DE19910955A1 - Determining sensitivity of cells to apoptotic agents, useful e.g. for selecting compounds for treating cancer, using externalization of phosphatidyl serine as apoptotic marker - Google Patents

Abstract

Determining the chemical sensitivity of cells towards at least one substance (I) comprising measuring (I)-induced apoptosis, is new. The cells are incubated, simultaneously, with at least one marker (II) that has a detectable interaction with phosphatidyl serine (PS), and (I), and the (II)-PS interaction is determined in a time-resolved manner. An Independent claim is also included for a kit containing at least one cytostatic agent and at least one (II), as a solid material, solution or in presence of a matrix substance.

Description

The invention relates to a method for determining the chemosensitivity of Cells against at least one substance by measuring the through the at least a substance triggered apoptosis.

Chemosensitivity tests

The treatment and healing success of tumor diseases have since Introduction of chemotherapy increased significantly. For example, the Überle Rate of childhood acute lymphoblastic leukemia (ALL) in the mid-1960s at less than 10%. Nowadays the chance of recovery is over 70%. The medi certain, so-called cytostatics, are administered alone or in accordance with certain therapy plans Combined with other active ingredients. In the meantime there is an unruly one manageable number of different therapy plans that are empirically determined and constant be further developed. The main criterion for an improved therapy plan is a better survival rate (clinical outcome). This criterion applies to the Majority of patients too. But each individual has a different one Equipment on cells with different properties. This is particularly true towards the properties of tumor cells. Some studies have shown who that the therapy works extremely well for a subpopulation while it works for  other patients little or over due to drug-resistant tumors is not effective at all. (Lacombe et al., Blood, 84, 716-723 (1994), Smit et al. International Journal of Cancer 51, 72-78 (1992)). It could be shown that not only the effectiveness of various drugs, but also the effectiveness same dose of a drug can be individually different. The investigators The individual dosage of a medication is for the patient of Significance so that on the one hand he receives as much medication as for his Healing is necessary and, on the other hand, the toxic effects of Therapy and the risk of secondary chemotherapy-induced Cancer can be kept as low as possible. As good as the current Common therapy plans are also stagnating, the progress of recent years in terms of mean survival. Another significant improvement in Chemotherapy should be done by creating customized therapy plans to be possible.

To address this problem, some researchers have tried the individual Measure the sensitivity of patient tumors to cytostatics in vitro. The Most chemosensitivity tests currently used are based at least partially based on that of Salmon (Salmon et al. Science, 197, 461-463 (1977)) developed an agar tumor culture test. These tests measure cell prolifera tion.

A second type of chemosensitivity test involves the exclusion of (fluorescence) dyes or the release of the radioactive chromium isotope ⁵¹ Cr, which measures the destruction of the cell membrane by direct or indirect cytostatic effects. A modification of the previous dye exclusion test is the so-called DiSC assay (Weisenthal, Kern Oncology 5: 92-103 (1991)), which additionally differentiates between normal and tumor cells through a second staining process.

The third type of chemosensitivity test determines parameters of the cellular me Tabolism as a measure of the damage caused by cytostatics. This type of test includes the radiometric BACTEC test (from Hoff D., Forseth B., Warfel L., in Salmon Trent (Eds.) Human tumor cloning, pp. 656-657, Grune & Stratton, Orlando (1984)), the MTT test (Freund A. et al. European Journal of Cancer 34: 895-901 (1998), Kaspers G.J.L. Blood 92: 259-266 (1998), Pieters R. et al. Leukemia 12, 1344-1348 (1998), Klumper E. et al. British Journal of Haematology 93: 903-910 (1996), Hwang W.S. et al. Leukemia Research 17: 685-688 (1993)) and its variations, the ATP test (Kangas L., Gronroos M., Nieminen A., Medical Biology 62: 338- 343 (1984)) and the so-called FCA test (Meitner P :, Oncology 5: 75-81, (1991), Rotman B, Teplitz C., Dickinson Kl., Cozzolino J., Cellular and Develop mental Biology 24: f1137-1146 (1988)).

Agar culture assays have the major disadvantage that far from all tumors cells grow in the agar cultures. This is especially true for lymphatic leu combs and lymphomas (Veerman A.J.P., Pieters R. British Journal of Haematology 74: 381-384 (1990)). The most famous representative of this type of assay, the clono gene assay, the percentage of evaluable cell populations is only 30- 40%. The cells must grow very long (10-20 days) before the evaluation he follows. The workload is also immense.

The dye exclusion tests as well as the ⁵¹ Cr release test determine the proportion of cells with a defective cell membrane. These tests often include fixation and subsequent microscopic evaluation of the cells or measurement of the radioactivity in the supernatant. Such tests are non-specific due to the measurement of a rather rough parameter, the cell membrane integrity, and cannot distinguish between a specific anti-tumor effect of a substance or a physical or z. B. distinguish cell damage caused by a strong chemical burn or oxidation by a substance. Tests of this type are consequently also positive for substances that generally damage all cells and are therefore not suitable as antitumor drugs. Probably the most meaningful test of the dye exclusion type is the DiSC test. By staining twice, in contrast to other dye exclusion tests, he can differentiate between the cell damage of tumor cells and normal cells. The double staining and the subsequent microscopic evaluation are extremely time consuming. In addition, the results vary depending on the person who performs the evaluation and the image section that is evaluated under the microscope.

For radiometric tests such as B. the ⁵¹ Cr release test applies very generally: The use of radioactive isotopes in diagnostic tests is increasingly being avoided these days because of the potential hazard and disposal problems. In addition, fluorescence and luminescence techniques can now achieve the same sensitivities with a frequently shorter overall test duration.

From chemosensitivity tests that measure a cell's metabolism as a measure of The most common use of proliferation is the MTT test and its variation used. The MTT test has after a 4-day incubation of the cells various cytostatics a relatively short test duration of about 4 hours. Moreover 96 samples can be processed in parallel in a microtiter plate using an ELISA Readout device can be evaluated. This ensures a reasonable throughput at rela Guaranteed low personnel costs. The MTT test is based on the implementation of 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide in a blue it formazan product. The reaction is catalyzed by dehydrogenases that  are only active in living cells. The MTT test depends on a constitution ven basic activity of the cells examined. However, it has been found that some ge cell types, e.g. For example, some FAB subtypes of acute myeloid are particularly common Leukemia, have a greatly reduced dehydrogenase activity (Santini V. et al. Hematological Oncology 7: 287-293 (1989)). The chemosensitivity of this tumor cells cannot therefore be assessed using an MTT test. Another pro The resulting water-insoluble formazan crystals are a problem. These must by adding organic solvents e.g. B. dimethyl sulfoxide or isopro panol are brought into solution. Experience has shown that this creates problems occur in the reproducibility of the test.

Apoptosis

Through the work of various research groups (e.g. Sen, S. et al. FEBS Lett. 307: 122- 127, Darzynkiewicz et al. Cytometry 13: 795-808 (1992), Fulda S., Los M., Friesen C., Debatin K.M. Int. J. Cancer 76: 105-114 (1998)) it appears likely that Cell death caused by cytostatics generally via the mechanism of apoptosis se runs. Apoptosis is a cell death programmed by the cell itself represents that can be induced in the organism by physiological factors or can be triggered by chemicals such as cytostatics. The programmed Cell death plays an extremely important role in cytostasis e.g. B. the Immunsy stems. For example, T cells that act against the body's own structures are removed by apoptosis from the organism. Otherwise it can symptoms of an autoimmune disease occur, e.g. B. lupus erythematosus, arthritic diseases or the disease caused by a T cell tumor. Substan Zen like the tumor necrosis factor alpha synthesized by macrophages are natural che inducers of apoptosis.  

Apoptosis follows a programmed, uniform scheme, in which in a very early phase of phosphatidylserine, which is normally exclusive located on the inside of the cell membrane, is presented on the outside (Inversion). This event occurs much earlier than the hours later fragmentation of the DNA and dissolution of the cell membrane in the late Phase of apoptosis.

In addition to cell death from apoptosis, there is another form of cell death that z. B. physically triggered by osmotic shock, chemical burns or oxidation Necrosis. This manifests itself in severely degenerative damage to the cell.

Medenica (US 5736129) determines the extent of cell damage by cytostats ka by staining the cells with propidium iodide and then staining the cells counted cells in the FACS (fluorescence activated cell sorter). This approach has but the big disadvantage of not being exactly between apoptosis and necrosis divorce, and therefore the specific mechanism of action of cytostatics do not record. In addition, the cell requirement for a meaningful analysis is very high high.

The detection of phosphatidylserine presented on the cell surface is ideally suited for the specific detection of apoptosis, since the inversion of phosphatidylserine is a very early step in apoptosis and the staining can be carried out quickly and easily on both fixed and suspended cells. In tests described so far, An nexin V is always used as the phosphatidylserine binding marker. This is characterized by a very low Kd of 5 × 10 ^-10 M. A disadvantage is the strong calcium-cation dependence of the bond. These tests all consist of an incubation phase with or without substances (e.g. cytostatics), sedimentation and washing in PBS and subsequent resuspension in calcium-containing staining buffer with labeled Annexin V. The stained cells can then be counted in the FACS or under the fluorescence microscope become. Since all previous test protocols contain washing steps and are therefore heterogeneous assays, these tests are relatively labor intensive and prone to errors. These tests are all characterized in that apoptosis is measured at a certain time after induction by substances. Since apoptosis is a dynamic process that goes through a maximum, which depends on both the substance being examined and the cell type, and then ends in a secondary necrosis, it is not known a priori at what point in time the extent of apoptosis is measured must become. It follows that, in principle, several samples with z. B. tumor cells after different incubation times with regard to chemosensitivity must be determined.

The phosphatidylserine tests described so far did not allow immediate Measurement of the course of apoptosis over time. With the previous test regulations needed for a certain combination of a cell type and a concentration a series of experiments can be carried out on a substance in order to chen course of apoptosis. The time and manpower, as well the cell material requirement for such a test would be so high that one would be high parallel testing of little cell material for chemosensitivity to ver different concentrations of many substances was impossible.

The object of the present invention was to overcome the disadvantages of the prior art overcoming technology.

The problem is solved by a method for determining the chemosensitivity cells against at least one substance by measuring the Mostly a substance triggered apoptosis, whereby the cells in the presence  at least one marker whose interaction with phosphatidylserine is detectable, are incubated with the at least one substance and the interaction time Lich resolved is detected.

The cells to be examined with regard to their chemosensitivity are described in Ge presence of the substances, d. H. simultaneously or with submitted marker molecules, incubated. This turns the heterogeneous test protocols used so far a homogeneous test "Mix and Measure", which measures the immediate kinetics of the apoptotic process. When Annexin V is used as a marker , it must be ensured that the calcium ion concentration during the entire measuring time remains constant and in the optimal concentration. This can e.g. B. can be achieved by buffering the calcium ion concentration becomes. The calcium ion dependency can be avoided entirely, if instead of Annexin V as a marker, other Annexins, Annexin derivatives, Annexin muteins, antibodies, Fab fragments, single-chain antibodies, aptamers and / or other proteins with binding sites for phosphatidylserine can be used.

Miniaturization is possible to further reduce material consumption. It could be shown that cells in less than 10 µl medium in a few days Culture can be kept. By reducing the number of cells per sample chamber to ≦ 1000 per test are highly parallel analyzes of samples with extremely little Patient cells, such as B. from fine needle biopsies. The low cell requirement also enables high throughput screening (HTS) from unknown substances on anti-tumor effects.

If the apoptosis of cells of pathological tissue via the Phophatidylseri ninversion is tested, it is advantageous to reference healthy cells of the tissue bes as well as the same cells with no added substance. In particular, it is  useful as a control a permanent cell line, whose chemosensitivity is known is to be tested in order to document the functionality of the test and errors recognizing the test execution.

As substances such. B. the following substances in question:
A) Cytostatics
Abrin, amethopterin, acivin, aclacinomycin A; Alanine mustard, altretamine, aminoglutethimide, aminopterin, amsacrine (mAMSA), various anabolic steroids, an thrapyrazole, L-asparaginase, 5-axacytidine, Bacillus Calmette-Guerin, bisantrene, buserelin, busulfan, butyryloxyiosglytamamatazalamatezamatezamatezalamatez esters, Carzinophyllin, CCNU, chlorambucil, Chlorethylmethylcyclohe xyl nitrosohamstoff, Chlorethylcyclohexylnitrosoharnstoff, chlorodeoxyadenosine, corticotropin, cyproterone acetate, chlorotrianisene, Chlorozotozin, chromomycin A, cytosine arabinoside (Ara-C), BCNU, bleomycin, cis-platinum, carbo-platinum, Cladribi ne, cyclophosphamide, dactinomycin, daunomycin, daunorubicin, Decarbacin, doxorubicin, DTIC, Dehydroemitin, 4-demethoxydaunorubicin, Demothydoxoru bicine, deoxydoxorubicin, dexamethasone, Dibromodulcitol, Dichloromethotrexat, diethylstilbestrol, bis- (2-chloropropyl) -DL-serine, doxifluridine, elliptinium , 4'-epidoxorubicin, epirubicin, epoetin alpha, erythropoeitin, esorubicin, estradiol, etoposide, fluoxymesterone, F lutamide, folic acid, Fotemustin, Ftorafur, 4-FU, flu darabine phosphate, 5-FU, floxuridine, galactitol, gallium nitrate, goserelin, G-CSF, GM-CSF, hydrea, hexamethylmelamine (HMM), hydrocortisone, hydroxyproge sterone, 4 -Hydroperoxycyclophosphamide, ICRf 159, Idamycin, Ifosfamide, Immune lobulin IGIV, Interferon, Cobalt-Proporphyrincomplex, Leucovorin Calcium, Leu prolid, Levadopa, Levothyroxine, Lindane, Liothyronine, Liotrix, Lomustin, Levamaristol, Masopinolinole, Masoprocolinole, Masoprocolinophenol Methosalen, methyl esterone, methyl lomustine, mithracin, mithramycin, mitotane, mitoxantrone, methotrexate (MTX), 6 MP, mechlorethamine hydrochloride, medroxyprogesterone, megestrol acetate, melphalan, mesna, mitomycin-C, nandroloc, navphosphinate, P32 , nHuIFNa, nHuIFNb, nHuIFNp, octreofide acetate, ondansetrone hydrochloride, disodium pamidronate, pentamethyl melamine (PMM), pentostatin, peptiochemio, plicamycin, prednimustine, probroman, procarbazine, profiromycin, paraplat in, prednisolone, prednisone, razoxan rIFNa-2a, rubidazone, rIFNa-2b, rIFNb-1b, rIFNt-1b, rIL-2, rTNF, Semustin, SPG 827 (podophyllin derivative), spirogermanium, streptonigrin, somatostatin, tamoxinatin, streptozoz Thio-TEPA, 6-thioguanine, tenoposide, testolactone, testosterone, 3-TGDR, rTNF, thyroglobuliri, thyrotropin, trilostane, uracil-mustard, VP-16, vincristine (VCR), vinblastine (VIBL), verapamil, vindesin, vinzesin Vitamin A acid, vitamin A analogues, zinostatin.
B) peptides and peptoids
C) Nucleic acids and derivatives
D) Peptide Nucleic Acids (PNA's)
E) Hybrid from RNA's, DNA's, PNA's and the like Derivatives

To detect extracellular phosphatidylserine, cells must be present of a marker are incubated with the substances to be examined. This Markers can be antibodies, Fab fragments, single-chain antibodies, aptamers and / or other proteins with binding sites for phosphatidylserine such as annexins be used. These markers can contain a dye, colloidal Edelme tall, have a radioactive isotope and / or rare earth metal chelates.

The detection is preferably carried out using fluorescence detection methods, in particular based on confocal or conventional microscopy. The intact fluores  cells labeled with zenate phosphatidylserine are e.g. B. with a CCD camera recorded and then using an image processing system programs counted. Cells that are characterized by a DNA dye, such as. B. Propidi Umiodid staining are considered necrotic and the number of subtracted fluorescence-labeled cells over phosphatidylserine, so that the number of apoptotic cells is determined. This is then related to the total cell ge set by automatic image evaluation in the phase contrast microscope is determined. Fluorescence correlation spectroscopy would also be suitable (FCS) and confocal fluorimetry, e.g. B. the concentration of fluorescent Marker molecules in the supernatant and / or on the cell surface is measured. At Multiple staining e.g. B. with a second marker or the same marker another fluorescent dye can be found in the supernatant as well as on the Cell surface cross-correlation or two-color coincidence fluorimetric measurements solutions are carried out. In this case, two methods are used simultaneously different fluorescent dyes detected. In the cross-correlation, the cross-time-dependent fluorescence fluctuation signals of the two measuring channels liert. With two-color coincidence fluorimetry there is a positive one Signal when both fluorescence signals appear at the same place at the same time. By Such two-color analyzes can determine the specificity of the detection for the phosphate dylserine inversion can be increased. The An is of particular use Use of FIDA and PIDA as in WO 98/16814 and PCT / EP 98/06165 described. This allows multiple binding of fluorescent marker moles cool easily on apoptotic cells due to the increased fluorescence emission be measured. The specificity can go even further by using 2D-FIDA be increased. A combination of the mentioned has proven to be very powerful Fluorescence measurement techniques with a relative movement of sample and measurement vo lumens shown against each other. Because this way a lot more cells per Unit of time can be observed, the signal statistics improve and thus  the signal-to-noise ratio drastically. In particular, there is also a quantifle Binding of fluorescent marker molecules to cells is possible.

By labeling phosphatidylserine, apoptotic cells, as well Stained cells with defective cell membranes, because then the phosphatidylserines become accessible on the inside of the cell membranes. If you have the temporal The course of apoptosis is followed, so initially intact, aptoptotic Cells stained externally. Smaller also colored apopti are laced vesicles (apoptotic bodies). In the final phase, apoptotic cells go in the secondary necrosis over and decay. Necrotic cells are known records that the cell membranes disintegrate into fragments. Apoptosis must be from Nekro se are differentiated so the effect of effective cytostatics, all pass through an apoptosis mechanism, is specifically detected. At two Differentiating between apoptotic and necrotic cells can additionally Marker to rule out necrosis, together with the phosphatidylserine dendem markers are used, for example dyes containing nucleic acid interact with one another, but cannot penetrate intact cell membranes nen (e.g. propidium iodide, BOBO ™ (Molecular Probes)).

The course of apoptosis over time can thus be marked by means of the labeling of phosphate dylserin "Online" will be followed.

It is important to determine the extent of apoptosis based on the total number of tried to normalize cells. This can e.g. B. by measuring light absorption, Scattered light, conductivity measurement or microscopic evaluation happen. The Standardization is necessary to determine the extent of cell damage caused by various To be able to compare cytostatics.  

example

A powerful cytostatic that immediately stops cell growth and all cells converted to apoptosis can be clear due to the small number of cells have less apoptosis than in the case of a weaker acting cytostatic cum, in which the cells can still multiply at first, and only later to go into apoptosis to a small extent. Only standardization is taken into account the apoptosis in relation to the total cell number and evaluates the more effective Cytostatic correct.

The chemosensitivity test of cells can be carried out particularly advantageously with a Perform kit. This kit includes a sample rack with multiple samples chambers, where a sample chamber at least one substance and a marker for phosphatidylserine detection and possibly a marker for exclusion of necrosis contains. In the sample holder it can be, for. B. a Handelselsübli act microtiter plate. The substances to be tested and the marker can either as a dry substance, in solution or in the presence of matrix sub punch like B. salts, buffers, carbohydrates, carboxylic acids, pyrimidines, anor ganic or organic nanoparticles up to 1 µm in diameter.  

example Material for performing the chemosensitivity test

• - Sterile bench (Holten, Antares)
• - fluorescence microscope (Carl Zeiss Jena)
• - CO ₂ incubator (WTC Binder)
• - Vortex mixer (Bender + Hobein AG)
• - refrigerator (2-10 ° C) (Bosch)
• - Freezer (-20 ° C) (Liebherr)
• - autoclave (H + P Labortechnik GmbH)
• - centrifuge (Eppendorf)
• - Pipettes (Eppendorf)
• - Pipette tips (ratiolab)
• - Microtiter plates (96 chambers) (Falcon)
• - 12.50 ml tubes (Falcon, Greiner)
• - RPMI 1640 medium (Gibco)
• - patient blood or bone marrow
• - Annexin V-Alexa 568 ™ (Boehringer.Mannheim)
• - BOBO ™ (Molecular Probes)
• - Dulbecco's PBS (Gibco)

Test execution Cell count determination

10 µl of a cell suspension are filled into a new building chamber. Under a Using a light microscope, the cells are counted in the 16 smallest squares. By Mul tlication of the cell number with 10000 gives the cell number per ml.

Preparations of some cytostatics solutions

Mix and incubate with cytostatics

The cells are mixed with 0.5 µg / ml BOBO ™ and 50 µM Annexin V- Alexa 568 ™ in the presence of a constant 3 mM calcium ion concentration with the Incubated cytostatics.

Quantification in the fluorescence microscope

Every hour, samples are taken with a CCD camera and image extractor fluorescence microscope equipped with the annexin V cells marked red by fluorescence, then by cells marked green by BOBO Cells and finally all of the cells in phase contrast mode counts. The necrotic cells stained by BOBO are determined by the number of Annexin V-positive cells subtracted and in relation to the total number of cells set. This gives the normalized percentage of apoptotic cells. With As the incubation period increases, the proportion of apoptotic cells initially increases by decrease again after a few hours when more and more cells in the end phase of apoptosis, the secondary necrosis. The maximum extent Apoptosis is a measure of the effectiveness of a cytostatic.

Claims (14)

1. Method for determining the chemosensitivity of cells against at least a substance by measuring the triggered by the at least one substance Most apoptosis, the cells being present in the presence of at least one marker Interaction with phosphatidylserine is detectable with which at least ei incubate a substance and the interaction is resolved in time is tect. 2. The method according to claim 1, characterized in that the at least one Markers before and / or during the incubation of the cells with the at least one egg NEN substance is added. 3. The method according to claim 1 and / or 2, characterized in that the cells animal or human cells, especially leukemia cells, are more solid cells Tumors, cells of pathological organs and / or reference cells such as as cells from other than the pathological organs or cells from ge healthy areas of pathological organs. 4. The method according to any one of claims 1 to 3, characterized in that a Reference measurement carried out without adding the at least one substance becomes. 5. The method according to any one of claims 1 to 4, characterized in that as Substances active pharmaceutical substances, chemotherapeutic agents, environment pollutants, peptides, nucleic acids or derivatives thereof, PNAs and / or nu Small acid hybrids are used.   6. The method according to any one of claims 1 to 5, characterized in that as Marker antibodies, Fab fragments, single-chain antibodies, aptamers and / or other proteins with binding sites for phosphatidylserine such as annexins be set. 7. The method according to any one of claims 1 to 6, characterized in that the Marker a dye component, a colloidal precious metal, a radioactive isotope and / or rare earth metal chelates. 8. The method according to any one of claims 1 to 7, characterized in that Apoptotic cells can be distinguished from necrotic cells, in particular by co-incubation with a marker for necrotic cells, in particular with a dye that interacts with nucleic acids and is intact Can not penetrate cell membranes. 9. The method according to any one of claims 1 to 8, characterized in that for Detection of fluorescence detection methods, in particular methods based on confocal or conventional microscopy. 10. The method according to any one of claims 1 to 9, characterized in that the the number of cells identified as apoptotic based on the total number of cells is standardized. 11. The method according to any one of claims 1 to 10, characterized in that the Detection with a temporal resolution of hours or longer stands.   12. The method according to any one of claims 1 to 11, characterized in that as Marker Annexin V in the presence of calcium in the concentration range of 0.1 to 30 mM, preferably 1 to 10 mM, is used. 13. The method according to any one of claims 1 to 12 for the search for apoptotic we substances. 14. Kit containing at least one cytostatic and at least one marker, the interaction with phosphatidylserine is detectable, whereby this escapes that as a dry substance, in solution or in the presence of matrix substances available.