WO2013011513A1 - Methods of determinig enzyme activity in preserved cell samples - Google Patents

Methods of determinig enzyme activity in preserved cell samples Download PDF

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Publication number
WO2013011513A1
WO2013011513A1 PCT/IL2012/050258 IL2012050258W WO2013011513A1 WO 2013011513 A1 WO2013011513 A1 WO 2013011513A1 IL 2012050258 W IL2012050258 W IL 2012050258W WO 2013011513 A1 WO2013011513 A1 WO 2013011513A1
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Prior art keywords
cells
cell
cancer
preserved
enzyme
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PCT/IL2012/050258
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French (fr)
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WO2013011513A9 (en
Inventor
Yosi SHACHAM-DIAMAND.
Sefi Vernick
Yaron NIV
Amihay Freeman
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Ramot at Tel- Aviv University Ltd.
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Publication of WO2013011513A1 publication Critical patent/WO2013011513A1/en
Publication of WO2013011513A9 publication Critical patent/WO2013011513A9/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention in some embodiments thereof, relates to methods of determining enzyme activity in preserved cell samples.
  • Cancer is responsible for the majority of morbidity and mortality worldwide, despite recent advances in medical technology.
  • Current therapeutic strategies focus predominantly on achieving the removal or death of cancer cells within the patient, through a diverse array of surgical and non-surgical techniques; the most widely used are chemotherapy and gamma irradiation.
  • Those methods have a number of prominent disadvantages, in particular the culling of healthy cells/tissues within the patient, and the rather toxic side-effects of the current generation of chemotherapeutic drugs utilized in cancer treatment.
  • Differentiation therapy is an alternative approach which promotes reversion of phenotype from malignant to normal.
  • Differentiation therapy is based on the concept that cancer cells are normal cells that have been arrested at an immature or less differentiated state, lack the ability to control their own growth, and thus multiply at an abnormally fast rate.
  • Differentiation therapy aims to force the cancer cell to resume the process of maturation. Although differentiation therapy does not kill the cancer cells, it restrains their growth and allows the application of more conventional therapies (such as chemotherapy) to eradicate the remaining malignant cells.
  • Differentiation therapy has a number of advantages over conventional therapeutic strategies that target death of cancer/tumor cells.
  • the culling of healthy cells/tissues within the patient with chemotherapeutic drugs or gamma irradiation would be eliminated, together with their associated adverse side-effects.
  • the killing of cancer cells through gamma irradiation or chemotherapeutic eliminates most, but not completely all cancer cells within the patient, thereby leading to remission of the disease.
  • differentiation therapy it is speculated that by inducing some of the cancer cells into the pathway of terminal differentiation and eventual senescence, this would somehow signal other cancer cells to follow suit through a variety of mechanisms.
  • alkaline phosphatase a marker that has been used as a means of monitoring patients undergoing differentiation therapy.
  • One such marker is alkaline phosphatase, wherein expression of same was shown to correlate with the differentiation status of a cell [Patnaik A, et al., Clin. Can Research, 2002 Jul;8(7):2142-8; Rephaeli A, Zhuk R, Nudelman A, 2000, Drug Develop. Res. Vol 50:379-391].
  • U.S. Pat. App. No. 20060100488 teaches detection of cancerous cells by directly monitoring the electrical response of the cells following application of an alternating current.
  • WO 91/15595 teaches analysis of electrical conductivity of cancer cells for monitoring responsiveness to therapy and drug screening. Specifically, WO 91/15595 teaches monitoring the effectiveness of a particular agent to inhibit increases in the volume and number of cancer cells by analyzing electrical conductivity thereof. Accordingly, both these patent applications teach that the intrinsic electrical properties of a cancer cell may be used as markers for detection and monitoring of cancer cells.
  • U.S. Pat. Appl. No. 20040053425 teaches amperometric analysis of an analyte in a fluid, wherein the electrode comprises the current producing enzyme.
  • U.S. Pat. Appl No. 20040053425 does not teach amperometric detection of intracellular markers.
  • U.S. Pat. No. 5,149,629 teaches amperometric analysis of markers, including cancer cell markers, wherein the electrode comprises antibodies capable of binding the markers thereto. The analysis is by substrate competition. U.S. Pat. No. 5,149,629 does not detect endogenous amperometric features of cancer cells.
  • a method of monitoring an anti cancer treatment in a subject comprising:
  • step (b) detecting a presence or level of cancer cells in a sample of the subject following step (a) according to the method of the present invention, wherein the presence or level is indicative of a state of the cancer.
  • a method of determining an anti-cancer treatment for a subject comprising:
  • a method of identifying an agent capable of reversing a malignant phenotype of a cell comprising,
  • the enzyme is selected from the group consisting of beta-galactosidase, alkaline phosphatase, secreted alkaline phosphatase (SEAP), glucose oxidase, chloramphenicol acetyl transferase (CAT) and beta-glucoronidase.
  • the substrate is selected from the group consisting of p-aminophenyl-.beta.-D- galactopyranoside (PAPG), p- aminophenol-phosphate (PAPP), glocuse-6-phosphate and chloramphenicol.
  • the preserved cells are comprised in a tissue slice.
  • the preserved cells were fixed in a fixative for at least one week.
  • the method comprises repeating step (a) following step (b).
  • the measuring is performed using means for high output.
  • the means is selected from the group consisting of an automated sampling device, a liquid handling equipment, a dispenser, an electrode array, a robot, or any combination thereof.
  • the sample comprises between 10-500 cells.
  • the tissue slice is about 2 mm 3 .
  • the preserved cells are derived from a suspected malignant polyp of a colon or an adenomatous of a colon.
  • the preserved cancer cells are derived from a malignant polyp of a colon.
  • the enzyme is alkaline phosphatase.
  • the substrate is 4- aminophenyl phosphate (p-APP).
  • the cells comprise colon cells.
  • the measuring is effected using an electrochemical cell.
  • the measuring is effected in a multiwell array.
  • each well of the multiwell array comprises an electrochemical cell.
  • each well of the multiwell array is a nano-volume well.
  • the agent comprises a test composition.
  • the test composition is selected from the group consisting of a polynucleotide a polypeptide, a small molecule chemical, a carbohydrate, a lipid and a combination of same.
  • the agent comprises a test condition.
  • the test condition is a radiation condition.
  • the cells are intact.
  • the cells are mammalian cells.
  • a fixative for the preserved cells comprises formaldehyde.
  • FIG. 1 is a graph illustrating chrono-amperometric measurement of biopsies removed from HCT116 tumors and from healthy colon tissue, and suspended in formalin for a period of two months. Current signals correspond to enzymatic activity of alkaline phosphatase reflecting cell differentiation state. Measurements were performed with in-house fabricated silicon based chips
  • FIG. 2 is a graph illustrating the average current signal and standard deviation obtained 800 seconds and 1500 sec (for tumor average currents and healthy colon average currents, respectively) following substrate addition.
  • FIG. 3 is a bar graph illustrating chrono-amperometric measurement of polyp biopsies of healthy and neoplastic tissue removed from a human patient.
  • FIG. 4 is a bar graph illustrating chrono-amperometric measurement of polyp biopsies of healthy and neoplastic tissue removed from a human patient.
  • FIGs. 5A-B are a bar graphs illustrating chrono-amperometric measurement of polyp biopsies of healthy and neoplastic tissue removed from two human patients.
  • the present invention is of a method of determining enzyme activity in preserved cell samples. Specifically, the present invention can be used detect cancer in tumor biopsies preserved in a fixative such as formaldehyde. The method may be used to diagnose cancer, monitor treatment, determine treatment regimens and develop novel treatment modalities for the disease.
  • hydrolases such as acid and alkaline phosphatase, b-glucoronidase, catalase, ATPase, ADPase, PNPase, BGPase only retain 25-47 % of their enzymatic activity following 2- 24 hours of fixation (22-27).
  • the presently described methods may be used to determine enzyme activity in preserved cellular samples. Using the presently described methods, the inventors have shown that it is possible to provide accurate diagnostic results within a few minutes from formalin preserved biopsy slices by determining alkaline phosphatase activity in suspected malignant cells.
  • a method of determining enzyme activity in preserved cells comprising:
  • the enzyme is one which catalyzes a reaction giving rise to a product which is permeable or which can be transported through the cell membrane and which can then undergo redox reaction at one of the electrodes of the electrochemical cell.
  • Example of such enzymes include beta-galactosidase, which can catalyze a reaction in which the p-aminophenyl-beta-D- galactopyranoside (PAPG) is convened into p- aminophenol (PAP). PAP can be transported through the cell membrane and then oxidized at the electrode.
  • PAPG p-aminophenyl-beta-D- galactopyranoside
  • PAP p- aminophenol
  • enzymes and relevant substrates include; The enzyme alkaline phosphatase (AP) or the enzyme secreted alkaline phosphatase (SEAP) with the substrate PAPP (p-aminophenol-phosphate); the enzyme glucose oxidase with the substrate e glocuse-6-phosphate, the enzyme chloramphenicol acetyl transferase (CAT) and the substrate chloramphhenicol, the enzyme b-glucuronidase and any glycosaminoglycans or other glycoconjugates that after the removal of the b-glucorunic acid residue become electrochemicaly active.
  • AP alkaline phosphatase
  • SEAP enzyme secreted alkaline phosphatase
  • PAPP p-aminophenol-phosphate
  • glucose oxidase with the substrate e glocuse-6-phosphate
  • CAT chloramphenicol acetyl transferase
  • CAT chloramphenicol acetyl transferase
  • the cells may be from any source including freshly isolated cells, ex vivo cells; part of a cell line; stem cells, ex vivo differentiated stem cells.
  • the cells may be eukaryotic (e.g. mammalian, plant, insect) or prokaryotic e.g. bacterial.
  • the cells may be derived from a healthy subject (i.e. non-diseased cells) or from a diseased subject (i.e. diseased cells), as further described herein below.
  • the enzyme is a marker for a particular disease.
  • the disease may be cancer and the enzyme alkaline phosphatase.
  • a method of detecting cancerous cells comprises:
  • detecting refers to the act of detecting, diagnosing, perceiving, uncovering, exposing, visualizing or identifying a cell.
  • the term "cell” refers to a mammalian cell, preferably a human cell.
  • Single cells may be used in accordance with the teachings of the present invention as well as plurality of cells.
  • the plurality of cells comprises no less than 10 cells and no more than 500 cells.
  • tissue slices are analyzed.
  • the cells that are analyzed are suspected of being malignant.
  • the cells may be obtained from a colon biopsy (e.g. from a malignant colon polyp or a colon adenomatous) and the enzyme which is assayed is alkaline phosphatase.
  • Analysis of placental alkaline phosphatase in fixed testicular tissue may be performed as part of a diagnosis of testicular cancer.
  • a down- regulation in an activity of the alkaline phosphatase is indicative of testicular cancer.
  • Analysis of acid phosphatase in fixed biopsies of the prostate or bladder may be performed as part of a diagnosis of cancer.
  • a down-regulation in an activity of the alkaline phosphatase is indicative of the cancer.
  • the cells that are analyzed are liver cells (e.g. a fixed liver tissue).
  • An increase or decrease in a level of alkaline phosphatase may be indicative of a liver disease.
  • the cells that are analyzed are cells of a fixed stomach tissue biopsy cells.
  • An increase or decrease in a level of acid phosphatase may be indicative of a gastric cancer or other diseases related to the stomach.
  • the cells are not peripheral blood cells.
  • the plurality of cells may be from any biological sample such as cell-lines, primary cultures and cellular samples, e.g. biopsies (surgical biopsies including incisional or excisional biopsy, fine needle aspirates and the like), complete resections or body fluids. Methods of biopsy retrieval are well known in the art.
  • a tumor sample is sliced.
  • the tumor slices may be weighed.
  • the cells in the biological sample are preferably intact (i.e. whole).
  • the cells in the biological sample are assayed for a functional enzyme following pre-treatment with a preservative e.g. a fixative.
  • a preservative e.g. a fixative
  • the fixative is a chemical fixative.
  • An exemplary chemical fixative is a crosslinking fixative.
  • fixatives include formaldehyde (e.g. a solution comprising 3-5 % formaldehyde) and glutaraldehyde
  • An exemplary fixative solution contemplated by the present invention is one which is a 10 % Neutral Buffered Formalin (NBF), that is approximately 3.7 % formaldehyde in phosphate buffered saline.
  • NBF Neutral Buffered Formalin
  • fixatives include precipitating fixatives, including, but not limited to ethanol, methanol and acetone; oxidizing agents, including but not limited to osmium tetroxide, Potassium dichromate, chromic acid, and potassium permanganate; Mercurials such as B-5 and Zenker's; Picrates; Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE).
  • precipitating fixatives including, but not limited to ethanol, methanol and acetone
  • oxidizing agents including but not limited to osmium tetroxide, Potassium dichromate, chromic acid, and potassium permanganate
  • Mercurials such as B-5 and Zenker's
  • Picrates Picrates
  • the present invention also contemplates other forms of fixation such as frozen sections and heat fixation.
  • the cell sample may remain in the chemical fixative for one day, one week, one month, two months, three months or even longer.
  • more than 50 % of the enzymes in the cell sample are nonfunctional.
  • more than 60 % of the enzymes in the cell sample are non-functional.
  • more than 70 % of the enzymes in the cell sample are non-functional.
  • more than 80 % of the enzymes in the cell sample are non-functional.
  • more than 90 % of the enzymes in the cell sample are nonfunctional.
  • a “cancer cell”, also referred to herein as a “malignant cell”, is a cell which has been released from normal cell division control, and is thus characterized by an abnormal growth and a tendency to proliferate in an uncontrolled way and, in some cases, to metastasize.
  • the cancer cell may be a neoplastic cell, a pre- malignant cell, a metastatic cell, a tumor cell, an oncogenic cell, a cell with a cancer genotype, a cell of malignant phenotype, an oncogene transfected cell, a virus transformed cell, a cell which expresses an oncogene, a cell which expresses a marker for cancer, or a combination thereof.
  • Non-limiting examples of a cancer cell which may be detected by the method of the present invention is: an adenocarcinoma cell, an adrenal gland tumor cell, an ameloblastoma cell, an anaplastic cell, anaplastic carcinoma of the thyroid cell, an angiofibroma cell, an angioma cell, an angiosarcoma cell, an apudoma cell, an argentaffmoma cell, an arrhenoblastoma cell, an ascites tumor cell, an ascitic tumor cell, an astroblastoma cell, an astrocytoma cell, an ataxia-telangiectasia cell, an atrial myxoma cell, a basal cell carcinoma cell, a benign tumor cell, a bone cancer cell, a bone tumor cell, a brainstem glioma cell, a brain tumor cell, a breast cancer cell, a Burkitt's lymphoma cell, a cancerous cell, a carcinoid cell, a
  • a melanoma cell a meningioma cell, a mesothelioma cell, a metastatic cell, a metastasis cell, a metastatic spread cell, a Morton's neuroma cell, a multiple myeloma cell, a myeloblastoma cell, a myeloid leukemia cell, a myelolipoma cell, a myeloma cell, a myoblastoma cell, a myxoma cell, a nasopharyngeal carcinoma cell, a neoplastic cell, a nephroblastoma cell, a neuroblastoma cell, a neurofibroma cell, a neurofibromatosis cell, a neuroglioma cell, a neuroma cell, a non-Hodgkin's lymphoma cell, an oligodendroglioma cell, an optic glioma cell, an osteochond
  • the cancer cell is a colonrectal cancer cell.
  • the method of the present invention is effected by contacting an enzyme substrate with a cell, to bring about a reaction of the cell, wherein the product of the enzymatic reaction is capable of generating an electrical signal.
  • reaction of the cell refers to a reaction that occurs between the substrate and an endogenous enzyme expressed by the cell, and not to a reaction that occurs with an exogenous enzyme.
  • the substrate is selected according to the enzyme, (also referred to herein as "marker enzyme") that is required to be detected.
  • the enzyme may be situated inside the cell (i.e. intracellular) or on the cell membrane (i.e. membrane bound). It will be appreciated that when the enzyme to be detected is intracellular, the substrate is preferably membrane permeable. Furthermore, the substrate is preferably selected such that following catalysis, the product formed is also membrane permeable such that it may diffuse away from the cell and on to the detector electrode.
  • the enzyme required to be detected is alkaline phosphatase (or secreted enzyme alkaline phosphatase, (SEAP)) and the substrate is, 4- aminophenyl phosphate (p-APP).
  • Alkaline phosphatase converts p-APP to the electrochemical product, /7-aminophenol (PAP).
  • P-APP is widely commercially available from such Companies as Sigma-
  • substrates which may be used, and are specific to the enzyme intestinal alkaline phosphatase, and also yield electrochemical product are, for example: 1- Naphthyl phosphate (1-NP) which is converted by the enzyme to the electrochemical product 1-naphthol.
  • 1-NP is commercially available.
  • contemplated enzymes include catalases, peroxidases and hydrolases.
  • Alkaline phosphatase is present in normal cells, but is reduced (or even absent) in cancerous cells. Therefore, analysis of the alkaline phosphatase activity level of cells, may be used as a marker for evaluating the efficiency of a particular drug for treating cancer and even for diagnostic purposes, wherein a decrease in the level of alkaline phosphatase compared to a control healthy sample of cells is indicative of cancer.
  • the term "contacting" refers to bringing the substrate into the vicinity of a cell under conditions such that the substrate may be catalyzed by the enzyme.
  • the contacting should by effected under buffer conditions, at a temperature and time sufficient to allow catalysis of the substrate and generation of sufficient product that it may be detected by an electrochemical cell.
  • the contacting is effected for at least 5 minutes.
  • the contacting may be effected in vitro or ex vivo.
  • the contacting may be effected in a vessel which is also capable of detecting the product of the enzymatic reaction (i.e., in the electrochemical cell), such that the electrical signal is detected online.
  • the contacting may be effected in a separate vessel from where the detection takes place such that it is possible to continuously withdraw samples at particular time points and place such samples within the electrochemical cells.
  • the contacting may be effected in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like.
  • the cells may be placed on a vibrating plate following the addition of the substrate for continuous thorough mixing of the contents of the cells.
  • electrochemical measurement of products capable of undergoing a redox reaction i.e. capable of electron transfer
  • an electrical signal i.e. electrochemical products
  • electrical signal refers to electrons or electrochemically active species.
  • electrochemical measurement refers to a measurement performed by the use of electrodes in a solution, typically in an electrochemical cell.
  • the measurement may be performed, for example, by chrono- amperometry, chrono-potentiometry, cyclic voltammetry, chrono-coulometry or square wave voltammetry.
  • a signal detectable in such a measurement is one that differs in such electrochemical measurement from the control.
  • the electrochemical cells of the present invention comprise a measurement (working) electrode, a counter (auxiliary) electrode, a reference electrode and a chamber to hold the cells.
  • the working electrode may be of a variety of different kinds, for example, it may be made of carbon, including glassy carbon, activated carbon cloth electrode, carbon felt, platinized carbon cloth, plain carbon cloth), may be made of gold, platinum or silver.
  • the counter electrode may also be made of the same material as the working electrode.
  • the reference electrode may for example be saturated calomel electrode, may be an Ag/AgCl electrode.
  • the electrodes may be of a screen printed electrode which can be inserted into the vessel comprising the cells without the need to withdraw a sample and transport it into a separate electrochemical cell.
  • the electrodes used to detect the product according to the method of the present invention may be reusable electrodes or disposable ones.
  • Reusable electrodes may for example be electrodes made of glassy carbon in a disk or rod shape which are embedded in teflon.
  • Disposable electrodes may for example be electrodes in the form of carbon paper, carbon cloth, carbon felts, or the screen printed electrode of the kind noted above.
  • the electrochemical cell is a three-electrode cell. According to another embodiment, the electrochemical cell is a two-electrode cell. According to a preferred embodiment the electrochemical cells are provided as an array (i.e. chip) comprising a plurality of such cells i.e. a multiwell array where each well is of a nano-volume size.
  • the system for measuring the electrical signal generated by the reaction product may further comprise a control module which may be a computer, a potentiostat and a multiplexer module which is needed in case of a typical embodiment for simultaneous measurement from a plurality of electrochemical cells.
  • a control module which may be a computer, a potentiostat and a multiplexer module which is needed in case of a typical embodiment for simultaneous measurement from a plurality of electrochemical cells.
  • the computer scans all the electrodes via the parallel port, and the background response to the potential application of each electrode is recorded by the computer.
  • the entire electrochemical measurement sequence can be performed over a long period of time while measuring the currents resulting from the changes in the concentration of the products.
  • the system can be calibrated by measuring the oxidation or reduction of an electroactive species, typically the same species which is the product of the enzymatic reaction in the electrochemical cell and comparison of the results of all the electrodes.
  • the electrodes may be connected to the potentiostat and at the same time also connected via the multiplexer to a parallel port of the microcomputer.
  • Each electrode is inserted in an electrochemical cell containing a reference electrode and a counter electrode which are also connected to the potentiostat.
  • a specific potential is applied by the potentiostat on the electrodes (which can be the same for all the electrodes or can be a different potential to each electrode) and the current in each electrode is detected.
  • the electrical signals are visualized in real-time on the computer screen.
  • the signals generated by the electrochemical products of the enzymatic reactions reflect the level of enzyme in the cell, and the enzymes (presence, absence or level of same) are markers for cancer
  • the signals may be used to determine whether a cell is cancerous (i.e. malignant) or not. Specifically, if the level of the generated electrical signal is different to a predetermined threshold, this would indicate that the cell is cancerous.
  • the predetermined threshold is determined by the electrical signal generated by a control cell.
  • the control cell can be a normally differentiated cell, non-cancerous cell, preferably of the same tissue and specimen as the tested cell suspicious of a cancerous or undifferentiated phenotype.
  • the difference is at least 10 , 20 , 30 , 40 , 50 , 80 , 100 % (i.e., two-fold), 3 fold, 5 fold or 10 fold different as compared to a control cell.
  • the amount of enzyme (and accordingly electrical signal) in a cancer cell is higher than the amount of enzyme (and accordingly electrical signal) in a non-cancer cell.
  • an increase in the amount of enzyme in the test sample above a predetermined threshold is indicative of cancer or non-differentiated tissue.
  • the method of the present invention may be used for diagnosing a subject with cancer.
  • diagnosis refers to classifying a cancer, determining a severity of cancer (grade or stage), monitoring cancer progression, forecasting an outcome of the cancer and/or prospects of recovery.
  • the subject may be a healthy animal or human subject undergoing a routine well-being check up.
  • the subject may be at risk of having cancer (e.g., a genetically predisposed subject, a subject with medical and/or family history of cancer, a subject who has been exposed to carcinogens, occupational hazard, environmental hazard] and/or a subject who exhibits suspicious clinical signs of cancer [e.g., blood in the stool or melena, unexplained pain, sweating, unexplained fever, unexplained loss of weight up to anorexia, changes in bowel habits (constipation and/or diarrhea), tenesmus (sense of incomplete defecation, for rectal cancer specifically), anemia and/or general weakness).
  • cancer e.g., a genetically predisposed subject, a subject with medical and/or family history of cancer, a subject who has been exposed to carcinogens, occupational hazard, environmental hazard
  • a subject who exhibits suspicious clinical signs of cancer
  • the present has a variety of applications pertaining to individually optimizing a treatment for cancer, monitoring an-anti cancer treatment in a subject, determining an anti cancer treatment for a subject and identifying an agent capable of reversing a malignant phenotype of a cell.
  • a method of identifying an agent capable of reversing a malignant phenotype of a cell comprises subjecting at least one preserved cancer cell to an agent and determining the efficiency of the anti cancer agent by monitoring the activity or expression of the marker enzyme (e.g. alkaline phosphatase) according to the method of the present invention.
  • the marker enzyme e.g. alkaline phosphatase
  • reversing a malignant phenotype refers to at least partially reversing the proliferative and/or invasive characteristics of the malignant cell.
  • agent refers to a test composition comprising a biological agent or a chemical agent
  • biological agents examples include, but are not limited to, nucleic acids, e.g., polynucleotides, ribozymes, siRNA and antisense molecules (including without limitation RNA, DNA, RNA/DNA hybrids, peptide nucleic acids, and polynucleotide analogs having altered backbone and/or bass structures or other chemical modifications); proteins, polypeptides (e.g. peptides), carbohydrates, lipids and "small molecule" drug candidates.
  • nucleic acids e.g., polynucleotides, ribozymes, siRNA and antisense molecules (including without limitation RNA, DNA, RNA/DNA hybrids, peptide nucleic acids, and polynucleotide analogs having altered backbone and/or bass structures or other chemical modifications)
  • proteins polypeptides (e.g. peptides)
  • carbohydrates e.g. lipids and "small molecule" drug candidates.
  • Small molecules can be, for example, naturally occurring compounds (e.g., compounds derived from plant extracts, microbial broths, and the like) or synthetic organic or organometallic compounds having molecular weights of less than about 10,000 daltons, preferably less than about 5,000 daltons, and most preferably less than about 1,500 daltons.
  • the agents are differentiation agents including, but not limited to butyric acid and its derivatives.
  • conditions that may be tested as potential anti cancer agents according to the method of the present invention include, but are not limited to, radiation exposure (such as, gamma radiation, UV radiation, X-radiation).
  • the "marker enzyme" is also assayed prior to contact with the agent so that a comparison may be made prior to and following treatment.
  • the agent is subjected to the preserved cancer cells for a period long enough to have an anti cancer effect.
  • these agents are subjected to the preserved cancer cells for at least 1 day and more preferably 3 days.
  • the agent may be contacted with cancer cells either in vitro or ex vivo.
  • the present invention can, in theory, be practiced with a single electrochemical cell, such a method is not efficient nor is it desirable.
  • the method of the present invention is used for high throughput screening of agents using a plurality of electrochemical cells to simultaneously screen a variety of agents.
  • the cells may be part of a chip, for example a silicon chip.
  • the method of the present invention is performed using means for high output. Accordingly, the method may be performed, for example, using an automated sampling device, a liquid handling equipment, a dispenser, an electrode array, a robot, or any combination thereof.
  • tumor treatment response cannot be predicted only from its type and anatomical location. It will be appreciated that the method of identifying an agent capable of reversing a malignant phenotype of a cell may be modified such that particular patient's cells may be used in the assay system, thereby tailoring therapeutic agents to specific patients. Furthermore, it will be appreciated that not only may the specific agent be selected using the method of the present invention, but the optimal dose and optimal treatment regimen may also be identified according to the method of the present invention. In this way a therapeutically effective amount of an agent may be determined.
  • the patient may be treated according to the optimal treatment conditions selected with the aid of the method of the present invention and optionally retested after a suitable time period. In this way a patient's response may be continually monitored whilst undergoing therapy.
  • the analyzing enzyme levels and administering steps may be repeated a number of times during the course of a treatment.
  • the alkaline phosphatase levels may be analyzed one week following administration of the agent. If the alkaline phosphatase levels are higher than those compared with a control, the dose of the agent may be decreased. If the alkaline phosphatase levels remain lower than those compared with a control, the dose of the agent may be increased.
  • Tumors were induced in mice by two cancer cell lines injected to nude mice, representing two different subtypes of colon cancer. Specifically, HCT116 and HM7 colon adenocarcinoma cell lines were injected separately to athymic nude mice subcutaneously and abdominally.
  • biopsy samples were suspended in 15 ml solution of 4 % (w/v) formaldehyde and kept at 4 °C for a period of 2.5 months. Prior to measurement the biopsies were suspended for 2 hours in a PBS solution, to remove free formaldehyde. Prior to measurements, biopsy samples were dissected to 2 mm slices. Biopsy samples were weighed and placed in temperature controlled (37 °C) PBS and tested for electrochemical alkaline phosphatase level within 1 hour.
  • Chronoamperometry was performed in an eight-channel, highly sensitive multiple potentiostat allowing for the simultaneous measurement of eight electrochemical cells.
  • An in-house apparatus providing electrical contacts of the screen print electrodes combined with suction-expulsion-based efficient stirring was used (12).
  • the potentiostat was interfaced to a PC via an A/D converter employing visual basic software (12). Additional measurements were performed with a PalmSens portable potentiostat (by Palm Instruments BV, Netherlands) equipped with an eight channel multiplexer.
  • the electrochemical chamber was constructed as a 300 ⁇ chamber equipped with a screen printed electrode (SPE) at its bottom.
  • Electrode configuration was comprised of a gold working electrode, carbon counter and Ag/AgCl reference electrodes.
  • Each biopsy slice was suspended in 220 ⁇ PBS in the electrochemical chamber.
  • All electrodes were connected via the eight-channel multiplexer, continuously operating under mixing. A potential of 220mV vs Ag/AgCl reference electrode was applied. Following a short equilibration time, allowing the stabilization of the system and determination of the background signal emerging from background electrochemical and biochemical reactions, the substrate pAPP (p-amino phenyl phosphate) was added (25 ⁇ to make a final concentration of 0.1 mg/ml).
  • pAPP p-amino phenyl phosphate
  • alkaline phosphatase activity was measured directly on the suspended biopsy samples.
  • Alkaline phosphatase activity from 2mm biopsy slices was measured by the addition of pAPP and monitoring of the current generated. Samples were allowed to equilibrate at 0.22V for a period of 3 minutes prior to the addition of the substrate pAPP. An electrochemical response was clearly visible within a few seconds following substrate addition, presenting a gradually increasing current.
  • Alkaline phosphatase expression in formaldehyde fixed healthy colon biopsies produced a current signal which may be easily distinguished from the current observed in the preserved samples derived from tumor biopsies.
  • the biopsy samples were suspended in the formalin solution and kept at 4 °C for a prolonged time periods, ranging from between 20 days - 1 month. These time periods allow for a complete cross linking of the tissue slices.
  • the tissue slices placed in formalin were thicker, ranging from between 3 mm-20 mm.
  • the fixation times were considerably longer than that reported in the literature, thus allowing sufficient time for complete binding and crosslinks creation.
  • FIGS 5A-B illustrate the results obtained from patients #6 samples ( Figures 5A-B).

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Abstract

A method of determining enzyme activity in preserved cells is disclosed. The method comprises: (a) contacting preserved cells with a substrate for an enzyme under conditions wherein the enzyme catalyzes a reaction of the cells with the substrate, so as to generate a product capable of producing an electrical signal; and (b) measuring a level of the electrical signal, wherein a level of the electrical signal correlates with enzyme activity. Uses of same for diagnosing diseases are also disclosed.

Description

METHODS OF DETERMINING ENZYME ACTIVITY IN PRESERVED CELL
SAMPLES
FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to methods of determining enzyme activity in preserved cell samples.
Cancer is responsible for the majority of morbidity and mortality worldwide, despite recent advances in medical technology. Current therapeutic strategies focus predominantly on achieving the removal or death of cancer cells within the patient, through a diverse array of surgical and non-surgical techniques; the most widely used are chemotherapy and gamma irradiation. Those methods have a number of prominent disadvantages, in particular the culling of healthy cells/tissues within the patient, and the rather toxic side-effects of the current generation of chemotherapeutic drugs utilized in cancer treatment.
'Differentiation therapy' is an alternative approach which promotes reversion of phenotype from malignant to normal. Differentiation therapy is based on the concept that cancer cells are normal cells that have been arrested at an immature or less differentiated state, lack the ability to control their own growth, and thus multiply at an abnormally fast rate. Differentiation therapy aims to force the cancer cell to resume the process of maturation. Although differentiation therapy does not kill the cancer cells, it restrains their growth and allows the application of more conventional therapies (such as chemotherapy) to eradicate the remaining malignant cells.
Differentiation therapy has a number of advantages over conventional therapeutic strategies that target death of cancer/tumor cells. For a start, the culling of healthy cells/tissues within the patient with chemotherapeutic drugs or gamma irradiation would be eliminated, together with their associated adverse side-effects. In many cases, the killing of cancer cells through gamma irradiation or chemotherapeutic eliminates most, but not completely all cancer cells within the patient, thereby leading to remission of the disease. With differentiation therapy, it is speculated that by inducing some of the cancer cells into the pathway of terminal differentiation and eventual senescence, this would somehow signal other cancer cells to follow suit through a variety of mechanisms.
Various markers have been used as a means of monitoring patients undergoing differentiation therapy. One such marker is alkaline phosphatase, wherein expression of same was shown to correlate with the differentiation status of a cell [Patnaik A, et al., Clin. Can Research, 2002 Jul;8(7):2142-8; Rephaeli A, Zhuk R, Nudelman A, 2000, Drug Develop. Res. Vol 50:379-391].
Rapid and easy detection of such markers with high sensitivity, selectivity and accuracy paves the way for tailoring therapeutic agent to specific patients - 'personalized medicine'. This is of great importance in cancer therapy where it is now known that tumor treatment response cannot be predicted only from its type and anatomical location.
Until presently, there is no detection system for such markers which meets all these demands.
U.S. Pat. App. No. 20060100488 teaches detection of cancerous cells by directly monitoring the electrical response of the cells following application of an alternating current. WO 91/15595 teaches analysis of electrical conductivity of cancer cells for monitoring responsiveness to therapy and drug screening. Specifically, WO 91/15595 teaches monitoring the effectiveness of a particular agent to inhibit increases in the volume and number of cancer cells by analyzing electrical conductivity thereof. Accordingly, both these patent applications teach that the intrinsic electrical properties of a cancer cell may be used as markers for detection and monitoring of cancer cells.
U.S. Pat. Appl. No. 20040053425 teaches amperometric analysis of an analyte in a fluid, wherein the electrode comprises the current producing enzyme. U.S. Pat. Appl No. 20040053425 does not teach amperometric detection of intracellular markers.
U.S. Pat. No. 5,149,629, teaches amperometric analysis of markers, including cancer cell markers, wherein the electrode comprises antibodies capable of binding the markers thereto. The analysis is by substrate competition. U.S. Pat. No. 5,149,629 does not detect endogenous amperometric features of cancer cells.
U.S. Patent Application No. 20090232740 teaches amperometric detection for diagnosing cancer in cancer cell samples and biopsy samples by analyzing cellular enzymatic activities. SUMMARY OF THE INVENTION
According to an aspect of some embodiments of the present invention there is provided a method of determining enzyme activity in preserved cells comprising:
(a) contacting preserved cells with a substrate for an enzyme under conditions wherein the enzyme catalyzes a reaction of the cells with the substrate, so as to generate a product capable of producing an electrical signal; and
(b) measuring a level of the electrical signal, wherein a level of the electrical signal correlates with enzyme activity.
According to an aspect of some embodiments of the present invention there is provided a method of detecting cancerous cells comprising:
(a) contacting preserved cells with a substrate for an enzyme under conditions wherein the enzyme catalyzes a reaction of the cells with the substrate, so as to generate a product capable of producing an electrical signal; and
(b) measuring a level of the electrical signal, wherein a difference in a level of the electrical signal compared to a predetermined threshold is indicative of the cancerous cells.
According to an aspect of some embodiments of the present invention there is provided a method of diagnosing a subject with cancer comprising:
(a) contacting preserved cells of a sample of a subject with a substrate for an enzyme under conditions wherein the enzyme catalyzes a reaction of the cells with the substrate, so as to generate a product capable of producing an electrical signal; and
(b) measuring a level of the electrical signal, wherein a difference in a level of the electrical signal compared to a predetermined threshold is indicative of cancer. According to an aspect of some embodiments of the present invention there is provided a method of optimizing a treatment for cancer, the method comprising:
(a) contacting preserved cancer cells of a sample of a subject with at least one anti cancer agent;
(b) contacting the preserved cells with a substrate for an enzyme, under conditions wherein the enzyme catalyzes a reaction of the cells with the substrate, so as to generate a product capable of producing an electrical signal; and (c) measuring a level of the electrical signal produced by the preserved cells, wherein the level is indicative of an efficiency of the anti cancer agent to treat the cancer of the subject.
According to an aspect of some embodiments of the present invention there is provided a method of monitoring an anti cancer treatment in a subject, the method comprising:
(a) administering at least one anti cancer agent to the subject;
(b) detecting a presence or level of cancer cells in a sample of the subject following step (a) according to the method of the present invention, wherein the presence or level is indicative of a state of the cancer.
According to an aspect of some embodiments of the present invention there is provided a method of determining an anti-cancer treatment for a subject, the method comprising:
(a) analyzing a presence or level of cancer cells in a sample of the subject according to the method of the present invention;
(b) administering to the subject a therapeutic effective amount of an anti cancer agent according to the presence or level of cancer cells in the sample of the subject.
According to an aspect of some embodiments of the present invention there is provided a method of identifying an agent capable of reversing a malignant phenotype of a cell, the method comprising,
(a) contacting preserved cancer cells with an agent;
(b) measuring a malignant phenotype of the preserved cancer cells following (a) and optionally prior to (a) according to the method of the present invention, wherein a reversion of phenotype is indicative of an agent capable of reversing a malignant phenotype of a cell.
According to some embodiments of the invention, the enzyme is selected from the group consisting of beta-galactosidase, alkaline phosphatase, secreted alkaline phosphatase (SEAP), glucose oxidase, chloramphenicol acetyl transferase (CAT) and beta-glucoronidase. According to some embodiments of the invention, the substrate is selected from the group consisting of p-aminophenyl-.beta.-D- galactopyranoside (PAPG), p- aminophenol-phosphate (PAPP), glocuse-6-phosphate and chloramphenicol.
According to some embodiments of the invention, the preserved cells are comprised in a tissue slice.
According to some embodiments of the invention, the preserved cells were fixed in a fixative for at least one week.
According to some embodiments of the invention, the method comprises repeating step (a) following step (b).
According to some embodiments of the invention, the measuring is performed using means for high output.
According to some embodiments of the invention, the means is selected from the group consisting of an automated sampling device, a liquid handling equipment, a dispenser, an electrode array, a robot, or any combination thereof.
According to some embodiments of the invention, the sample comprises between 10-500 cells.
According to some embodiments of the invention, the tissue slice is about 2 mm3.
According to some embodiments of the invention, the preserved cells are derived from a suspected malignant polyp of a colon or an adenomatous of a colon.
According to some embodiments of the invention, the preserved cancer cells are derived from a malignant polyp of a colon.
According to some embodiments of the invention, the enzyme is alkaline phosphatase.
According to some embodiments of the invention, the substrate is 4- aminophenyl phosphate (p-APP).
According to some embodiments of the invention, the cells comprise colon cells. According to some embodiments of the invention, the measuring is effected using an electrochemical cell.
According to some embodiments of the invention, the measuring is effected in a multiwell array. According to some embodiments of the invention, each well of the multiwell array comprises an electrochemical cell.
According to some embodiments of the invention, each well of the multiwell array is a nano-volume well.
According to some embodiments of the invention, the agent comprises a test composition.
According to some embodiments of the invention, the test composition is selected from the group consisting of a polynucleotide a polypeptide, a small molecule chemical, a carbohydrate, a lipid and a combination of same.
According to some embodiments of the invention, the agent comprises a test condition.
According to some embodiments of the invention, the test condition is a radiation condition.
According to some embodiments of the invention, the cells are intact.
According to some embodiments of the invention, the cells are mammalian cells.
According to some embodiments of the invention, a fixative for the preserved cells comprises formaldehyde.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
In the drawings:
FIG. 1 is a graph illustrating chrono-amperometric measurement of biopsies removed from HCT116 tumors and from healthy colon tissue, and suspended in formalin for a period of two months. Current signals correspond to enzymatic activity of alkaline phosphatase reflecting cell differentiation state. Measurements were performed with in-house fabricated silicon based chips
FIG. 2 is a graph illustrating the average current signal and standard deviation obtained 800 seconds and 1500 sec (for tumor average currents and healthy colon average currents, respectively) following substrate addition.
FIG. 3 is a bar graph illustrating chrono-amperometric measurement of polyp biopsies of healthy and neoplastic tissue removed from a human patient.
FIG. 4 is a bar graph illustrating chrono-amperometric measurement of polyp biopsies of healthy and neoplastic tissue removed from a human patient.
FIGs. 5A-B are a bar graphs illustrating chrono-amperometric measurement of polyp biopsies of healthy and neoplastic tissue removed from two human patients.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention is of a method of determining enzyme activity in preserved cell samples. Specifically, the present invention can be used detect cancer in tumor biopsies preserved in a fixative such as formaldehyde. The method may be used to diagnose cancer, monitor treatment, determine treatment regimens and develop novel treatment modalities for the disease.
The principles and operation of the method according to the present invention may be better understood with reference to the drawings and accompanying descriptions.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
In routinely practiced clinical procedures, cell samples are suspended in formalin (4 % formaldehyde) following biopsy removal and prior to further pathological examinations. Formalin treatment affects chemical fixation of the tissue resulting in a prolonged preservation of cellular structure of the specimen; most enzymes are, however, inactivated by exposure to formaldehyde (21).
Upon exposure, formaldehyde molecules react with various protein groups, particularly the amino groups of lysine and the nitrogen atoms of peptide linkages. Subsequently, the chemically bound hydroxymethyl groups react with other nitrogen atoms of the same or adjacent protein molecules. The resulting cross-links, known as methylene (-CH2-) bridges, are stable and account for the insolubility and rigidity of protein-containing tissues that have been fixed by formaldehyde (21). It is these crosslinks which are responsible for the associated protein denaturation, and for the inevitable loss of enzymatic activity (22). The various effects of aldehyde fixation on enzyme activity has been extensively studied and it has been suggested that most hydrolases, such as acid and alkaline phosphatase, b-glucoronidase, catalase, ATPase, ADPase, PNPase, BGPase only retain 25-47 % of their enzymatic activity following 2- 24 hours of fixation (22-27).
The present inventors had previously developed an electrochemical method for sensitive and high-throughput detection of a cancer cell's response to differentiation therapy based on expression of tumor cell alkaline phosphatase. The present inventors had shown that tissue biopsies which contain malignant cells may be distinguished from normal healthy tissue biopsies using an electrochemical method, thus detecting cancer.
Whilst reducing the present invention to practice, the present inventors have now shown that unexpectedly, preserved cell samples may be analyzed in the same way as fresh tumor cell samples with surprisingly accurate results.
The presently described methods may be used to determine enzyme activity in preserved cellular samples. Using the presently described methods, the inventors have shown that it is possible to provide accurate diagnostic results within a few minutes from formalin preserved biopsy slices by determining alkaline phosphatase activity in suspected malignant cells.
Thus, according to one aspect of the present invention there is provided a method of determining enzyme activity in preserved cells comprising:
(a) contacting preserved cells with a substrate for an enzyme under conditions wherein the enzyme catalyzes a reaction of the cells with the substrate, so as to generate a product capable of producing an electrical signal; and
(b) measuring a level of the electrical signal, wherein a level of the electrical signal correlates with an amount of the enzyme.
Typically, the enzyme is one which catalyzes a reaction giving rise to a product which is permeable or which can be transported through the cell membrane and which can then undergo redox reaction at one of the electrodes of the electrochemical cell. Example of such enzymes include beta-galactosidase, which can catalyze a reaction in which the p-aminophenyl-beta-D- galactopyranoside (PAPG) is convened into p- aminophenol (PAP). PAP can be transported through the cell membrane and then oxidized at the electrode.
Other non limiting examples of enzymes and relevant substrates include; The enzyme alkaline phosphatase (AP) or the enzyme secreted alkaline phosphatase (SEAP) with the substrate PAPP (p-aminophenol-phosphate); the enzyme glucose oxidase with the substrate e glocuse-6-phosphate, the enzyme chloramphenicol acetyl transferase (CAT) and the substrate chloramphhenicol, the enzyme b-glucuronidase and any glycosaminoglycans or other glycoconjugates that after the removal of the b-glucorunic acid residue become electrochemicaly active.
The cells may be from any source including freshly isolated cells, ex vivo cells; part of a cell line; stem cells, ex vivo differentiated stem cells.
The cells may be eukaryotic (e.g. mammalian, plant, insect) or prokaryotic e.g. bacterial.
The cells may be derived from a healthy subject (i.e. non-diseased cells) or from a diseased subject (i.e. diseased cells), as further described herein below.
According to a particular embodiment, the enzyme is a marker for a particular disease. Thus for example, the disease may be cancer and the enzyme alkaline phosphatase. Thus, according to another aspect of the present invention, there is provided a method of detecting cancerous cells. The method comprises:
(a) contacting preserved cells with a substrate for an enzyme under conditions wherein the enzyme catalyzes a reaction of the cells with the substrate, so as to generate a product capable of producing an electrical signal; and
(b) measuring a level of the electrical signal, wherein a difference in a level of the electrical signal compared to a predetermined threshold is indicative of the cancerous cells.
The term "detecting", as used herein, refers to the act of detecting, diagnosing, perceiving, uncovering, exposing, visualizing or identifying a cell.
As used herein, the term "cell" refers to a mammalian cell, preferably a human cell. Single cells may be used in accordance with the teachings of the present invention as well as plurality of cells. According to an exemplary embodiment, the plurality of cells comprises no less than 10 cells and no more than 500 cells. According to another exemplary embodiment tissue slices are analyzed.
According to a specific embodiment, the cells that are analyzed are suspected of being malignant. For example, the cells may be obtained from a colon biopsy (e.g. from a malignant colon polyp or a colon adenomatous) and the enzyme which is assayed is alkaline phosphatase. Analysis of placental alkaline phosphatase in fixed testicular tissue may be performed as part of a diagnosis of testicular cancer. Typically a down- regulation in an activity of the alkaline phosphatase is indicative of testicular cancer. Analysis of acid phosphatase in fixed biopsies of the prostate or bladder may be performed as part of a diagnosis of cancer. Typically a down-regulation in an activity of the alkaline phosphatase is indicative of the cancer.
According to another embodiment, the cells that are analyzed are liver cells (e.g. a fixed liver tissue). An increase or decrease in a level of alkaline phosphatase may be indicative of a liver disease.
According to still another embodiment, the cells that are analyzed are cells of a fixed stomach tissue biopsy cells. An increase or decrease in a level of acid phosphatase may be indicative of a gastric cancer or other diseases related to the stomach.
According to another embodiment, the cells are not peripheral blood cells. The plurality of cells may be from any biological sample such as cell-lines, primary cultures and cellular samples, e.g. biopsies (surgical biopsies including incisional or excisional biopsy, fine needle aspirates and the like), complete resections or body fluids. Methods of biopsy retrieval are well known in the art.
According to one embodiment, following biopsy, a tumor sample is sliced. In order to calibrate the system, such that comparison between healthy and non-healthy slices is accurate, the tumor slices may be weighed.
The cells in the biological sample are preferably intact (i.e. whole).
As mentioned, the cells in the biological sample are assayed for a functional enzyme following pre-treatment with a preservative e.g. a fixative.
According to one embodiment, the fixative is a chemical fixative. An exemplary chemical fixative is a crosslinking fixative. Such fixatives include formaldehyde (e.g. a solution comprising 3-5 % formaldehyde) and glutaraldehyde
An exemplary fixative solution contemplated by the present invention is one which is a 10 % Neutral Buffered Formalin (NBF), that is approximately 3.7 % formaldehyde in phosphate buffered saline.
Other contemplated fixatives include precipitating fixatives, including, but not limited to ethanol, methanol and acetone; oxidizing agents, including but not limited to osmium tetroxide, Potassium dichromate, chromic acid, and potassium permanganate; Mercurials such as B-5 and Zenker's; Picrates; Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE).
Besides chemical fixation, the present invention also contemplates other forms of fixation such as frozen sections and heat fixation.
According to this aspect of the present invention, the cell sample may remain in the chemical fixative for one day, one week, one month, two months, three months or even longer. Typically, more than 50 % of the enzymes in the cell sample are nonfunctional. According to another embodiment, more than 60 % of the enzymes in the cell sample are non-functional. According to another embodiment, more than 70 % of the enzymes in the cell sample are non-functional. According to another embodiment, more than 80 % of the enzymes in the cell sample are non-functional. According to another embodiment, more than 90 % of the enzymes in the cell sample are nonfunctional. A "cancer cell", also referred to herein as a "malignant cell", is a cell which has been released from normal cell division control, and is thus characterized by an abnormal growth and a tendency to proliferate in an uncontrolled way and, in some cases, to metastasize. Accordingly, the cancer cell may be a neoplastic cell, a pre- malignant cell, a metastatic cell, a tumor cell, an oncogenic cell, a cell with a cancer genotype, a cell of malignant phenotype, an oncogene transfected cell, a virus transformed cell, a cell which expresses an oncogene, a cell which expresses a marker for cancer, or a combination thereof.
Non-limiting examples of a cancer cell which may be detected by the method of the present invention is: an adenocarcinoma cell, an adrenal gland tumor cell, an ameloblastoma cell, an anaplastic cell, anaplastic carcinoma of the thyroid cell, an angiofibroma cell, an angioma cell, an angiosarcoma cell, an apudoma cell, an argentaffmoma cell, an arrhenoblastoma cell, an ascites tumor cell, an ascitic tumor cell, an astroblastoma cell, an astrocytoma cell, an ataxia-telangiectasia cell, an atrial myxoma cell, a basal cell carcinoma cell, a benign tumor cell, a bone cancer cell, a bone tumor cell, a brainstem glioma cell, a brain tumor cell, a breast cancer cell, a Burkitt's lymphoma cell, a cancerous cell, a carcinoid cell, a carcinoma cell, a cerebellar astrocytoma cell, a cervical cancer cell, a cherry angioma cell, a cholangiocarcinoma cell, a cholangioma cell, a chondroblastoma cell, a chondroma cell, a chondrosarcoma cell, a chorioblastoma cell, a choriocarcinoma cell, a colon cancer cell, a common acute lymphoblastic leukemia cell, a craniopharyngioma cell, a cystocarcinoma cell, a cystofbroma cell, a cystoma cell, a cytoma cell, a ductal carcinoma in situ cell, a ductal papilloma cell, a dysgerminoma cell, an encephaloma cell, an endometrial carcinoma cell, an endothelioma cell, an ependymoma cell, an epithelioma cell, an erythroleukemia cell, an Ewing's sarcoma cell, an extra nodal lymphoma cell, a feline sarcoma cell, a fibro adenoma cell, a fibro sarcoma cell, a follicular cancer of the thyroid cell, a ganglioglioma cell, a gastrinoma cell, aglioblastoma multiform cell, a glioma cell, a gonadoblastoma cell, an haemangioblastomacell, an haemangioendothelioblastoma cell, an haemangioendothelioma cell, an haemangiopericytoma cell, an haematolymphangioma cell, an haemocytoblastoma cell, an haemocytoma cell, a hairy cell leukemia cell, a hamartoma cell, an hepatocarcinoma cell, an hepatocellular carcinoma cell, an hepatoma cell, an histoma cell , a Hodgkin's disease cell, an hypernephroma cell, an infiltrating cancer cell, an infiltrating ductal cell carcinoma cell, an insulinoma cell, a juvenile angioforoma cell, a Kaposi sarcoma cell, a kidney tumor cell, a large cell lymphoma cell, a leukemia cell, a chronic leukemia cell, an acute leukemia cell, a lipoma cell, a liver cancer cell, a liver metastases cell, a Lucke carcinoma cell, a lymphadenoma cell, a lymphangioma cell, a lymphocytic leukemia cell, a lymphocytic lymphoma cell, a lymphoeytoma cell, a lymphoedema cell, a lymphoma cell, a lung cancer cell, a malignant mesothelioma cell, a malignant teratoma cell, a mastocytoma cell, a medulloblastome. cell, a melanoma cell, a meningioma cell, a mesothelioma cell, a metastatic cell, a metastasis cell, a metastatic spread cell, a Morton's neuroma cell, a multiple myeloma cell, a myeloblastoma cell, a myeloid leukemia cell, a myelolipoma cell, a myeloma cell, a myoblastoma cell, a myxoma cell, a nasopharyngeal carcinoma cell, a neoplastic cell, a nephroblastoma cell, a neuroblastoma cell, a neurofibroma cell, a neurofibromatosis cell, a neuroglioma cell, a neuroma cell, a non-Hodgkin's lymphoma cell, an oligodendroglioma cell, an optic glioma cell, an osteochondroma cell, an osteogenic sarcoma cell, an osteosarcoma cell, an ovarian cancer cell, a Paget' s disease of the nipple cell, a pancoast tumor cell, a pancreatic cancer cell, a phaeochromocytoma cell, a pheoehromocytoma cell, a plasmacytoma cell, a primary brain tumor cell, a progonoma cell, a prolactinoma cell, a renal cell carcinoma cell, a retinoblastoma cell, a rhabdomyosarcoma cell, a rhabdosarcoma cell, a solid tumor cell, sarcoma cell, a secondary tumor cell, a seminoma cell, a skin cancer cell, a small cell carcinoma cell, a squamous cell carcinoma cell, a strawberry haemangioma cell, a T-cell lymphoma cell, a teratoma cell, a testicular cancer cell, a thymoma cell, a trophoblastic tumor cell, a tumorigenic cell, a tumor initiation cell, a tumor progression cell, a vestibular schwannoma cell, a Wilm's tumor cell, or a combination thereof.
According to a preferred embodiment of this aspect of the present invention, the cancer cell is a colonrectal cancer cell.
As mentioned hereinabove, the method of the present invention is effected by contacting an enzyme substrate with a cell, to bring about a reaction of the cell, wherein the product of the enzymatic reaction is capable of generating an electrical signal. As used herein, the phrase "reaction of the cell" refers to a reaction that occurs between the substrate and an endogenous enzyme expressed by the cell, and not to a reaction that occurs with an exogenous enzyme.
The substrate is selected according to the enzyme, (also referred to herein as "marker enzyme") that is required to be detected. The enzyme may be situated inside the cell (i.e. intracellular) or on the cell membrane (i.e. membrane bound). It will be appreciated that when the enzyme to be detected is intracellular, the substrate is preferably membrane permeable. Furthermore, the substrate is preferably selected such that following catalysis, the product formed is also membrane permeable such that it may diffuse away from the cell and on to the detector electrode.
According to one embodiment, the enzyme required to be detected is alkaline phosphatase (or secreted enzyme alkaline phosphatase, (SEAP)) and the substrate is, 4- aminophenyl phosphate (p-APP). Alkaline phosphatase converts p-APP to the electrochemical product, /7-aminophenol (PAP).
P-APP is widely commercially available from such Companies as Sigma-
Aldrich (worldwidewebdotsigmaaldrichdotcom), Bio- world (worldwidewebdotBio- worlddotcom) and many others.
Other substrates, which may be used, and are specific to the enzyme intestinal alkaline phosphatase, and also yield electrochemical product are, for example: 1- Naphthyl phosphate (1-NP) which is converted by the enzyme to the electrochemical product 1-naphthol. 1-NP is commercially available.
Other contemplated enzymes include catalases, peroxidases and hydrolases.
Alkaline phosphatase is present in normal cells, but is reduced (or even absent) in cancerous cells. Therefore, analysis of the alkaline phosphatase activity level of cells, may be used as a marker for evaluating the efficiency of a particular drug for treating cancer and even for diagnostic purposes, wherein a decrease in the level of alkaline phosphatase compared to a control healthy sample of cells is indicative of cancer.
As used herein, the term "contacting" refers to bringing the substrate into the vicinity of a cell under conditions such that the substrate may be catalyzed by the enzyme. Thus, for example, the contacting should by effected under buffer conditions, at a temperature and time sufficient to allow catalysis of the substrate and generation of sufficient product that it may be detected by an electrochemical cell. For example, when p-APP is used as a substrate, preferably the contacting is effected for at least 5 minutes.
The contacting may be effected in vitro or ex vivo. The contacting may be effected in a vessel which is also capable of detecting the product of the enzymatic reaction (i.e., in the electrochemical cell), such that the electrical signal is detected online. Such vessels are further described herein below. Alternatively, the contacting may be effected in a separate vessel from where the detection takes place such that it is possible to continuously withdraw samples at particular time points and place such samples within the electrochemical cells. Thus, the contacting may be effected in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like. The cells may be placed on a vibrating plate following the addition of the substrate for continuous thorough mixing of the contents of the cells.
As mentioned hereinabove, electrochemical measurement of products capable of undergoing a redox reaction (i.e. capable of electron transfer) at an electrode of a chemical cell to yield an electrical signal (i.e. electrochemical products) is typically effected in electrochemical cells.
As used herein, the phrase "electrical signal" refers to electrons or electrochemically active species.
The phrase "electrochemical measurement" as used herein, refers to a measurement performed by the use of electrodes in a solution, typically in an electrochemical cell. The measurement may be performed, for example, by chrono- amperometry, chrono-potentiometry, cyclic voltammetry, chrono-coulometry or square wave voltammetry. A signal detectable in such a measurement, is one that differs in such electrochemical measurement from the control.
For on-line measurement, the electrochemical cells of the present invention comprise a measurement (working) electrode, a counter (auxiliary) electrode, a reference electrode and a chamber to hold the cells.
The working electrode may be of a variety of different kinds, for example, it may be made of carbon, including glassy carbon, activated carbon cloth electrode, carbon felt, platinized carbon cloth, plain carbon cloth), may be made of gold, platinum or silver. The counter electrode may also be made of the same material as the working electrode. The reference electrode may for example be saturated calomel electrode, may be an Ag/AgCl electrode. Furthermore, the electrodes may be of a screen printed electrode which can be inserted into the vessel comprising the cells without the need to withdraw a sample and transport it into a separate electrochemical cell.
The electrodes used to detect the product according to the method of the present invention may be reusable electrodes or disposable ones. Reusable electrodes may for example be electrodes made of glassy carbon in a disk or rod shape which are embedded in teflon. Disposable electrodes may for example be electrodes in the form of carbon paper, carbon cloth, carbon felts, or the screen printed electrode of the kind noted above.
According to one embodiment, the electrochemical cell is a three-electrode cell. According to another embodiment, the electrochemical cell is a two-electrode cell. According to a preferred embodiment the electrochemical cells are provided as an array (i.e. chip) comprising a plurality of such cells i.e. a multiwell array where each well is of a nano-volume size.
The system for measuring the electrical signal generated by the reaction product may further comprise a control module which may be a computer, a potentiostat and a multiplexer module which is needed in case of a typical embodiment for simultaneous measurement from a plurality of electrochemical cells.
The electrochemical measurement performed in the cell will now be described in reference to the chrono-amperometric mode. As will be appreciated, it applies, mutatis, mutandis also to the other electrochemical measurement modes mentioned above. Furthermore, the description will be made with reference to the use of a multi-electrode system (the system comprising an array of electrodes) and it is clear that it applies to a system comprising a single cell as well.
In the beginning of the electrochemical measurement all the electrodes are operated together, and the computer scans all the electrodes via the parallel port, and the background response to the potential application of each electrode is recorded by the computer. The entire electrochemical measurement sequence can be performed over a long period of time while measuring the currents resulting from the changes in the concentration of the products. In cases where the electrodes' surfaces are not identical due to natural variability, the system can be calibrated by measuring the oxidation or reduction of an electroactive species, typically the same species which is the product of the enzymatic reaction in the electrochemical cell and comparison of the results of all the electrodes.
In performing the assay, the electrodes may be connected to the potentiostat and at the same time also connected via the multiplexer to a parallel port of the microcomputer.
Each electrode is inserted in an electrochemical cell containing a reference electrode and a counter electrode which are also connected to the potentiostat. A specific potential is applied by the potentiostat on the electrodes (which can be the same for all the electrodes or can be a different potential to each electrode) and the current in each electrode is detected. The electrical signals are visualized in real-time on the computer screen.
Since the electrical signals generated by the electrochemical products of the enzymatic reactions reflect the level of enzyme in the cell, and the enzymes (presence, absence or level of same) are markers for cancer, the signals may be used to determine whether a cell is cancerous (i.e. malignant) or not. Specifically, if the level of the generated electrical signal is different to a predetermined threshold, this would indicate that the cell is cancerous. Typically, the predetermined threshold is determined by the electrical signal generated by a control cell.
The control cell can be a normally differentiated cell, non-cancerous cell, preferably of the same tissue and specimen as the tested cell suspicious of a cancerous or undifferentiated phenotype. Preferably, the difference is at least 10 , 20 , 30 , 40 , 50 , 80 , 100 % (i.e., two-fold), 3 fold, 5 fold or 10 fold different as compared to a control cell.
According to one embodiment of the present invention, the amount of enzyme
(and accordingly electrical signal) in a cancer cell is lower than the amount of enzyme (and accordingly electrical signal) in a non-cancer cell. In this case a decrease in the amount of enzyme in the test sample below a predetermined threshold is indicative of cancer or non-differentiated tissue.
According to another embodiment of the present invention, the amount of enzyme (and accordingly electrical signal) in a cancer cell is higher than the amount of enzyme (and accordingly electrical signal) in a non-cancer cell. In this case an increase in the amount of enzyme in the test sample above a predetermined threshold is indicative of cancer or non-differentiated tissue.
It will be appreciated that the method of the present invention may be used for diagnosing a subject with cancer.
As used herein the term "diagnosing" refers to classifying a cancer, determining a severity of cancer (grade or stage), monitoring cancer progression, forecasting an outcome of the cancer and/or prospects of recovery.
The subject may be a healthy animal or human subject undergoing a routine well-being check up. Alternatively, the subject may be at risk of having cancer (e.g., a genetically predisposed subject, a subject with medical and/or family history of cancer, a subject who has been exposed to carcinogens, occupational hazard, environmental hazard] and/or a subject who exhibits suspicious clinical signs of cancer [e.g., blood in the stool or melena, unexplained pain, sweating, unexplained fever, unexplained loss of weight up to anorexia, changes in bowel habits (constipation and/or diarrhea), tenesmus (sense of incomplete defecation, for rectal cancer specifically), anemia and/or general weakness).
It will be appreciated that the present has a variety of applications pertaining to individually optimizing a treatment for cancer, monitoring an-anti cancer treatment in a subject, determining an anti cancer treatment for a subject and identifying an agent capable of reversing a malignant phenotype of a cell.
Thus, according to another aspect of the present invention, there is provided a method of identifying an agent capable of reversing a malignant phenotype of a cell. The method comprises subjecting at least one preserved cancer cell to an agent and determining the efficiency of the anti cancer agent by monitoring the activity or expression of the marker enzyme (e.g. alkaline phosphatase) according to the method of the present invention.
As used herein the phrase "reversing a malignant phenotype" refers to at least partially reversing the proliferative and/or invasive characteristics of the malignant cell.
As used herein, the term "agent" refers to a test composition comprising a biological agent or a chemical agent
Examples of biological agents that may be tested as potential anti cancer agents according to the method of the present invention include, but are not limited to, nucleic acids, e.g., polynucleotides, ribozymes, siRNA and antisense molecules (including without limitation RNA, DNA, RNA/DNA hybrids, peptide nucleic acids, and polynucleotide analogs having altered backbone and/or bass structures or other chemical modifications); proteins, polypeptides (e.g. peptides), carbohydrates, lipids and "small molecule" drug candidates. "Small molecules" can be, for example, naturally occurring compounds (e.g., compounds derived from plant extracts, microbial broths, and the like) or synthetic organic or organometallic compounds having molecular weights of less than about 10,000 daltons, preferably less than about 5,000 daltons, and most preferably less than about 1,500 daltons.
According to a preferred embodiment of this aspect of the present invention the agents are differentiation agents including, but not limited to butyric acid and its derivatives.
Examples of conditions that may be tested as potential anti cancer agents according to the method of the present invention include, but are not limited to, radiation exposure (such as, gamma radiation, UV radiation, X-radiation).
According to an embodiment of this aspect of the present invention, the "marker enzyme" is also assayed prior to contact with the agent so that a comparison may be made prior to and following treatment.
According to another embodiment of this aspect of the present invention, the agent is subjected to the preserved cancer cells for a period long enough to have an anti cancer effect. Thus, for example if butyric acid and/or its derivatives are being analyzed, preferably these agents are subjected to the preserved cancer cells for at least 1 day and more preferably 3 days.
It will be appreciated that the agent may be contacted with cancer cells either in vitro or ex vivo.
Although the present invention can, in theory, be practiced with a single electrochemical cell, such a method is not efficient nor is it desirable. Preferably, the method of the present invention is used for high throughput screening of agents using a plurality of electrochemical cells to simultaneously screen a variety of agents. The cells may be part of a chip, for example a silicon chip.
Thus, according to one embodiment, the method of the present invention is performed using means for high output. Accordingly, the method may be performed, for example, using an automated sampling device, a liquid handling equipment, a dispenser, an electrode array, a robot, or any combination thereof.
It is now known that tumor treatment response cannot be predicted only from its type and anatomical location. It will be appreciated that the method of identifying an agent capable of reversing a malignant phenotype of a cell may be modified such that particular patient's cells may be used in the assay system, thereby tailoring therapeutic agents to specific patients. Furthermore, it will be appreciated that not only may the specific agent be selected using the method of the present invention, but the optimal dose and optimal treatment regimen may also be identified according to the method of the present invention. In this way a therapeutically effective amount of an agent may be determined.
The patient may be treated according to the optimal treatment conditions selected with the aid of the method of the present invention and optionally retested after a suitable time period. In this way a patient's response may be continually monitored whilst undergoing therapy.
Conceivably the analyzing enzyme levels and administering steps may be repeated a number of times during the course of a treatment. For instance the alkaline phosphatase levels may be analyzed one week following administration of the agent. If the alkaline phosphatase levels are higher than those compared with a control, the dose of the agent may be decreased. If the alkaline phosphatase levels remain lower than those compared with a control, the dose of the agent may be increased.
It is expected that during the life of this patent many relevant substrates will be developed and the scope of the term substrate is intended to include all such new technologies a priori.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
EXAMPLE 1
Detection of Alkaline phosphatase in mouse biopsy slices MATERIALS AND METHODS
Tumors were induced in mice by two cancer cell lines injected to nude mice, representing two different subtypes of colon cancer. Specifically, HCT116 and HM7 colon adenocarcinoma cell lines were injected separately to athymic nude mice subcutaneously and abdominally.
Tumors were allowed to develop and mice were sacrificed following 6 weeks. Subcutaneous and abdominal HCT116 and HM7 tumors were collected. As control, healthy tissues were simultaneously collected from the small and large intestine (colon) of same mice.
Following removal, biopsy samples were suspended in 15 ml solution of 4 % (w/v) formaldehyde and kept at 4 °C for a period of 2.5 months. Prior to measurement the biopsies were suspended for 2 hours in a PBS solution, to remove free formaldehyde. Prior to measurements, biopsy samples were dissected to 2 mm slices. Biopsy samples were weighed and placed in temperature controlled (37 °C) PBS and tested for electrochemical alkaline phosphatase level within 1 hour.
Chronoamperometry was performed in an eight-channel, highly sensitive multiple potentiostat allowing for the simultaneous measurement of eight electrochemical cells. An in-house apparatus providing electrical contacts of the screen print electrodes combined with suction-expulsion-based efficient stirring was used (12). The potentiostat was interfaced to a PC via an A/D converter employing visual basic software (12). Additional measurements were performed with a PalmSens portable potentiostat (by Palm Instruments BV, Netherlands) equipped with an eight channel multiplexer. The electrochemical chamber was constructed as a 300 μΐ chamber equipped with a screen printed electrode (SPE) at its bottom. Electrode configuration was comprised of a gold working electrode, carbon counter and Ag/AgCl reference electrodes. Additional measurements were performed with silicon based electrochemical cells with similar dimensions planar gold working, gold counter and Ag/AgCl reference electrodes fabricated in-house by photolithography process followed by gold sputtering and Ag electroplating (Ag/AgCl). During measurements continuous mixing was affected.
Each biopsy slice was suspended in 220 μΐ PBS in the electrochemical chamber.
All electrodes were connected via the eight-channel multiplexer, continuously operating under mixing. A potential of 220mV vs Ag/AgCl reference electrode was applied. Following a short equilibration time, allowing the stabilization of the system and determination of the background signal emerging from background electrochemical and biochemical reactions, the substrate pAPP (p-amino phenyl phosphate) was added (25 μΐ to make a final concentration of 0.1 mg/ml).
RESULTS
In order to distinguish between cancer tissue and healthy tissue samples on the basis of electrochemical detection of the expression level of alkaline phosphatase, the level of alkaline phosphatase was measured directly on the suspended biopsy samples. Alkaline phosphatase activity from 2mm biopsy slices was measured by the addition of pAPP and monitoring of the current generated. Samples were allowed to equilibrate at 0.22V for a period of 3 minutes prior to the addition of the substrate pAPP. An electrochemical response was clearly visible within a few seconds following substrate addition, presenting a gradually increasing current. Alkaline phosphatase expression in formaldehyde fixed healthy colon biopsies produced a current signal which may be easily distinguished from the current observed in the preserved samples derived from tumor biopsies.
Standard deviation was calculated from the average current signal obtained following 800 seconds and 1500 sec (for tumor average currents and healthy colon average currents, respectively) from substrate addition. Despite the differences in cell types of the samples tested, it is evident from our results that preserved tumor derived samples yielded reproducible results reflecting substantially lower levels of alkaline phosphatase activity detected by the electrochemical measurements. Thus, within 3 minutes following substrate addition HCT116 tumors samples could be clearly distinguished from healthy tissues samples as illustrated in Figures 1 and 2. It is evident from Figures 1 and 2 that biopsies kept in formalin for prolonged period of time yield a current signal corresponding to a high level of ALP enzyme activity which may be electrochemically measured as described herein. EXAMPLE 2
Detection of Alkaline phosphatase in human biopsy slices MATERIALS AND METHODS
Briefly, 16 subjects, male and female above age 18 were recruited for an open labeled study. The study was designed to provide comparison of results obtained by the described method and by the standard biopsy pathology examination of same biopsies. Each patient enrolled in the study, had undergone a regular colonoscopy. Three samples from polyps of >0.5cm were taken by either using the endoscope forceps or by a sterile splinter after polyp removal. In addition, two samples from a— healthy, normal colon tissue were taken by the forceps. All five samples were placed, separately in buffered formalin (10 % formaldehyde), marked by the enrolled patient number and roman letter, e.g., patient#-A, patient#-B, patient#-C, etc. The biopsy samples were suspended in the formalin solution and kept at 4 °C for a prolonged time periods, ranging from between 20 days - 1 month. These time periods allow for a complete cross linking of the tissue slices. Formalin is traditionally considered "fast to penetrate, slow to fixate" (Fox, Johnson et al. 1985) and its rate of penetration may be calculated from the formula: d=K^t where d = depth of penetration, K = a constant specific for the fixative, t = time (Hopwood 1967). In the case of 4 % formaldehyde the constant is about 5.5, which means that for a 24 hour immersion, formaldehyde may penetrate 20 mm or more.
In this example, the tissue slices placed in formalin were thicker, ranging from between 3 mm-20 mm. However, the fixation times were considerably longer than that reported in the literature, thus allowing sufficient time for complete binding and crosslinks creation.
Prior to measurement, the biopsies were suspended in a PBS solution for 4 hours in room temperature (or overnight in 4 °C), to remove unbound formaldehyde. The remaining experimental procedure was as described above for Example 1. RESULTS
The results presented herein describe the current response generated by the oxidation of the electroactive products: p-Aminophenol or 1-Naphthol. These products were generated via dephosphorylation of their corresponding monophosphates, p- aminopehnyl phosphate or 1-Naphthyl phosphate, respectively. Dephosphorylation was catalyzed by intestinal alkaline phosphatase, as previously mentioned.
In Figure 3, the average current values obtained from all measured biopsy samples removed from both healthy and neoplastic tissue of patient #3, are presented. Results were calculated and presented as 'AF(the background current prior to substrate addition subtracted from the maximum current value). The average ΔΙ currents, denoted 'ΔΙ average' was calculated, as well as the standard deviation.
As expected, currents obtained from biopsies originated from healthy tissues were consistently higher indicating up regulated ALP activity, in comparison to their neoplastic counterparts.
In Figure 4, results of the amperometric detection of ALP from both healthy and tumor tissue samples of patient #4, are similarly presented. Current results obtained from patient #4 samples demonstrate once more the ability of the described electrochemical detection method to detect the activity of enzymes from a chemically fixed tissue.
Figures 5A-B illustrate the results obtained from patients #6 samples (Figure
5 A) and from patient #2 samples (Figure 5B).
The results obtained from measurements of 32 samples removed from four different human patients indicate that a higher residual enzymatic activity of ALP may be observed in 'healthy' tissue samples removed from normal colonic mucosa, whereas significantly lower activities where recorded for samples removed from neoplastic tissues. The electrochemical detection method is sensitive enough to detect these activities. In light of the known ALP fixation-related loss of activity, which was widely reported in the literature, these results are unexpected and may be explained by the ultra-sensitivity of the method. CONCLUSIONS
The feasibility of EC detection of ALP enzymatic activity in biopsy samples, removed from polyps of human patients, which were kept in formaldehyde for a prolonged period of time (up to 1 month), was demonstrated.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
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Claims

WHAT IS CLAIMED IS:
1. A method of determining enzyme activity in preserved cells comprising:
(a) contacting preserved cells with a substrate for an enzyme under conditions wherein said enzyme catalyzes a reaction of said cells with said substrate, so as to generate a product capable of producing an electrical signal; and
(b) measuring a level of said electrical signal, wherein a level of said electrical signal correlates with enzyme activity.
2. The method of claim 1, wherein said enzyme is selected from the group consisting of beta-galactosidase, alkaline phosphatase, secreted alkaline phosphatase (SEAP), glucose oxidase, chloramphenicol acetyl transferase (CAT) , beta- glucoronidase, catalase, peroxidase, and a hydrolase.
3. The method of claim 1 wherein said substrate is selected from the group consisting of p-aminophenyl-.beta.-D- galactopyranoside (PAPG), p-aminophenol- phosphate (PAPP), glocuse-6-phosphate and chloramphenicol.
4. A method of detecting cancerous cells comprising:
(a) contacting preserved cells with a substrate for an enzyme under conditions wherein said enzyme catalyzes a reaction of said cells with said substrate, so as to generate a product capable of producing an electrical signal; and
(b) measuring a level of said electrical signal, wherein a difference in a level of said electrical signal compared to a predetermined threshold is indicative of the cancerous cells.
5. A method of diagnosing a subject with cancer comprising:
(a) contacting preserved cells of a sample of a subject with a substrate for an enzyme under conditions wherein said enzyme catalyzes a reaction of said cells with said substrate, so as to generate a product capable of producing an electrical signal; and
(b) measuring a level of said electrical signal, wherein a difference in a level of said electrical signal compared to a predetermined threshold is indicative of cancer.
6. A method of optimizing a treatment for cancer, the method comprising:
(a) contacting preserved cancer cells of a sample of a subject with at least one anti cancer agent;
(b) contacting said preserved cells with a substrate for an enzyme, under conditions wherein said enzyme catalyzes a reaction of the cells with said substrate, so as to generate a product capable of producing an electrical signal; and
(c) measuring a level of said electrical signal produced by said preserved cells, wherein said level is indicative of an efficiency of said anti cancer agent to treat the cancer of said subject.
7. A method of monitoring an anti cancer treatment in a subject, the method comprising:
(a) administering at least one anti cancer agent to the subject;
(b) detecting a presence or level of cancer cells in a sample of the subject following step (a) according to the method of claim 4, wherein said presence or level is indicative of a state of the cancer.
8. A method of determining an anti-cancer treatment for a subject, the method comprising:
(a) analyzing a presence or level of cancer cells in a sample of the subject according to the method of claim 4;
(b) administering to the subject a therapeutic effective amount of an anti cancer agent according to said presence or level of cancer cells in said sample of the subject.
9. A method of identifying an agent capable of reversing a malignant phenotype of a cell, the method comprising,
(a) contacting preserved cancer cells with an agent;
(b) measuring a malignant phenotype of said preserved cancer cells following (a) and optionally prior to (a) according to the method of claim 4, wherein a reversion of phenotype is indicative of an agent capable of reversing a malignant phenotype of a cell.
10. The method of any of claims 1, 4, 5 or 6, wherein said preserved cells are comprised in a tissue slice.
11. The method of any of claims 1, 4, 5 or 6, wherein said preserved cells were fixed in a fixative for at least one week.
12. The method of claim 8, further comprising repeating step (a) following step (b).
13. The method of claims 1, 4, 5 or 6, wherein said measuring is performed using means for high output.
14. The method of claim 13, wherein said means is selected from the group consisting of an automated sampling device, a liquid handling equipment, a dispenser, an electrode array, a robot, or any combination thereof.
15. The method of claims 1, 4, 5 or 6, wherein said sample comprises between 10-500 cells.
16. The method of claim 10, wherein said tissue slice is about 2 mm .
17. The method of claims 4 or 5, wherein said preserved cells are derived from a suspected malignant polyp of a colon or an adenomatous of a colon.
18. The method of claims 6 or 9, wherein said preserved cancer cells are derived from a malignant polyp of a colon.
19. The method of claims 4, 5 or 6, wherein said enzyme is alkaline phosphatase.
20. The method of claims 4, 5 or 6, wherein said substrate is 4-aminophenyl phosphate (p-APP).
21. The method of claims 4 or 5, wherein said cells comprise colon cells.
22. The method of claims 1, 4, 5 or 6, wherein said measuring is effected using an electrochemical cell.
23. The method of claim 22, wherein said measuring is effected in a multiwell array.
24. The method of claim 23, wherein each well of said multiwell array comprises an electrochemical cell.
25. The method of claim 23, wherein each well of said multiwell array is a nano-volume well.
26. The method of claim 6, wherein said agent comprises a test composition.
27. The method of claim 26, wherein said test composition is selected from the group consisting of a polynucleotide a polypeptide, a small molecule chemical, a carbohydrate, a lipid and a combination of same.
28. The method of claim 6, wherein said agent comprises a test condition.
29. The method of claim 28, wherein said test condition is a radiation condition.
30. The method of claims 1, 4, 5 or 6, wherein said cells are intact.
31. The method of claims 1, 4, 5 or 6, wherein said cells are mammalian cells.
32. The method of any of claims 1, 4, 5 or 6, wherein a fixative for said preserved cells comprises formaldehyde.
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