LU86721A1 - METHOD FOR IN VITRO DETERMINATION OF THE SENSITIVITY OF TUMORS AND CELL LINES TO THE SPECIFIC ACTION OF ANTI-CANCER DRUGS - Google Patents

Description

METHOD FOR IN VITRO DETERMINATION OF THE SENSITIVITY OF TUMORS AND CELL LINES TO THE SPECIFIC ACTION OF

ANTI-CANCER DRUGS.

Technological background

Cancer is the second leading cause of morbidity and mortality in western Europe and North America, immediately after cardiovascular disease. In addition to local treatments (surgery and radiation therapy), hormonal or cytotoxic drugs are currently available which make it possible to halt its development in a certain number of patients. Applied intensively and early, they reduce mortality, as for example in the case of breast cancer. However, the spectrum of action of these .j g drugs is relatively small and their side effects are not negligible.

Numerous attempts have been made to date to predict the real effectiveness of these treatments, for each patient individually, in order to select the most active products and avoid unnecessary administration of toxic compounds. The most promising technique developed to date is undoubtedly that developed by Hamburger and Salmon in the USA. It is based on the cultivation in agarose or in 2 ^ synthetic medium of a cell suspension prepared from the tumor freshly operated. In a few weeks, if the culture succeeds (+ 60% uptake), one can assist in the development of cell colonies that can be clearly identified by microscopic examination. In this system, the inhibition of colony formation caused by the addition of drugs into the medium can be measured. A similar technique can be used, replacing the semi-solid medium that represents agarose with a solid support (Petry dish or derivative) covered with liquid medium, the cells adhering to the bottom of the dish. A recent trend, notably in the screening of new drugs potentially useful in oncology, is to replace "wild" tumors with a certain number of lines established from human tumors, and which we hope are representative of the cancers of which they are derived.

In order to reliably assess the effects of a drug, the culture must have resulted in the development of a sufficient number of colonies in the controls cultivated without drug, which reduces the applicability of the test to approximately 30 % of cases. The predictive value of clonogenic cultures with respect to the therapeutic response observed in the patient is far from absolute, and many false positives or false negatives have been described, suggesting that the suspension procedure cell followed by prolonged culture profoundly alters the biological characteristics of the material tested. Finally, this technique requires a time of the order of 3 weeks before obtaining a response, which constitutes a waste of precious time for the application of the treatment to the patient.

OBJECT OF THE INVENTION The present invention aims to provide a method making it possible to determine in vitro the sensitivity of tumors and cell lines to the specific action of anti-cancer drugs, both drugs of the hormonal type and of the cytotoxic type.

2 5 It aims in particular to provide a process which, by its nature, is applicable practically to all cases of tumors, which is more reliable, in particular by reducing the risk of false answers, and which allows a specific observation of the activity on different cell populations from the same tumor and which ultimately is faster than the techniques of the prior art. The envisaged technique should also be transposable to a partially or fully automated processing of the tests.

35 Character-defining elements of the invention

The principle underlying the invention is that which consists in monitoring the development of the proliferating fraction originating from a fragment of fresh tissue or from a cell suspension of this tissue, by means of labeling.

Specifically, the invention relates to a method for in vitro determination of the sensitivity of tumors and cell lines to the specific action of anti-cancer drugs comprising labeling of the cell fraction in an appropriate manner, incubation in presence of the drug to be tested for a sufficient time to observe the toxic effect of said drug and a measurement of the proliferating cell fraction.

10 The measurement of the tumor can be carried out by simple cutting of the fresh tissue into fragments of the order of a cubic millimeter or can be carried out in the conventional manner in the form of a cell suspension, although this procedure offers less practical information or make it more difficult and less complete to interpret the results.

The preparation of the test drug can be brought into contact before labeling, but to avoid any interference or possible effect of this drug with the labeling, it is preferable that this contacting occurs after labeling. .

Said marking can be carried out according to techniques known per se. According to a first embodiment of the invention, this labeling can be carried out using tritiated thymidine, in which case the measurement of the proliferating cell fraction is advantageously carried out by autoradiography. Another embodiment includes labeling with biotin and the measurement is advantageously carried out in this case by measuring the integrated optical density.

The measurement of the proliferating cell fraction can therefore be a measurement of the percentage of cells in phase S or in phase M or both a measurement of the percentage of cells in phase S and M, which provides valuable information on the mode of action of the drug tested.

In practical terms, to obtain a statistically reliable answer, the time required to observe “4 the toxicity of the drug tested is equal to or greater than 24 h / but is however significantly lower (one week at most) than the delays of the order of several weeks which are required in the prior art techniques.

According to a particular embodiment, it is possible to proceed to the measurement of the proliferating cell fraction using an automated apparatus of the type of Microscopic Analysis System with Automatic Scanning 10 (SAMBA 200).

According to the technique of the invention, it becomes possible to considerably improve the yield and the reliability of the hormochemogram and the technique is applicable to all cancers (solid tumors, lines, hematological cancers). It makes it possible to evaluate the effects of a drug in all cases and types of tumors. The results are reliable because the biological properties of the tumor (environment, architecture of the tissue) are preserved since it is not necessary to carry out a homogenization of the preparation. The quantity of available tissue does not represent a limiting factor, insofar as, from 10 mm 3, it is already possible at present by the technique of the invention to test a medicament at a determined concentration. If you have 25 cm3, you can test at least three drugs at one concentration, or one drug at three different concentrations. It is likely that it will be possible to further refine the technique in the future to use less starting material.

Since, unlike the methods of the prior art, the method of the invention does not involve prior homogenization (grinding) of the preparation with destruction of the cells, examination and histological observation remains possible and it is it becomes for the first time possible to determine for two drugs which would have an identical cytotoxic effect, that which exerts the most reduced toxic effect on healthy cells.

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Preferred forms of execution.

The invention will be described in more detail with the aid of the illustrations which follow execution techniques applied on the one hand to solid tumors and on the other hand to "monolayers" or cell suspensions.

A. Solid tumors.

The material used consists of tumors freshly removed, sterile.

The operating steps are: 1. The tumors are fragmented (scalpel, scissors, etc.) into pieces of approximately 1 mm 3. These are incubated during the whole procedure in a synthetic culture medium (for example MEM or DMEM, supplemented with antibiotics) and maintained in survival under adequate conditions of temperature, humidity and oxygenation (for example oven ).

2. Labeling of the DNA of the proliferating cell fraction with a purine or pyrimidine base which is itself labeled (for example with tritium, carbon-14, biotin, or by a substrate recognizable by specific anti-bodies ... ). This labeling is carried out for 1 to 3 hours and the operation is followed by several washes in order to get rid of the unincorporated excess of labeled nucleic base.

25 3. Exposure of the tumor material to drugs whose cytotoxic or antiproliferative action is to be tested. The duration of exposure (a few minutes to a few hours) and the concentrations depend on the drugs studied. The operation is followed or not by washing in order to possibly get rid of the excess drug (s).

Part of the tumor material, used as a control, is not subjected to the action of the drug (s).

4. Incubation for variable durations (from 1 hour to 7 days), depending on the drugs studied and the amount of material available. This is optionally distributed in individual wells (one per condition) suitable for organotypic culture.

5. Histological fixation, dehydration, inclusion, microtome section of the material.

6. Revelation of the labeled base by an appropriate method (autoradiography, immunohistochemistry, avidin-biotin technique, protein A, etc.) and possible counter staining.

| 7. Reading of the slides in order to define for the controls (C), and for each experimental condition (E) the proportion of labeled cells at the end of incubation. The W / C ratio defines the proportion of surviving cells within the initially labeled proliferating fraction; it makes it possible to assess the cytotoxic or antiproliferative effect of the drug (s) considered). The reading is done using an optical microscope, either manually or automatically, using for example the Microscopic Analysis System at | Automatic scanning (SAMBA 200).

Alternatively, it is possible to proceed in such a way that the exposure to the drug (s) precedes the labeling operation.

B. Monolayers or cell suspensions

The material can be constituted by cell lines, established or not, or cell suspensions (cytopuncture or dispersion of solid tumors, blood, marrow or effusion fluid).

During the first four steps, the cells are treated as indicated for steps 1 to 4 of the procedure intended for solid tumors. The monolayers are cultivated on a histological slide; the suspensions are centrifuged at the end of incubation, either on a slide or in the form of a pellet.

The fifth step consists of histological fixation, dehydration, inclusion, if necessary, of the pellets and section of the microtome thereof.

For steps 6 and 7 the procedure is identical to that described in the procedure for solid tumors.

Also in this case, it is possible to proceed so that the exposure to the drug or drugs 7 precedes the marking operation.

The invention will be further described by way of illustration with the aid of the following embodiment example. Example: 5 Illustration of a chemogram or a hormonogram established from organotypic culture of human tumor tissue.

1. Transport medium

Culture of human tissue requires trans-10 port from the operating room to the culture chamber.

The minimum essential medium (MEM) supplied by the company Gibco, devoid of serum, but supplemented with L-glu-tamine (0.6 mg / ml, Gibco), gentamycin '(40 pg / ml, She-15 ring Corporation ), penicillin (100 U / ml, Difco) and streptomycin (100 pg / ml, Difco) was used as “transport medium”. This medium is contained in sterile, single-use vials, kept at 4 ° C on ice The time elapsed between the time of aseptic removal of the tumor and the setting in culture is on average from 2 to 12 hours.

2. Handling medium

The cutting of the tumor into fragments of 1 x 1 x 2 mm ("survival explants"), using a scalpel, must be done in a liquid medium where the tissues are immersed in order to avoid their deterioration and their desiccation. For the manipulations the above-mentioned "transport medium" was used, but with a concentration of antibiotics five times higher.

The handling medium is poured into a sterile 60 x 10 mm Petri dish. At the end of cutting, the explants are rinsed three times in the medium rich in antibiotics.

3. The labeling of the proliferating cell fraction.

As soon as the cutting (+ rinses) is complete, the tumor fragments are incubated for 2 hours in a Petri dish (60 x 10 mm) containing 20 ml of MEM medium (defined in §1) to which 1 pc of tritiated thymidine 8 (^ H-TdR: specific activity 40 Ci / mmol) per ml of MEM medium. This step represents the "labeling of the proliferating cell fraction" of the tumor. When the two hours of labeling have elapsed, the fragments are rinsed four times in order to remove the tritiated thymidine which has not incorporated.

4. Chemotherapeutic and / or hormone therapy treatment of tumor fragments.

The tumor fragments are distributed in diver-10 its PETRI boxes (60 × 10 mm), each corresponding to an individual experimental condition. The attached figure shows the following hormonogram: 25 tumor fragments were incubated in 20 ml of "transport medium" only (control condition "Ct" in attached figure 15), while 3 x 25 fragments were respectively incubated in 20 ml of "transport medium" containing either 0.1 μg of adriamycin (ADM) / ml of MEM medium (condition "ADM 0.1 pg / ml" in the attached figure), or 1 pg ADM / ml MEM (condition "ADM 1 pg / ml"), ie 10 pg ADM / ml 20 MEM (condition "ADM 10 pg / ml). The incubation in the presence of 11 ADM was one hour in the The tumor fragments were then rinsed four times in the "transport medium".

Then, the tumor fragments originating from the same experimental condition were then distributed in 5 35 x 10 mm Petri dishes, containing 2 ml of "transport medium", at a rate of 5 fragments per Petri dish. These tumor fragments were fixed for histology respectively 2 hours, 1, 3, 5 and 7 days after their cultivation in these 35 x 10 mm Petri dishes.

5. Evaluation of the proliferating cell fraction by autoradiography.

The fragments are fixed for histology for 24 hours, in a liquid composed of 75 parts of 96 ° ethanol, 20 parts of neutral formalin (40%) and 5 parts of pure acetic acid. The pieces are then dehydrated, coated with paraffin, cut to 4 μm thick, dewaxed and soaked in liquid nuclear emulsion / 9 (Ilford K5).

These histological slides are exposed for 2 weeks then revealed and fixed for autoradiography, slightly counter-stained with hematoxylin and finally 5 mounted with DPX (BDH Chemicals).

The evaluation of the proliferating cell fraction consists in establishing the percentage of cells whose nucleus is marked by tritiated thymidine, that is to say the TLI (Thymidine Labeling Index) in each of the 5 tumor fragments corresponding to the same experimental condition. This percentage is established on 1000 tumor cells counted per fragment.

The results can for example be presented as an average of TLI + the standard error over the average (SEM). The Fischer F test (analysis of variance with one criterion) was used to assess the effect of a drug or hormone on the proliferating cell fraction of the tumor studied.

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Claims (9)

10 1. Method for in vitro determination of the sensitivity of tumors and cell lines to the specific action of anti-cancer drugs, characterized in that it comprises the labeling of the proliferating cell fraction in an appropriate manner, the incubation in the presence of the drug to be tested for a time sufficient to observe the toxic effect of said drug and a measurement of the labeled cell fraction after an incubation of an appropriate duration, 2. Method according to claim 1 characterized in that the preparation of the tumor is carried out by simple cutting of the fresh tissue into fragments of the order of a cubic millimeter. 15 · Method according to claim 1 characterized in that the preparation of the tumor is carried out in the form of a cell suspension. 4. Method according to any one of the preceding claims, characterized in that the preparation of the preparation and of the drug to be tested is carried out after labeling. 5. Method according to any one of claims 1 to 4 characterized in that the labeling of the DNA of the proliferating cell fraction is carried out with a purine or pyrimidine base itself labeled. 6. Method according to claim 5 characterized in that the labeling of the purine or pyrimidine base is carried out with tritium, carbon 14, biotin, or by a substrate recognizable by specific antibody 30 for 1 to 3 hours and that the this is followed by several washes in order to get rid of the unincorporated excess of labeled nucleic base and that the revelation of the labeled base is obtained by an appropriate method such as autoradiography, measurement of integrated optical density, immunohistochemistry, Avidin-biotin, protein A, followed by an optional counterstaining. 7. Method according to any one of the preceding claims, characterized in that the measurement of the proliferating cell fraction is a total or partial measurement of the percentage of cells in the different phases of the mitotic cycle, in particular of cells in phase S or in phase M or both a measure of the percentage of cells in phase S and M. 8. Method according to any one of the preceding claims, characterized in that the duration for observing the toxicity of the test drug is equal to or greater than 24 h. S · Method according to any one of the preceding claims, characterized in that the proliferating cell fraction is measured using an automated apparatus of the type: Analysis System 15 Automatic Scanning Microscopic (SAMBA 200). 20 25 1 35