Method for culturing a subpopulation of circulating epithelial tumour cells from a body fluid

Abstract

The invention relates to a method for culturing a subpopulation of circulating epithelial tumour cells from a body fluid of a human or animal suffering from an epithelial tumour, wherein cells contained in the body fluid each containing at least one cell nucleus are separated from the body fluid and cultured over at least 24 hours in suspension, with formation of spheroids.

Claims

1. A method of testing sensitivity of tumor cells with respect to a medicament or a therapeutic measure for treating a tumor disease underlying the occurrence of circulating epithelial tumor cells in a human or animal, comprising: (i) separating cells present in a body fluid from the human or the animal and containing at least one nucleus in each case from the body fluid without selection of certain of these cells; (ii) transferring said separated cells to a cell culture medium; (iii) culturing said transferred separated cells under cell culture conditions in said cell culture medium containing at least an animal serum, a growth stimulator and one growth factor, wherein the growth stimulator is insulin and hydrocortisone and wherein the growth factor is Epidermal growth factor, and wherein the cells are cultured for at least 24 hours in suspension at least until a subpopulation of tumor cells, which does not require adherence to a surface for proliferation, has formed spheroids by proliferation, and wherein tumor cells which have formed the spheroids during culturing are separated from the cultured tumor cells, by separating the spheroids formed during culture; and (iv) testing of sensitivity of tumor cells in the spheroids obtained in step (iii) with respect to the medicament or the therapeutic measure by exposing the spheroids obtained in step (iii) to the medicament or the therapeutic measure and analyzing the effect of the exposure.

2. The method as claimed in claim 1, wherein the tumor cells present in the spheroids are fluorescently labeled using antibodies directed against the human epithelial antigen EpCAM (epithelial cell adhesion molecule) prior or after exposing the spheroids to the medicament or the therapeutic measure and then analyzed using an image analysis method.

3. The method as claimed in claim 2, wherein the tumor cells present in the spheroids are stained with a substance, which is detectable by fluorescence and stains dead cells after exposing the spheroids to the medicament or the therapeutic measure.

4. The method as claimed in claim 2, wherein labeling, and/or analyzing are carried out in the presence of a Ca.sup.2+ chelator.

5. The method as claimed in claim 1, wherein the therapeutic measure is a physical measure, or a pharmaceutical measure.

6. The method as claimed in claim 1, wherein the cells are cultured in a cell culture vessel that does not allow adherence to a surface thereof.

Description

(1) The invention will now be described in more detail on the basis of an exemplary embodiment and the figures. What are shown are:

(2) FIG. 1a, b, c spheroids of tumor cells from peripheral blood from a patient suffering from colon carcinoma after culturing for 7 days (FIG. 1a), 14 days (FIG. 1b) and 21 days (FIG. 1c) and

(3) FIG. 2 a fluorescence microscopy image of a tumor spheroid fluorescently stained with antibodies and propidium iodide.

(4) The methods described below were carried out using the following chemicals, reagents, buffers, solutions, antibodies, instruments and consumables and also the indicated medium:

(5) TABLE-US-00001 Chemicals and reagents Chemicals and reagents Company Ammonium chloride Sigma-Aldrich, USA (NH.sub.4Cl) (155 mM) Potassium bicarbonate Sigma-Aldrich, USA (KHCO.sub.3) (10 mM) Ethylenediaminetetraacetate Sigma-Aldrich, USA (EDTA) (0.5M) Dulbecco's Phosphate GIBCO, USA Buffered Saline (D-PBS) Flow-Check 770 Fluorospheres Beckman Coulter, Ireland Propidium iodide solution (1 mg) Sigma-Aldrich, USA RPMI 1640 (1X) 500 ml Invitrogen GmbH, Germany Hepes buffer solution (1M) Invitrogen GmbH, Germany Penicillin/streptomycin Invitrogen GmbH, Germany (10000 U/10000 μg) L-Glutamine (200 mM) Biochrom AG, Germany Fetal bovine serum (FBS) Invitrogen GmbH, Germany Insulin (100 U/mL) Sanofi-Aventis, USA Hydrocortisone 100 mg Pfizer, USA EGF Sigma-Aldrich, USA

(6) TABLE-US-00002 Buffers and solutions Buffers and solutions Constituents Amount D-PBS CaCl.sub.2 0.901 mM MgCl.sub.2—6H.sub.2O 0.493 mM KCl 2.67 mM KH.sub.2PO.sub.4 1.47 mM NaCl 137.93 mM Na.sub.2HPO.sub.4—7H.sub.2O 8.06 mM D-PBS-EDTA D-PBS 500 ml EDTA 2 ml Erythrocyte lysis buffer (dissolve NH.sub.4Cl 8.3 g in 1 L of distilled water) KHCO.sub.3 1 g EDTA 2 ml Propidium iodide solution PI 3.5 μl (in 1 ml of distilled water)

(7) TABLE-US-00003 Antibodies Article Antibodies Company number CD 326 (EpCAM) conjugated with Miltenyi Biotec 130-080-301 fluorescein isothiocyanate (FITC) GmbH, Germany

(8) TABLE-US-00004 Instruments Instruments Company Centrifuge 5810 R Eppendorf, Germany Laser scanning microscope iCys.sup. ™ CompuCyte Corporation, USA Vortexer Bender und Hobein GmbH, Germany

(9) TABLE-US-00005 Consumables Consumables Company 15 ml Falcon tubes Labor Schubert, Germany 1.5 ml Eppendorf tubes Labor Schubert, Germany Micropipettes Eppendorf, Germany Disposable Pasteur pipettes, graduated ROTH, Germany (3.2 ml) Microtiter plates (MTP) with glass base Greiner bio-one, Germany Culture flasks (25 cm.sup.2, 65 ml) neoLab, Germany

(10) TABLE-US-00006 Composition of the medium Chemicals Final concentration A culture medium, preferably RPMI 1640 A serum additive, 5% preferably fetal calf serum L-Glutamine 4 mM Hepes 15 mM Growth stimulator, 5 μg/ml preferably insulin Hydrocortisone 0.5 μg/ml Antibiotics, preferably 100 U/ml; penicillin/streptomycin 100 μg/ml Growth factors, 40 ng/ml preferably EGF

(11) Blood samples were collected from the peripheral vein in 2-7 ml tubes containing EDTA as anticoagulant. The viability of the circulating epithelial tumor cells was on average 95%.

(12) In a sample tube, 1 ml of the particular blood sample was topped up to a total volume of 15 ml using the erythrocyte lysis buffer and incubated in the refrigerator for 15 min at a temperature of 4° C. Next, the sample was centrifuged at 2000 rpm for 7 min and at a temperature of 18° C. The supernatant was then decanted. Thereafter, the pellet was resuspended with 2 ml of medium and transferred to a culture flask into an initially charged 3 ml of medium (depending on the leukocyte count: 5 ml of medium per 10 000 leukocytes). This was incubated for 21 days at 5% CO.sub.2 and 37° C. Every 5 days, 2 ml of fresh medium were added. Every 7 days, i.e., on the 7th, 14th, 21st and 28th day, the tumor spheroids were examined under the microscope. For the analysis, the tumor spheroids were collected by means of a gentle centrifugation and then the pellet was resuspended with 500 μl of D-PBS-EDTA. 50 μl of this mixture were then transferred to a 1.5 ml Eppendorf tube. Thereafter, 5 μl of a (FITC)-monoclonal antibody against the human epithelial antigen (EpCAM) were pipetted thereinto. This was followed by another 15-minute cooling at 4° C. Lastly, 430 μl of D-PBS-EDTA were added and the samples, now completed, were stored overnight in the refrigerator at 4° C.

(13) On the following day, 100 μl of cell suspension of the sample to be measured and 5 μl of propidium iodide (PI) were pipetted in each case into a well of an ELISA plate. The plate was then covered and allowed to rest for about 20 min so that the cells could sediment on the base. Subsequently, the cells were measured using the laser scanning cytometer and the measurement results were evaluated.

(14) What were obtained were nonadherent, three-dimensional spheroids, which are typical of the presence of tumor stem cells. FIG. 1a shows the spheroids after 7 days of culturing, FIG. 1b shows them after 14 days of culturing, and FIG. 1c shows them after 21 days of culturing. FIG. 2 shows a red-stained necrotic tumor cell in the middle of a tumor spheroid cell aggregate. The death of this tumor cell may possibly be due to a lack of diffusion of growth factors through the tight cell packing surrounding said cell.

(15) It was possible to culture a subpopulation of the circulating epithelial tumor cells which forms spheroids and has a high capacity for proliferation.