an anti-DNA peroxidase conjugated antibody were used.
For flow cytometry analysis, antibodies against CD4 (1 :50), CD8a (1:100), TCR P (1:50), CD19 (1:50), CD45 (1:50), NKp46 (1 :20), F4/80 (1 :20), CDl lb (1 :50), CDl lc (1 :50), Ly6C (1:50), Ly6G (1:50), and CD107a (1 :20) were used.
Immunostaining of tissue samples For paraffin-embedded tissue, the lungs were fixed overnight at 4°C in 4% PF A, rinsed with PBS and transferred into 70% ethanol, processed using conventional methods, embedded in paraffin, and sectioned at 8 pm. Paraffin-embedded tissue sections were deparaffinized and rehydrated, and antigen retrieval was performed in EDTA buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0). Sections were blocked with Fc Receptor blocker and incubated with IX blocking buffer (5% donkey serum, 2.5% BSA, 0.1% Triton X-100 in PBS). Then, sections were incubated overnight at 4°C with anti-myeloperoxidase (1: 100) and anti-citrullinated histone H3 antibodies (1:250) in 0.5X blocking buffer. After three washes with PBS, the sections were incubated with the suitable fluorochrome-conjugated secondary antibodies (1:150) in 0.5X blocking buffer for 45 min in the dark at room temperature. After two washes with PBS and one with water, sections were counterstained with DAPI and rinsed in water, and the slides were mounted onto coverslips using mounting media).
Hematoxylin and Eosin staining
Paraffin-embedded tissue sections were first deparaffinized and rehydrated. The slides were then incubated with hematoxylin (15 min), an ammonia solution (0.08 % in water), and eosin (30 sec) and washed with tap water between each step. After dehydration, the slides were mounted onto cover slips using mounting media. Metastasis areas and foci were then quantified with Imaged software.
Plasma sample collection from mice
Plasma samples were collected from cardiac blood using a syringe with a 25 G needle and placed into EDTA tubes. Whole blood was centrifuged at 4°C at 1300g for 10 min, and the top plasma layer was collected.
Enzyme-linked immunosorbent assay (ELISA)
For NETs, 96-well Enzyme Immuno As say /Radio ImmunoAssay (EIA/RIA) plates were coated overnight at 4°C with an anti-elastase antibody (1:250) in 15 mM of Na2CO3, 35 mM of NaHCO3, at pH 9.6. The next day, the wells were washed three times with PBS, blocked in 5% BSA for two hours at room temperature, and washed three times with PBS. Then, 50 mL of plasma samples were added to the wells, incubated for two hours at room temperature on a shaker, and plates were washed three times with wash buffer (1% BSA, 0.05% Tween 20 in PBS). Next, an anti-DNA-peroxidase conjugated antibody (1:50) in 1% BSA in PBS was added to the wells for 2 hours at room temperature, and the wells were washed five times with wash buffer before the addition of 2,2Z -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid). Optical density was read 40 min later at 405 nm using a plate reader. An IL-1β kit and a TGF-pi kit were used on CM as indicated and following the manufacturer’s instructions.
For creatinine and urea analysis, ELISA kits were used to evaluate the plasma concentration of creatinine and urea, following the manufacturer’s instructions.
ATP quantification
ATP was quantified in CM using an ATP determination kit following the manufacturer’s instructions.
Cell culture reagents
The following reagents were used as stated, unless otherwise mentioned in figure legends. Phorbol 12-myristate 13-acetate (PMA) was used at 20 nM; recombinant mouse complement 5a was used at 100 ng/mL. Recombinant IL-1β and TGFβ-1 were used at 1 ng/mL. Cisplatin was used at 10 pM and Adriamycin/Cyclophosphamide (referred as AC) were used at 0.1 pM and 1 pM respectively. The PAD4 inhibitor GSK484 was used at 10 pM to inhibit NET formation, and 1.5 units/mL of DNase I was used to digest NET scaffolds. The NRPL3 inhibitor MCC950 was used at 5 pM; AC-YVAD-CMK was used at 100 pM to inhibit caspase 1 and Bayl 1-7082 was used at 10 pM to inhibit NF-kB activity; Sivelestat, MMP9 inhibitor 1, Cathepsin G inhibitor 1, 4-Aminoebnzoic Acid hydrazide were used at 10 pM to inhibit NE, MMP9, Cathepsin G and MPO activity respectively, RGD peptide was used at 1 mM to counteract integrin pi-dependant adhesion; aVpi integrin-IN-1 TFA (HY-100445A, Medchem Express) was used at 100 pM to inhibit ITGavpi activity; anti -Integrin pi antibodies clone Ha2/5 and anti-integrin Dv were used at 10 pg/mL to inhibit integrin pi and av activity respectively; anti-mouse CXCL5, CXCL1 and IL-1β were used at 10 pg/mL.
Western blot
For immunoblotting analysis, cells grown on plastic plates were lysed on ice in lysis buffer (25 mM Tris [pH 6.8], 2% sodium dodecyl sulfate (SDS), 5% glycerol, 1% P- mercaptoethanol, 0.01% bromophenol blue) and the samples were sonicated. Equal amounts of protein from each sample were loaded on SDS-polyacrylamide gel electrophoresis, separated, and transferred onto nitrocellulose The immunoblots were incubated in blocking buffer (5% BSA, 10 mM Tris-HCl [pH 7.5], 500 mM NaCl) for 30 min at room temperature and probed with specific antibodies overnight at 4°C. Then, the immunoblots were washed three times for 10 min in Tris-buffered saline Tween 20 (TBST, 10 mM Tris-HCl [pH 7.5], 500 mM NaCl, 0.1% Tween 20), incubated with secondary antibodies for one hour at room temperature in blocking buffer, and washed three times in TBST again. Immunodetection was performed using chemiluminescent horseradish peroxidase (HRP) substrate. Immunofluorescence of cell cultures
Cells were fixed with 4% paraformaldehyde (PF A) for 20 min. After fixation, they were rinsed twice in PBS, incubated in 50 mM of NH4C1 for 10 min and permeabilized with 0.5% Triton X-100 for 5 min. Cells were next blocked in PBS containing 1% bovine serum albumin (BSA) for 60 min and incubated with anti-H2B (1:200), anti-myeloperoxidase (1:400) antibodies in blocking buffer overnight at 4°C. After two washes in PBS, cells and matrices were incubated in the presence of fluorochrome-conjugated secondary antibodies (1:250) for 40 min, rinsed twice in PBS, stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min, rinsed in water, and the coverslips were mounted onto glass slides using mounting media. mRNA analysis
RNA was isolated from total cell lysates using a RNeasy Mini kit according to the manufacturer's instructions. Reverse transcription was performed using 500 ng cytoplasmic RNA using Superscript II reverse transcriptase. Real time PCR was performed using Fast SYBR Green Master Mix in duplicates according to the recommendations of the manufacturer on an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA).
Relative expression of the respective gene was determined after normalization to GAPDH and calculated using the following formula: relative expression level = 2ddCT.
Statistical analysis
Data from lung metastasis burden and foci were analyzed using two-way ANOVA followed by Tukey’s procedure, and a two-way ANOVA followed with Sidak’s procedure.
Data from in vitro BLI were analyzed using two-way ANOVA followed by Tukey’s procedure.
Data from plasma NETs were analyzed using two-way ANOVA followed by Tukey’s procedure, and ANOVA followed by Tukey ’ s procedure and another result which was analyzed using two-way ANOVA followed by Sidak’s procedure.
Data from IL-1 P and TGF P ELISA were analyzed using two-way ANOVA followed by Tukey’s procedure, and using one-way ANOVA followed by Dunnett’s procedure, where the veh. group was used as the reference group.
Quantification of neutrophils and NETs from immunofluorescence images were analyzed using two-way ANOVA followed by Tukey’s procedure, and one-way ANOVA followed by Dunnett’s procedure, where the veh. group was used as the reference group and and using two-way ANOVA followed by Sidak’s procedure. ATP quantification was analyzed using one-way ANOVA followed by Dunnett’s procedure, where the veh. group was used as the reference group. ATP quantification was analyzed using two-way ANOVA followed by Tukey’s procedure.
Data from RT-qPCR where analyzed using one-way ANOVA followed by Dunnett’s procedure, where the veh. group was used as the reference group, and a two-sided t-test was used.
Data from neutrophil migration assays were analyzed using two-way ANOVA followed by Tukey’s procedure, and one-way ANOVA followed by Dunnet’s procedure where the veh. group was used as the reference group.
Data from plasma creatinine and BUN were analyzed using two-way ANOVA followed by Tukey’s procedure and one-way ANOVA followed by Dunnett’s procedure where the veh. group was used as the reference group.
Data from primary tumor volume and data comparing primary tumor and metastasis volume were analyzed using two-way ANOVA followed by Tukey’s procedure.
Data from Sytox green quantification (NET quantification) were analyzed using one- way ANOVA followed by Dunnet’s procedure where the veh. group or the indicated group was used as the reference group and a two-sided t-test was used and two-way ANOVA followed by Tukey’s procedure.
A p-value less than 0.05 was considered significant, and p-values are indicated in figures as ***P<0.001, **P<0.01, and *P<0.05. All statistical analyses were performed using GraphPad Prism software version 7, unless otherwise stated.
Results
Neutrophil recruitment triggered by chemotherapy reduced treatment response of lung metastases
To determine how chemotherapy modifies the metastatic lung environment, syngeneic Balb/c mice were injected intravenously with 410.4 murine breast cancer cells and metastases were allowed to form for 7 days. Then, mice were treated with cisplatin or Adriamycin/Cyclophosphamide (AC) chemotherapies (data not shown). While chemotherapy caused no significant changes in the lungs of non-tumor bearing mice, we observed marked effects of chemotherapy of inflammatory cell infiltrate in the metastatic lungs: it triggered an increased neutrophil recruitment as determined by flow cytometry (data not shown) and confirmed by immunofluorescence (data not shown). The marked increase in neutrophil infiltration, combined with prior research showing that neutrophils promote cancer progression [16], led us to hypothesize that neutrophil recruitment might modulate therapy response. To test this hypothesis, we depleted neutrophils using Ly6G antibodies (data not shown) [29], Depletion of neutrophils dramatically improved both cisplatin and AC treatment responses, reducing the metastatic burden after treatment from 20% to less than 5% (Figures ID, 1A and data not shown). Neutrophil chemotaxis is mediated by the expression of C-X-C motif Chemokine Receptor 2 (CXCR2). We found that in vitro cisplatin treatment upregulated two CXCR2 ligands, CXCL1 and CXCL5, in cancer cells (data not shown). Furthermore, conditioned media (CM) from cisplatin-treated 410.4 cancer cells (410.4 Cis CM) promoted neutrophil chemotaxis in vitro and this was abrogated by a CXCR2 inhibitor, and by CXCL1 and CXCL5 blocking antibodies (data not shown). In mice, the pharmacological inhibition of CXCR2 reduced chemotherapy-induced neutrophil recruitment in the lungs and improved chemotherapy treatment response (Figure IB and data not shown).
NETs promoted chemoresistance of breast cancer lung metastases
To determine how neutrophils modulated chemotherapy response, we designed a co- culture system where cancer cells were cultured with freshly isolated bone-marrow derived neutrophils (data not shown). In vitro co-culturing the breast cancer cells with primary neutrophils reduced the response to chemotherapy (Figure 1C). We have previously reported that some cancer cells (data not shown) induce neutrophils to form NETs while others do not (e g., D2.0R and 4T07) [18, 30], and noticed that neutrophils formed NETs when co-cultured with the 410.4 cells, but only in the co-cultures treated with chemotherapy (data not shown). However, neutrophils cultured alone did not form NETs when treated with chemotherapy (data not shown). Due to prior research showing that NETs promote cancer progression [31], we hypothesized that NETs modulated the therapy response. Indeed, treatment with a PAD4 inhibitor to block NET formation or DNase I to digest the NET-DNA scaffold overcame neutrophil-mediated chemoresistance but had no effect on cancer cell numbers in the untreated conditions (Figure ID). Further supporting a direct role of the NETs on chemoresistance, the addition of NET-containing conditioned media (CM, induced with Phorbol Myristate Acetate [PMA] and henceforth referred to as NET CM) also caused breast cancer cell resistance to chemotherapy (Figure ID). In contrast, CM from unstimulated neutrophils (PMN CM) and CM from degranulated neutrophils (obtained using complement 5a; PMN C5a CM) had no effect on response to chemotherapy (Figure ID). As observed for the co-culture experiments, inhibiting the formation of NETs with a PAD4 inhibitor or digesting the NET-DNA scaffold with DNase I during PMA-induced NET formation also overcame the chemoresistance induced by the NET CM in vitro (Figure ID). In vivo, chemotherapy treatment also led to NET formation in the lungs (as detected by co-localized staining for citrullinated histone H3 and myeloperoxidase [MPO]) at day 21, four days after the last treatment (data not shown). We next collected plasma and lungs at different time points after chemotherapy treatment: in the lungs, increased neutrophil infiltration was first evident two days after chemotherapy, while NETs were detected in both lungs and plasma after three days, and present until five days after treatment (data not shown). These results support that chemotherapy did not directly act on neutrophils to cause NETs. To determine whether NETs contributed to chemoresistance in vivo, we targeted NETs in our mouse model of lung metastasis (data not shown). In accordance with our in vitro results, targeting of NETs with either a PAD4 inhibitor or DNase I enhanced chemotherapy efficacy (Figures IE and data not shown). PAD4 inhibition and DNase I treatment not only eliminated NETs in the metastatic lungs and in the plasma, but also reduced neutrophil recruitment to the lungs (data not shown), consistent with previous reports suggesting that the presence of NETs promotes further neutrophil recruitment [18, 21, 32], Inhibiting NETs in the absence of chemotherapy had no effect on the metastatic burden of mice bearing 410.4 cancer cells (Figures 1E-F). This is consistent with the inability of 410.4 cells to directly induce pro-metastatic NETs, while inhibiting NETs reduces metastases of cancer cells that induce NETs (e g., the murine breast cancer cell line 4T1) [30, 33],
The dramatic inhibitory effects of neutrophils and NETs on chemotherapy response at the lung metastatic site, prompted us to assess the effects of NETs on chemotherapy response of primary tumors. Only a few neutrophils were present in the primary tumors formed by 410.4 cells (data not shown). We detected no NETs regardless of whether the mice were treated with chemotherapy (data not shown) and targeting NETs with a PAD4 inhibitor or DNase I did not affect chemotherapy response at the primary site (data not shown). Thus, the effects of NETs on chemotherapy response are specific to lung metastatic disease.
Chemotherapy triggers NLRP3-mediated IL-1β secretion in cancer cells, leading to NET formation and chemoresistance
We next sought to determine the mechanism driving NET formation after chemotherapy. Neither cisplatin nor AC chemotherapy directly induced primary neutrophils to form NETs (data not shown). However, CM from chemotherapy-treated cancer cells (410.4 Cis CM and 4104 AC CM) promoted NET formation (data not shown), suggesting the presence of a NET-inducing, secreted factor released by cancer cells after chemotherapy. To identify candidate factors, we used a proteome cytokine array on the CM from chemotherapy- treated cancer cells (data not shown). As before (data not shown), our analysis showed upregulation of the neutrophil chemoattractants CXCL1 and CXCL5 but also revealed upregulation of interleukin- 10 (IL- Iβ), specifically in the CM of chemotherapy-treated cancer cells (data not shown). Recombinant IL-1β triggered NET formation in vitro (data not shown), consistent with prior reports [35-37], Furthermore, secretion of IL-1β by chemotherapy-treated cancer cells was confirmed by enzyme-linked immunoabsorbent assay (ELISA) and neutralization of IL-1β with an IL-1β blocking antibody inhibited NET formation induced by 410.4 Cis CM and 410.4 AC CM (data not shown). Additionally, IL-1β blocking antibodies improved chemotherapy response in the co-culture assay in vitro (data not shown). In contrast, IL-1β blocking antibodies did not increase the chemotherapy response when using NET CM, where the NETs had already been formed (data not shown). These data support a role for IL-1β in NET formation but not in the downstream effects causing chemotherapy resistance. Also, in the context of lung metastasis, the IL-1β blocking antibodies inhibited NET formation, reduced neutrophil recruitment (Figures 2A-C), improved chemotherapy response (Figures 2D and 2E) and improved kidney function following cisplatin treatment (data not shown).
It was unclear why chemotherapy-treated cancer cells would secrete IL-1β. IL-1β is generally secreted after activation of inflammasomes, large protein complexes assembled when cells sense danger, e.g., components such as those from dead cells [38], The assembly of the inflammasome complex activates caspase- 1, which then cleaves pro-IL-1β to generate mature IL-1β [39], We determined that NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and caspase-1 inhibition inhibited IL- Iβ secretion by chemotherapy-treated cancer cells (data not shown). NLRP3 inflammasome activation requires two signals: (i) a priming signal, which leads to Nuclear Factor-K-light-chain-enhancer of activated B cells (NF- KB)-mediated inflammasome and pro-IL- Iβ transcription; and (ii) a danger signal, which leads to inflammasome assembly and caspase-1 activation. Constitutive activation of NF-KB signaling pathway in cancer cells has been well described in the past [40] and was identified in 410.4 cancer cells with the constitutive phosphorylation of IKB in the absence of any external signal (data not shown). Adenosine triphosphate (ATP) binding to its purigenic receptor P2X 7 (P2RX7) is a well-known NLRP3 danger signal and cancer cell death induced by chemotherapy causes the release of ATP into the extracellular space [41], We confirmed that chemotherapy treated 410.4 cancer cells released ATP (data not shown) In addition, we determined that both NF-KB and P2RX7 inhibition blocked chemotherapy-induced IL- Iβ secretion (data not shown). We therefore hypothesized that ATP released by dying chemotherapy-treated tumor cells triggers NLRP3 inflammasome activation in neighboring non-dying cancer cells. To test this hypothesis, we first generated the CM from cisplatin-treated cancer cells in which we identified the presence of ATP (data not shown). We next used this CM to treat naive cancer cells and used immunofluorescence for Apoptosis-associated Speck- like protein containing a CARD (ASC) assembly as a readout of inflammasome activation (data not shown) [42], CM from cisplatin-treated cancer cells triggered ASC assembly, and second, it was dependent on NF-κB, P2RX7, and NLRP3 activity. As expected, caspase 1 inhibition did not block ASC assembly as caspase-1 only mediates the cleavage of pro-IL-1β into IL-1β following ASC assembly. Similarly, to the blocking of IL-1β secretion, pharmacological inhibition of NF-kB, NRLP3, P2RX7 and caspase- 1 counteracted the ability of cisplatin-treated cancer cells to promote NET formation (data not shown). In vitro, NLRP3 inhibition improved chemotherapy efficacy in the co-culture system (data not shown) but had no effect when NETs were already formed (data not shown). Additionally, NLRP3 inhibition blocked chemotherapy-induced NET formation in vivo and significantly improved both treatment efficacy (data not shown). Altogether, our results suggest that as chemotherapy induces cancer cell death, the dying cancer cells respond by releasing CXCL1 and CXCL5, which promote neutrophil recruitment, and ATP, which triggers NLRP3 inflammasome activation and IL-1β secretion from neighboring cancer cells. IL-1β then promotes NET formation and these NETs inhibit chemotherapy efficacy.
NETs activate TGFβ signaling pathway in cancer cells, causing EMT and chemoresistance
In vitro, we had observed that resistant cancer cells found in the proximity of NETs displayed an elongated shape, reminiscent of Epithelial-to-Mesenchymal Transition (EMT) (data not shown). EMT causes chemoresistance in several cancer types, including breast cancer, and Transforming Growth Factor P (TGFp) is a major inducer of EMT [43], qRT-PCR analysis revealed that NETs and TGFβ induced a similar upregulation of most mesenchymal markers analyzed in the cancer cells, but NETs and TGFβ induced the downregulation of only Claudin 1 among the epithelial markers tested (data not shown). Accordingly, NETs induced SMAD family member 2 (SMAD2) phosphorylation in cancer cells, indicating TGFp signaling activation, and TGFβ Receptor 1 (TGFβR1) inhibition inhibited NET-mediated SMAD2 phosphorylation and EMT gene expression (data not shown). In addition, TGFβRl inhibition improved chemotherapy response both in vitro (data not shown) and in vivo data not shown) Our results also indicated that TGFβ-blocking antibodies (targeting isoforms 1, 2 and 3) sensitized cancer cells to chemotherapy in vivo (data not shown). Interestingly, while recombinant TGFβ had no effect on NET formation in vitro, targeting TGFβ led to decreased neutrophil recruitment and NET formation in vivo (data not shown). This suggests that targeting TGFβ signaling indirectly limits lung inflammation which further improves chemotherapy treatment. This is in accordance with the pleiotropic role of TGFβ signaling in immunity and cancer [44], Altogether, these results indicate that NETs induce a TGFβ- dependent EMT in cancer cells which correlates with chemoresistance.
NET-associated MMP9 activates latent TGFβ which counteracts chemotherapy efficacy
Our results suggested that TGFβ played a role in NET-mediated chemoresistance. TGFβ is synthesized and secreted as a latent complex which is then processed to its active form [45], Using an ELISA test to detect both total- and active- TGFβ, we found a large amount of the latent form of TGFβ in the CM from 410.4 cancer cells, but not in the CM from neutrophils including after NET formation. The active form of TGFβ was barely detectable in the CMs (data not shown). Chemotherapy did not change the levels of active and total TGFβ present in the CM from cancer cells (data not shown). However, when we incubated the CM from 410.4 cells with the CM from neutrophils (unstimulated, degranulating or induced to form NETs), the NETs activated latent TGFβ released by 410.4 cells (data not shown). NETs did not modulate transcription of TGFβ family members or receptors in 410.4 cells (data not shown) but targeting of NETs abrogated the activation of latent TGFβ (data not shown).
Latent TGFβ is secreted either as a Small Latent Complex (SLC), associated with Latency Associated Protein (LAP) or as a Large Latent Complex (LLC), associated with LAP and Latent TGFβ Binding Protein (LBTP) [46], LBTP was not detected in cell lysate, ECM, or CM from 410.4 cancer cells, but the LAP-TGFp complex was detectable in all three isolates of the cancer cells (data not shown). In agreement with the ELISA results (data not shown), Western Blot revealed that incubation of cancer cell CM with NETs led to the degradation of LAP and the release of active TGFp and targeting NETs abrogated LAP degradation (data not shown). In contrast, CM from unstimulated and degranulated neutrophils had no effect (data not shown). Activation of latent TGFβ into active TGFβ was also observed when culturing cancer cells with NET CM or when using a co-culture system and targeting NETs abrogated the activation of TGFp also under these conditions (data not shown).
NETs are characterized by the association of neutrophil proteases with the DNA scaffold [47, 48], MMP9 can proteolytically activate TGFp and is present within the NET-DNA scaffold (data not shown) [18, 49], Using inhibitors against some of the major NET-associated proteases - Neutrophil Elastase (NE), MMP9 and Cathepsin G - and MPO (which indirectly can activate TGFβ activation [50]), we found that only MMP9 inhibition inhibited NET-mediated TGFβ activation (data not shown) and chemoresistance in vitro (data not shown). MMP9 inhibition also sensitized cancer cells to cisplatin and AC chemotherapy in vivo (data not shown). MMP9 inhibition additionally decreased the number of neutrophils and NETs in the lungs (data not shown) but did not block the ability of chemotherapy-treated cancer cells to promote NET formation in vitro (data not shown).. Altogether, our results show that NET- associated MMP9 can activate latent TGFβ, secreted e.g., by cancer cells, and that this activation induces EMT in cancer cells and correlates with chemoresistance.
NET-associated ITGavp traps latent TGFβ
NET-mediated proteolytic activation of TGFβ was required for cancer cell resistance to treatment and digesting NETs with DNase I improved treatment efficacy in vivo and in vitro (data not shown). Yet, we previously showed that DNase I digestion of NETs does not reduce NET-MMP9 activity [18], Therefore, we tested whether the NET-DNA scaffold would contribute to TGFβ activation through a different means. Integrins are large transmembrane proteins, but parts of them are also found in NETs [51], Moreover, integrins, including integrin (ITG) ocvpi, have been shown to bind latent TGFβ through the LAP-RGD domain [45, 46], We hypothesized that latent TGFβ was trapped and then processed within the NET-DNA scaffold. We found that ITGavp 1 and MMP9 were both present within the NET-DNA scaffold by immunofluorescence (data not shown). A potent and highly specific small-molecule inhibitor of ITGavp 1 has been developed [52], and this inhibitor, as well as blocking antibodies against ITGαvβ1 and a RGD peptide inhibited TGFβ activation and LAP degradation (data not shown), indicating that NET-associated ITGαvβ1 is necessary for TGFβ activation by NET- associated MMP9. Accordingly, TGFβ, released by cancer cells co-localized with ITGp 1 within the NET-DNA scaffold (data not shown). Next, using NETs incubated with CM from chemotherapy treated cancer cells, we showed that TGFp, secreted by cancer cells, was trapped within the NET-DNA scaffold and that this trapping was prevented when targeting ITGαvβ1 (data not shown). Targeting ITGαvβ1 improved chemotherapy response both in vitro (data not shown) and in vivo (data not shown) and reduced neutrophil recruitment and NET formation (data not shown). However, ITGαvβ1 targeting did not block NET formation induced by chemotherapy-treated cancer cell CM in vitro (data not shown). Altogether, our results suggest that NETs act as a scaffold that traps cancer-cell derived latent TGFp, which can then be cleaved efficiently by NET-associated MMP9 and released as active TGFβ to promote chemoresistance. The inventors also show that targeting NETs ameliorates chemotherapy efficacy against breast cancer lung metastasis (Figures 3A and 3B) and against breast cancer liver metastasis (Figures 4A to 4C).
Finally, the inventors show that resistant metastatic breast cancer patients exhibit high level of NETs after chemotherapy (Figures 5A to 5C).
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57. Zhu, T., et al., Neutrophil extracellular traps promote gastric cancer metastasis by inducing epithelialmesenchymal transition. Int J Mol Med, 2021. 48(1). 58. Pandolfi, L., et al., Neutrophil Extracellular Traps Induce the Epithelial- Mesenchymal Transition: Implications in Post-COVID-19 Fibrosis. Front Immunol, 2021. 12: p. 663303.
59. Jin, W., et al., Neutrophil extracellular DNA traps promote pancreatic cancer cells migration and invasion by activating EGFR/ERK pathway. J Cell Mol Med, 2021. 25(12): p. 5443-5456.
60. Xia, X., et al., Neutrophil extracellular traps promote metastasis in gastric cancer patients with postoperative abdominal infectious complications. Nat Commun, 2022. 13(1): p. 1017.
61. Akhurst, R.J. and A. Hata, Targeting the TGFbeta signalling pathway in disease. Nat Rev Drug Discov, 2012. 11(10): p. 790-811.
62. Clancy, D.M., et al., Neutrophil extracellular traps can serve as platforms for processing and activation of IL-1 family cytokines. FEBS J, 2017. 284(11): p. 1712-1725.
63. Jin, M.Z. and W.L. Jin, The updated landscape of tumor microenvironment and drug repurposing. Signal Transduct Target Ther, 2020. 5(1): p. 166.
64. Nakazawa, D., et al., Histones and Neutrophil Extracellular Traps Enhance Tubular Necrosis and Remote Organ Injury in Ischemic AKI. J Am Soc Nephrol, 2017. 28(6): p. 1753-1768.
65. Lin, Q., et al., Inhibiting NLRP3 inflammasome attenuates apoptosis in contrast- induced acute kidney injury through the upregulation of HIF1A and BNIP3 -mediated mitophagy. Autophagy, 2021. 17(10): p. 2975-2990.
66. Anders, H.J., Of Inflammasomes and Alarmins: IL-lbeta and IL-lalpha in Kidney Disease. J Am Soc Nephrol, 2016. 27(9): p. 2564-75.

Claims

CLAIMS:
1. A method for identifying whether a subj ect suffering from a cancer is at risk of having or developing a resistance to chemotherapy treatment comprising following steps: i) determining in a biological sample obtained from said subject the level of neutrophil extracellular traps (NETs); ii) comparing the level and/or the number determined in step i) with a predetermined reference value and iii) concluding that the subj ect has or is at risk of having or developing a resistance to chemotherapy treatment when the level and/or the number of NETs is higher than the predetermined reference value or concluding that the subject has not or is not at risk of having or not develop a resistance to the chemotherapy treatment when the level and/or the number of NETs is lower than the predetermined reference value.
2. The method according to claim 1 wherein the biological sample is tissue or plasma sample.
3. The method according to claim 1 wherein the cancer is breast cancer.
4. The method according to claim 1 wherein the subject has breast cancer with lung metastasis, brain metastasis, liver metastasis and/or melanoma metastasis.
5. A method for treating a subject suffering from a cancer resistant to a chemotherapeutic agent comprising a step of administering said subject with a therapeutically effective amount of an inhibitor of NETs.
6. The method according to claim 5 wherein the subject has breast cancer with lung metastasis.
7. The method according to claim 5 wherein the inhibitor of NETs is PAD4 inhibitor, CXCR2 inhibitor, IL-1β inhibitor, TGFβ inhibitor, MMP9 inhibitor or ITGαvβ1 inhibitor
8. The method according to claims 5 to 7 wherein the inhibitor of NETs is DNase I.
9. The method according to claims 5 to 7 wherein the inhibitor of NETs is GSK484.
10. The method according to claims 5 to 7 wherein the inhibitor of NETs is a compound of Formula II and its derivatives.
11. A chemotherapeutic agent and (ii) a NETs inhibitor, as a combined preparation for simultaneous, separate or sequential use for treating a cancer.
12. A kit for use in the method according to claims 1 to 4, wherein said kit comprising a reagent that specifically recognizes NETs and instructions.
13. The kit according to claim 12 wherein the antibody recognizes histone H2B and myeloperoxidase.
14. The kit according to claim 12, wherein the reagent is an antibody.
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