Patent Application
20210148916
Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled “WST165US_ST25.txt”, created Aug. 3, 2017, and having 4 KB.
Myeloid-derived suppressor cells (MDSC) represent a heterogeneous population of immature myeloid cells. These cells accumulate to a great extent in cancer patients and play a major role in regulating immune responses in cancer42. MDSC suppress T cells activation and proliferation as well as function of natural killer (NK) cells14, 15. Ample evidence links these cells with tumor progression and outcome of the disease in cancer patients34,15. The accumulation of relatively immature and pathologically activated myeloid-derived suppressor cells (MDSC) with potent immunosuppressive activity is common in tumors. MDSC have the ability to support tumor progression by promoting tumor cell survival, angiogenesis, invasion of healthy tissue by tumor cells, and metastases4. There is now ample evidence of the association of accumulation of immune suppressive MDSC with negative clinical outcomes in various cancers32. MDSC have been implicated in resistance to anticancer therapies with kinase inhibitor11, chemotherapy9, 47, 23, 8, and immune therapy33,44,50,12,20.
MDSC have been divided in two large sub-populations52, monocytic myeloid-derived suppressor cells (M-MDSC) and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC). About 20-30% of MDSC consists of monocytic cells, i.e., M-MDSC, and are generally associated with high activity of Arginase-1 and iNOS10. Two different phenotypes (CD11b+ CD14− CD15− and CD33+ or CD11b+ CD14+ CD33+ and HLA-DRlo) are used to characterize these M-MDSC cells depending on the type of cancer.
The second population, i.e., PMN-MDSC, are comprised of granulocytic cells and are usually associated with high level of ROS production36. PMN-MDSC represent the major population of MDSC (about 60-80%) and represent the most abundant population of MDSC in most types of cancer. PMN-MDSC are phenotypically and morphologically similar to neutrophils (PMN)36 and share the CD11b+CD14−CD15+/CD66b+ phenotype. The may also be characterized as CD33+. PMN-MDSC are important regulators of immune responses in cancer and have been directly implicated in promotion of tumor progression. However, the heterogeneity of these cells and lack of distinct markers hampers the progress in understanding of the biology and clinical significance of these cells. One of the major obstacles in the identification of PMN-MDSC is that they share the same phenotype with normal polymorphonuclear cells (PMN).
Distinction between PMN-MDSC and PMN in tumor tissues is not possible. Currently, these cells can be separated only in peripheral blood (PB) and only by density gradient. Since gradient centrifugation may enrich not only for true PMN-MDSC, but also for activated PMN without suppressive activity, the heterogeneity of PMN-MDSC population raised the questions of whether PMN-MDSC and PMN are truly cells with distinct features. It is not clear what defines the specific functional state of human PMN-MDSC vis-à-vis PMN in the same patient. More importantly, the mechanisms responsible for acquisition of pathological activity by human neutrophils in cancer remained unclear.
Current methods for separating populations of PMN-MDSC from populations of PMN in biological samples are complicated, time-consuming and inaccurate, requiring multiple gradient separation as well as multi-color flow cytometry analysis. Normal PMN have high density and pass through the gradient, whereas PMN-MDSC have lower density become trapped on the gradient together with mononuclear cells. This process of distinguishing between the two sets of PMN has two major shortcomings. The density of the cells depends on many parameters, such as conditions for collection, time of storage, etc., which affect the proportion of the cells obtained on the gradient regardless of their PMN-MDSC true state. These conditions thus introduce errors into the analysis. Additionally, these processes are inconvenient and difficult to standardize. Thus, there are no useful methods currently exist that allow for discrimination of these two populations in blood and tissues.
In one aspect, a method for monitoring the population of polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) in a mammalian subject involves contacting a biological sample from the subject containing polymorphonuclear neutrophils (PMNs) and PMN-MDSC with a ligand that specifically binds or forms a complex with LOX-1 on the cell surface. Detecting and distinguishing the complexes of ligand-bound LOX-1-cells from other cells not bound to the ligand in the sample enables the tracking of the number or changes in the number of PMN-MDSCs substantially free of PMN.
In another aspect, a method of differentiating polymorphonuclear myeloid derived, suppressor cells (PMN-MDSCs) from polymorphonuclear neutrophils (PMNs) in a biological sample containing both types of cells involves contacting the sample with a ligand that specifically binds or forms a complex with LOX-1 on the cell surface. The LOX-1-bound cells can be detected, identified, or measured apart from other cells not bound to the ligand in the sample. The LOX-1-bound cells are PMN-MDSCs substantially free of PMN.
In another aspect, a method of obtaining a population of PMN-MDSC from a biological sample containing other cell types comprises isolating from a cell suspension those cells which express LOX-1 to provide a population of cells enriched with PMN-MDSCs.
In another aspect, a method for differential diagnosis of cancer comprises contacting a biological sample of a subject with reagents capable of complexing or binding with LOX-1 on the surface of a cell; and detecting or measuring any cells that complex with the reagent. Cells that form a complex with the LOX-1 reagent indicate the presence of cancer cells in the sample.
In another aspect, a substantially pure population of PMN-DMSCs is produced by isolating LOX-1+ cells from a biological sample by contacting the sample with a reagent that forms a complex or binds to LOX-1.
In a further aspect, a pharmaceutical composition is provided that reduces or inhibits ER stress in mammalian neutrophils or reduces or inhibits LOX-1 expression on LOX-1+ neutrophil populations, LOX-1+ PMN and/or PMN-MDSC in a pharmaceutically acceptable carrier or excipient. In certain embodiments, the composition comprises an antagonist or inhibitor of the expression, activity or activation of one or more of sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI or NOS-2. In other embodiments, the composition comprises an antagonist or inhibitor of LOX-1 or an antagonist or inhibitor of the expression, activity, or activation of one or more of MYCN, CSF3, IL3, TGFβ1, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, NFkB, IL13, AGT, IL1β, ERBB2, MAP2K1, VEGFα, CSF1, FLI1, or IFNγ.
In another aspect, a method for reducing or inhibiting LOX-+ PMN-MDSC accumulation in a cancer patient comprises administering a composition as described herein.
A method of diagnosing a mammalian subject with a cancer comprises detecting and distinguishing the complexes of antibody-bound LOX-1-cells from other cells not bound to the antibody in the sample, and determining the size of a tumor in the subject by correlation with the number of LOX-1+ PMN or PMN-MDSC detected.
In another embodiment, a method of diagnosing and treating a cancer comprises diagnosing the subject with cancer when the presence of LOX-1+ is detected at a level that indicates PMN-MDSC are present; and administering an effective amount of a composition that reduces or inhibits ER stress response in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations.
Other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof.
As disclosed herein, methods and compositions are described which are useful in the isolation of certain cells indicative of cancer in a mammalian subject. Cell preparations that are substantially purified PMN-DMSCs are prepared by methods involving the use of reagents that complex with or bind the LOX-1 biomarker on the surface of cells, thereby discriminating between PMN cells and PMN-DMSCs. The methods described herein are also useful for the diagnosis and/or monitoring of cancer and tumor cells, i.e., both malignant and benign tumors, so long as the cells to be treated carry the LOX-1 cell surface antigen. Further, the inventors, using partial enrichment of PMN-MDSC with gradient centrifugation, determined that low density PMNMDSC and high-density neutrophils from the same cancer patients had a distinct gene profile.
Most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Surprisingly, low-density lipoprotein (LDL) was one of the most increased regulators and its receptor oxidized LDL receptor 1 (OLR1) was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor 1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5-15% of total neutrophils in cancer patients and 15-50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1− counterparts, LOX-1+ neutrophils had gene signature, potent immune suppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSC. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSC. As described in the specification and examples herein by evaluating populations of PMN-MDSC and PMN from the same patients, genomic signature of PMN-MDSC and certain significant surface markers specific for these cells were identified. Induction of ER stress response was sufficient to convert neutrophils to PMN-MDSC.
These discoveries by the inventors in identifying specific markers of human PMN-MDSC associated with ER stress and lipid metabolism, permit the development of novel diagnostic and therapeutic methods and compositions for cancer.
Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the fields of biology, biotechnology and molecular biology and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. The definitions herein are provided for clarity only and are not intended to limit the claimed invention.
“Patient” or “subject” as used herein means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject of these methods and compositions is a human.
The term “LOX-1” as used herein is a cell surface receptor, oxidized low density lipoprotein (lectin-like) receptor 1, first identified in endothelial cells as one of the main receptors for oxidized-LDL (ox-LDL)40. Besides ox-LDL, this receptor has been shown to bind many different ligands including other modified lipoproteins, advanced glycosylation end products, aged red blood cells, apoptotic cells and activated platelets45. LOX-1 has been involved in many different pathological conditions including atherogenesis, myocardial ischemia, hypertension, vascular diseases and thrombosis45. Expression of LOX-1 can be induced by a wide array of stimuli including pro-inflammatory factor (TNF-α, IL-1ρ or IFN-γ), angiotensin II, endothelin-1, modified lipoproteins and free radicals35. Engagement of LOX-1 can lead to induction of oxidative stress, apoptosis, endothelial dysfunction, fibrosis and inflammation through the activation of the NF-κB pathway. LOX-1 has also been described to play a role in tumorigenesis24. Indeed, LOX-1 up-regulation has been observed during cellular transformation into cancer cell and can have a pro-oncogenic effect by activating the NF-κB pathway, by increasing DNA damage through increase ROS production and by promoting angiogenesis and cell dissemination24,16.
The nucleic acid sequence for the gene encoding LOX-1 (gene name OLR1) can be found in databases such as NCBI, i.e., NCBI gene ID: 4973 or Gene sequence: Ensembl:ENSGW0000173391. The LOX-1 protein sequence is found at Hugo Gene Nomenclature Committee 8133, Protein Sequence HPRD:04003. It should be understood that the term LOX-1 can also represent the receptor protein in various species, and with conservative changes in the amino acid or encoding sequences, or with other naturally occurring modifications that may vary among species and between members of the same species, as well as naturally occurring mutations thereof.
The term “cancer” or “tumor” as used herein refers to, without limitation, refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. By cancer as used herein is meant any form of cancer, including hematological cancers, e.g., leukemia, lymphoma, myeloma, bone marrow cancer, and epithelial cancers, including, without limitation, breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, endometrial cancer, esophageal cancer, stomach cancer, bladder cancer, kidney cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, leukemia, myeloma, lymphoma, glioma, Non-Hodgkin's lymphoma, leukemia, multiple myeloma and multidrug resistant cancer.
A “tumor” is an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, and is also referred to as a neoplasm. The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Whenever the term “lung cancer” is used herein, it is used as a representative cancer for demonstration of the use of the methods and compositions described herein.
“Sample” as used herein means any biological fluid or suspension or tissue from a subject, including samples that contains cells carrying the LOX-1+ biomarker or PMN-MDSC signature biomarkers identified herein. The sample in one embodiment contains cells that are both PMN and PMN-MDSC. The sample in one embodiment contains cells carrying one or more other biomarkers or cell surface antigens indicative of polymorphonuclear cells or neutrophils. In one embodiment, cells (neutrophils) in the sample express CD66b+. In another embodiment, cells (neutrophils) in the sample express CD15+. In still another embodiment, cells in the sample express CD11b+ or CD33+. The most suitable samples for use in the methods and with the diagnostic compositions or reagents described herein are samples or suspensions which require minimal invasion for testing, e.g., blood samples, including whole blood, and any samples containing shed or circulating tumor cells. It is anticipated that other biological samples that contain cells at a sufficiently detectable concentration, such as peripheral blood, serum, saliva or urine, vaginal or cervical secretions, and ascites fluids or peritoneal fluid may be similarly evaluated by the methods described herein. In one embodiment, the sample is a tumor secretome, i.e., any fluid or medium containing the proteins secreted from the tumor. These shed proteins may be unassociated, associated with other biological molecules, or enclosed in a lipid membrane such as an exosome. Also, circulating tumor cells or fluids or tissues containing them are also suitable samples for evaluation in certain embodiments of this invention. In another embodiment, the biological sample is a tissue or tissue extract. e.g., biopsied material, containing the PMN-MDSC. In one embodiment, such samples may further be diluted with or suspended in, saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are tested neat. In another embodiment, the samples are concentrated by conventional means.
In one embodiment, the biological sample is whole blood, and the method employs the PaxGene Blood RNA Workflow system (Qiagen). That system involves blood collection (e.g., single blood draws) and RNA stabilization, followed by transport and storage, followed by purification of Total RNA and Molecular RNA testing. This system provides immediate RNA stabilization and consistent blood draw volumes. The blood can be drawn at a physician's office or clinic, and the specimen transported and stored in the same tube. Short term RNA stability is 3 days at between 18-25° C. or 5 days at between 2-8° C. Long term RNA stability is 4 years at −20 to −70° C. This sample collection system enables the user to reliably obtain data on gene expression and miRNA expression in whole blood. In one embodiment, the biological sample is whole blood. While the PAXgene system has more noise than the use of PBMC as a biological sample source, the benefits of PAXgene sample collection outweighs the problems. Noise can be subtracted bioinformatically.
By “ER stress response” is meant a response mediated by the endoplasmic reticulum to protect cells from various stress conditions including hypoxia, nutrient deprivation, low pH, etc. and includes three major signaling cascades initiated by three protein sensors: PERK (protein kinase RNA (PKR)-like ER kinase), IRE-1 (inositol-requiring enzyme 1) and ATF6 (activating transcription factor 6)17. Antagonists or inhibitors of ER stress include ligands, e.g., antibodies, fragments thereof, small molecules that can block the activity, function or activation of the regulators of ER stress, identified herein.
The term “biomarker” as described in this specification includes any physiological molecular form, or modified physiological molecular form, isoform, pro-form, naturally occurring forms or naturally occurring mutated forms of LOX-1 and peptide fragments of LOX-1, expressed on the cell surface, unless otherwise specified. Other biomarkers that may be useful to detect neutrophils to assist in distinguishing the two subsets PMN and PMN-MDSCs according to the teachings herein include CD66b, CD11b, CD33, CD15 and/or CD14 as well as the biomarkers that have been shown to be part of the PMN-MDSC signature, e.g., those of
By “PMN-MDSC gene signature”, as discovered by the inventors and as used throughout this specification, is meant a compilation of genes whose expression differs significantly when comparing the expression in normal neutrophils to their respective expression in PMN-MCSC, i.e., PMNs to PMN-MDSC. For example,
cerevisiae) (LSM4), mRNA. (S)
cerevisiae) (SAMM50), mRNA. (S)
cerevisiae) (LSM5), mRNA. (S)
cerevisiae) (SIL1), transcript variant 1, mRNA. (S)
By “isoform” or “multiple molecular form” is meant an alternative expression product or variant of a single gene in a given species, including forms generated by alternative splicing, single nucleotide polymorphisms, alternative promoter usage alternative translation initiation small genetic differences between alleles of the same gene, and posttranslational modifications (PTMs) of these sequences.
By “related proteins” or “proteins of the same family” are meant expression products of different genes or related genes identified as belonging to a common family. Related proteins in the same biomarker family, e.g., LOX-1, may or may not share related functions. Related proteins can be readily identified as having significant sequence identity either over the entire protein or a significant part of the protein that is typically referred to as a “domain”. Proteins with at least 20% sequence homology or sequence identity can be readily identified as belonging to the same protein family.
By “homologous protein” is meant an alternative form of a related protein produced from a related gene having a percent sequence similarity or identity of greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, or greater than 99%.
The term “ligand” with regard to protein biomarkers refers to a molecule that binds or complexes, with the PMN-MDSC biomarker protein, e.g., LOX-1. Thus, a ligand can be an amino acid sequence or protein sequence, or a molecular form or peptide, such as an antibody, antibody mimic or equivalent, or a fragment thereof. The ligand can be a naturally occurring peptide that binds to a portion of the LOX-1 receptor or a synthetically or recombinantly produced chimeric peptide having a portion that binds to the LOX-1 receptor and a portion designed for other purposes, e.g., to assist in the detection of the binding. Similarly, the peptide may be designed, or a small molecule designed, to bind to LOX-1 by mimicking the three-dimensional physical structure of the LOX-1 receptor. The term ligand as used with respect to the neutrophil biomarkers, e.g., CD15 and CD66b, and the PMN-MDSC signature biomarkers identified herein refers to similar amino acid sequences, peptides, chimeric proteins, etc, which can bind with the respective cell proteins.
The term “ligand” with regarding to a nucleic acid sequence encoding a biomarker, refers to a molecule that binds or complexes, with the indicated biomarker nucleic acid, e.g., LOX-1 DNA or RNA. Such a ligand can itself be an antibody or antibody fragment, a nucleotide sequence, e.g., a polynucleotide or oligonucleotide, primer or probe, which can be complementary to the biomarker-encoding sequence.
As used herein for the described methods and compositions, the term “antibody” refers to an intact immunoglobulin having two light and two heavy chains or fragments thereof capable of binding to a biomarker protein or a fragment of a biomarker protein. Thus a single isolated antibody or an antigen-binding fragment thereof may be a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, or a bi-specific antibody or multi-specific construct that can bind two or more target biomarkers.
The term “antibody fragment” as used herein for the described methods and compositions refers to less than an intact antibody structure having antigen-binding ability. Such fragments, include, without limitation, an isolated single antibody chain or an scFv fragment, which is a recombinant molecule in which the variable regions of light and heavy immunoglobulin chains encoding antigen-binding domains are engineered into a single polypeptide. Other scFV constructs include diabodies, i.e., paired scFvs or non-covalent dimers of scFvs that bind to one another through complementary regions to form bivalent molecules. Still other scFV constructs include complementary scFvs produced as a single chain (tandem scFvs) or bispecific tandem scFvs.
Other antibody fragments include an Fv construct, a Fab construct, an Fc construct, a light chain or heavy chain variable or complementarity determining region (CDR) sequence, etc. Still other antibody fragments include monovalent or bivalent minibodies (miniaturized monoclonal antibodies) which are monoclonal antibodies from which the domains non-essential to function have been removed. In one embodiment, a minibody is composed of a single-chain molecule containing one VL, one VH antigen-binding domain, and one or two constant “effector” domains. These elements are connected by linker domains. In still another embodiment, the antibody fragments useful in the methods and compositions herein are “unibodies”, which are IgG4 molecules from with the hinge region has been removed. See, reference 56 and the documents cited thereon for other forms of antibodies useful in these methods and compositions. For example, a LOX-1 antibody is available from commercial sources, such as Biolegend Inc., San Diego, Calif. Anti-LOX-1 antibodies or the antagonists or inhibitors referred to herein for the ER stress targets or other targeted biomarkers may also be any of these forms of antibody or antibody fragments.
As used herein, “labels” or “reporter molecules” or “detectable label components” are chemical or biochemical moieties that do not naturally occur in association with a ligand, but that are useful when manipulated into association with a ligand, that alone or in concert with other components enable the detection of a target, e.g., the biomarker LOX-1. Such labels or components include, without limitation, fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionucleotides, enzymes, enzymatic substrates, cofactors, inhibitors, radioactive isotopes, magnetic particles, and other moieties known in the art. In certain embodiments, the “labels” or “reporter molecules” are covalently attached or associated with the ligand. In certain other embodiments, the “labels” or “reporter molecules” are non-covalently attached or associated with the ligand. Such labels are capable of generating a measurable signal alone, e.g., radioactivity, or in association with another component, e.g., an enzymatic signal in the presence of a substrate.
By “physical substrate is meant a substrate upon which said polynucleotides or oligonucleotides or ligands are immobilized. The physical substrate can be e.g., a glass slide, a plastic support, or a microchip. The term “macroarray” refers to an ordered arrangement of binding/complexing array elements or ligands, e.g. antibodies, probes, etc. on a physical substrate.
By “significant change in expression” is meant an upregulation in the expression level of a nucleic acid sequence, e.g., genes or transcript, encoding a selected biomarker, in comparison to the selected reference standard or control; a downregulation in the expression level of a nucleic acid sequence, e.g., genes or transcript, encoding a selected biomarker, in comparison to the selected reference standard or control; or a combination of a pattern or relative pattern of certain upregulated and/or down regulated biomarker genes. The degree of change in biomarker expression can vary with each individual as stated above for protein biomarkers.
The term “polynucleotide,” when used in singular or plural form, generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term “polynucleotide” specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. In general, the term “polynucleotide” embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
The term “oligonucleotide” refers to a relatively short polynucleotide of less than 20 bases, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
One skilled in the art may readily reproduce the compositions and methods described herein by use of the amino acid sequences of the biomarkers and other molecular forms, which are publicly available from conventional sources.
Throughout this specification, the words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively. The words “consist”, “consisting”, and its variants, are to be interpreted exclusively, rather than inclusively. It should be understood that while various embodiments in the specification are presented using “comprising” language, under various circumstances, a related embodiment is also be described using “consisting of” or “consisting essentially of” language.
The term “a” or “an”, refers to one or more, for example, “a biomarker,” is understood to represent one or more biomarkers. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.
As used herein, the term “about” means a variability of 10% from the reference given, unless otherwise specified.
A method for differentiating polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) from polymorphonuclear neutrophils (PMNs) or monocytic myeloid derived suppressor cells (M-MDSCs) in a biological sample containing these types of cells involves the following steps. The biological sample, e.g., whole blood or a cell suspension, or a tumor exudate, or tissue, e.g., biopsy material, is contacted with a ligand that specifically binds or forms a complex with LOX-1 receptor on the cell surface. As described in the example below, the ligand is an antibody that binds to LOX-1. Thus, by contacting the sample with an anti-LOX-1 antibody, one may detect antibody-conjugate complexes in the sample. However, other ligands can be used in a similar fashion. The resulting complexes of ligand-bound LOX-1-cells in the sample are detected. Such detection can be based upon separation of the ligand-bound cells from unbound cells in the sample. The LOX-1-bound cells are PMN-MDSCs substantially free of PMN. In certain embodiments, the ligand is an anti-LOX-1 antibody, or an anti-LOX-1 antibody fragment. In certain embodiments, the ligands are associated with a detectable label component. In still other embodiments, the ligand is immobilized on a substrate.
In samples containing red blood cells, such as whole blood, one embodiment of the method involves killing or lysing the red blood cells to permit their elimination from the sample and possible interference with the results of the assay. In one aspect, the methods described herein comprise combining the whole blood sample with a lytic reagent system. This step can occur before contact of the sample with the ligand. In another embodiment, this step can occur after contact of the sample with the ligand. In still another embodiment, this step can occur simultaneously or substantially simultaneously with contact with the ligand. In such embodiments, the lytic reagent system is used to lyse red blood cells and to preserve the integrity of the remaining cells in the sample. Exemplary lytic reagents, stabilizing reagents and the method of use have been described, e.g., in U.S. Pat. Nos. 6,573,102 and 6,869,798. Alternatively, the reagent system can also be an isotonic lysing reagent as described in U.S. Pat. No. 5,882,934. Other lytic reagents known in the art can also be used for the purpose of the present methods.
The detection and separation of the ligand bound LOX-1 cells in the sample may be accomplished by a physical characteristic, such as the difference in size or weight of the bound LOX-1 cells vs. the unbound cells which do not have LOX-1 on their surfaces. Such detection and/or separation techniques can thus employ appropriately sized filtration units, or the use of flow cytometry, or chromatographic or centrifugation techniques (size exclusion or weight exclusion), among others known to the art.
Alternatively, where the ligand is associated with a detectable label component, the detection and separation may employ methods of detecting independently detectable labels by radioactivity, light wavelength, etc. Where the ligand is associated with a label which is capable of generating a measurable detectable signal when contacted with another label component, these methods employ the addition of such components and suitable detection methods dependent upon the signal generated. The separated, collected ligand-bound LOX-1+ cells are then collected and counted.
Where the ligand is immobilized on a physical substrate, the separating step can include washing the unbound cells and other debris in the sample from the substrate and counting or collecting the bound PMN-MDSCs from the substrate. In another embodiment, the separating step comprises treating the sample with a reagent, such as an enzymatic substrate, where the label is an enzyme. The interaction of the label and enzymatic substrate or cofactor identifies LOX-1-PMN-MDSC complexes from unbound cells to permit enumeration of PMN-MDSC.
The method of identifying and separating PMN-MDSCs from a sample can also include contacting the biological sample with the other biomarkers forming the distinguishing signature of PMN-MDSC or other biomarkers that identify as a single population both PMN-MDSCs and PMNs and/or M-MDSCs and isolating a cell suspension containing PMN-MDSCs and PMNs (and/or M-MDSCs) prior to, or simultaneously with, contacting the cell suspension with the LOX-1 ligand. In still other embodiments of the methods, the sample may be contacted (with or without RBC lysis) with a LOX-1 ligand and a ligand that identifies neutrophils, i.e., other PMN that are not LOX-1+. In one embodiment, the sample is contacted with a LOX-1 ligand and a CD15 ligand. In still other embodiments of the methods, the sample may be contacted with a LOX-1 ligand and a CD66b ligand. Still other ligands that identify neutrophils generally may be useful in this context.
In one embodiment, therefore, the method involves contacting the biological sample with the ligand for CD15 prior to, or simultaneously with, the use of the LOX-1 ligand. In one embodiment, therefore, the method involves contacting the biological sample with a ligand for CD66b prior to, or simultaneously with, the use of the LOX-1 ligand. In one embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14 prior to, or simultaneously with, the use of the LOX-1 ligand. In one embodiment, therefore, the method involves contacting the biological sample with a ligand for CD11b prior to, or simultaneously with, the use of the LOX-1 ligand. In one embodiment, therefore, the method involves contacting the biological sample with the ligand for CD33, prior to, or simultaneously with, the use of the LOX-1 ligand. In one embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14 and a ligand for CD15 prior to, or simultaneously with, the use of the LOX-1 ligand. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14, and a ligand for CD11b prior to, or simultaneously with, the use of the LOX-1 ligand. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14 and a ligand for CD33 prior to, or simultaneously with, the use of the LOX-1 ligand. In another embodiment, therefore, the method involves contacting the biological sample a ligand for CD15 and a ligand for CD11b prior to, or simultaneously with, the use of the LOX-1 ligand. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD15 and a ligand for CD33 prior to, or simultaneously with, the use of the LOX-1 ligand. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD15, a ligand for CD11b and a ligand for CD33 prior to, or simultaneously with, the use of the LOX-1 ligand. In another embodiment, therefore, the method involves contacting the biological sample with a ligand for CD14, a ligand for CD11b and a ligand for CD33 prior to, or simultaneously with, the use of the LOX-1 ligand.
In one embodiment of the method, any of these biomarkers may be detected prior to, or simultaneously with, the detection of the LOX-1 biomarker. The use of these other ligands assists in identifying all PMNs from other cells in the sample. Subsequent exposure of this population of cells from the sample with the LOX-1 ligands enables further separation of the PMN-MDSCs from the PMN population.
In one embodiment, following contact with the LOX-1 ligand and a second neutrophil specific biomarker ligand, such as a CD15 ligand or CD66b ligand, one may calculate the number of LOX-1+ vs. CD15+ or the number of LOX-1+ vs. CD66b+ cells are present in the sample. Such calculation can involve cell counting systems known to those of skill in the art.
In another embodiment, the method involves collecting as a second population, the cells which did not form complexes with the ligands, e.g., are not providing a detectable signal or are not immobilized on the substrate. This second population contains PMNs and other cells substantially free from PMN-MDSCs.
In still another embodiment, the methods described herein permit the obtaining of a population of cells enriched in human polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) by isolating from a cell suspension those cells which express LOX-1 to provide a population of cells enriched with PMN-MDSCs.
In still another embodiment, the methods involve measuring the amount of soluble LOX-1+ in the serum and correlating that number with the number of LOX-1 PMN-MDSC.
These methods also permit the removal of human PMN-MDSCs from a cell population, comprising isolating from the cell population those cells which express LOX-1. These methods are useful in one embodiment for monitoring of the progression or metastasis of a cancer or the monitoring of therapy in a cancer patient by permitting the evaluation of an increase in the LOX-1 cell surface receptor in a biological sample of a patient having a cancer or under treatment for cancer. The increase of LOX-1+ cell number is indicative of metastasizing cancer or a progression of cancer. In other embodiments, this method may be useful diagnostically to initially detect the presence of cancer.
These methods depend initially upon obtaining an accurate enumeration or concentration of a PMN-MDSC cell population, substantially free of any PMNs, from a suitable biological sample of a subject. In one embodiment, these methods of determining an accurate cell count/concentration of cells expressing LOX-1 in a subject having a cancer or being treated for a cancer can be used to monitor the progression of the cancer (with or without treatment).
In still another embodiment, the use of these methods to determine an accurate measurement of LOX-1+ cells enable the monitoring of metastasis in a cancer, e.g., an increase in the LOX-1+ cell number indicates metastatic cancer. In another embodiment, these methods are useful to monitor and/or influence cancer treatment. For example, where the LOX-1+ cell number is increasing prior to cancer therapy, and subsequent performance of the method on a similar sample in the subject does not show a decrease in LOX-1+ cell number, the method can indicate that a change in therapeutic method or dosage is necessary.
In another embodiment, these methods of determining an accurate cell count/concentration of cells expressing LOX-1 in a subject suspected of having cancer, can diagnose the presence of cancer. In another embodiment, these methods can diagnose the aggressiveness of a cancer. In another embodiment, these methods can diagnose the stage of a cancer. According to the inventors' early studies, in most healthy individuals the proportion of LOX-1+ PMN is less than between 0.5% to 1% PMN. Patients with stage II diseases usually have between about 3 about 5% of LOX-1+ PMN and patients at stages III-IV have over 5% to about 12% PMN.
In still another aspect, the method of measuring the LOX-1*population in a sample, such as whole blood, can be employed as a research method to determine the cause of the increase in such cells during the progression of a cancer.
In still other aspects of the diagnostics methods identified above, additional diagnostics steps include contacting the sample with a reagent that identifies activators or regulators of ER stress response in said cells. In one embodiment, the activators or regulators so identified are one or more of sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI or NOS-2. In another embodiment, the regulators are one or more of one or more of MYCN, CSF3, IL3, TGFβ1, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, NFkB, IL13, AGT, IL1β, ERBB2, MAP2K, VEGFα, CSF1, FLI1, or IFNγ.
Yet another embodiment of a diagnostic method for a mammalian subject with a cancer comprises the additional step of determining the size of a tumor in the subject by correlation with the number of LOX-1+ PMN or PMN-MDSC detected. This method step is further described in detail in the examples, but can include obtaining a biological sample from the subject; detecting whether soluble LOX-1 is present in the sample by contacting the sample with an antibody or functional antibody fragment that specifically binds or forms a complex with LOX-1 on the cell surface; and detecting and distinguishing the complexes of antibody-bound LOX-1-cells from other cells not bound to the antibody in the sample. The size of the tumor is then determined based upon the increase of LOX-1+ PMNs or PMN-MDSCs over a baseline level. The baseline level is readily determined based upon enumeration of patient samples to create a standard.
Still another method combines diagnosing and treating a cancer and combines the steps, such as obtaining a biological sample from a subject; detecting whether PMN-MDSC are present in the sample; diagnosing the subject with cancer when the presence of LOX-1+(or any other of the PMN-MDSC signature biomarkers identified herein) is detected at a level that indicates PMN-MDSC are present; and administering an effective amount of a composition that reduces or inhibits ER stress response in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations.
The presence of LOX-1 (or any of the PMN-MDSC signature biomarkers) in the sample (or a LOX-1-ligand complex) may be detected using any assay format known in the art or described herein. There are a variety of assay formats known to the skilled artisan for using a ligand to detect a target molecule in a sample. (For example, see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, the presence or absence of LOX-1 in a sample may be determined by (a) contacting the sample with a ligand that interacts with LOX-1: and (b) determining the presence or level of LOX-1 in the sample, wherein the presence of LOX-1 in the sample is indicative of cancer or where an increase in the level of LOX-1 in the sample as compared to a control, is indicative of cancer. The various assay methods employ one or more of the LOX-1-binding ligands described herein, e.g., polypeptide, polynucleotide, and/or antibody, which detect the LOX-1 protein or mRNA encoding the same (including fragments or portions thereof).
Methods of detection, diagnosis, monitoring, and prognosis of cancer, or the status of cancer, and for the identification of subjects with an increased risk of cancer metastasis by detecting the presence of, or measuring the level of LOX-1 protein or another biomarker described herein, are provided herein. Such methods may employ polypeptides and/or antibodies as described herein. The particular assay format used to measure the LOX-1 in a biological sample may be selected from among a wide range of immunoassays, such as enzyme-linked immunoassays, sandwich immunoassays, homogeneous assays, immunohistochemistry formats, or other conventional assay formats. One of skill in the art may readily select from any number of conventional immunoassay formats to perform this invention. Other reagents for the detection of protein in biological samples, such as peptide mimetics, synthetic chemical compounds capable of detecting LOX-1 may be used in other assay formats for the quantitative detection of LOX-1 protein in biological samples, such as high pressure liquid chromatography (HPLC), immunohistochemistry, etc.
Methods of detection, diagnosis, monitoring, and prognosis of cancer, or the status of cancer, and for the identification of subjects with an increased risk of cancer metastasis by detecting the presence of, or measuring the level of LOX-1 mRNA, are provided herein. Such methods include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, proteomics-based methods or immunochemistry techniques. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization; RNAse protection assays: and PCR-based methods, such as reverse transcription polymerase chain reaction (RT-PCR) or qPCR.
Such PCR-based method may employ a primer or primer-probe set capable of identifying and/or amplifying a LOX-1 nucleic acid sequence or a portion thereof. An example of a primer set capable of identifying and/or amplifying a LOX-1 nucleic acid sequence or a portion thereof is described in Example 1E. Such primers include those described in the examples or other suitable primers can be designed by the person of skill in the art and/or obtained commercially based on the LOX-1 nucleic acid sequence.
The methods described herein are not limited by the particular techniques selected to perform them. Exemplary commercial products for generation of reagents or performance of assays include TRI-REAGENT, Qiagen RNeasy mini-columns, MASTERPURE Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), Paraffin Block RNA Isolation Kit (Ambion, Inc.) and RNA Stat-60 (Tel-Test), the MassARRAY-based method (Sequenom, Inc., San Diego, Calif.), differential display, amplified fragment length polymorphism (iAFLP), and BeadArray™ technology (Illumina, San Diego, Calif.) using the commercially available Luminex100 LabMAP system and multiple color-coded microspheres (Luminex Corp., Austin, Tex.) and high coverage expression profiling (HiCEP) analysis.
The diagnostic methods described herein can employ contacting a patient's sample with a diagnostic reagent, as described above, which forms a complex or association with LOX-1 in the patients' sample. Detection or measurement of the sample LOX-1 may be obtained by use of a variety of apparatus or machines, such as computer-programmed instruments that can transform the detectable signals generated from the diagnostic reagents complexed with the LOX-1 or other biomarker in the biological sample into numerical or graphical data useful in performing the diagnosis. Such instruments may be suitably programmed to permit the comparison of the measured LOX-1 in the sample with the appropriate reference standard and generate a diagnostic report or graph.
The selection of the polynucleotide sequences, their length and labels used in the composition are routine determinations made by one of skill in the art in view of the teachings of which genes can form the gene expression profiles suitable for the diagnosis and prognosis of cancer. For example, useful primer or probe sequences can be at least 8, at least 10, at least 15, at least 20, at least 30, at least 40 and over at least 50 nucleotides in length. For example, such probes and polynucleotides can be complementary to portions of mRNA sequences encoding LOX-1 or another of the biomarkers identified herein. The probes and primers can be at least 70%, at least 80%, at least 900, at least 95%, up to 100% complementary to sequences encoding.
In any of the methods described herein, in one embodiment, the sample comprises blood, plasma or cells. Such sample may be derived from a tissue biopsy. In some of the methods described herein, a control level is used as a reference point. The control level can be any of those described herein. In one embodiment, the control level is the level obtained from an individual, or a population of individuals, who are healthy (i.e., who do not have cancer). In another embodiment, the control level is the level obtained from an individual, or a population of individuals, who have cancer that has not metastasized.
Compositions
In yet another embodiment, the methods described above result in a composition of cells, i.e., a substantially pure population of PMN-DMSCs produced by isolating LOX-1+ cells from a biological sample by contacting the sample with a reagent that forms a complex or binds to LOX-1. The methods described above can also result in a population of PMNs which contain substantially no PMN-DMSCs. These cell populations are useful in research.
In a further aspect, diagnostic composition or kit is provided by the disclosures and experiments described herein. In one embodiment, such a composition comprises a ligand that specifically binds or forms a complex with LOX-1 on the cell surface. Such a composition may include ligands and antibodies and small molecules that can detecting or isolate a population of human polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs). Such useful compositions include anti-LOX-1 antibodies or small molecules that can bind thereto. Also useful are antibodies or ligands that bind other of the genes that form the genetic signature of the PMN-DMSCs, such as the genes identified in
Included among them are markers and regulators of pathways for ER stress response, such as sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI, NOS-2, MYCN, CSF3, IL3, TGFβ1, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG. NFkB, IL13, AGT, IL1β, ERBB2, MAP2K1, VEGFα, CSF1, FLI1, or IFNγ. Still other likely biomarkers for pathways involved or activated in PMN-MDSC production are described in the Examples below. This composition/kit containing ligands/antibodies or small molecules that bind to one or a combination of any of these biomarkers may be used in diagnosing the presence, progression or metastasis of a cancer.
A variety of compositions and methods can be employed for the detection, diagnosis, monitoring, and prognosis of the relevant cancer, or the status of cancer, and for the identification of subjects with an increased risk of cancer metastasis. The cancer may be any one of the cancers described in Tables 3 and 4 below, for example. In one aspect, a diagnostic composition useful in diagnosing and/or treating cancer is provided. In one embodiment, the composition includes a ligand which is capable of specifically complexing with, or identifying, LOX-1 or the other biomarkers that together with LOX-1 can differentiate non-immunosuppressive neutrophils or LOX-1− PMNs from immunosuppressive PMN-MDSCs or subsets thereof, or the mRNA encoding the same, including a fragment or portion thereof.
There are a variety of assay formats known to the skilled artisan for using a binding agent to detect a target molecule in a sample. Any ligand which is capable of specifically complexing with, or identifying, the relevant biomarker, the mRNA encoding the same, including a fragment or portion thereof, which is useful in one or more of the various assay methods, is contemplated herein. In one embodiment, the ligand is a polynucleotide or oligonucleotide sequence, which sequence binds to, complexes with or identifies LOX-1 (or any of the biomarkers forming the PMN-MDSC signature) or the mRNA encoding the same, or a fragment thereof. In another embodiment, the ligand is a protein or peptide, which protein or peptide binds to, complexes with or identifies LOX-1 (or any of the biomarkers forming the PMN-MDSC signature) or the mRNA encoding the same or a portion or fragment thereof. In another embodiment, the ligand is an antibody or fragment thereof which binds to, complexes with or identifies LOX-1 (or any of the biomarkers forming the PMN-MDSC signature) or the mRNA encoding the same or a portion or fragment thereof.
The terms antibody and antibody fragment are defined above. A recombinant molecule bearing the binding portion of an anti-LOX-1 antibody (or another molecule designed similarly to target one or more of the PMN-MDSC biomarkers referred to herein, or the ER stress response proteins or genes), e.g., carrying one or more variable chain CDR sequences that bind LOX-1 or the other target, may also be used in a diagnostic assay. As used herein, the term “antibody” may also refer, where appropriate, to a mixture of different antibodies or antibody fragments that bind to LOX-1 or another selected target disclosed herein. Such different antibodies may bind to different biomarkers in the PMN-MDSC signature or different portions of LOX-1 protein than the other antibodies in the mixture.
Similarly, the antibodies may be tagged or labeled with reagents capable of providing a detectable signal, depending upon the assay format employed. Such labels are capable, alone or in concert with other compositions or compounds, of providing a detectable signal. Where more than one antibody is employed in a diagnostic method. e.g., such as in a sandwich ELISA, the labels are desirably interactive to produce a detectable signal. Most desirably, the label is detectable visually, e.g. colorimetrically. A variety of enzyme systems operate to reveal a colorimetric signal in an assay, e.g., glucose oxidase (which uses glucose as a substrate) releases peroxide as a product that in the presence of peroxidase and a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized TMB that is seen as a blue color. Other examples include horseradish peroxidase (HRP) or alkaline phosphatase (AP), and hexokinase in conjunction with glucose-6-phosphate dehydrogenase that reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 nm wavelength.
Other label systems that may be utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles (Bangs Laboratories, Indiana) in which a dye is embedded may be used in place of enzymes to provide a visual signal indicative of the presence of the resulting selected biomarker-antibody complex in applicable assays. Still other labels include fluorescent compounds, radioactive compounds or elements. Preferably, an anti-biomarker antibody is associated with, or conjugated to a fluorescent detectable fluorochromes, e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 (PC5), and PE-Texas Red (ECD). Commonly used fluorochromes include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and also include the tandem dyes, PE-cyanin-5 (PC5), PE-cyanin-7 (PC7), PE-cyanin-5.5, PE-Texas Red (ECD), rhodamine, PerCP, fluorescein isothiocyanate (FITC) and Alexa dyes. Combinations of such labels, such as Texas Red and rhodamine, FITC+PE, FITC+PECy5 and PE+PECy7, among others may be used depending upon assay method.
In yet another embodiment, the reagent is a primer set or primer-probe set capable of identifying and/or amplifying LOX-1 or a portion thereof or any of the other biomarkers discussed herein. An example of a primer set capable of identifying and/or amplifying such a biomarker or a portion thereof is described in the examples below. Other suitable primers can be designed by the person of skill in the art and/or obtained commercially.
In one embodiment, the reagent forms a complex with LOX-1. In one embodiment, the reagent-LOX-1 complex is capable of being detected. Various methods of detection of the reagent-LOX-1 complex are known in the art. In some embodiments, such methods include the use of labels as described herein.
In one embodiment, the ligand is associated with a detectable label or a substrate. The ligand may be covalently or non-covalently joined with the detectable label or substrate. In one embodiment, the comprises a substrate upon which said ligand is immobilized. For these reagents, the labels may be selected from among many known diagnostic labels, including those described above. Selection and/or generation of suitable ligands with optional labels for use in this invention is within the skill of the art, provided with this specification, the documents incorporated herein, and the conventional teachings of the art. Ligands may be labeled using conventional methods with a detectable substance. Examples of detectable substances include, but are not limited to, the following: radioisotopes (e.g., 3H, 14C, 35S, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol, enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
Similarly, the substrates for immobilization may be any of the common substrates, glass, plastic, a microarray, a microfluidics card, a chip or a chamber. The reagent itself may be labeled or immobilized. For example, a ligand or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies, etc. Suitable carriers or supports may comprise nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite. The support material may have any possible configuration including spherical (e.g. bead), cylindrical (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test strip). Immobilization typically entails separating the binding agent from any free analytes (e.g. free markers or free complexes thereof) in the reaction mixture.
Still another diagnostic reagent includes a composition or kit comprising at least one reagent that binds to, hybridizes with or amplifies LOX-1 or any of the other PMN-MDSC signature biomarkers. Such diagnostic reagents and kits containing them are useful for the measurement and detection of the biomarkers in the methods described herein for diagnosis/prognosis of cancer or metastasis of cancer. In addition to the reagents above, alternatively, a diagnostic kit thus also contains miscellaneous reagents and apparatus for reading labels, e.g., certain substrates that interact with an enzymatic label to produce a color signal, etc., apparatus for taking blood samples, as well as appropriate vials and other diagnostic assay components.
In yet another aspect, a pharmaceutical composition is provided that reduces or inhibits ER stress in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations in a pharmaceutically acceptable carrier or excipient. In one embodiment, this composition comprises an antagonist or inhibitor of the expression, activity or activation of one or more of sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI or NOS-2. In one embodiment, the composition comprises an antagonist or inhibitor of LOX-1. In still further embodiments, the composition contains additional antagonist or inhibitor of the expression, activity or activation of one or more of MYCN, CSF3, 1L3, TGFβ1, TNF, LDL. RAF1, APP, IL6 PDGFBB. EPO, CD40LG, NFkB, IL13, AGT, IL1β, ERBB2, MAP2K1, VEGFα, CSF1, FLI1, or IFNγ, or of the pathways leading to the production of the immunosuppressive PMN-MDSC populations in vivo.
In one embodiment, the antagonist or inhibitor of the selected mediator of ER stress is an antibody, functional antibody fragment or equivalent as defined herein, or a similarly functioning small molecule that binds to and thus prevents the normal activity of the particular gene/protein described above, leading to a reduction of the ER stress induction to which the neutrophils are exposed.
As another aspect, a novel pharmaceutical composition comprises the antagonist or inhibitors and immunotherapeutics described above in a pharmaceutically acceptable carrier or excipient in an effective amount to reduce, inhibit, retain or suppress growth of the PMN-MDSC population. In one aspect, the pharmaceutical composition contains, e.g., about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, to about 90% of the antagonist or inhibitors in combination with a pharmaceutical carrier or excipient.
By “pharmaceutically acceptable carrier or excipient” is meant a solid and/or liquid carrier, in in dry or liquid form and pharmaceutically acceptable. The compositions are typically sterile solutions or suspensions. Examples of excipients which may be combined with the antagonist or inhibitor include, without limitation, solid carriers, liquid carriers, adjuvants, amino acids (glycine, glutamine, asparagine, arginine, lysine), antioxidants (ascorbic acid, sodium sulfite or sodium hydrogen-sulfite), binders (gum tragacanth, acacia, starch, gelatin, polyglycolic acid, polylactic acid, poly-d,l-lactide/glycolide, polyoxaethylene, polyoxapropylene, polyacrylamides, polymaleic acid, polymaleic esters, polymaleic amides, polyacrylic acid, polyacrylic esters, polyvinylalcohols, polyvinylesters, polyvinylethers, polyvinylimidazole, polyvinylpyrrolidon, or chitosan), buffers (borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (mannitol or glycine), carbohydrates (such as glucose, mannose, or dextrins), clarifiers, coatings (gelatin, wax, shellac, sugar or other biological degradable polymers), coloring agents, complexing agents (caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin), compression aids, diluents, disintegrants, dyes, emulsifiers, emollients, encapsulating materials, fillers, flavoring agents (peppermint or oil of wintergreen or fruit flavor), glidants, granulating agents, lubricants, metal chelators (ethylenediamine tetraacetic acid (EDTA)), osmo-regulators, pH adjustors, preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, hydrogen peroxide, chlorobutanol, phenol or thimerosal), solubilizers, sorbents, stabilizers, sterilizer, suspending agent, sweeteners (mannitol, sorbitol, sucrose, glucose, mannose, dextrins, lactose or aspartame), surfactants, syrup, thickening agents, tonicity enhancing agents (sodium or potassium chloride) or viscosity regulators. See, the excipients in “Handbook of Pharmaceutical Excipients”, 5′ Edition, Eds.: Rowe, Sheskey, and Owen, APhA Publications (Washington, D.C.), 2005 and U.S. Pat. No. 7,078,053, which are incorporated herein by reference. The selection of the particular excipient is dependent on the nature of the compound selected and the particular form of administration desired.
Solid carriers include, without limitation, starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, calcium carbonate, sodium carbonate, bicarbonate, lactose, calcium phosphate, gelatin, magnesium stearate, stearic acid, or talc. Fluid carriers without limitation, water, e.g., sterile water, Ringer's solution, isotonic sodium chloride solution, neutral buffered saline, saline mixed with serum albumin, organic solvents (such as ethanol, glycerol, propylene glycol, liquid polyethylene glycol, dimethylsulfoxide (DMSO)), oils (vegetable oils such as fractionated coconut oil, arachis oil, corn oil, peanut oil, and sesame oil; oily esters such as ethyl oleate and isopropyl myristate; and any bland fixed oil including synthetic mono- or diglycerides), fats, fatty acids (include, without limitation, oleic acid find use in the preparation of injectables), cellulose derivatives such as sodium carboxymethyl cellulose, and/or surfactants.
By “effective amount” is meant the amount or concentration (by single dose or in a dosage regimen delivered per day) of the antagonist or inhibitor sufficient to retard, suppress or inhibit the PMN-MDSC, while providing the least negative side effects to the treated subject. One of skill in the art would be able to determine the amount of these antagonist or inhibitors to administer alone or in combination with an additional reagent, e.g., chemotherapeutic, antibiotic or the like. In a further embodiment, the combination of the antagonist or inhibitors with another pharmacological agent or treatment protocol permits lower than usual amounts of the agonist and additional chemotherapeutic agent to achieve the desired therapeutic effect. In another embodiment, the combination of the antagonist or inhibitors with another chemotherapy treatment protocol permits adjustment of the additional protocol regimen to achieve the desired therapeutic effect.
In one embodiment, the effective amount of the antagonist or inhibitors is within the range of 1 mg/kg body weight to 100 mg/kg body weight in humans including all integers or fractional amounts within the range. In certain embodiments, the effective amount is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg body weight, including all integers or fractional amounts within the range. In one embodiment, the above amounts represent a single dose. In another embodiment, the above amounts define an amount delivered to the subject per day. In another embodiment, the above amounts define an amount delivered to the subject per day in multiple doses. In still other embodiments, these amounts represent the amount delivered to the subject over more than a single day.
In another embodiment, the pharmaceutical composition contains a LOX-1 or ER response antagonist or inhibitor and a chemotherapeutic. Alternatively, the active compound is formulated with a chemotherapeutic for treatment of the cancers described herein. In one embodiment, the chemotherapeutic is selected from among those described above. Alternatively, the composition is formulated with another effective compound or reagent for treatment of the cancers described herein, such as an antibiotic or bactericide, a surfactant, or other reagent commonly used in formulation of anti-cancer compositions.
The forms of the pharmaceutical compositions may be liquid, solid or a suspension or semi-solid and designed for use with a desired administrative route, such as those described herein. The doses and dosage regimens are adjusted for the particular cancer, and the stage of the cancer, physical status of the subject. Such doses may range from about 1 to about 100 mg/kg subject body weight of the antagonist or inhibitor, as discussed above and include dosage regimens designed to administer the effective amount in smaller repeated doses.
These compositions are useful in methods for treating any cancer including the cancers described herein and in the examples. In still another embodiment, a therapeutic method for reducing or inhibiting LOX-1+ PMN-MDSC accumulation in a cancer patient comprises administering a composition such as described herein at a suitable dosage. This reduction can be for the treatment of cancer alone. In still other embodiments, the treatment step may be combined with the diagnostic steps in a combined method.
The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only. The compositions, experimental protocols and methods disclosed and/or claimed herein can be made and executed without undue experimentation in light of the present disclosure. The protocols and methods described in the examples are not considered to be limitations on the scope of the claimed invention. Rather this specification should be construed to encompass any and all variations that become evident as a result of the teaching provided herein. One of skill in the art will understand that changes or variations can be made in the disclosed embodiments of the examples, and expected similar results can be obtained. For example, the substitutions of reagents that are chemically or physiologically related for the reagents described herein are anticipated to produce the same or similar results. All such similar substitutes and modifications are apparent to those skilled in the art and fall within the scope of the invention.
In order to identify specific markers discriminating between these two populations, we performed genome-wide microarrays (Human HT-12 v4 expression Beadchip, Illumina) to compare the gene expression profiles between PMN-MDSC and PMN from the same cancer patients (7 patients) as well as age matching healthy donors (4 donors). All samples of peripheral blood (PB) were collected from patients at the Helen F. Graham Cancer Center and were analyzed within 3 hours of collection. PMN-MDSCs were evaluated in mononuclear fraction of PB after ficoll density gradient. PMN were evaluated from the cell fraction remaining after removal of mononuclear cells. Cells were resuspended in PBS and loaded on a step density gradient (Percoll 63% on top of Percoll 72%) to separate PMNs in a monolayer between the two Percoll phases. In an attempt to minimize the number of potential candidates and to identify true marker of PMN-MDSC, we analyzed the gene expression profiles of PMN-MDSC from head and neck cancer patients (4 samples) as well as lung cancer patients (3 samples).
The analysis was performed using SAM analysis (significant analysis of microarray) and the false discovery rate set at 5% (analysis was performed by the Wistar bioinformatics core facility). This analysis allowed us to identify more than 1500 genes showing a significant differential expression between PMN-MDSC and PMN. The vast majority of the differentially regulated genes were up-regulated in PMN-MDSC compared to PMN. After filtering for molecules expressed on the surface of the cells, we ended with a relatively small list of specific biomarkers for PMN-MDSC. One of these biomarkers is the Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a 50 kDa transmembrane glycoprotein encoded by the gene olr1 (oxidized LDL receptor 1). According to the microarray, LOX-1 was increased by 5.75-fold in PMN-MDSC compared to PMN.
To confirm the validity of LOX-1 as a potential biomarker of PMN-MDSC, we analyzed the expression of this receptor by flow cytometry using an anti-LOX-1 monoclonal antibody (clone 15C4; Biolegend Inc., San Diego, Calif.) in blood samples from patients with 4 different types of cancer: head and neck, breast, non-small lung, or colon cancer.
We first analyzed the expression of LOX-1 using the classical definitions of PMN-MDSC (CD11b+ CD14− CD5+ and CD33+ from the low density mononuclear cells fraction) and PMN (cells with the same phenotype from high density fraction). The results of this experiment are reported graphically when healthy donors (HD) were compared with all cancer patients in
The results of this experiment are reported by separating the results for cancer types as shown in the graphs of
Preliminary data also suggest that the percentage of PMN-MDSC expressing LOX-1 could correlate with the stage of the disease. As shown graphically in the preliminary analysis of
We also performed an analysis of unseparated whole blood samples. As shown in
To assess possible functional relevance of these findings. LOX-1+ and LOX-1− PMN were isolated from peripheral blood of three patients with head and neck cancer using magnetic beads separation as follows: Samples of whole blood were collected from patient with HNC. Red cells were lysed, and PMN were highly enriched by negative selection using Mitlenyi bead kit. Cells were then labeled with biotinylated LOX-1 antibody followed by streptavidin beads. LOX-1+ and LOX-1− PMN were added to mixed allogeneic reaction at ratios if 1:2, 1:4 and 1:8, and T-cell proliferation was measured 5 days later by 3H-thymidin uptake. Experiments were performed in triplicate. Cells were used in allogeneic mixed leukocyte reactions where dendritic cells from healthy donors were cultured with T cells from unrelated healthy donors. Mixing cells from unrelated donors stimulated potent T-cell proliferation. As in shown in
Reactive oxygen species (ROS) are considered as major mechanism responsible for immune suppressive activity of PMN-MDSC. We evaluated the level of ROS in LOX-1+ and LOX-1− PMN in patients with head and neck cancers (HNC) as follows. Samples of whole blood were collected from head and neck cancer patients. Red cells were lysed and PMN were labeled with CD15, LOX-1 antibodies, and with the cell permeant reagent 2′,7′-dichlorofluorescin diacetate (DCFDA). DCFDA is a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. After diffusion in to the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2′, 7′-dichlorofluorescein (DCF). DCF is a highly fluorescent compound which can be detected by fluorescence spectroscopy with maximum excitation and emission spectra of 495 nm and 529 nm respectively. As shown in the histograms of
LOX-1 is known to be cleaved from the surface of the cells and can be detected in sera of patients. We hypothesized that LOX-1 may be cleaved from PMN-MDSC and therefore, the presence of soluble LOX-1 (sLOX-1) may correlate with the amount of LOX-1− PMN-MDSC. Concentrations of sLOX-1 were measured in sera of 16 lung cancer patients and 6 colon cancer patients using ELISA. Samples of whole blood were collected; PBMC were purified using Ficoll gradient: and the proportion of PMN-MDSC out of total live PBMC was measured by flow cytometry using antibodies to CD11b, CD33, CD14, and CD15. The correlation between the presence of PMN-MDSC and soluble LOX-1 in sera of the lung cancer patients is shown in
Highly significant correlation between these two parameters was found (correlation of coefficient in lung cancer patients 0.65, p=0.007: in patients with colon cancer 0.98, p=0.0005).
The following Examples 8-13 employ one or more of these methods and materials:
Human Samples: Samples of peripheral blood and tumor tissues were collected from patients at Helen F. Graham Cancer Center and University of Pennsylvania. The study was approved by Institutional Review Boards of the Christiana Care Health System at the Helen F. Graham Cancer Center, University of Pennsylvania and The Wistar Institute. All patients signed approved consent forms. Peripheral blood was collected from:
All patients were either previously untreated or received treatment (chemotherapy or radiation therapy) at least 6 months before collection of blood. In some patients, tumor tissues were collected during the surgery. In addition, 6 patients with eosinophilic colitis, 3 patients with ulcerative colitis, and 8 patients with Crohn's disease were evaluated.
Peripheral samples of blood from 18 healthy volunteers 12 females, 6 males age 35-56 (median 42 years) were used as control.
Lung cancer tumor microarrays were produced from formalin-fixed paraffin embedded tissue. Each block was examined by a pathologist and three cores were obtained from tumor-containing areas and three blocks from non-tumor involved lung regions. Samples were obtained from 32 patients with adenocarcinoma. Clinical data obtained included tumor histology, size, stage, and time to recurrence (all patients followed for 5 years).
De-identified samples from normal colonic biopsies colon were obtained from St. Mark's Hospital, Harrow, UK. Samples were taken from patients after obtaining informed consent and with the approval of the Outer West London Research Ethics Committee (UK). Paraffin-embedded tissue blocks of samples of normal skin, lymph nodes, and melanoma were retrieved using an approved IRB protocol for de-identified archived skin biopsies through the Department of Dermatology NIH SDRC Tissue Acquisition Core (P30-AR057217), Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.
Cell isolation and culture: PMN-MDSC and PMN were isolated by centrifugation over a double density gradient Histopaque (Sigma) (1.077 to collect PBMC and 1.119 to collect PMN) followed by labeling with CD15-PE mAb (BD Biosciences) and then separated using anti-PE beads and MACS column (Miltenyi). Tissues were first digested with human tumor dissociation kit (Miltenyi) and then red blood cell lysed. Cells were then culture in RPMI (Biosource International) supplemented with 10% FBS, 5 mM glutamine, 25 mM HEPES, 50 μM β-mercaptoethanol and 1% antibiotics (Invitrogen). In some experiments, recombinant GM-CSF (Peprotech) was added to the culture media at a concentration of 10 ng/mL.
Isolation of LOX1+ PMN from peripheral blood and suppression assay: Whole blood was enriched for PMNs using MACSxpress® Neutrophil Isolation Kit (Miltenyi) following the protocol provided by the manufacturer. Cells were then labeled with anti-Lox1-PE mAb (Biolegend) and then separated using anti-PE beads and MACS column (Miltenyi). For the three-way allogenic MLR suppression assay, T lymphocytes from one healthy donor were purified using the Human CD3+ T Cell Enrichment Column Kit (R&D Systems) and used as responder cells. Dendritic cells were generated from adherent monocytes from another healthy donor in the presence of 25 ng/mL GM-CSF and 25 ng/mL IL-4 (Peprotech) for 6 days and used as stimulator cells. Responder and stimulator cells were then mixed at a 10:1 ratio followed by the addition of Lox1+ or Lox1− PMNs. T lymphocyte proliferation was assessed after 5 days of culture by thymidine incorporation.
Concurrently, T-lymphocytes were isolated from the PBMC of the same patient as LOX-1+ PMN using human CD3+ T Cell Enrichment Column Kit. PMNs were plated at different ratios with 105 T lymphocytes in a 96-well plate coated with 10 μg/ml anti-CD3 (clone UCHT1; BD Biosciences) followed by the addition of 1 μg/ml of soluble anti-CD28 (clone CD28.2; BD Biosciences). T lymphocyte proliferation was assessed after 3 days of culture by thymidine incorporation.
In some experiments, 1 μM N-acetyl cysteine (NAC; Sigma) or 20 μM of Nω-hydroxy-norarginine (nor-NOHA; Cayman Chemical) was added to the culture media to block ROS or agrinase 1 activity, respectively. T lymphocyte proliferation was assessed after 5 days of culture by thymidine incorporation.
In vitro PMN LOX-1 induction: PMNs from healthy donors were isolated on a Histopaque gradient. 5×105 cells/ml were cultured for 12 hrs with 10 ng/ml of GM-CSF in the presence of dithiothreitol (DTT) (0.5, 1, 2 mM: Sigma), tunicamycin (0.5, 1, and 2 μg/ml; Sigma-Aldrich), or thapsigargin (0.5, 1, and 2 M; Sigma). In some instances, 20 μM of the XBP-1 inhibitor BIO9 was added 3 hours prior to culture. Cells were then stained for flow cytometry or used for functional assays as described above.
Flow cytometry: Flow cytometry data were acquired using a BD LSR II flow cytometer and analyzed using FlowJo software (Tree Star). Immunofluorescent microscopy: Following deparaffinization and rehydration, heat induced antigen retrieval was performed using Tris-EDTA buffer pH 9. Followed by blocking with 5% BSA, tissues were stained with Lox1 antibody (Abcam: Cat no ab126538) and CD15 antibody (BD biosciences: Cat no 555400) at 1:200 dilution in 5% BSA each for 1 hour at room temperature. The following secondary antibodies were used Alexa Fluor anti-rabbit A647 (1:200 dilution in 5% BSA, Life technologies) for Lox1 and anti-mouse A514 (1:400 dilution in 5% BSA, Life technologies) for CD15 staining. CD15 staining was pseudo colored red and Lox1 staining was pseudo colored green. Nucleus was stained using DA PI (1:5000 dilution in PBS, Life technologies). Images were obtained using Leica TCS SP5 Confocal microscope. Cell counts from 16 frames were used to calculate counts per sq. mm.
Microarray analysis: For sample preparation and hybridization, total RNA from purified cells was isolated with TRIzol reagent according to the manufacturer's recommendations. RNA quality was assessed with a Bioanalyzer (Agilent). Only samples with RIN numbers>8 were used. Equal amount (400 ng) of total RNA was amplified as recommended by Illumina and was hybridized to the Illumina HumanHT-12 v4 human whole-genome bead arrays.
For data preprocessing, Illumina GenomeStudio software was used to export expression values and calculated detection p-values for each probe of each sample. Signal-intensity data were log 2 transformed and quantile-normalized. Only probes with a significant detection p-value (p<0.05) in at least one of sample were considered. The data was submitted to GEO and is accessible using accession number GSE79404. Differential expression for probes was tested using SAM (‘significance analysis of microarrays’) method55. Multiple groups were compared using “Multiclass” option and matched patient samples groups were compared using “Two sample paired” option. False discovery rate was estimated using Storey et. al procedure41. Genes with a false-discovery rate of <5% were considered significant unless stated otherwise.
Hierarchical cluster was performed using standardized Euclidean distance with average linkage. Genes that had GO annotation GO:0005886 (plasma membrane) and either GO:0004872 (receptor activity) or GO:0009897 (external side of plasma membrane) were considered as a candidate for a surface molecule marker. For expression heatmaps samples from the same patient were additionally normalized to the average between them, and samples from healthy donors were normalized to average between all patient samples.
Enrichment analyses were done using QIAGEN's Ingenuity Pathway Analysis software (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity). Pathway results with FDR<5% and p<10-5 were considered significant. Only regulators that passed p<0-8 threshold with significantly predicted (Z>2) activation state in PMN-MDSCs were reported.
For OLR1 gene expression association with cancer, Oncomine (https://www.oncomine.org) was used with “Cancer vs. Normal” gene report without any additional filters. Additionally, TCGA RNAseqV2 level 3 data (https://tcga-data.nci.nih.gov) was used and RPKM expression values were compared between cancer and normal tissues (where available) using t-test. Association with survival was done using univariate cox regression and Kaplan-Meier curves were plotted for patients split into two groups using median expression. Results with p<0.05 were considered significant.
qRTPCR: Total RNA was prepared with E.Z.N.A total RNA isolation Kit I (Omega Biotek) and cDNA was synthesized with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative RT-PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems). The relative amount of mRNA was estimated by the comparative threshold cycle method with GAPDH as reference gene. For the analysis of gene expression, the following primers of Table 2 were used:
Statistics: Statistical analysis was performed using a 2-tailed Student's t-test or Mann-Whitney test and GraphPad Prism 5 software (GraphPad Software Inc.), with significance determined at p<0.05.
To compare PMN-MDSC and PMN from PB of the same patients with non-small cell lung cancer (NSCLC) and head and neck cancer (HNC) we used dual-density Histopaque gradient, the standard method of isolation of PMN-MDSC28. Low density PMN-MDSC are co-purified with PBMC, whereas high density PMN are collected from lower gradient5. As a control, PMN from healthy donors were used. Both, low-density PMN-MDSCs and high-density PMN were purified further with CD15 magnetic beads to achieve similar high purity of both cell populations (data not shown).
A typical phenotype of PMN-MDSC and PMN was isolated from peripheral blood of cancer patients using gradient centrifugation and CD15 beads. Immune suppressive activity of PMN-MDSC, the main characteristics of these cells, was confirmed in allogeneic mixed leukocyte reaction (MLR) (
To study overall differences and similarities between patients' PMN and PMN-MDSC as well as PMNs from healthy donors, we performed whole-genome analysis using Illumina HumanHT-12 v4 bead arrays.
A relative expression heatmap and gene/sample clustering was generated based on expression of 985 genes significantly differentially expressed (p<0.05, fold>2) between cancer patients' PMN, PMN-MDSCs and PMN of healthy donors (data not shown). Hierarchical clustering of the samples using expression of the 985 most differentially expressed genes revealed that PMN-MDSC samples have a unique expression profile and a distinct genetic signature. PMN from cancer patients are very similar to healthy donor PMN samples, as they grouped within the same cluster for HNC and NSCLC patients (
Specifically, of the 985 genes different between any pair of groups (see Table 1), the majority (74%) showed significant differences (false discovery rate, FDR<5%) between patients' PMN-MDSC and PMN, while no genes were significantly different when corrected for multiple testing (best FDR=19%) between PMN from healthy donors and PMN from cancer patients, with only 12% of the genes significantly different at nominal p<0.05. This result indicates a high similarity of PMN samples between cancer patients and healthy donors. The direct pair-wise comparison identified 1870 array probes significantly differentially expressed (FDR<5%) between PMN-MDSC and PMN in the same patients and 36 probes showed difference of at least 5-fold. See,
Using Ingenuity Pathway Analysis, we identified 14 pathways significantly enriched in PMN-MDSCs, including eukaryotic Translation Initiation Factors 2 and 4 (eIF2 and eIF4) pathways and mTOR signaling, as shown in the following Table 3.
4E−46
5E−16
The regulators of genes enriched in PMN-MDSC included regulators of ER stress response, MAPK pathway, CSF1, IL-6, IFN-γ, NF-κB. These molecules were previously directly implicated in MDSC biology, primarily PMN-MDSC, as discussed in Reference 4, incorporated herein by reference. Surprisingly, one of the most significant changes was associated with low density lipoprotein (LDL) as shown in Table 4 below. Table 4 lists the upstream Regulators identified by Ingenuity Pathway Analysis (IPA) among genes significantly differentially expressed between PMN-MDSC and PMN cells. N=number of genes from the category, Z=z-score of predicted activation state calculated by IPA.
Thus. PMN-MDSC had a distinct genomic profile from PMN isolated from the same cancer patients and PMN from healthy donors. Genes associated with ER stress response were among the most up-regulated in PMN-MDSC.
To search for potential markers of PMN-MDSC we evaluated differentially expressed genes, which encoded surface molecules and compared expression of various surface molecules between PMN-MDSC and PMN from the same patients and PMN from healthy donors. More than 20 genes encoded surface molecules were found to be differentially expressed in PMNMDSC and PMN (
We evaluated LOX-1 expression in high density PMN and low-density PMN-MDSC in cancer patients. LOX-1 was practically undetectable in PMN but expressed in about ⅓ of PMN-MDSC fraction (
These results suggested that LOX-1 could be associated with PMN-MDSC. We asked whether LOX-1 can be a marker of PMN-MDSC. To test this hypothesis, it was important to avoid the use of gradient centrifugation and labeled cells in PB directly with granulocyte-specific CD15 antibody and evaluated expression of LOX-1 among all CD15+ cells. In preliminary experiments, we found no differences in the results obtained with CD15 or CD66b antibodies. We referred to CD15+ cells as PMN since Siglec-8+ eosinophils represented very small proportion of CD15+ cells and no differences in the presence of eosinophils between CD15+LOX-1+ and CD15+LOX-1− cells was seen (data not shown). The proportion of LOX-1+ cells among all PMN in healthy donors was very low (range 0.1-1.5, mean 0.7%). In patients with NSCLC it increased to 4.9% (p<0.001), in patients with HNC to 6.4% (p<0.0001), and in patients with colon cancer (CC) to 6.5% (p=0.0035) (
We also assessed the changes in LOX-1+ PMN in tumor-free patients with inflammatory conditions: eosinophilic esophagitis, ulcerative colitis and Crohn's disease. Only patients with Crohn's disease had a small increase in the proportion of these cells (
Next, we addressed the question whether LOX-1 can be considered as marker of human PMN-MDSC. LOX-1+ and LOX-1− PMN were sorted directly from PB of the same patients. LOX-1− PMN had the typical morphology of mature neutrophils, whereas LOX-1+ PMN displayed more immature morphology with band shape nuclei (data not shown). Whole genome array was performed on LOX-1+ and LOX-1− PMN and compared with that of PMN and PMN-MDSC. Analysis of gene expression revealed 639 genes significantly different between LOX-1+ and LOX-1− (FDR<5%, fold>2) and based on expression of those genes LOX-1+ PMN clustered together with PMN-MDSC, whereas LOX-1− PMN were very similar to patients' and healthy donor's PMN (
The hallmark of PMN-MDSC is their ability to suppress T-cell function. We isolated LOX-1− and LOX-1+ PMN directly from PB of cancer patients and used them in T-cell suppression assay. LOX-1+ PMN suppressed T-cell proliferation, whereas LOX-1− PMN did not (
We then evaluated possible mechanisms responsible for LOX-1+ PMN-MDSC suppression. We tested several common mechanisms implicated in PMN-MDSC function. LOX-1+ PMN-MDSC had significantly higher production of reactive oxygen species (ROS) than LOX-1− PMN (
Then, we investigated the possible role of LOX-1 as marker of mouse PMN-MDSC. Similar to human PMN, CD11b+Ly6CloLy6G+ mouse PMN had very low expression of LOX-1. However, in contrast to human PMN-MDSC, spleen, BM, or tumor PMN-MDSC from mice bearing EL-4 lymphoma, Lewis Lung Carcinoma (LLC) or transgenic Ret melanoma did not up-regulate LOX-1 (data not shown). To evaluate the possible role of LOX-1 in PMN-MDSC function we used bone marrow (BM) cells from LOX-1 knockout (olr1−/) mice31. Lethally irradiated wildtype recipients were reconstituted with congenic bone marrow cells isolated from wild-type or olr1−/− mice. Ten weeks after reconstitution donor's cells represented more than 95% of all myeloid cells. LLC tumor was implanted s.c. and mice evaluated 3 weeks later. No differences in the presence of PMN-MDSC in spleens or tumors were observed between mice reconstituted with WT and LOX-1 KO BM (data not shown). WT and olr1−/− PMN-MDSC suppressed T-cell proliferation equally well (data not shown). Gene expression profile demonstrated no differences between WT and olr1−/− PMN-MDSC. Most importantly WT PMN-MDSC had the same undetectable level of olr1 expression as olr1−/− PMN-MDSC (data not shown). Thus, in contrast to humans, mouse LOX-1 is not associated with PMN-MDSC.
What could induce LOX-1 up-regulation in PMN-MDSC? Based on the fact that in endothelial cells LOX-1 can be induced by pro-inflammatory cytokines35, we tested the effect of several cytokines as well as tumor-cell conditioned medium (TCM) on LOX-1 expression in PMN isolated from healthy donors. None of the tested pro-inflammatory cytokines (IL-1β, TNFα, IL-6) or TCM induced upregulation of LOX-1 in PMN after 24 hr culture (GM-CSF was added to protect PMN viability) (
Our previous observations5 and data obtained in this study demonstrated that PMN-MDSC in cancer patients displayed signs of ER stress response. LOX-1+ and LOX-1− PMN were isolated from PB of cancer patients and expression of genes associated with ER-stress were evaluated. LOX-1+ PMN-MDSC had significantly (p<0.001) higher expression of sXBP1 (
Overnight THG treatment of PMN also caused acquisition of potent immune suppressive activity by the PMN (
It is known that LOX-1 is shed from the surface of the cells and can be detected in plasma39. We evaluated correlation between the presence of PMN-MDSC in cancer patients and soluble LOX-1 in plasma. In NSCLC and CC patients, the proportion of PMN-MDSC strongly correlated with soluble LOX-1 (
There is now sufficient evidence demonstrating that tumor MDSC are more suppressive than cells in PB21. We asked whether the population of PMN-MDSC is more prevalent among all PMN in tumors than in PB. The proportion of LOX-1+ cells in CD15+ PMN isolated from tumors of patients with HNC and NSCLC was >3-fold higher than in CD15+ PMN from PB of the same patients (p<0.001) (
To evaluate the presence of LOX-1+ PMN-MDSC in tumor tissues, we have developed a method of immune fluorescent staining of paraffin-embedded tissues with combination of LOX-1 and CD15 antibody (data not shown). Control tissues from normal skin, colon and lymph nodes had similar low numbers of LOX-+CD15+ PMN-MDSC (
Using ONCOMINE and TCGA databases we evaluated the association of OLR1 expression in tumor tissues with clinical parameters in different types of cancer. Significant upregulation of OLR1 was observed in many types of cancer. As shown in Table 5, there is a clinical association of OLR1 expression and LOX-1+ PMN-MDSC accumulation in cancer patients. The following Table 5 shows the number of independent data sets from Oncomine database that showed OLR1 upregulated (up) or downregulated (down) in Cancer vs Normal tissues. P-value and fold change for Cancer/Normal comparison from TCGA database. na=data not available:
The notable exception was lung cancer, where normal lung tissues showed dramatically higher expression of OLR1 than other normal tissues, apparently due to cells with high expression of OLR1 (possibly lung epithelium). OLR1 expression positively correlated with clinical stage in patients with bladder cancer, colon adenocarcinoma, and clear cell kidney cancer. The positive correlation with tumor size was found in patients with prostate adenocarcinoma and rectal adenocarcinoma (data not shown). Higher expression of OLR1 was associated with worse survival in patients with HNC (
Although these results are suggestive, their interpretation as reflecting PMN-MDSC presence has some limitation due to the fact that OLR1 can be expressed on different cells in the tumor microenvironment. We focused on evaluation of LOX-1+ PMN-MDSC in tumor tissues and PB.
In patients with NSCLC we evaluated the possible link between stage of the disease and the proportion of LOX-1+ PMN-MDSC in PB. Patients with both early (I/II) and late (III/IV) stages of NSCLC had significantly higher proportion of LOX-+ PMN-MDSC than in healthy donors (p<0.01 and p<0.0001 respectively). There was no statistical significant difference between these two groups of patients (
Significant association of the presence of LOX-1+ PMN in PB of cancer patients was found with size of the tumors. Only patients with large tumors (T2-T3) had significantly (p<0.001) higher proportion of LOX-1+ PMN than healthy donors, whereas patients with small tumors (T1) had similar very low level of LOX-+ PMN as healthy donors. Patients with large tumors had significantly more LOX-+ PMN (p<0.05) than patients with small tumors (
As revealed by these examples, PMN-MDSC have a unique gene expression profile, which is substantially different from that of PMN from the same patients and from healthy donors. This directly supports that PMN-MDSC represent a distinct functional state of pathological activation of neutrophils in cancer15,29 and is consistent with the analysis of gene expression performed in mice, which demonstrated differences in transcriptome between granulocytes isolated from naïve mice and PMN-MDSC from tumor-bearing mice13.
Up-regulation of genes associated with ER stress response was one of the most prominent features of PMN-MDSC. The ER stress response is developed to protect cells from various stress conditions including hypoxia, nutrient deprivation, low pH, etc. and includes three major signaling cascades initiated by three protein sensors: PERK (protein kinase RNA (PKR)-like ER kinase), IRE-1 (inositol-requiring enzyme 1) and ATF6 (activating transcription factor 6)17. PERK phosphorylates eukaryotic protein synthesis initiation factor 2 alpha (eIF2α), which controls the initiation of mRNA translation and inhibits the flux of synthesized proteins. eIF2α induces the expression of ATF4 and its downstream targets, including the pro-apoptotic transcription factor CHOP. IRE1 cleaves the mRNA encoding for the transcription factor X-box binding protein-1 (XBP1)38. Spliced XBP1 (sXBP1) mRNA is then ligated by a RNA ligase and translated to produce sXBP1 transcription factor that regulates many target genes including SEC61a3.
Factors implicated in LOX-1 up-regulation include Angiotensin II (Ang II), C-reactive protein (CRP), Endothelin-1 (ET-1), Glucose, Histamine, Homocysteine, Human cytomegalovirus (HCMV), Interferon-γ (IFN-γ), Interleukin-1β (IL-1β), Oxidant species, Oxidized-low density lipoprotein (ox-LDL), Phorbol ester, Shear stress, Transforming growth factor-β (TGF-β) and Tumor necrosis factor-α (TNF-α).
ER stress response was previously shown to be transmitted to dendritic cells and macrophages from tumor cells and was associated with up-regulation of arginase-1 in macrophages25,26,27. Constitutive activation of XBP1 in tumor-associated dendritic cells promoted ovarian cancer progression by blunting anti-tumor immunity48. We have recently found activation of ER stress response in MDSC5. We demonstrated that MDSC isolated from tumor-bearing mice or cancer patients overexpressed sXBP1 and CHOP, and displayed an enlarged endoplasmic reticulum, one of the hallmarks of the ER stress5. Other study implicated CHOP in the suppressive activity of MDSC in tumor site46. Consistent with these observations administration of an ER stress inducer to tumor-bearing mice increased the accumulation of MDSC and their suppressive activity22.
We have discovered that expression of LOX-1 receptor was associated with PMN-MDSC. LOX-1 is a class E scavenger receptor expressed on macrophages and chondrocytes, as well as endothelial and smooth muscle cells45. Expression of this receptor on neutrophils previously was not described. We have found that neutrophils from healthy donors and cancer patients have practically undetectable expression of LOX-1. Our data indicated that LOX-1 expression is not just associated with, but actually defines, the population of PMN-MDSC in cancer patients. This is supported by several lines of evidence. LOX-1+ PMN had a gene expression profile similar to that of enriched PMN-MDSC isolated using gradient centrifugation.
In contrast, LOX-1− PMN had a profile similar to neutrophils. LOX-1+ but not LOX-1− PMN potently suppressed T-cell response. Finally, LOX-1+ PMN had significantly higher expression of ARG1 and production of ROS, typical characteristics of PMN-MDSC. We found that in tumor tissues, only LOX-1+ PMN were immune suppressive and could be considered as PMN-MDSC. This permits a direct identification of PMN-MDSC in PB and tumor tissues.
These observations, although unexpected, fit the overall concept of a critical role of ER stress response in MDSC biology. It was recently demonstrated that in human endothelial cells oxLDL induced expression of LOX-1 through activation of ER stress sensors IRE1 and PERK19. Ox-LDL induces LOX-1-dependent ER stress19. In contrast, ER stress induced by tunicamycin in hepatic L02 cells caused down-regulation of LOX-1. Knock down of IRE1 or XBP-1 restored LOX-1 expression in these cells18.
It is likely that signaling through LOX-1 is responsible for, or at least contributes to, acquisition of immune suppressive activity by neutrophils. Engagement of LOX-1 can lead to induction of oxidative stress, apoptosis, and activation of the NF-κB pathway1. These pathways are known to be important for PMN-MDSC function. The ER stress response pathway has been shown to regulate inflammation by activating the NF-κB pathway3,2,54. LOX-1 up-regulation has been observed during cellular transformation into cancer cell and can have a pro-oncogenic effect by activating the NF-κB pathway, by increasing DNA damage through increase ROS production and by promoting angiogenesis and cell dissemination16,24. It is possible that LOX-1 signaling may drive pathological activation of PMN towards PMN-MDSC. Cell surface LOX-1 expression can be elevated by multiple stimuli including reactive oxygen species (ROS), inflammatory cytokines (TNF-α, TGF-β) as well as oxLDL49. These factors are produced in cancer and it is possible that they can affect differentiation of granulocytes from precursors leading to acquisition of LOX-1 expression.
Our data demonstrated that patients have variable amount of LOX-1+ PMN-MDSC, which at least in patients with NSCLC was associated with size of the tumors. It is theorized that the presence of these cells in tumor tissues can predict clinical outcome. Expression of LOX-1 on PMN-MDSC opens an opportunity for selective targeting of these cells, since antibody targeting LOX-1 have been already tested in cardiovascular diseases in mice7,30. Average expression values (FPKM values) for cancer and normal tissues for different cancers indicates high baseline expression level of OLR1 in normal lung tissues, as shown in Table 6.
Specific embodiments of the methods and compositions described herein include:
A method for monitoring the population of polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) in a mammalian subject comprising: contacting a biological sample from the subject containing polymorphonuclear neutrophils (PMNs) and PMN-MDSC with a ligand that specifically binds or forms a complex with LOX-Ion the cell surface; and detecting and distinguishing the complexes of ligand-bound LOX-1-cells from other cells not bound to the ligand in the sample wherein the LOX-1-bound cells are PMN-MDSCs substantially free of PMN. Also included in an embodiment of the method further comprising counting the cells bound to the ligand to obtain a LOX-1+ population. Also included in an embodiment of the method wherein said ligand is an anti-LOX-1 antibody, an anti-LOX-1 antibody fragment, optionally associated with a detectable label component. Also included in an embodiment of the method further comprising contacting the sample with a ligand that specifically binds or forms a complex with a neutrophil biomarker to identify PMN in the sample.
In certain embodiments of these methods, the neutrophil biomarker is CD15 or CD66b. In certain embodiments of these methods, the ligand is an anti-CD15 antibody, an anti-CD15 antibody fragment, optionally associated with a detectable label component. In other embodiment of these methods, the ligand is an anti-CD66b antibody, an anti-CD66b antibody fragment, optionally associated with a detectable label component.
Another embodiment of the method employs the ligand immobilized on a substrate or associated with a detectable label component. In such embodiments, the detectable label component is independently detectable or is capable of generating a measurable detectable signal when contacted with another label component. In certain embodiments, the separating step comprises washing the unbound cells and other debris in the sample from the substrate and counting or collecting the bound PMN-MDSCs from the substrate. In other embodiments, the separating step comprises treating the sample with a reagent which identifies LOX-1-PMN-MDSC complexes from unbound cells to permit enumeration of PMN-MCSC. Other specific embodiments comprise separating bound cells from unbound cells in the sample based on size exclusion.
In still other embodiments, the method further comprises a step of contacting the sample with biomarkers that identify as a single population both PMN-MDSCs and PMNs and isolating a cell suspension containing both PMN-MDSCs and PMNs prior to contacting the cell suspension with the LOX-1 ligand.
In other specific embodiments of these methods, the biological sample is whole blood and the method further comprises destroying or lysing any red blood cells in the sample. In yet other embodiments, the methods involve collecting a second population which is not immobilized on the substrate, this second population containing PMNs and being substantially free from PMN-MDSCs.
Another specific embodiment is a method of differentiating polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) from polymorphonuclear neutrophils (PMNs) in a biological sample containing both types of cells comprising: contacting the sample with a ligand that specifically binds or forms a complex with LOX-Ion the cell surface; and detecting and separating the complexes of ligand-bound LOX-1-cells from other cells not bound to the ligand in the sample, wherein the LOX-1-bound cells are PMN-MDSCs substantially free of PMN.
Still another specific method is designed for obtaining a population of cells enriched in human polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) and comprises isolating from a cell suspension those cells which express LOX-1 to provide a population of cells enriched with PMN-MDSCs. In certain embodiment, these methods also comprise detecting a population of LOX-1+ cells greater than 1% of the total neutrophil population in the sample of a subject, wherein said population of LOX-1+ cells indicates the presence, progression or metastasis of a cancer. Thus, other embodiments involve measuring the concentration of soluble LOX-1 in the serum of the subject and correlating that concentration with the concentration of PMN-MDSC in the subject.
Other embodiments involve a composition comprising a ligand that specifically binds or forms a complex with LOX-1 on the cell surface for use in detecting or obtaining a population of human polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) and diagnosing the presence, progression or metastasis of a cancer.
In still other embodiments, any of these methods can further comprise contacting the sample with a reagent that identifies activators or regulators of ER stress response in said cells or a reagent that identifies other biomarkers that distinguish PMN-MDSC from PMN. In certain embodiment, the activators are one or more of sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI or NOS-2. In other embodiments, the regulators or biomarkers are one or more of one or more of MYCN, CSF3. IL3. TGFβ1, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, Nek, IL13, AGT, IL1β, ERBB2, MAP2K1, VEGFα, CSF1, FLI1, or Fin or the biomarkers of Table 1,
In still other specific embodiments, a composition comprises a ligand that specifically binds or forms a complex with LOX-1 on the cell surface for use in detecting or obtaining a population of human polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs) and diagnosing the presence, progression or metastasis of a cancer.
In yet other embodiments, a pharmaceutical composition that reduces or inhibits ER stress in mammalian neutrophils or reduces or inhibits LOX-1 expression on neutrophil populations in a pharmaceutically acceptable carrier or excipient is provided. In some embodiment, such a composition comprises an antagonist or inhibitor of the expression, activity or activation of one or more of sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI or NOS-2. In other embodiments, such a composition contains an antagonist or inhibitor of LOX-1. In other embodiments, such a composition contains an antagonist or inhibitor of the expression, activity or activation of one or more of MYCN, CSF3, IL3, TGFβ1, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, Nek, IL13, AGT, IL1β, ERBB2, MAP2K1, VEGFα, CSF1, FL11, or Fin. In any of these compositions, the antagonist or inhibitor is an antibody, functional antibody fragment, single chain antibody, or equivalent.
In yet another specific embodiment a method for reducing or inhibiting LOX-1+ PMN-MDSC accumulation in a cancer patient comprises administering a composition as described herein.
In another specific embodiment, a method of diagnosing a mammalian subject with a cancer comprises obtaining a biological sample from the subject: detecting whether soluble LOX-1 is present in the sample by contacting the sample with an antibody or functional antibody fragments that specifically binds or forms a complex with LOX-1 on the cell surface; detecting and distinguishing the complexes of antibody-bound LOX-1-cells from other cells not bound to the antibody in the sample, and correlating the size of a tumor in the subject with the number of LOX-+ PMN or PMN-MDSC detected.
Yet another method of treating a cancer comprises obtaining a biological sample from a subject; detecting whether PMN-MDSC are present in the sample; diagnosing the subject with cancer when the presence of LOX-1+ is detected at a level that indicates PMN-MDSC are present: and administering an effective amount of a composition that reduces or inhibits ER stress response in mammalian LOX-1+ neutrophils, LOX-1+ PNM or PMN-MDSC or reduces or inhibits LOX-1 expression on LOX-1+ neutrophils. LOX-1+ PNM or PMN-MDSC.
Yet another method of treating a cancer comprises the use of immunotherapeutics. The patient having cancer is administered an antibody or functional antigen-binding fragment that binds to LOX-1. In another embodiment, a method of treating cancer involves administering an antibody or functional antigen-binding fragment that inhibits that inhibits the expression, activity or activation of at least one of sXBP1, DDIT3 (CHOP), ATF4, ATF3, SEC61A ARGI, MYCN, CSF3, IL3, TGFβ1, TNF, LDL, RAF1, APP, IL6 PDGFBB, EPO, CD40LG, Nek, IL13, AGT, IL1β, ERBB2, MAP2K1, VEGFα, CSF1, FLI1, or Fin. In another embodiment the method involves inhibiting the expression, activity or activation of one or more of the biomarkers of
Each and every patent, patent application and any document listed herein, particularly references 4, 6, 7, 21, 28 and 29, and the sequence of any publically available nucleic acid and/or peptide sequence cited throughout the disclosure, is/are expressly incorporated herein by reference in its entirety. Embodiments and variations of this invention other than those specifically disclosed above may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include such embodiments and equivalent variations.
This application is a continuation of pending U.S. patent application Ser. No. 15/668,867, filed Aug. 4, 2017, which claims the benefit of the priority of US Provisional Patent Application No. 62/371,493, filed Aug. 5, 2016, which applications are incorporated herein by reference.
This invention was made with government support under grant numbers. CA084488, CA100062 and CA010815, awarded by the National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62371493 | Aug 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15668867 | Aug 2017 | US |
| Child | 16943947 | US |
