Methods of selecting treatment for cancer

Abstract

A method of assessing responsiveness to gaseous nitric oxide (gNO) treatment in a subject diagnosed with cancer is provided. The method comprising determining in a tissue of a subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with an effective amount of gNO, the increase being indicative of an efficacious treatment with the gNO. Also provided is a method of determining a likelihood of a subject diagnosed with a primary tumor to develop a secondary tumor.

Description

[0001] METHODS OF SELECTING TREATMENT FOR CANCER

[0002] RELATED APPLICATION

[0003] This application claims the benefit ofU.S. Provisional Application No. 63 / 317,133 filed March 7, 2022. The entire contents of the above-referenced application are incorporated by reference herein.

[0004] FIELD OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to methods of selecting treatments for cancer.

[0006] BACKGROUND OF THE INVENTION

[0007] Metastases are responsible for the majority of cancer-related deaths. Therefore, the primary therapeutic goals are preventing initial metastasis formation, targeting established metastases and preventing additional ones. However, despite the high mortality from metastatic cancers, therapeutic targets to prevent metastases are limited. Furthermore, survival among metastatic cancer patients has not improved within the past decade.

[0008] Immunotherapeutic treatment options may overcome the challenge of metastases via an anti-tumor memory response of the adaptive immune system. Tumor ablation is a minimally invasive technique that is commonly used in the treatment of solid tumors. Several ablation methods exist, such as radiofrequency, cryoablation, focused ultrasound, and microwave ablation. These ablation methods have been shown to trigger an immune response. Moreover, image-guided tumor ablation for early-stage hepatocellular carcinoma (HCC) is an accepted non-surgical treatment that provides local tumor control and favorable survival benefit.

[0009] Local and in situ tumor ablation methods have been shown to enhance anti -tumor immune responses resulting in the destruction of residual malignant cells in primary tumors and distant metastases. Preclinical studies testing the effect of exogenously administered nitric oxide (NO) demonstrated anti-cancer properties and suggested that NO may serve as a potent tumoricidal agent. While NO at low doses may possess pro-oncogenic properties, at high doses, NO may have a role in cancer therapy either as a single agent or in combination with other antineoplastic compounds.

[0010] More specifically, high NO doses may have anti-cancer properties by promoting oxidative / nitrosative stress and DNA damage. The generation of reactive nitric oxide species, including peroxynitrite can oxidize the DNA and induce single-strand breaks. In addition, NO can induce cell death via both i) necrosis, and ii) apoptosis. Apoptosis can occur via the upregulation of tumor suppressor proteins and the degradation of antiapoptotic proteins. Further, changes in the mitochondrial permeability transition pore cause intracellular calcium release and the subsequent release of cytochrome c resulting in dow nstream apoptosis.

[0011] Interestingly, NO generation in the blood vessels adjacent to the tumor may prevent cancer cells from invading the circulation, through DNA damage and apoptosis of these cells. Importantly, NO can sensitize resistant cancer cells to standard cancer therapies, such as chemotherapy and radiotherapy, as NO can improve the tumor blood flow and, therefore, drug delivery for these agents. Specifically, increasing DNA double-strand breaks mediated by NO causes cancer cells to become more sensitive. Thus, high dose NO can serve as an antiinvasion, anti-metastatic, and anti-angiogenic agent.

[0012] Use of gaseous NO (gNO) in cancer treatment has been previously described in W02021 / 105901.

[0013] Therefore, there remains a need in the art for methods of assessing responsiveness to cancer treatment using gNO. There also remains a need for methods of determining the likelihood of a subject diagnosed with a primary tumor will develop a secondary tumor following treatment with gNO.

[0014] SUMMARY OF THE INVENTION

[0015] According to an aspect of some embodiments of the present invention there is provided a method of assessing responsiveness to gaseous nitric oxide (gNO) treatment in a subject diagnosed with cancer, the method comprising locally administering an effective amount of gNO; determining in a tissue of the subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO, the increase being indicative of an efficacious treatment with the gNO.

[0016] According to an aspect of some embodiments of the present invention there is provided a method of using gaseous nitric oxide (gNO) in the treatment of a subject diagnosed with cancer, the method comprising locally administering an effective amount of gNO; determining in a tissue of the subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO, wherein when a presence of the increase is indicated, the treatment is terminated, repeated or decreased, while when an absence of the increase is indicated the treatment is elevated or repeated. According to an aspect of some embodiments of the present invention there is provided a method of determining a likelihood of a subject diagnosed with a primary tumor to develop a secondary tumor following treatment with gaseous nitric oxide (gNO), the method comprising locally administering an effective amount of gNO; determining in a tissue of a subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO, the increase being indicative of a reduced likelihood to develop a secondary tumor. In embodiments, when a presence of said increase is indicated said administering is terminated, repeated or decreased, and wherein when an absence of said increase is indicated said administering is elevated or repeated.

[0017] An indication that there is an increase in in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO can be determined by a reduction in systemic and / or tumor levels of inhibitory myeloid-derived suppressor cells (MDSCs), Regulatory T-cells(Tregs), expression of immune check point molecules (PD-1, Lag-3, TIGIT, Tim-3) in CD4 / 8 T-cells and / or increase in Ml macrophages, expression of MHC-I on tumor cells and expression of co-stimulatory molecules(ICOS,GITR) in CD4 / 8 T-cells.

[0018] According to some embodiments of the invention, the tissue is selected from the group consisting of a tumor, a peripheral blood, lymphatic fluid or tissue, thymus and a spleen.

[0019] According to some embodiments of the invention, the cells are selected from the group consisting of T cells, B cells, dendrocytes and macrophages.

[0020] According to some embodiments of the invention, the cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, B220+ B cells, CD19+, CD38+, CD20+, CD138+ B cells, CDl lb+ dendrocytes, CDla dendrocytes, CD68 macrophages and F4 / 80 macrophages.

[0021] According to some embodiments of the invention, the cells comprise activated T cells. According to some embodiments of the invention, the cancer is a solid tumor.

[0022] According to some embodiments of the invention, the solid tumor is a colon cancer.

[0023] According to some embodiments of the invention, the method further comprises determining in the tissue of the subject a decrease in myeloid-derived suppressor cells (MDSCs) or other suppressor cells below a predetermined threshold.

[0024] According to some embodiments of the invention, the effective amount of gNO is at a dose from about 1,000 ppm to about 1,000,000 ppm for a time period from about 1 second to about 60 minutes at a volumetric flow from about 0.00001 liter per minute (LPM) to about 1 LPM

[0025] According to some embodiments of the invention, the effective amount of gNO is at a dose from about 10,000 ppm to 200,000 ppm or from about 20,000 ppm to about 200,000 ppm or from about 20,000 ppm to about 100,000 ppm.

[0026] According to some embodiments of the invention, the effective amount of gNO is administration for a time period that ranges from about 1 second to about 10 minutes or from about 30 seconds to about 10 minutes.

[0027] According to some embodiments of the invention, the effective amount of gNO is administration at a volumetric flow from about 0.001 LPM to about 2 LPM, or from about 0.01 to about 2 LMP about 0.001 LPM to about 0.5 LPM.

[0028] According to some embodiments of the invention, the local treatment is intratumoral treatment.

[0029] According to some embodiments of the invention, the local administration comprises two or more administration sites.

[0030] According to some embodiments of the invention, a distance between the two or more administration sites is, independently, from about 2.5 mm to about 1 cm, or from about 0.25 cm to about 0.5 cm.

[0031] According to some embodiments of the invention, the effective amount is at a dose from about 0. 1 mg to about 300 mg, per cm3tumor.

[0032] According to some embodiments of the invention, the effective amount is a dose from about 0.01 mg to about 100 mg, or from 0.1 mg to about 10 mg, per a tumor of 20 mm3or less.

[0033] According to some embodiments of the invention, the local administration comprises scavenging excess gNO from the administration site.

[0034] According to some embodiments of the invention, the local administration comprises pulsing gNO from about 2 to about 20 times.

[0035] According to some embodiments of the invention, the local administration is performed at least once.

[0036] According to some embodiments of the invention, local administration is performed at least twice, and wherein a time interval between the two administrations is at least one week.

[0037] According to some embodiments of the invention, the determining comprises ex vivo determining.

[0038] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0039] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0040] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0041] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken wings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0042] In the drawings:

[0043] Fig. 1A - Fig. ID show the immune response in primary tumor following 5-minute administration of 20,000 - 50,000 ppm NO or nitrogen 14 days post-treatment. Fig. 1 A depicts an exemplary Assay scheme. Fig. IB depicts an example of a level of inflammation and lymphocytes infiltration 14 days post-gas delivery. Fig. 1C depicts an exemplary analysis of B cell, T-cell, macrophages and dendrocytes of treated tumors 14 days post gas delivery. Fig. ID depicts exemplary representative pictures of tumors stained with anti-CD3, where tumor cells are stained in blue and T cells are stained in brown.

[0044] Fig. 2A - Fig. 2E show T cells, B cells and MDSCs changes in the spleen following 5- minute administration of 20,000 - 50,000 ppm 21 days post treatment. Fig. 2A depicts an example of IFNY positive T cells out of total T cells in the spleen. Fig. 2B depicts an example of IFNY positive helper T cells out of total helper T cells in the spleen. Fig. 2C depicts an example of IFNY positive cytotoxic T cells out of total cytotoxic T cells in the spleen. Fig. 2D depicts an example of B cells in the spleen. Fig. 2E depicts an example of polymorphonuclear MDSCs out of total monocytes in the spleen.

[0045] Fig. 3A - Fig. 3B show T cells and B cells upregulation in the blood following 5-minute administration of 20,000 - 50,000 ppm 21 days post treatment. Fig. 3A depicts an example of T cells in the blood. Fig. 3B depicts an example B cells in the blood. Fig. 4A - Fig. 4E show T cell analysis one day after 5-minute 50,000 ppm NO treatment. Fig. 4A depicts an example of tumor CD8+T cells that express GITR. Fig. 4B depicts an example of tumor CD8+ T cells that express ICOS. Fig. 4C depicts an example of tumor CD8+T cells that have higher levels of CD44hlghexpression. Fig. 4D depicts an example of tumor CD8+T cells that express PD-1. Fig. 4E depicts an example of Blood T regulatory levels.

[0046] Fig. 5 shows an example of M l macrophages in the blood of CT26 tumor bearing mice 1 day after NO treatment.

[0047] Fig. 6 shows an example of mononuclear MDSCs in the blood of CT26 tumor bearing mice 1 day after NO treatment.

[0048] Fig. 7A - Fig. 7C shows examples of Treg frequency in CT26 tumors and Treg proliferation in blood 1 day following treatment.

[0049] Fig. 8A and Fig. 8B show examples of tumor viability 5 days post treatment.

[0050] Fig. 9A - Fig. 9C show examples of MHC-I expression on tumor cells in CT26 tumors following treatment.

[0051] Fig. 10 shows an example of the expression of the co-stimulatory molecule GITR on CD8 T-cells 1 day post treatment.

[0052] Fig. 11A - Fig. 1 IF show examples of the expression of immune checkpoint markers on tumor infiltrating T-cells 5 days post treatment in CT26 tumors.

[0053] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0054] The present invention, in some embodiments thereof, relates to methods of selecting treatments for cancer.

[0055] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0056] In situ tumor ablation via delivery of an exogenous ultra-high concentration of gaseous nitric oxide (gNO) to the solid tumor has been recently described. Because typically not ail the patients respond to any drug, efforts have been made to devise strategies to personalize care. The present inventors have now discovered that such treatment alters the innate and / or immune system profile in the tumor and in related tissues, i.e., peripheral blood and spleen. Overall, a more innate immunity environment is evident with reduced suppressive immunity. These findings can be harnessed towards predicting the efficacy of treatment and the likelihood to develop secondary tumors, e.g., metastases.

[0057] As is described hereinbelow and in the Examples section which follows, 1 day following gNO intra-tumor treatment, T-cell activation in tumor was promoted, where gNO treatment strongly induces ICOS expression and significantly induces GITR on CD8+ CTLs, and where increased T-cell activation can be reflected in increased expression of and CD44. Further, the inhibitory molecule PD-1 is also upregulated on T cells. In contrast, T-cell proliferation is mildly reduced and Tregs proliferation specifically is reduced. Ml macrophages and mononuclear MDSCs are also increased in the blood, while regulatory T cell (Treg) numbers in tumors and Treg proliferation in blood are reduced. Further, expression of the costimulatory molecule ICOS and GITR on tumor infiltrating CD8 T-cells may also increase. In addition, 5 days following treatment, cell death can be induced in tumors and tumor viability is reduced, with gNO having shown to maintain MHC-I expression on tumor cells. Further, positive response to the treatment is associated with reduced expression of immune checkpoint markers on tumor infiltrating T-cells.

[0058] 14 days following treatment, B cells, T cells, macrophages and dendrocytes penetrate the tumor mass, which may be correlated with the shrinkage of the tumor. It is suggested that this correlates with efficacy of treatment, a finding that was not observed in the nitrogen-treated tumors (control). In addition, T cells and B cells were elevated in both spleen and blood 21 days post gNO treatment. In stark contrast to the increase in adaptive immune cells, polymorphonuclear MDSCs were reduced in the spleen at this timepoint.

[0059] These findings of upregulation of adaptive immune cells on the one hand, and downregulation of immune suppressor cells might explain the results seen in a previous challenge study, in which 64%-100% of 20,000 ppm and 50,000 ppm gNO treated mice (respectively) rejected a secondary tumor cell inoculation up to 75 days post challenge tumor induction.

[0060] Overall provided herein are biomarkers, i.e., indicators for the efficacy of treatment, dosing, and forthcoming prognosis following treatment with gNO.

[0061] Thus, according to an aspect of the invention there is provided a method of assessing responsiveness to gaseous nitric oxide (gNO) treatment in a subject diagnosed with cancer, the method comprising locally administering an effective amount of gNO; determining in a tissue of the subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO, said increase being indicative of an efficacious treatment with said gNO. In embodiments, when a presence of said increase is indicated said administering is terminated, repeated or decreased, and wherein when an absence of said increase is indicated said administering is elevated or repeated.

[0062] An indication that there is an increase in in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO can be determined by a reduction in systemic and / or tumor levels of inhibitory myeloid-derived suppressor cells (MDSCs), Regulatory T-cells(Tregs), expression of immune check point molecules (PD-1, Lag-3, TIGIT, Tim-3) in CD4 / 8 T-cells and / or increase in Ml macrophages, expression of MHC-I on tumor cells and expression of co-stimulatory molecules(ICOS,GITR) in CD4 / 8 T-cells.

[0063] As used herein “responsiveness” refers to the extent to which a patient improves following or during treatment, irrespectively of the presence or absence of symptoms.

[0064] As used herein throughout, the term "subject" and "patient" are used interchangeably herein and refer to both human and nonhuman organisms, i.e., animals. The term "nonhuman animals" of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like, for medical and / or laboratory research purposes. Preferably, the subject is a human subject. More preferably, the subject is a human patient who has a pre-malignant or malignant tumor, e.g., has been diagnosed with cancer.

[0065] As used herein throughout, the term “tumor” describes a plurality of cells or a tissue composed of the plurality of cells that are characterized by abnormal cell growth and which serve no physiological function.

[0066] By “abnormal cell growth” it is meant uncontrolled, progressive proliferation of the cells, which is no longer under normal bodily control. The growth of a tumor tissue typically exceeds, and is uncoordinated with, that of the normal cells or tissues around it.

[0067] "Abnormal cell growth” also describes cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition), including, for example, abnormal growth of: (1) cancerous (or cancer) cells that proliferate by expressing a mutated tyrosine kinase or over-expression of a receptor tyrosine kinase; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs; (3) any tumors that proliferate by receptor tyrosine kinases; (4) any tumors that proliferate by aberrant serine / threonine kinase activation; and (5) benign and malignant cells of other proliferative diseases in which aberrant serine / threonine kinase activation occurs.

[0068] A tumor as described herein can be a primary tumor or a secondary tumor. The term “malignant tumor” describes a tumor that is not self-limited in its growth, is capable of invading into adjacent tissues, and may be capable of spreading to distant tissues (metastasizing). The term “benign tumor” describes a tumor which is not malignant (i.e. does not grow in an unlimited, aggressive manner, does not invade surrounding tissues, and does not metastasize).

[0069] The term “primary tumor” describes a tumor that is at the original site where it first arose.

[0070] The term “secondary tumor” describes a tumor that has spread from its original (primary) site of growth to another site, close to or distant from the primary site, and is also referred to herein and in the art as metastasis, or as metastasizing tumor. The term “secondary tumor” as used herein also describes recurrent tumor, which can be at the original site as the primary tumor and / or at another site, as a metastasizing tumor.

[0071] According to some of any of the embodiments described herein, the tumor is a malignant tumor, for example, a malignant cancerous tumor, and the tumor cells are cancer or cancerous cells.

[0072] According to these embodiments, the methods and uses as described herein in any of the respective embodiments are for treating and / or controlling cancer or a cancerous tumor is a subject in need thereof.

[0073] The methods and uses as described herein are in the context of subjects having a primary cancer tumor, a metastasizing cancer and / or a recunent cancer, as described herein.

[0074] The term “cancer” encompasses malignant and benign tumors as well as disease conditions evolving from primary or secondary tumors, as described herein.

[0075] Examples of benign tumors include, without limitation, lipomas, chondromas, adenomas, pilomatricomas, teratomas, and hamartomas.

[0076] Cancers treatable according to embodiments of the invention include, but are not limited to, carcinomas, sarcomas, blastomas, and germ cell tumors. Carcinomas include, without limitation, adenocarcinomas (e.g., small cell lung cancer, kidney, uterus, prostate, bladder, ovary and / or colon adenocarcinoma) and epithelial carcinomas.

[0077] Examples of cancers treatable according to embodiments of the invention include, without limitation, adenocarcinoma, adrenal tumors (e g., hereditary' adrenocortical carcinoma), biliary tract tumors, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., ductal breast cancer, invasive intraductal breast cancer, sporadic breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer-1, breast cancer-3, and / or breast-ovarian cancer), bronchogenic large cell carcinoma, cervical cancer (e.g., cervical carcinoma), carcinosarcoma, choriocarcinoma, cystadenocarcinoma, dermatofibrosarcoma protuberans, ductal carcinoma, Ehrlich-Lettre ascites, embryonal rhabdomyosarcoma, endocrine neoplasia, endometrial cancer (e.g., endometrial carcinoma), ependimoblastoma, epidermoid carcinoma, epithelial adult tumor, epithelioma, erythroleukemia (e.g., Friend and / or lymphoblast), extraskeletal myxoid chondrosarcoma, fibrosarcoma, gallbladder carcinoma, ganglioblastoma, gastrointestinal tract tumors (e.g., colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6, hereditary nonpolyposis type 7, small and / or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, and / or pancreatic endocrine tumors), germ cell tumor (male germ cell tumor, and / or testicular and / or ovarian dysgerminoma), giant cell tumor, glial tumor, glioma, glioblastoma (e.g., glioblastoma multiforme, astrocytoma), head & neck cancer, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B-cell), hypernephroma, insulinoma, islet tumor, keratoma, large cell carcinoma, leiomyoblastoma, leiomyosarcoma, leukemia (e.g., acute lymphatic leukemia, acute lymphoblastic leukemia, acute lymphoblastic pre-B cell leukemia, acute lymphoblastic T cell leukemia, acute megakaryoblastic leukemia, monocytic leukemia, acute myelogenous leukemia, acute myeloid leukemia, acute my eloid leukemia with eosinophilia, B- cell leukemia, basophilic leukemia, chronic myeloid leukemia, chronic B-cell leukemia, eosinophilic leukemia, Friend leukemia, granulocytic or myelocytic leukemia, hairy' cell leukemia, lymphocytic leukemia, mast cell leukemia, megakaryoblastic leukemia, monocytic leukemia, monocytic-macrophage leukemia, myeloblastic leukemia, myeloid leukemia, myelomonocytic leukemia, plasma cell leukemia, pre-B cell leukemia, promyelocytic leukemia, subacute leukemia, T-cell leukemia, lymphoid neoplasm, predisposition to myeloid malignancy, and / or acute nonlymphocytic leukemia), Li-Fraumeni syndrome, liposarcoma, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer, and / or hepatoma), lung cancer (e.g., Lewis lung carcinoma, small cell carcinoma and / or non-small cell carcinoma) lymphoma (e.g., Hodgkin’s disease, non-Hodgkin’s lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, cutaneous T-cell lymphoma, histiocytic lymphoma, lymphoblastic lymphoma, T-cell lymphoma, and / or thymic lymphoma), lymphosarcoma, lynch cancer family syndrome II, mammary tumor, mastocytoma, medulloblastoma, medullary carcinoma, melanoma, mesothelioma, metastatic tumor, monocyte tumor, mucoepidermoid carcinoma, multiple glomus tumors, multiple meningioma, myelodysplastic syndrome, myeloma (e.g., multiple myeloma), nasopharyngeal cancer, nephroblastoma, nervous tissue glial tumor, nervous tissue neuronal tumor, neurinoma, neuroblastoma, neurogenic tumor, non-melanoma skin cancer, oat cell carcinoma, oligodendroglioma, osteochondroma, osteomyeloma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma, serous ovarian cancer, and / or ovarian sex cord tumors), papillary carcinoma, papilloma, paraganglioma (e.g., familial nonchromaffin), pheochromocytoma, pituitary tumor (invasive), placental site trophoblastic tumor, plasmacytoma, prostate cancer (e.g., prostate adenocarcinoma), renal cancer (e.g., Wilms’ tumor type 2 or type 1), retinoblastoma, rhabdoid tumors (e.g., rhabdoid predisposition syndrome), rhabdomyosarcoma, sacrococcygeal tumor, sarcoma (e.g., Ewing’s sarcoma, histiocytic cell sarcoma, Jensen sarcoma, myxosarcoma, osteosarcoma, reticulum cell sarcoma, soft tissue sarcoma and / or synovial sarcoma), schwannoma, small cell carcinoma, spindle cell carcinoma, spinocellular carcinoma, squamous cell carcinoma (e.g., in head and neck), subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma (e.g., immature teratoma of ovary), testicular cancer (e g. testicular germ cell tumor), transitional cell carcinoma, Turcot syndrome with glioblastoma, thymoma, thyroid cancer (e.g., follicular, medullary and / or papillary thyroid cancer), trichoepithelioma, trophoblastic tumor, undifferentiated carcinoma, uterine cancer, uterine cervix carcinoma.

[0078] According to a specific embodiment, the cancer is colon cancer.

[0079] Methods and uses of the present embodiments can be used to treat one or more solid tumors.

[0080] As used herein, the term "solid tumor" refers to those conditions, such as cancer, that form an abnormal tumor mass, such as sarcomas, carcinomas, and lymphomas. For example, solid tumors can include, but are not limited to, ovarian tumors, prostate tumors, skin tumors, lung tumors, breast tumors, liver tumors, brain tumors, CNS tumors, kidney tumors, colon tumors, bladder tumors, intestinal tumors, melanomas, gliomas, ependymomas, oligodendrogliomas, oligoastrocytomas, astrocytomas, glioblastomas, and medulloblastomas. Suitable examples of solid tumor diseases include, but are not limited to, non-small cell lung cancer (NSCLC), neuroendocrine tumors, thyomas, fibrous tumors, metastatic colorectal cancer (mCRC), and the like. In certain embodiments, the solid tumor disease is an adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and the tike.

[0081] According to some embodiments, the cancer is or comprises a solid tumor, and can be, for example, adenocarcinoma, adrenal tumors (e.g., hereditary adrenocortical carcinoma), biliary tract tumors, bladder cancer, bone cancer, brain cancer, breast cancer (e.g., ductal breast cancer, invasive intraductal breast cancer, sporadic breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer-1, breast cancer-3, and / or breast-ovarian cancer), bronchogenic large cell carcinoma, cervical cancer (e.g., cervical carcinoma), carcinosarcoma, choriocarcinoma, cystadenocarcinoma, dermatofibrosarcoma protuberans, ductal carcinoma, Ehrlich-Lettre ascites, embryonal rhabdomyosarcoma, endocrine neoplasia, endometrial cancer (e.g., endometrial carcinoma), ependimoblastoma, epidermoid carcinoma, epithelial adult tumor, epithelioma, extraskeletal myxoid chondrosarcoma, fibrosarcoma, gallbladder carcinoma, ganglioblastoma, gastrointestinal tract tumors (e.g., colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6, hereditary nonpolyposis type 7, small and / or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, and / or pancreatic endocrine tumors), germ cell tumor (male germ cell tumor, and / or testicular and / or ovarian dysgerminoma), giant cell tumor, glial tumor, glioma, glioblastoma (e.g., glioblastoma multiforme, astrocytoma), head & neck cancer, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B-cell), hypernephroma, insulinoma, islet tumor, keratoma, large cell carcinoma, leiomyoblastoma, liposarcoma, liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer, and / or hepatoma), lung cancer (e.g., Lewis lung carcinoma, small cell carcinoma and / or non-small cell carcinoma) lymphoma (e.g., Hodgkin’s disease, non-Hodgkin’s lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, cutaneous T-cell lymphoma, histiocytic lymphoma, lymphoblastic lymphoma, T-cell lymphoma, and / or thymic lymphoma), lymphosarcoma, lynch cancer family syndrome II, mammary tumor, mastocytoma, medulloblastoma, medullary carcinoma, melanoma, mesothelioma, metastatic tumor, monocyte tumor, mucoepidermoid carcinoma, multiple glomus tumors, multiple meningioma, myelodysplastic syndrome, myeloma (e.g., multiple myeloma), nasopharyngeal cancer, nephroblastoma, nervous tissue glial tumor, nervous tissue neuronal tumor, neurinoma, neuroblastoma, neurogenic tumor, non-melanoma skin cancer, oat cell carcinoma, oligodendroglioma, osteochondroma, osteomyeloma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma, serous ovarian cancer, and / or ovarian sex cord tumors), papillary carcinoma, papilloma, paraganglioma (e.g, familial nonchromaffin), pheochromocytoma, pituitary tumor (invasive), placental site trophoblastic tumor, plasmacytoma, prostate cancer (e.g., prostate adenocarcinoma), renal cancer (e.g., Wilms’ tumor type 2 or type 1), retinoblastoma, rhabdoid tumors (e.g., rhabdoid predisposition syndrome), rhabdomyosarcoma, sacrococcygeal tumor, sarcoma (e.g., Ewing’s sarcoma, histiocytic cell sarcoma, Jensen sarcoma, myxosarcoma, osteosarcoma, reticulum cell sarcoma, soft tissue sarcoma and / or synovial sarcoma), schwannoma, small cell carcinoma, spindle cell carcinoma, spinocellular carcinoma, squamous cell carcinoma (e.g., in head and neck), subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma (e.g., immature teratoma of ovary), testicular cancer (e g. testicular germ cell tumor), transitional cell carcinoma, Turcot syndrome with glioblastoma, thymoma, thyroid cancer (e.g., follicular, medullary and / or papillary thyroid cancer), trichoepithelioma, trophoblastic tumor, undifferentiated carcinoma, uterine cancer, uterine cervix carcinoma.

[0082] According to some of any of the embodiments described herein, the gNO is locally administered to the primary tumor and / or to a metastasizing tumor.

[0083] According to some of any of the embodiments described herein, depending on the site of the primary or metastasizing tumor, the gNO is locally administered to an organ that hosts the tumor cells or to an organ that is in close proximity (e.g., up to 5 cm or up to 2 cm) to the tumor cells. Exemplary organs to which gNO can be locally administered according to some of any of the respective embodiments as described herein include, but are not limited to, the adrenal gland, bladder, bones, brain, breast, cervix, colon, colorectum, esophagus, gastrointestinal tract, heart, kidney, liver, large intestine, lungs, mouth, ovaries, pancreas, parathyroid, pituitary gland, prostate, salivary gland, skin, small intestine, spleen, stomach, thymus, thyroid, testicles, urinary tract, uterus, or vagina. According to some embodiments, the gNO is locally administered to the liver, in case of a primary liver cancer or of liver metastases.

[0084] According to some embodiments, the gNO is locally administered to the colon, in case of a primary colon cancer or of colon metastases.

[0085] The methods and uses provided herein for inhibiting abnormal growth of tumor cells or tissue, and are for treating, controlling, or preventing cancer may be generally applicable to all known or to-be-discovered cancerous cell phenotypes and cancerous growths.

[0086] The present embodiments relate to any size and shape of tumors, including large, spread and amorphic cancerous outgrowths.

[0087] The methods and uses provided herein may be especially useful for the treatment, control, and / or prevention of tumors (e.g., cancerous tumors) at localized sites, including inoperable tumors, tumors where localized treatment would be beneficial, and solid tumors.

[0088] According to some of any of the embodiments described herein, the methods and uses of the present embodiments are for inhibiting growth of cells of a primary tumor. According to some of these embodiments, a subject as described herein, which has been diagnosed as being afflicted by a tumor, is subjected to the high dose local administration of gNO as described herein in any of the respective embodiments.

[0089] According to a specific embodiment, an efficacious treatment with gNO is considered when between about 25 % and about 100 % (e.g., 30-100 %, 35-100 %, 40-100 %, 45-100 %, 50-100 %, 55-100 %, 60-100 %, 65-100 %, 70-100 %, 75-100 %, 80-100 %, 85-100 %, 90- 100 %) of the cancerous cells may be killed by the gaseous nitric oxide over the course of one or more administrations, as described herein in any of the respective embodiments.

[0090] According to some embodiments, the subject is exposed or has been exposed to local / intratumoral gNO treatment.

[0091] The present invention provides methods to treat tumors or cancers by local administration of gNO, preferably of high dose gNO as described in further detail hereinunder.

[0092] The present invention provides methods to treat tumors or cancers by local administration of a gas, preferably under a high pressure and for a sufficient time period, as described in further detail hereinunder.

[0093] High dose gNO is defined as the delivery of gNO in a preferably inert gas such as nitrogen at a concentration of between about 1,000 and 1,000,000 ppm, as is described in further detail hereinafter.

[0094] The methods of delivering gNO may include administration of gNO in a continuous or pulsed (e.g., intermittent) manner.

[0095] According to some embodiments of the present invention, the methods are affected by locally administering to the tumor gaseous NO (gNO).

[0096] By “locally administering” it is meant directly contacting the tumor cells or tissue with gNO, such that gNO is applied directly to the tumor and / or its close vicinity. In some embodiments, local administration of gNO is affected mtratumorally, by applying gNO directly into the tumor cells or tissue. In some embodiments, local administration (intratumoral) of gNO is affected by applying gNO to the surface of the tumor tissue, for example, by contacting the surface of the tumor with gNO. In some embodiments, local administration is affected by bringing gNO in close vicinity to the tumor cells or tissue, for example, directly or up to 2 cm, or up to 1 cm, from at least one and preferably all of the tumor surfaces.

[0097] In some of any of the embodiments described herein, local administration is affected intratumorally, such that gNO is injected or otherwise delivered into the tumor. In some of any of the embodiments described herein, local administration is affected by spraying or otherwise applying gNO onto at least one of the tumor’s surface. At least some of the gNO then enters into the tumor via, e.g., diffusion.

[0098] In some of any of the embodiments described herein, local administration is affected by exposing the tumor to gNO in a close container, such that gNO is contacted with the tumor and enters into the tumor via e.g., diffusion. The container can be open or closed and can be sized to conform to the contours of the tumor.

[0099] In some of any of the embodiments described herein, local administration is affected by delivering gNO to a physiological space or cavity which contains at least a portion of the tumor tissue, such that the tumor is contacted with the gNO, and the gNO enters the tumor via e.g., diffusion.

[0100] According to some of any of the embodiments described herein for local administration of gNO, a high dose of gNO is administered locally, as described herein. These embodiments are also referred to herein as “high dose local administration of gNO”, “high dose gNO treatment”, and grammatical diversions of the foregoing, or simply as “high dose gNO”.

[0101] In some of any of the embodiments described herein, the high dose of gNO is reflected by its relatively high concentration in the total amount of gas that is locally administered to the tumor, and is presented by ppm (part per million) units.

[0102] In some of any of the embodiments described herein, the high dose of gNO is presented as its fraction, in ppm units, in the gas carrier. The gas carrier can be air, and preferably an inert gas such as nitrogen or argon, preferably nitrogen.

[0103] In some of any of the embodiments described herein, the high dose of gNO is reflected by the mass of gNO that is locally administered to the tumor, per a volumetric unit of the tumor.

[0104] According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration from about 1,000 ppm to about 1,000,000 ppm (0.1% to 100%), including any intermediate values and subranges therebetw een, for example, from about 1,000 ppm to about 200,000 ppm, or from about 1,000 ppm to about 100,000 ppm, preferably from about 10,000 ppm to about 500,000 ppm, or from about 10,000 ppm to about 200,000 ppm, or from about 10,000 ppm to about 100,000 ppm, or from about 20,000 ppm to about 100,000 ppm, or from about 25,000 ppm to about 100,000 ppm, or from about 25,000 ppm to about 75,000 ppm, or from about 10,000 ppm to about 50,000 ppm, or from about 50,000 ppm to about 100,000 ppm, including any intermediate values and subranges between any of the foregoing, or is about 50,000 ppm. According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration from about 10,000 ppm to about 200,000 ppm.

[0105] In some of any of the embodiments described herein in the context of high dose of gNO, the high dose can be obtained by locally administering gNO at a dose of at least 10,000 ppm, or at least 20,000 ppm, or at least 50,000 ppm, and optionally up to about 1,000,000 ppm.

[0106] In exemplary embodiments, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration of about 25,000 to 200,000 ppm.

[0107] In some embodiments the gNO is provided at a dose from about 1,000 ppm to about 1,000,000 ppm for a time period from about 1 second to about 60 minutes at a flow volume from about 0.00001 LPM to about 1 LPM.

[0108] In some embodiments the gNO is provided for a time period that ranges from about 1 second to about 10 minutes, at about 10,000 ppm to about 200,000 ppm, at about 0.01 LPM to about 2 LPM.

[0109] In some embodiments the gNO is provided for a time period that ranges from about 30 seconds to about 10 minutes, at about 10,000 ppm to about 100,000 ppm.

[0110] In exemplary embodiments, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration of about 25,000 ppm. In exemplary embodiments, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration of about 20,000 ppm. In exemplary embodiments, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration of about 10,000 ppm. In exemplary embodiments, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration of about 100,000 ppm. In exemplary embodiments, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration from about 100,000 ppm to about 200,000 ppm, or from about 200,000 ppm to about 500,000 ppm, or from about 500,000 ppm to about 1 ,000,000 ppm or from about 50,000 ppm or about 100,000 ppm or about 200,000 ppm or about 500,000 ppm or about 1,000,000 ppm.

[0111] According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, at a volumetric flow rate from about 0.00001 LPM to about 10 LPM, preferably from about 0.0001 LPM and about 1 LPM, or from about 0.001 LPM and 0.5 LPM, including any intermediate values and subranges therebetween. For example, the volumetric flow rate can be from about 0.001 LPM to about 0.01 LPM, or from about 0.01 LPM to about 0.1 LPM, or from about 0.1 LPM to about 0.25 LPM, or from about 0.25 LPM to about 0.5 LPM, or from about 0.5 LPM to about 1 LPM, or from about 1 LPM to about 2 LPM, or from about 2 LPM to about 3 LPM, or from about 3 LPM to about 4 LPM, or from about 4 LPM to about 5 LPM, or from about 5 LPM to about 6 LPM, or from about 7 LPM to about 8 LPM, or from about 8 LPM to about 9 LPM, or from about 9 LPM to about 10 LPM, including any intermediate values and subranges therebetween, or it can be, for example, about 0.0001 LPM, or about 0.001 LPM, or about 0.01 LPM, or about 0. 1 LPM, or about 1 LPM or about 10 LPM.

[0112] According to some of any of the embodiments described herein, the gNO is administered for a time period that ranges from about 0.1 seconds to about 10 hours, per administration, including any intermediate values and subranges therebetween. For example, the time period can be from about 0. 1 second to about 1 hour, or from about 1 second to about 10 minutes, or from about 1 minute to about 10 minutes, or from about 10 seconds to about 10 minutes, or from about 0.1 second to about 10 minutes, or from about 30 seconds to about 3 minutes, or from about 1 minute to about 30 minutes, or from about 10 minutes to about 60 minutes, or from about 60 minutes to about 180 minutes, or from about 180 minutes to about 600 minutes, including any intermediate values and subranges between any of foregoing, or it can be about 30 seconds, about 10 minutes, about 30 minutes, or about 60 minutes.

[0113] In some of any of the embodiments related to methods and uses as described herein in the context of high dose of gNO, the high dose can be obtained by locally administering gNO, as described herein in any of the respective embodiments, at a dose from about 1,000 ppm to about 1,000,000 ppm for a time period from about 1 second to about 60 minutes at a volumetric flow (flow volume) from about 0.00001 liter per minute (LPM) to about 1 LPM, including any intermediate values and subranges between any of the foregoing.

[0114] In some of any of the embodiments related to methods and uses as described herein in the context of high dose of gNO, the high dose can be obtained by locally administering gNO, as described herein in any of the respective embodiments, at a dose of at least 10,000 ppm, and optionally up to about 1,000,000, or up to about 500,000 ppm, or up to about 200,000 ppm, or up to about 100,000 ppm, for a time period from about 1 second to about 60 minutes at a volumetric flow (flow volume) from about 0.0001 liter per minute (LPM) to about 1 LPM, including any intermediate values and subranges between any of the foregoing.

[0115] In some of any of the embodiments described herein in the context of high dose of gNO, the high dose can be obtained by locally administering gNO at a dose of at least 10,000 ppm, or at least 20,000 ppm, or at least 50,000 ppm, and optionally up to about 1,000,000 ppm, for a time period of at least 1 second, or at least 10 seconds, or at least 30 seconds, or at least 1 minute, and optionally up to about 60 minutes, at a volumetric flow rate (flow volume) of at least 0.0001 liter per minute (LPM), or at least 0.001 LPM, or at least 0.01 LPM, and optionally up to about 1 LPM, including any intermediate values and subranges between any of the foregoing.

[0116] Alternatively or in addition, in any of the methods and uses described herein in the context of high dose of gNO, the amount of gNO locally administered to the tumor ranges from about 0.1 mg to about 300 mg, per cm3tumor, per administration.

[0117] The parameters of the gNO concentration (ppm), volumetric flow rate (LPM) and time to achieve a desired mass of gNO, and vice versa, the gNO mass achieved by locally administering gNO at a concentration, volumetric flow and time, can be calculated using the know n ideal gas equation, PV=nRT, wherein P is the pressure, V is the volume, n is the number of moles, R is the gas constant and T is the temperature.

[0118] More specifically, these relations can be calculated or converted one to the other using the following equations:

[0119] 1) X = y x 10“6

[0120] 2) V = V x t

[0121] V x 10“3x 101325

[0122] 3) n = -

[0123] 38.314 x 298

[0124] 4) m = n x X x 30.01 x 103

[0125] (V x t) x 10“3x 101325

[0126] 5) m = (- - —— - — - ) X (y X 10’6) X 30.01 X 1033 v8.314 x 2987 3y is the concentration in ppm units;

[0127] X is the concentration in molar fraction units, and equation 1 presents the relation between molar fraction and ppm (y);

[0128] V is the volume, V is the volumetric flow in LPM and t is the time in minutes, and equation 2 presents the relation between Volume, volumetric flow rate and time;

[0129] Equation 3 is the Ideal gas equation, and the 10'3factor is added for transformation from Liters to m3;

[0130] Equation 4 presents the relation between the mass (m) the mole number («), the molar fraction X and Nitric Oxide molar mass (30.01 grams / mol); and

[0131] Equation 5 is a combination of equations 1 -4 into a single equation, reflecting the relation between the mass of gNO and the molar fraction, volumetric flow and time. The 103is for obtaining the mass m on a milligram (mg) scale.

[0132] Thus, for example, at a volumetric flow of 0.1 LPM, time of 1 minute and concentration of 50,000 ppm, using equation 5 above, about 6.1-6.2 mg gNO is administered. According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration from about 1,000 ppm to about 1,000,000 ppm (0.1% to 100%), preferably from about 10,000 ppm to about 500,000 ppm, or from about 10,000 ppm to about 100,000 ppm, or at about 50,000 ppm; at a volumetric flow rate from about 0.0001 LPM to about 10 LPM, preferably from about 0.001 LPM and 1 LPM; and during a time period that ranges from about 1 second to about 30 minutes, per administration.

[0133] According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, at a concentration (dose) from about 20,000 ppm to about 100,000 ppm or from about 20,000 ppm to about 50,000 ppm; for a time period that ranges from about 30 seconds to about 10 minutes; at a volumetric flow (flow volume) from about 0.001 LPM to about 0.5 LPM, per administration.

[0134] According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, in an amount of no more 1 mg gNO per 100 mm3tumor volume, per administration, so as to avoid possible damage to healthy tissues adjacent to, or surrounding, the treated tumor.

[0135] According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, in an amount of about 250 mg per cm3tumor, per administration.

[0136] According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, in an amount from about 0. 1 to about 10 mg per a tumor of 20 mm3or less, including any intermediate values and subranges therebetween.

[0137] According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, in an amount from about 0.1 to about 300 mg including any intermediate values and subranges therebetween, per 1 cm3tumor, per administration. For example, the gNO is locally administered in an amount from about 0.1 mg to about 250 mg, or from 0.1 mg to about 100 mg, or from 1 mg to about 50 mg, or from about 1 mg to about 100 mg, or from about 1 mg to about 300 mg, from about 50 mg to about 100 mg, or from about 50 mg to about 300 mg, or from about 100 mg to about 150 mg, or from about 100 mg to about 300 mg, or from about 10 mg to about 100 mg, or from about 10 mg to about 250 mg, or from about 0.1 mg to about 10 mg, or from about 10 mg to 200 mg, including any intermediate values and subranges of any of the foregoing, per 1 cm3tumor, per administration. According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, to a tumor having a volume of up to 20 mm3, and an amount of gNO that is locally administered as described herein in any of the respective embodiments is from about 0.001 mg to about 10 mg, or from about 0.01 mg to about 20 mg, or from about 0.01 mg to about 2 mg, or from about 0. 1 mg to about 1.0 mg, or from about 0.2 mg to about 0.8 mg, including any intermediate values and subranges between any of the foregoing, per administration.

[0138] Without being bound by any particular theory, it is assumed that a high dose (concentration or amount) as described herein in any of the respective embodiments, inhibits the growth of tumor cells, reduces tumor volume and / or stimulates an anti-tumor immune response, as described herein in any of the respective embodiments, without causing a harmful effect to healthy tissues in the vicinity of the tumor.

[0139] For any of the embodiments described herein for local administration of gNO, the administration can be either continuous or pulsed (e.g., intermittent), such that for each administration, the indicated dose of gNO is administered either continuously or in a pulsed manner. When the high dose is referred to in ppm units, each pulse is at the indicated high dose concentration, as described herein in any of the respective embodiments. When the high dose is referred to as the total mass per administration, the indicated dose is divided into pulses.

[0140] According to some of any of the embodiments described herein, gNO is pulsed from about 2 to about 50 times, or from about 2 to about 30 times, or from about 2 to about 20 times, or from about 2 to about 15 times, or from about 5 to about 15 times, including any intermediate values and subranges therebetween, or about 10 times, per administration.

[0141] According to some of any of the embodiments described herein, each pulse is between about 1,000 ppm and about 1,000,000 ppm, or between 4000 ppm and about 1,000,000 ppm gNO, or between 10,000 ppm and about 1,000,000 ppm gNO, at a volumetric flow (flow volume) from about 0.00001 LPM to about 0.5 LPM, wherein each pulse is, independently, between about 0.1 second and about 10 minutes per pulse with a break from about 0.1 second to about 10 minutes between pulses.

[0142] According to some of any of the embodiments described herein, each pulse of gNO is, independently, from about 10 seconds per pulse to about 45 seconds per pulse, including any intermediate values and subranges therebetween.

[0143] According to some of any of the embodiments described herein, each pulse of gNO is about 30 seconds per pulse. According to some of any of the embodiments described herein, gNO is not administered between pulses and the time between each two pulses is, independently, from about 1 second to about 300 seconds, or from about 1 second to about 200 seconds, or from about 1 second to about 100 seconds, or from about 1 second to about 50 seconds, or from about 10 seconds to about 50 seconds, including any intermediate values and subranges therebetween, or is about 20 seconds.

[0144] According to some of any of these embodiments, the ratio between the time of gNO pulsed administration and the resting time between pulses ranges from 1:2 to 1:5. For example, for each pulse of gNO administration during 5 seconds, a following resting time is independently from 10 to 50 seconds. Preferably, the gNO is pulsed such that about 33 % of the time gNO is delivered and 66 % of the time is resting or waiting time between pulses.

[0145] According to some of any of the embodiments described herein, the gNO is locally administered, as described herein in any of the respective embodiments, at two or more administration sites in or on the tumor (depending in the administration mode). In some of these embodiments, the distance between the two administration sites is, independently, from about 2.5 mm to about 1 cm, or from about 0.25 cm to about 0.5 cm, including any intermediate values and subranges therebetween.

[0146] When gNO is administered to two or more tumor sites, each administration is at the ppm dose or mass amount indicated herein in any of the respective embodiments, or, the total mass (amount) administered to all tumor sites is as indicated herein in any of the respective embodiments.

[0147] According to some of any of the embodiments described herein, the method further comprises scavenging excess gNO from the one or more administration sites, as described in further detail hereinunder. In exemplary embodiments, the scavenging comprises applying a reduced pressure (vacuum) around the administration site(s).

[0148] According to some of any of the embodiments described herein for high dose local administration of gNO, the administration is performed one or more times per a treatment session.

[0149] In some embodiments, it is performed once during a treatment session. In some embodiments, it is performed twice, thrice or more times during a treatment session. In some of these embodiments, the administration is performed once daily during the treatment session. Preferably, the administration is performed such that a time interval between the two administrations is at least one week, during a treatment session. The duration of a treatment session can be determined by skilled persons such as physicians, in accordance with the subject’s response to the treatment, that is, in accordance with the effect of the treatment on the growth of the cells of the primary and / or secondary tumor, as described herein.

[0150] According to some of any of the embodiments described herein for high dose local administration of gNO, the administration is performed once a day, although two or more times a day are also contemplated.

[0151] According to some embodiments, the local administration comprises injecting, or exposing the outer layer of a tumor to, or filling a space or cavity containing a tumor with, a high dose gNO, and in some embodiments, the gNO is at a concentration ranging from about 1,000 ppm to about 1,000,000 ppm, preferably 50,000 ppm, as described herein in any of the respective embodiments. The local administration to the tumor is for a period of time from about 1 second to about 3 hours, depending on the size and location of the tumor, with a very low volume in the order of up to 0. 1 LPM and preferably 0.01 LPM.

[0152] According to some of any of the embodiments described herein, the gNO local administration treatment is also referred to as a high dose, low volume, local administration or treatment.

[0153] According to some of any of the embodiments described herein, in cases where the tumor is covered by skin, peritoneum, crust or any other thick layer, this layer can be removed prior to or during exposure of the tumor to the gNO local administration.

[0154] In any of the embodiments described herein in the context of gNO local administration, the gNO may be provided by an external source, for example, a reservoir of gNO or a chemical generator of gNO. In some embodiments, gNO is provided by a reservoir of gNO, preferably of a small volume of, for example, a single administration dosage (that is, the dose of gNO used per a single administration, as described herein in any of the respective embodiments). Such reservoirs are described in further detail hereinunder.

[0155] The gNO is preferably of medical purity, that is, preferably at least about 95 %, more preferably at least about 99 %, and even more preferably at least about 99.5 % pure gNO. The gNO is preferably provided as a mixture of gNO and other gases, such as air, nitrogen, oxygen, and so forth, preferably an inert gas such as, for example, nitrogen, and its ppm concentration is within the gas it is mixed with.

[0156] Systems and modes of administration:

[0157] Embodiments of the present invention further relate to a system, which is also referred to herein interchangeably as “device”, which is configured for locally administering gNO to a tumor as described herein in any of the respective embodiments. Such a system is also referred to herein as a delivery system. Such a system is also described in W02021 / 105900, which is fully incorporated herein by reference.

[0158] Generally, but not obligatory, a system for locally administering gNO at a high dose as indicated herein can include a pressure regulator, a flow meter, optionally an exposure box or container, one or more delivery lines, which are optionally terminated by or connected to a delivery device or configuration through which the gNO is administered and further optionally, aNO and / or NOx (as defined hereinunder) detector. Purging the gNO delivery system with an inert gas, such as nitrogen, may be desired.

[0159] The volume and / or flow rate of the administered (delivered) gas can be regulated by a digital flow controller, designed to deliver low volumes or flow rates of gas, of less than 0.1 LPM per cm3of tissue, in accordance with any of the respective embodiments as described herein. Purging of the gNO delivery system, including purging the pressure regulator, flow meter and delivery lines can be performed before and / or after gNO local administration. The gas purge can preferably last at least 1 minute or until the NO and NOx (as described below) detectors read no signal. In exemplary embodiments, nitrogen is used as the purging gas at a flow rate of at least 0.5 LPM.

[0160] According to some of any of the embodiments described herein, the delivery device is inserted into a body and advanced adjacent to an administration site, on or near tumor cells or tissue. When a delivery device or configuration is appropriately positioned, gNO is supplied and exits the device into the tumor or on or above a surface of the tumor, depending on the nature of the delivery device and the local administration mode of choice. In some embodiments, the delivery device extends against a tumor in order to form a seal, thus further reducing damage that may be caused by the gNO to adjacent normal cells that are outside of the area sealed off by the device.

[0161] The delivery device as described herein is meant to describe a component or configuration of the delivery system through which the gas exits the delivery system and contacts the administration site (e.g., the tumor or its close vicinity).

[0162] According to some of any of the embodiments described herein, locally administering gNO to a tumor as described herein in any of the respective embodiments can be accomplished by delivery device means such as one or more needles, including, for example, perforated spray needles, non-perforated and non-spray needles, umbrella needles, or other needles. The needles can optionally be nano-sized, micron-sized or macro-sized needles (having a diameter of 1 mm or higher). Other deliver}' devices are described hereinunder. Embodiments in which needles are used are typically used when the gNO is locally administered intra-tumorally, e.g., by intratumoral injection.

[0163] In some of any of the embodiments described herein in the context of a delivery system, the opening from which the gNO is delivered can be adjusted to the size of the tumor, so as to prevent damage to the area surrounding the cancer. In some embodiments, the opening does not exceed the size of the tumor.

[0164] The methods of locally administering gNO to a tumor can include contacting at least a portion of the tumor with the gaseous nitric oxide, and subsequently removing gaseous nitric oxide and NOx gas molecules from the treated site during or after the administration step. NOx encompasses NO, when x is 1, and oxidized forms of NO, which can be formed when gNO is in contact with a physiological environment and / or the subject’s environment, whereby x can be, for example, 2. Vacuuming the gas can be done in a pulsed (e.g., intermittent) or continuous manner, preferably synchronized with the gNO mode of administration.

[0165] A delivery system can include a full -body or a differently -sized chemical hood designed to evacuate excessive gNO or NOx during treatment, in a pulsed or continuous manner, preferably synchronized with the gNO mode of administration.

[0166] The delivery system can include an evacuation cylinder, which is connected directly to a regulator of the gNO tank. To purge the regulator safely, the evacuation cylinder can be filled with a gas accumulated in the regulator.

[0167] Another embodiment of controlling high dose gNO includes the use of a one-way valve where disconnecting the regulator or flow meter from the cylinder locks the valve, thereby preventing gas release from a gas tank.

[0168] According to some of any of the embodiments described herein, the methods, uses and delivery systems as described herein utilize a gNO cylinder, optionally equipped with a gas regulator, and one or more valves, and further optionally, the cylinder further comprises a delivery device for executing the local administration. The delivery device is in fluid communication with the cylinder, preferably via the valves and gas regulator (e.g., flow controller). Alternatively, the cylinder comprises means to connect the delivery device to the cylinder, to obtain fluid communication therebetween. The delivery device can be, for example, a scope with an annular-shape or a needle or a device configured for spraying the tumor, or else, as described in further detail hereinunder.

[0169] According to some of any of the embodiments described herein, the gNO cylinder is a miniature or at least portable cylinder. According to some embodiments, the cylinder is of a volume of less than 1 liter, or less than 0.8 liter, or less than 0.75 liter, or less than 0.5 liter, or less than 0.3 liter.

[0170] The cylinder is preferably under low pressure, such as less than about 40 bar (about 600 psi). Thus, the cylinder can deliver about 10 liters of high concentration gNO. A regulator can optionally limit output pressure to about 50 psi, for example, and can be connected to an emergency on / off valve. The gas flow, in a case of, for example, a delivery system configured for injection, can be less than about 0.1 LPM or about 0.05 LPM. The use of such low flow rates during local administration of gNO can assist in limiting the exposure to the tumor or cancerous cells and protect the surrounding area, or healthy cells and tissues.

[0171] According to some of any of the embodiments described herein, the methods and uses involve scavenging gNO and optionally other gases, and the scavenging can be performed by applying vacuum so as to remove gNO and other gases from the administration site. A delivery system as described herein is configured, according to some embodiments, as being capable of scavenging gNO from an administration site.

[0172] A delivery system according to some of the present embodiments can additionally or alternatively comprise one or more, preferably two, vacuum devices for scavenging gaseous nitric oxide and other gases that may form during the local administration. The vacuum devices can be placed or held above, or distal to, the tumor, or to the delivering device, during administration, for example, about 15 cm away. The vacuum devices can vacuum all the gases from the area at a rate of at least about 50 liters per minute. The vacuuming of the gas can be done in a pulsed or continuous manner, preferably synchronized with gNO administration. Purging the gNO delivery system, for example, with nitrogen, before and / or after can also be performed. Purging can also be performed intermittently during the procedure.

[0173] According to some embodiments, a chemical hood is placed above the tumor or tumor mass and used to apply vacuum and scavenge gNO. The hood can be placed about 15 cm above the tumor. The hood can vacuum gas at a flow rate of at least about 50 LPM.

[0174] A whole-body chemical hood that can accommodate the patient’s body can also be used. The patient’s head can be placed outside the hood to minimize the risk of breathing NOx molecules. gNO can be delivered as described herein. The vacuum system can exchange the gas at a flow rate of at least 50 titers per minute.

[0175] In each of the embodiments that relate to a vacuum application, a NO and NOx filtering pump can be placed in the discharge tine. A soda lime filter, such as a Sofnolime filter, or a similar filter that can absorb NOx molecule, can be used. In each instance, vacuuming can be done in a pulsed or continuous manner, preferably synchronized with gNO administration or not.

[0176] According to some of any of the embodiments described herein, the delivery system is configured for delivering gNO to one or more administration sites by a positive pressure gradient, and scavenging gNO from the one or more administration sites by a negative pressure gradient. In this way, the delivery system may deliver gNO to one or more administration sites with reduced or nullified damage to collateral host cells.

[0177] It is to be noted that a certain level of damage to collateral cells may be tolerated, and that the conditions under which the gNO gas is administered may be optimized to decrease damage to collateral cells while also providing the therapeutic effects described herein.

[0178] According to some of any of the embodiments described herein, the delivery system comprises a gas supply passage in fluid communication with gas supply openings. The delivery system can further comprise an exhaust passage in fluid communication with an exhaust opening. The delivery system can comprise one or more gas supply openings and / or one or more exhaust openings, and / or one or more gas supply passages and / or one or more exhaust passages.

[0179] An exemplary system for the local administration and scavenging of gaseous nitric oxide comprises a container or box, as illustrated, for example in Fig. 22 of W02021 / 105901 and described in further detail hereinunder, that can be filled with high dose (e.g., concentration) gNO at a volume of at least 0.5 Liter Per Minute (LPM) supplied from a tank (a gas reservoir). The container can have 2 or more holes, or ports. A first hole or port can be an input hole, sized to allow insertion of at least a portion of the tumor or of a bodily organ containing the tumor. An output hole can be connected to a discharge conduit or pipe that can remove or evacuate excessive NOx gases out from the box to the outside air, avoiding or minimizing the risk of contaminating the room and overexposing staff and the treated subjects (patients). Applying a vacuum can be in a pulsed or continuous manner, preferably synchronized with gNO administration.

[0180] An exemplary delivery system can comprise a small bore inner cannula that delivers an adequate dose of gNO to the target site, for example, a target site from about I mm2to about 2 cm2in size. The delivery system can further comprise an outer lumen through which a vacuum may be applied to scavenge excess gNO away from tumor cells and tissue that surround and border the tumor site. Such a configuration allows locally administering gNO to the target site (a tumor), without excessive damage to healthy host tissues. In an exemplary method, a tumor or a portion thereof is inserted into a hole that substantially matches the tumor’s diameters. An additional output pore in the box enables lowering the pressure. The excessive gas is cleared up from the box through this output pore as described above. For example, the gas can flow into the box containing the tumor for 2 seconds followed by a suction of the gas for 2 seconds.

[0181] An exemplary delivery system comprises an outer lumen or cannula, trocar, tube, etc. An inner lumen or cannula, tube, etc. can be disposed coaxially inside of the outer lumen. The inner lumen is disposed approximately centrally in the outer lumen, although other configurations are also contemplated.

[0182] In some embodiments of such an exemplary delivery system, a space, preferably an exhaust space, is between the outer lumen and inner lumen. The exhaust space can be annular or may take any other configuration and / or geometry. A tip can be attached to the inner lumen at the distal end and is in fluid communication with the inner lumen. In some embodiments, the tip comprises a wire mesh or screen that accesses the space inside of the inner lumen. The delivery device may be advanced to an administration site in the retracted configuration. In some embodiments, the tip is rounded and seals the outer lumen when it is in the retracted position, thus easing insertion of the tip into the body where it is brought adjacent to an administration site.

[0183] When the tip of the system is brought adjacent to an administration site, the system is adjusted to its extended configuration in order to affect the administration of the gNO. In the extended configuration, an exhaust path is opened between the distal end of the outer lumen and the tip. gNO is delivered through the inner lumen. The gNO exits the inner lumen at the tip that is in fluid communication with the inner lumen. A wire mesh or screen at the distal end of the tip may assist in diffusing the gNO gas as it exists the device. The exhausted gNO gas returns to the device at the exhaust path. In embodiments, a vacuum is applied to the exhaust space between the outer lumen and the inner lumen in order to attract the exhausted gNO. The exhausted gNO is then brought through the exhaust space to exit the body and be disposed of appropriately. In this way, the device is capable of scavenging gNO from an administration site.

[0184] In an alternative configuration, the flow of gNO could be reversed such that gNO is delivered through the space between the outer lumen and inner lumen and removed from the administration site through the inner lumen. In this alternative configuration, a vacuum can be applied to the inner lumen and a positive pressure of gNO is applied to the space defined by the inner and outer lumens. In another alternative, the tip is permanently secured to the outer lumen as well as the inner lumen, such that the tip does not have retracted and extended configurations. In this alternative, permanent passages are provided in the outer lumen for gNO to be expelled from the device or sucked into the device by vacuum. For example, the permanent passages could be small holes or slits radially disposed around the outer lumen, preferably near the distal end of the outer lumen so as to be near the tip of the device.

[0185] According to some of any of the embodiments described herein, a delivery device is attached to the end of an endoscope or bronchoscope (e.g. a blue-fluorescence endoscope or bronchoscope) so that the insertion of the delivery device into the body, advancement towards the administration site, and retraction from the body can be visually observed by or otherwise made known to the operator or someone working in concert with the operator. Alternatively, the delivery device can be attached to a guidewire in order to more effectively insert, advance, and retract the device. Additionally, the device can be coated with a fluoroscopic material or have one or more fluoroscopic tags attached to it so that its insertion, advancement, and retraction could be fluoroscopically observed.

[0186] In some embodiments, a system for delivering gNO to and scavenging gNOx from, one or more administration sites is intended to fit over the distal end or tip of an endoscope or bronchoscope. The device can be, for example, annular-shaped, having a hole in or about its center and is approximately circular in shape. The hole is preferably sized to accommodate the distal end of an endoscope or bronchoscope. For example, the hole is from about 0.5 cm to about 10 cm in diameter. The hole can be sized so as to fit snugly over the distal end of an endoscope or bronchoscope.

[0187] According to some of any of the embodiments described herein, the delivery system comprises a double-needle system in which a suction needle is located adjacent or proximal to the gas delivery needle (e.g., a distance of between 3 mm and 1 cm). The suction needle can decrease or maintain intra-tumoral pressure. Further, two or more needles can be spaced at least about 2 mm apart. gNO can be delivered at a flow rate of at least about 0.01 LPM as described herein. The needles can be designed to have holes along the length of the needle. In example, the diameter of the holes is about 1 mm and disposed every 2 mm. The needles can be disposed within a lumen, placed outside of the tumor mass, while the shaft of the needle can be placed inside the tissue. The length of the needles can be selected to be at least half the tumor’s longest dimension. Vacuuming gas through one or more suction needles or holes can be done in a pulsed or continuous manner, preferably synchronized with gNO administration. For example, the gNO can flow into the tumor for 2 seconds followed by applying a vacuum or suction for 2 seconds. One or more, such as a plurality, of needles or an array of needles, such as nano-sized or micron-sized needles can be used. In some embodiments, the gas is injected into the tumor by means of an array of needles with a spacing of about 0.5 cm. The ratio of suction needles and delivery needles can be 1: 10 to 10:1, preferably 1 :1. The suction needles can be designed to remove less than about 1 liter per minute per cm gas or fluid.

[0188] According to some of the any of the embodiments described herein, gNO is applied to a targeted tumor through one or more intra-tumoral channels. For example, a channel 2 mm in width respective to every 4 mm of tissue can be formed through which gas can be delivered, for example at a flow rate higher than 0.01 LPM, directly to the tumor mass by a needle that is placed at the center of each channel, while the gas is cleared up from a scavenging channel. The flow rate in the scavenging channel can be lower than the del i very channel, such as at least 0.001 LPM less than the gNO delivery flow rate. The vacuuming can be accomplished through suction needles or hoses and can be pulsed or continuous, preferably synchronized with gNO administration. For example, the gNO can flow into the tumor for 2 seconds followed by a suction of the gas for 2 seconds.

[0189] According to some of the any of the embodiments described herein, gNO is sprayed onto a targeted tumor, optionally when a method as described herein is used in combination with surgical treatment (e.g., tumor resection) and / or in combination with gNO intra-tumoral injection and / or when the tumor is inoperable, flat or amorphous.

[0190] Thus, as mentioned, the method comprises determining in a tissue of a subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following intratumoral treatment with an effective amount of gNO.

[0191] As used herein “following” refers to any time period following application of gNO. It may be concomitant with the treatment but preferably following termination of treatment.

[0192] As used herein “tissue” refers to the tissue in which the measurement of cellular immune response can be affected.

[0193] According to a specific embodiment, the tissue is in situ, i.e., the site of treatment, i.e., tumor or vicinity thereof as described above with respect to local administration.

[0194] According to another embodiment, the tissue is a blood or spleen tissue.

[0195] According to another embodiment, the tissue is a blood or spleen tissue, lymphatic fluid or tissue or thymus.

[0196] According to a specific embodiment, the tissue is a peripheral blood or portions thereof. It will be appreciated that measuring the response in the blood renders the test a non- invasive test and therefore the entire test highly desirable for use as a biomarker. Otherwise, a tissue biopsy is retrieved.

[0197] As used herein “adaptive immune response” relates to infiltrating immune cells which are either active against the tumor cells or present its antigens. Cells of the innate immune response are also contemplated herein.

[0198] According to some embodiments, the cells are selected from the group consisting of T cells, B cells, dendrocytes (also referred to as “dendritic cells”) and macrophages (e g., Ml).

[0199] According to some embodiments, the cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, B220+ B cells, CD19+, CD38+, CD20+, CD 138+ B cells, CD1 lb+ dendrocytes, CD la dendrocytes, CD68 macrophages and F4 / 80 macrophages.

[0200] According to a specific embodiment, the cells are activated T cells as determined by an interferon gamma activation assay described herein in the Examples section which follows.

[0201] More specific examples of cells that are considered as cells of the adaptive humoral immune response and / or innate immune response are provided in Table 1 A which follows.

[0202] As used herein, the terms "increase", "above", “up-regulation”, “elevation” or “elevated” (or any grammatical forms thereol as used herein, refer to at least about 10 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 % or at least about 100 % higher level of cells of the adaptive humoral immune response and / or innate immune response in the measured tissue of the subject following treatment as compared to a level of same in a subject prior to treatment or to corresponding literature data from healthy individuals (confirmed not having cancer) or sham treated individuals, collectively referred to as “a predetermined threshold”.

[0203] Typically, but not necessarily, along or alternately with an increase in cells of the adaptive humoral immune response and / or innate immune response there is a decrease in myeloid-derived suppressor cells (MDSCs) below a predetermined threshold, following local treatment with gNO.

[0204] Thus, according to some embodiments, the method further comprises determining in the tissue of the subject a decrease in myeloid-derived suppressor cells (MDSCs) below a predetermined threshold.

[0205] As used herein “suppressor cells” refers to polymorphonuclear myeloid-derived suppressor cells as well as regulatory T cells B cells, M2 macrophages and regulatory dendritic cells. As used herein, the terms "decrease", "below", “down-regulation” (or any grammatical forms thereof) as used herein, refer to at least about 10 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 % or even 100 % lower level of suppressor cells, e.g., MDSCs in the measured tissue of the subject following treatment as compared to a level of same in a subject prior to treatment or to corresponding literature data from healthy individuals (confirmed not having cancer) or sham treated individuals.

[0206] Exemplary of suppressor cells are provided in Table IB below. Table 1A - Example of adaptive and / or innate immune cells markers:

[0207] Table IB - Example of immune suppressor cells markers:

[0208] Table IC-Example of additional functional markers:

[0209] It is appreciated that other markers are known in the literature for these cells and are contemplated herein.

[0210] Methods of analyzing cell profiles are well known in the art.

[0211] Such methods are protein-based, RNA-based, genomic-based, epigenomic-based, morphology -based or combinations of same. The determination can be performed on a solid tissue (e.g., tumor or microenvironment thereof, spleen) or solution (e.g., on blood samples).

[0212] For example, analysis of tissue sections by traditional IHC- and immunofluorescencebased methods are useful in providing spatial information. Alternatively, protein-based assays can be used to determine cells in suspension, e.g., by the use of antibody-based assays, e.g., flow cytometry e g., FACS, and antibody-conjugated microbeads.

[0213] Fluorescence activated cell sorting (FACS): This method involves detection of a substrate in situ in cells by substrate specific antibodies. The substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.

[0214] Immunohistochemical analysis: This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies. The substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxylin or Giemsa stain.

[0215] In situ activity assay: According to this method, a chromogenic substrate is applied on the cells containing an active enzyme and the enzyme catalyzes a reaction in which the substrate is decomposed to produce a chromogenic product visible by a light or a fluorescent microscope.

[0216] In vitro activity assays: In these methods the activity of a particular enzyme is measured in a protein mixture extracted from the cells. The activity can be measured in a spectrophotometer well using colorimetric methods or can be measured in a non-denaturing acrylamide gel (i.e., activity gel). Following electrophoresis, the gel is soaked in a solution containing a substrate and colorimetric reagents. The resulting stained band corresponds to the enzymatic activity of the protein of interest If well calibrated and within the linear range of response, the amount of enzyme present in the sample is proportional to the amount of color produced. An enzyme standard is generally employed to improve quantitative accuracy.

[0217] In other embodiments, the cells are analyzed by nucleic acid content. In some embodiments, DNA and / or RNA (e.g., mRNA, rRNA, tRNA, etc.) from a sample may be analyzed. In some embodiments, RNA content of a sample is analyzed using RNA sequencing (RNA-seq). RNA-seq is a reliable, scalable, and comprehensive method to evaluate transcriptomic changes in the tumor immune microenvironment, blood or spleen.

[0218] Regardless of the method employed, when an increase in adaptive humoral immune response and / or innate immune response is indicated and / or a decrease in suppressive immune response is indicated, the subject is found responsive to the treatment. In such a case the treatment may be ceased (no further treatment with gNO is necessary) or repeated in the same or different regimen. When the results indicate no increase in adaptive humoral immune response and / or innate immune response and / or ano decrease in suppressive immune response, a different treatment or a change in dosing regimen may be administered.

[0219] As used herein “dosing regimen” refers to any of timing of treatment, amount of gNO, number of treatments, mode of administration.

[0220] Thus, according to another aspect there is provided a method of using gaseous nitric oxide (gNO) in the treatment of a subject diagnosed with cancer, the method comprising locally administering an effective amount of gNO; determining in a tissue of the subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO, wherein when a presence of the increase is indicated, the treatment is terminated, repeated or decreased, while when an absence of the increase is indicated the treatment is elevated or repeated.

[0221] The determination can be done at least following first treatment (where “following” is defined above) and / or subsequent treatments. An indication that there is an increase in in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO can be determined by a reduction in systemic and / or tumor levels of inhibitory myeloid-derived suppressor cells (MDSCs), Regulatory T-cells(Tregs), expression of immune check point molecules (PD-1, Lag-3, TIGIT, Tim-3) in CD4 / 8 T-cells and / or increase in Ml macrophages, expression of MHC-I on tumor cells and expression of co-stimulatory molecules(ICOS,GITR) in CD4 / 8 T-cells.

[0222] In any of the methods envisaged by the invention, the gNO treatments can be combined or in view of the method results e g., when monitoring efficacy of treatment, replaced by another anti-cancer treatment. Such include, but are not limited to chemotherapy, radiotherapy, phototherapy and photodynamic therapy, surgery, nutritional therapy, ablative therapy, combined radiotherapy and chemotherapy, brachytherapy, proton beam therapy, immunotherapy, immune-checkpoint therapy, cellular therapy and photon beam radio-surgical therapy.

[0223] As mentioned hereinabove, the present invention provides a method for determining the likelihood of a subject to develop a secondary tumor as defined hereinabove.

[0224] The term throughout "about" as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass variations of + / - 20% or + / - 10% from the specified amount, as such variations are appropriate to perform the disclosed method. In embodiments, the term “about” is meant to encompass variations of + / - 5%. In embodiments, the term “about” is meant to encompass variations of + / - 1 %. In embodiments, the term “about” is meant to encompass variations of + / - 0.1%.

[0225] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0226] The term “consisting of’ means “including and limited to”.

[0227] The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0228] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0229] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0230] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0231] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0232] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0233] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0234] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0235] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0236] EXPERIMENTAL PROCEDURES

[0237] Study Drug: NO is a colorless, nonflammable, oxidizing gas. NO was administered from 3.5-L cylinders, prepared and supplied by Gordon Gas and Chemical (Tel Aviv, Israel). As NO is a free radical that reacts within seconds, the inert gas nitrogen served as its stabilizing gas. NO concentrations used for this study were 0.015%-20% NO in nitrogen. Air or nitrogen supplied from 3.5-liter cylinders served as the control gases.

[0238] All procedures were performed in a chemical hood in a well-ventilated room. Appropriate personal protection was used at all times. During gas exposure, the cylinder was placed inside a stabilizing apparatus. The gas was delivered via a pressure regulator through a designated silicone hose (International Biomedical, USA). The flow rate was set to 0.2 liters per minute (LPM) using a manual flow meter.

[0239] Tumor cell lines: Mouse colon and breast cancer cell lines were purchased from an ATCC local distributor, Biological Industries (Beit HaEmek, Israel). The mouse colon carcinoma cell line, CT26.WT, was cloned from CT26, an N-nitroso-N-methylurethane- induced, undifferentiated colon carcinoma cell line.

[0240] Preparation of tumor cells: Tumor cell suspensions in cell culture medium or Hanks’ Balanced Salt Solution (HBSS) (Biological Industries, Israel), at appropriate concentration of 5* 106cells / ml for in vivo studies were freshly prepared. Cells were grown to 70 % confluence and harvested using trypsin (Biological Industries, Israel), and counted using a hemocytometer.

[0241] Mice: All in vivo experimental procedures were carried out in accordance with the protocol approved by the Ethics Committee on the Use and Care of Animals. Institutional Animal Care and Use Committee (IACUC): 67-09-2019 and IL-20-3-149. The in vivo work was performed on Balb / c or C57BL / 6J mice: 8-10 weeks of age, obtained from Envigo (Israel and the Netherlands).

[0242] Primary tumor induction: Cancer cell suspensions at a concentration of 2.5-10.0*106cells / ml were inoculated to the right flank of mice at a dose volume of 0.1 ml. More specifically, CT26 cells were inoculated subcutaneously (s.c.) on the right flank of male Balb / c mice at a concentration of 500,000 CT26 cells in lOOpl HBSS per mouse. Test article treatment: Mice were first anesthetized by an intraperitoneal (i.p.) injection of 100 mg / kg ketamine and 20 mg / kg xylazine hydrochloride solution. After 10 minutes, tumor-bearing mice were treated with 20,000 ppm - 50,000ppm NO.

[0243] 20,000 - 50,000 ppm NO was delivered into the tumor for 5 minutes using 23G hypodermic needles. Fourteen days post NO treatment, all tumors were excised. After treatment, all mice were closely monitored until complete recovery was observed.

[0244] Control treatment: Nitrogen was delivered into the tumor for 5 minutes using 23 G hypodermic needles. Fourteen days post gas treatment, all tumors were excised.

[0245] After treatment, all mice w ere closely monitored until complete recovery w as observed.

[0246] Tumor volume calculation: Local tumor growth was determined by measuring 3 mutually orthogonal tumor dimensions 2-3 times per week, according to the following formula: Tumor volume = -x[Diameter 1 x Diameter 2 x Diameter 3],

[0247] Histology and immunohistochemistry: Samples of colon tumors from mice were harvested, fixed in 4 % formaldehyde, arrived to Patho-Logica in the fixative and kept in the fixative for 48 hours for further fixation. The tissues were trimmed, put in embedding cassettes and processed routinely for paraffin embedding. One cassette was prepared per animal. Paraffin sections (4 microns thick) were cut, put on glass slides and stained with Hematoxylin & Eosm (H&E) for general histopathology and by immunohistochemistry (IHC) of the following markers: CD3 (T-lymphocytes), CD4 (helper T-lymphocytes), CD8 (effector / cytotoxic T-lymphocytes), B220 (B cells), CDl lb (dendrocytes), F4 / 80 (macrophages).

[0248] Immune cells level: 0: Without a positive reaction, 1 : Few positive cells (<5 cells), 2: Very mild staining (5-15 cells), 3: Mild staining (15-25 cells), 4: Moderate staining (25-50 cells), 5: Marked staining (>50 cells).

[0249] Inflammation (as assayed by a qualified pathologist on tissue slides): 0: Scant or absence of inflammatory cells, 1 : Inflammatory cells present but markedly less than tumor cells, 2: Inflammatory cells roughly equal to tumor cells, 3: Predominantly inflammatory cells.

[0250] Lymphocyte infiltration: 0: Absence of lymphocytes in the tumor, 1: < 5 lymphocytes per field, 2: 5-20 lymphocytes per field, 3: > 20 lymphocytes per field.

[0251] Flow cytometry analysis: Mouse spleens were dissociated with the GentleMACS Octo (Miltenyi Biotech). Mouse blood samples were processed using Red Blood Cell Lysis Solution (cat# 130-094-183, Miltenyi Biotech). Extracellular labeling of T cells was performed with FITC labeled anti-CD3 (cat# 130-119-758, Miltenyi Biotech), VioGreen labeled anti-CD4 (cat# 130-118-693, Miltenyi Biotech) and APC-Vio770 labeled anti-CD8 (cat# 130-120-806, Miltenyi Biotech) antibodies. Extracellular labeling of B cells was performed with PE-Vio615 anti-CD19 (cat# 130-111-890, Miltenyi Biotech) antibodies. Extracellular labeling of polymorphonuclear MDSCs cells was performed with FITC labeled anti-CD3 (cat# 130-119- 758, Miltenyi Biotech) and PE-Vio615 anti-CD19 (cat# 130-111-890, Miltenyi Biotech) for negative staining, VioGreen-labeled anti-CDl l (cat# 130-113-811, Miltenyi Biotech), PE- labeled Ly-6G (cat# 130-123-780, Miltenyi Biotech) antibodies for positive staining and VioBlue anti-Ly6C (cat# 130-111-921, Miltenyi Biotech) for low staining. Samples were analyzed using MacsQuant™ 16 Flow cytometer.

[0252] Extracellular labeling of Leukocytes was performed with APC-vio770 conjugated anti CD45 (cat # 130-110-662, Miltenyi Biotech). Extracellular labeling of myeloid cells was performed with PE conjugated anti-CDl lb (cat# 130-113-806, Miltenyi Biotech). Extracellular labeling of neutrophils was performed with Vioblue conjugated Ly6g (cat# 130- 119-902, Miltenyi Biotech). Extracellular labeling of dendritic cells was performed with APC conjugated anti CD11C (cat# 130-110-839, Miltenyi Biotech) and Viogreen conjugated anti MHCII (cat# 130-112-238, Miltenyi Biotech). Extracellular labeling of Macrophages was performed with PE-vio770 conjugated F4 / 80 (cat# 130-118-459, Miltenyi Biotech with Extracellular labeling of Macrophages Ml performed with APC conjugated anti CD11C (cat# 130-110-839, Miltenyi Biotech). Extracellular labeling of Macrophages M2 SB600 conjugated anti CD163 (cat# 63-1631-802, Thermo fisher scientific). Extracellular labeling of M-MDSCs was performed with FITC conjugated anti Ly6C (cat# 130-111-915, Miltenyi Biotech). Extracellular labeling of NK cells was performed with BV650 conjugated anti NK46P (cat# 137635, Biolegend). Extracellular labeling ofNKT cells was performed with was performed with BV650 conjugated anti NK46P (cat# 137635, Biolegend) and Percp- vio700 conjugated anti CD3 (cat# 130-120-826, Miltenyi Biotech).

[0253] Extracellular labeling of T-cells was performed with Percp-vio700 conjugated anti CD3 (cat# 130-120-826, Miltenyi Biotech). Extracellular labeling of CD4 helper cells was performed with APC-fire 810 conjugated anti CD4 (cat# 100479, Biolegend). Extracellular labeling of cytotoxic CD8 T-cells was performed with Spark574 conjugated anti CDS (cat# 100794, Biolegend). Labeling Tregs was performed with APC-fire 810 conjugated anti CD4 (cat# 100479, Biolegend), BV650 conjugated anti CD25 (cat# 564021, BD biosciences) and Intracellular BV421 conjugated anti Foxp3 (cat# 562996, BD biosciences). Extracellular labeling of co-inhibitory and co-stimulatory molecules on T-cells was performed with BV711 conjugated anti PD-1 (CD279) (cat# 748265, BD biosciences), BUV737 conjugated anti ICOS (CD278) (cat# 567918, BD biosciences), PE-Vio® 770 conjugated anti CD357 (GITR) (cat# 130-129-799. Miltenyi Biotech), VioBright FITC conjugated anti TIM-3 (cat# 130-120- 823, Miltenyi Biotech), and BV605 conjugated anti TIGIT (cat# 744212, BD biosciences). Intracellular labeling proliferation was performed with APC conjugated anti Ki-67 (cat# 130- 120-416, Milteny Biotech).

[0254] Extracellular labeling of MHC-I on tumor cells was performed with BUV395 conjugated anti H-2Kd SF1-1.1 (cat# 742437, BD biosciences).

[0255] Viability staining was performed with Zombie NIR (cat# 423105, Biolegend).

[0256] IFNY assay: Mouse spleens were dissociated with the GentleMACS Octo (Miltenyi Biotech). T cells were activated for overnight with TexMACS + 10% FBS medium. Then, cell stimulation cocktail (cat# TNB-4970-UL100, Tonbo biosciences) at a concentration of 2pl / ml was added for a 2-hour incubation at 37°C. Then, Brefeldin A solution (cat# TNB-4506-L001, Tonbo biosciences) at a final concentration of Ipl / ml was added for a 2-hour incubation at 37°C. Extracellular labeling of T cells was performed with FITC labeled anti-CD3 (cat# 130- 119-758, Miltenyi Biotech), VioGreen labeled anti-CD4 (cat# 130-118-693, Miltenyi Biotech) and APC-Vio770 labeled anti-CD8 (cat# 130-120-806, Miltenyi Biotech) antibodies. After fixation and permeabilization of cells, intracellular labeling of T-Cells was performed with APC labeled anti-IFNY antibody (cat# 130-123-283, Miltenyi Biotech). Samples were analyzed using MacsQuant™ 16 Flow cytometer.

[0257] EXAMPLE 1

[0258] B cells, T cells, macrophages and dendrocytes are recruited at a higher level to NO- treated tumors compared to nitrogen-treated tumors 14 days post NO treatment

[0259] The adaptive immune cells were assessed on days 14 and 21 post gas delivery to mouse colon tumors (Fig. 1 A). On day 14, lymphocytes were recruited to the primary tumor showing a dose dependence, which correlated with the level of inflammation (Fig. IB). Specifically, T cells penetrated the primary tumor site in a dose response manner. The level of T cell infiltration to 50,000 ppm NO-treated tumors was ~1.7 times higher as compared to the nitrogen-treated tumors. In the 20,000 ppm NO tumors, the level of T cells penetration was intermediate, but higher than in the nitrogen treated tumor. Similar trend was seen with helper T cells recruited to the tumor. In case of cytotoxic T cells, the highest level of recruitment was observed in the 50,000 ppm NO groups as compared with both 20,000 ppm and nitrogen - treated tumors (Fig. 1C). Representative slides show that in the NO-treated groups T cell (brown cells) penetrate to the tumor mass (highlighted in yellow), as opposed to the nitrogen treated group in which T cells are present at the margins of the tumor (Fig. ID). EXAMPLE 2

[0260] Systemic T cells and B cells are elevated and immune suppressor cells are downregulated in the spleen 21 days post NO treatment

[0261] T cells, B cells and Myeloid-derived suppressor cells were assessed in the spleen 21 days post treatment. Specifically, IFNY positive T cells out of total T cells were assessed in the spleen. Flow cytometry analysis showed that following 50,000 ppm NO treatment, the activated T cells (IFNY positive) out of total spleen T cells were elevated by 37 % as compared to the nitrogen control group. Dose-dependent effect is seen systemically as the activated T cells are increased by 23 % in the 20,000 ppm NO group as compared to the nitrogen control group (Fig. 2A). Similar trends were evident with IFNY positive helper and cytotoxic T cells out of total helper and cytotoxic T cells (respectively) in the spleen. In the 50,000 ppm NO and 20,000 ppm NO groups the activated helper T cells were higher by 38 % and 22 % (respectively) as compared to the nitrogen control group (Fig. 2B). In the 50,000 ppm NO and 20,000 ppm NO groups the activated cytotoxic T cells were higher by 25 % and 11 % (respectively) as compared to the nitrogen control group (Fig. 2C).

[0262] B cells in the spleen are elevated in both NO-treated groups compared to the nitrogen group, however, not showing a dose dependence between 20,000 ppm and 50,000 ppm NO (17% and 28% increase, respectively, Fig. 2D).

[0263] The immune suppressor cells, polymorphonuclear MDSCs, are decreased in the spleen 21 days post either 50,000 ppm or 20,000 ppm NO treatment (-25 % to -37 %, respectively) as compared to the nitrogen control group (Fig. 2E).

[0264] EXAMPLE 3

[0265] Systemic T cells and B cells are elevated in the blood 21 days post NO treatment

[0266] T cells (CD3+) and B cells were assessed in the blood 21 days post treatment. Both T cells and B cells are elevated in the NO groups as compared to the nitrogen control. Increase by 31 %-36 % in the blood T cells in 50,000 ppm and 20,000 ppm NO groups (respectively) were observed compared to the blood T cell level in the nitrogen control group (Fig. 3A). An increase by 9-20 % in blood B cells at 50,000 ppm and 20,000 ppm NO treatment groups (respectively) were observed compared to the blood B cell level in the nitrogen control group (Fig. 3B). Example 4

[0267] Immune profiling 1 day and 5 days post NO treatment

[0268] CT26 tumor-bearing mice were treated with 50,000 ppm NO or nitrogen as a control. Other CT26 tumor-bearing mice were untreated or were treated by insertion of a needle to the tumor for 5 minutes (a sham control). Immune profiling was evaluated 1 day and 5 days post treatment.

[0269] Intratumoral treatment promotes T-cell activation in tumors where gNO treatment strongly induces ICOS expression and significantly induces GITR on CD8+ CTLs (Fig. 4A and Fig. 4B). Increased T-cell activation is reflected in increased expression of CD44 (Fig. 4C). The inhibitory molecule PD-1 is also upregulated on T cells after gNO treatment. In contrast, T-cell proliferation is mildly reduced and Treg is specifically reduced. NO and to a lesser extent N2 may reduce Tregs infiltration, proliferation and systemic levels only frequency is depicted here but Fig. 7 provides additional details (Fig. 4E).

[0270] Ml macrophages (Fig. 5) and mononuclear MDSCs (Fig. 6) are increased in the blood of CT26 tumor bearing mice 1 day after NO treatment. Treg frequency in CT26 tumors and Treg proliferation in blood are reduced 1 day following treatment (Fig. 7A-Fig. 7C). gNO induces cell death in CT26 tumors and tumor viability is reduced 5 days (post treatment (Fig. 8A and Fig. 8B). gNO maintains MHC-I expression on tumor cells in CT26 tumors (Fig. 9A-Fig. 9C).

[0271] Expression of the co-stimulatory molecule GITR (Fig. 10) on CD8 T-cells may increase 1 day post treatment.

[0272] Positive response to gNO treatment is associated with reduced expression of immune checkpoint markers on tumor infiltrating T-cells 5 days post treatment in CT26 tumors. (Fig. HA-Fig. 11F).

[0273] In view of the above, following gNO treatment, an increase in the above-described cells of the adaptive humoral immune response and / or innate immune response can indicate an efficacious treatment and can also indicate a reduced likelihood in the development of a secondary tumor. After such an increase, the treatment can be terminated, repeated, or decreased. Further, in the absence of such an increase, the treatment can be elevated or repeated. An indication that there is an increase in in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO can be determined by a reduction in systemic and / or tumor levels of inhibitory myeloid- derived suppressor cells (MDSCs), Regulatory T-cells (Tregs), expression of immune check point molecules (PD-1, Lag-3, TIGIT, Tim-3) in CD4 / 8 T-cells and / or increase in Ml macrophages, expression of MHC-I on tumor cells and expression of co-stimulatory molecules (ICOS, GITR) in CD4 / 8 T-cells.

[0274] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0275] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

[0276] Leukocytes vary in their contribution to immune response against tumors. Cells that ty pically promote anti-tumor response include CD4 Thl and CD8 T-cells, dendritic cells, certain types of neutrophils, NK cells and Ml macrophages. In contrast, cells such as MDSCs, M2 macrophages, Tregs and certain types of neutrophils often dampen immune response.

[0277] As demonstrated herein, there is a reduction of immunosuppressive mononuclear MDSCs in the circulation and Tregs in tumors of gNO treated tumor-bearing animals whereas circulating immunostimulatory Ml -macrophages increased, indicating a shift towards antitumor immune profile. Analysis of circulating M-MDSCs and Ml -macrophages following gNO treatment can be informative of an acquisition of immunostimulatory profile and help define the relevant time window for immunotherapy. In the case of insufficient stimulation (e.g., high MDSCs / Tregs or low Ml macrophages) additional interventions can be dictated such as repeated gNO exposures or neutralization of specific immunosuppressive populations by additional means 1 Sampling data for these biomarkers can be utilized to determine cut-offs associated with favorable response to gNO. Integrating data for several markers can further be used to determine an overall score for response gNO to improve fidelity.

[0278] Increased expression of ICOS and GITR on tumor infiltrating T-cells are indicative of a heightened activation state of T-cells and addition of agonists for these molecules can elicit greater cytotoxic response. Evaluating these markers in tumor biopsies can identify an optimal time window and select for patients that can benefit from treatment with agonists.# It is also shown that gNO maintains MHC-I expression on tumor cells. Since MHC-I is essential for recognition of tumor antigens by cytotoxic T-cells, gNO treatment can provide means to elevate MHC-I in tumors, prompt greater antigen representation, maximize TCR engagement and increased cytotoxic activity of CD8 T-cells. Liquid biopsies or tumor biopsies (experimental endpoints) may inform of such change and define therapeutic window for immunotherapies including checkpoint blockade and inoculation of cytotoxic T-cells in adoptive transfer. gNO treatment inflicts considerable cell death in tumors. Imaging methods such as ultrasound can detect necrosis and thus may enable gross assessment of response to gNO. As cell death has been implicated as a stimulus for anti-tumor immunity, estimating tissue necrosis can provide a simple non-invasive marker for response to gNO and lack of sufficient cell killing can educate on a need for repeated exposure 1

[0279] Variability in immune checkpoints expression on CD4 and CD8 T-cells in CT26 tumors was observed and positively correlates with changes in tumor growth. Evaluating these markers on T-cells in tumor biopsies can segregate between patients that acquired a less immunosuppressive T-cell phenotype following treatment to those who did not.

[0280] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

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Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of assessing responsiveness to gaseous nitric oxide (gNO) treatment in a subject diagnosed with cancer, the method comprising locally administering an effective amount of gNO to the subject, determining in a tissue of the subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO, said increase being indicative of an efficacious treatment with said gNO.

2. A method of using gaseous nitric oxide (gNO) in the treatment of a subject diagnosed with cancer, the method comprising locally administering an effective amount of gNO to the subject, determining in a tissue of the subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO, wherein when a presence of said increase is indicated, said treatment is terminated, repeated or decreased, and wherein when an absence of said increase is indicated said treatment is elevated or repeated.

3. A method of determining a likelihood of a subject diagnosed with a primary tumor to develop a secondary tumor following treatment with gaseous nitric oxide (gNO), the method comprising locally administering an effective amount of gNO to the subject, determining in a tissue of the subject an increase in cells of the adaptive humoral immune response and / or innate immune response above a predetermined threshold following local treatment with gNO, said increase being indicative of a reduced likelihood to develop a secondary tumor.

4. The method of claim 1, wherein when a presence of said increase is indicated said administering is terminated, repeated or decreased, and wherein when an absence of said increase is indicated said administering is elevated or repeated.

5. The method of claim 3, wherein when a presence of said increase is indicated said administering is terminated, repeated or decreased, and wherein when an absence of said increase is indicated said administering is elevated or repeated.

6. The method of any one of claims 1-5, wherein said tissue is selected from the group consisting of a tumor, a peripheral blood, lymphatic fluid or tissue, thymus and a spleen.

7. The method of any one of claims 1-6, wherein said cells are selected from the group consisting of T cells, B cells, dendrocytes and macrophages.

8. The method of any one of claims 1-6, wherein said cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, B220+ B cells, CD19+, CD38+, CD20+, CD138+ B cells, CDl lb+ dendrocytes, CDla dendrocytes, CD68 macrophages and F4 / 80 macrophages.

9. The method of any one of claims 1-6, wherein said cells comprise activated T cells.

10. The method of any one of claims 1-9, wherein said cancer is a solid tumor.

11. The method of claim 10, wherein said solid tumor is a colon cancer.

12. The method of any one of claims 1-9, further comprises determining in said tissue of said subject a decrease in myeloid-derived suppressor cells (MDSCs) or other suppressor cells below a predetermined threshold.

13. The method of any one of claims 1-12, wherein said effective amount of gNO is at a dose from about 1,000 ppm to about 1,000,000 ppm for a time period from about 1 second to about 60 minutes at a volumetric flow from about 0.00001 LPM to about 1 LPM.

14. The method of any one of claims 1-12, wherein said effective amount of gNO is at a dose from about 20,000 ppm to about 200,000 ppm or from about 20,000 ppm to about 100,000 ppm.

15. The method of any one of claims 1-14, wherein said effective amount of gNO is administration for a time period that ranges from about 30 seconds to about 10 minutes.

16. The method of any one of claims 1-15, wherein said effective amount of gNO is administration at a volumetric flow from about 0.001 LPM to about 0.5 LPM.

17. The method of any one of claims 1-16, wherein said local treatment is intratumoral treatment.

18. The method of any one of claims 1-16, wherein said local administration comprises two or more administration sites.

19. The method of claim 18, wherein a distance between the two or more administration sites is, independently, from about 2.5 mm to about 1 cm, or from about 0.25 cm to about 0.5 cm.

20. The method of any one of claims 1-19, wherein said effective amount is at a dose from about 0.1 mg to about 300 mg, per cm3tumor.

21. The method of any one of claims 1-20, wherein said effective amount is a dose from about 0.01 mg to about 100 mg, or from 0.1 mg to about 10 mg, per a tumor of 20 mm3or less.

22. The method of any one of claims 1-21, wherein said local administration comprises scavenging excess gNO from the administration site.

23. The method of any one of claims 1-22, wherein said local administration comprises pulsing gNO from about 2 to about 20 times.

24. The method of any one of claims 1-23, wherein said local administration is performed at least once.

25. The method of any one of claims 1-23, wherein said local administration is performed at least twice, and wherein a time interval between said two administrations is at least one week.

26. The method of any one of claims 1-25, wherein said determining comprises ex vivo determining.

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