METHOD FOR ISOLATING AND DETECTING CANCER STEM CELLS

Abstract

Disclosed is the in vitro use of at least one lectin for marking cancer stem cells of hormone-dependent cancer target organs, selected from the lectins Maackia amurensis lectin II (MAH-II), Euonymus europaeus lectin (EEL), Psophocarpus tetragonolobus lectin I (PTL-I) and Griffonia simplicifolia lectin II (GSL-II), in particular at least two lectins selected from MAH-II, EEL, PTL-I and GSL-II, in particular the two lectins MAH-II and EEL, in order to obtain cancer stem cells of labeled hormone-dependent cancer target organs in a biological sample.

Claims

1-13. (canceled)

14. An in vitro method of labeling by using of at least one lectin for the labeling of cancer stem cells of hormone-dependent cancer target organs, chosen from the lectins Maackia amurensis lectin II (MAH-II), Euonymus europaeus lectin (EEL), Psophocarpus tetragonolobus lectin I (PTL-I) and Grijfonia simplicifolia lectin II (GSL-II), to obtain labeled cancer stem cells of hormone-dependent cancer target organs, in a biological sample.

15. The in vitro method according to claim 14, wherein at least two lectins are chosen from MAH-II, EEL, PTL-I and GSL-II.

16. The in vitro method according to claim 15, wherein at least two lectins are used, said at least two lectins being in equal amount or in unequal amount, in particular in unequal amount in a weight ratio of 2:1, and preferably both lectins being MAH-II/EEL in unequal amount in a 2:1 weight ratio.

17. The in vitro method according to claim 14, wherein the MAH-II lectin recognizes O-linked glycans, the PTL-I lectin recognizes-linked glycans, the EEL lectin recognizes galactosylated glycans, and the GSL-II lectin recognizes N-linked glycans.

18. The in vitro method according to claim 17, wherein the MAH-II lectin recognizes the disialyl-T group [NeuAc 2-3Gal 1-3 (NeuAc 2-6) GalNAc].

19. The in vitro method according to claim 17, wherein the PTL-I lectin recognizes the Gal 1-3 (Fuc 1-2) Gal and GalNAc 1-3 (Fuc 1-2) Gal groups of the B and A antigens.

20. The in vitro method according to claim 17, wherein the EEL lectin recognizes the Gal1-3(Fuc 1-2)Gal group of the B antigen and the Fuc1-2Gal1-3GlcNAc group of the H antigen.

21. The in vitro method according to claim 17, wherein the GSL-II lectin recognizes agalactosylated tri- or tetra-antennal N-linked glycans.

22. The in vitro method according to claim 14, wherein said cancer is a cancer chosen among the group of the following cancers: breast, uterus, prostate and ovaries, endometrium, thyroid or adrenal glands.

23. The in vitro method according to claim 14, wherein said labeling of cancer stem cells of hormone-dependent cancer target organs is carried out with a lectin conjugated to a marker chosen from: a fluorophore in particular chosen from rhodamine, FITC or Alexa Fluor a radioisotope in particular chosen from iodine 125, tritium or technetium, an enzyme using a chromogenic or luminescent substrate, said enzyme being in particular chosen from horseradish peroxidase (HRP), phosphatase alkaline, glucose oxidase or -galactosidase, gold beads or biotin.

24. The in vitro method according to claim 14, wherein said labeling of cancer stem cells of hormone-dependent cancer target organs is carried out with a conjugated lectin and is followed by the detection of said labeled cancer stem cells of hormone-dependent cancer target organs via the detection of the conjugated lectin.

25. The in vitro method according to claim 14, wherein said labeling of cancer stem cells of hormone-dependent cancer target organs with a lectin conjugated to a marker is followed by the isolation of said labeled cancer stem cells of hormone-dependent cancers, in which said marker is biotin and said isolation is carried out via a support functionalized with streptavidin or avidin consisting of magnetic beads and in the presence of a magnet, or in which said marker is a fluorophore and said isolation is carried out by flow cytometry.

26. The in vitro method according to claim 14, wherein said biological sample is a sample of target organs of hormone dependent cancers.

27. An in vitro method of diagnosis of the risk of recurrence of hormone-dependent or non-hormone-dependent cancers and/or the aggressiveness of hormone-dependent or non-hormone-dependent cancers to define a prognostic value for the therapeutic adaptation of hormone-dependent cancers dependent or non-hormone-dependent, comprising the steps of: (a) Labeling of the cancer stem cells of hormone-dependent cancer target organs of a biological sample with at least one lectin chosen from lectins MAH-II, EEL, PTL-I and GSL-II, to obtain cancer stem cells of hormone-dependent cancer target organs labeled with at least one lectin in said biological sample, said lectin being conjugated to a marker chosen from a fluorophore, a radioisotope, an enzyme, gold beads or biotin, and (b1) Isolation of cancer stem cells of hormone-dependent cancer target organs labeled with said at least one conjugated lectin: when labeling with a conjugated lectin, biotin, said isolation is carried out via a support functionalized with streptavidin or avidin, in particular said functionalized support consists of magnetic beads functionalized with streptavidin or avidin and said isolation is carried out by magnetic cell sorting in the presence of a magnet, or during labeling with a conjugated lectin a fluorophore, said isolation is carried out by cell sorting in flow cytometry (c1) New labeling of cancer stem cells of hormone-dependent cancer target organs isolated with at least one lectin selected from MAH-II, EEL, PTL-I and GSL-II lectins, to obtain cancer stem cells from target organs of hormone-dependent cancers isolated and marked by the new labeling, said lectin being conjugated to a marker chosen from a fluorophore, a radioisotope, an enzyme, gold beads or biotin, (d1) Detection of said cancer stem cells of hormone-dependent cancer target organs isolated and marked with the new labeling by: fluorescence microscopy or fluorescence reader when the lectin is conjugated to a fluorophore, or when the lectin is conjugated to biotin and is detected via a fluorophore conjugated to streptavidin or avidin; luminescence microscopy or luminescence reader when the lectin is conjugated to an enzyme using a chemiluminescent substrate, or when the lectin is conjugated to biotin and is detected via an enzyme using a chemiluminescent substrate conjugated to streptavidin or to the avidin; gamma camera when the lectin is conjugated to a radioisotope, or when the lectin is conjugated to biotin and is detected via a radioisotope conjugated to streptavidin or avidin; UV/visible microscopy or absorbance reader when the lectin is conjugated to an enzyme using a chromogenic substrate, or when the lectin is conjugated to biotin and is detected via an enzyme using a chromogenic substrate conjugated to streptavidin or to the avidin; or electron microscopy when the lectin is conjugated to gold beads, or when the lectin is conjugated to biotin and is detected via gold beads conjugated to streptavidin or avidin; (e1) Possibly quantification of cancer stem cells of hormone-dependent cancer target organs; (f1) Comparison of the intensity of the detection of cancer stem cells of hormone-dependent cancer target organs in said biological sample versus the intensity of the detection of cancer stem cells of hormone-dependent cancer target organs in a healthy sample adjacent to the biological sample, and optionally comparison of the quantification of cancer stem cells of hormone-dependent cancer target organs in said biological sample compared to the quantification of cancer stem cells of hormone-dependent cancer target organs dependent in a healthy sample adjacent to the biological sample; and (g1) Deduction of the risk of recurrence of hormone-dependent or non-hormone-dependent cancers and/or the aggressiveness of hormone-dependent or non-hormone-dependent cancers to define a prognostic value for the therapeutic adaptation of hormone cancers-dependent or non-hormone-dependent on the basis of the presence and possibly the quantity of cancer stem cells of hormone-dependent cancer target organs, or (b2) Detection of said labeled cancer stem cells of hormone-dependent cancer target organs by fluorescence microscopy or reader of fluorescence when the lectin is conjugated to a fluorophore or when the lectin is conjugated to biotin and is detected via a fluorophore conjugated to streptavidin or avidin; luminescence microscopy or luminescence reader when the lectin is conjugated to an enzyme using a chemiluminescent substrate, or when the lectin is conjugated to biotin and is detected via an enzyme using a chemiluminescent substrate conjugated to streptavidin or to avidin; gamma camera when the lectin is conjugated to a radioisotope, or when the lectin is conjugated to biotin and is detected via a radioisotope conjugated to streptavidin or avidin; UV/visible microscopy or absorbance reader when the lectin is conjugated to an enzyme using a chromogenic substrate, or when the lectin is conjugated to biotin and is detected via an enzyme using a chromogenic substrate conjugated to streptavidin or to the avidin; orelectron microscopy when the lectin is conjugated to gold beads, or when the lectin is conjugated to biotin and is detected via gold beads conjugated to streptavidin or avidin; (c2) Possibly quantification of cancer stem cells of hormone-dependent cancer target organs; (d2) Comparison of the intensity of the detection of cancer stem cells of hormone-dependent cancer target organs in said biological sample versus the intensity of the detection of cancer stem cells of hormone-dependent cancer target organs in a healthy sample adjacent to the biological sample, and optionally comparison of the quantification of cancer stem cells of hormone-dependent cancer target organs in said biological sample compared to the quantification of cancer stem cells of hormone-dependent cancer target organs dependent in a healthy sample adjacent to the biological sample; and (e2) Deduction of the risk of recurrence of hormone-dependent or non-hormone-dependent cancers and/or the aggressiveness of hormone-dependent or non-hormone-dependent cancers to define a prognostic value for therapeutic adaptation of hormone-dependent or non-hormone-dependent cancers on the basis of the presence and possibly the quantity of cancer stem cells of hormone-dependent cancer target organs.

28. An in vitro diagnostic kit for the risk of recurrence of hormone-dependent or non-hormone-dependent cancers and/or the aggressiveness of hormone-dependent or non-hormone-dependent cancers to define a prognostic value for the therapeutic adaptation of hormone-dependent cancers dependent or non-hormone-dependent, comprising the mixture of MAH-II and EEL lectins, and/or the mixture of MAH-II and PTL-I lectins, and/or the mixture of MAH-II and GSL-II lectins, and/or the mixture of EEL and PTL-I lectins, and/or the mixture of EEL and GSL-II lectins, and/or the mixture of PTL-I and GSL-II lectins, and/or the mixture of MAH-II, EEL and PTL-I, and/or the mixture of MAH-II, EEL and GSL-II lectins, and/or the mixture of MAH-II, PTL-I and GSL-II lectins and/or the mixture of EEL, PTL-I and GSL-II lectins and/or the mixture of MAH-II, EEL, PTL-I and GSL-II lectins, said lectins being conjugated to biotin, and magnetic beads functionalized with streptavidin, and optionally the mixture of lectins MAH-II and EEL, and/or the mixture of MAH-II and PTL-I lectins, and/or the mixture of MAH-II and GSL-II lectins, and/or the mixture of EEL and PTL-I lectins, and/or the mixture of lectin EEL and GSL-II, and/or the mixture of PTL-I and GSL-II lectins, and/or the mixture of MAH-II, EEL and PTL-I, and/or the mixture of lectin MAH-II, EEL and GSL-II, and/or the mixture of MAH-II, PTL-I and GSL-II lectins and/or the mixture of EEL, PTL-I and GSL-II lectins and/or the mixture of MAH-II, EEL, PTL-I and GSL-II lectins, said lectins being conjugated to a fluorophore, a radioisotope, an enzyme or gold beads, or comprising the mixture of MAH-II and EEL lectins, and/or the mixture MAH-II and PTL-I lectins, and/or the mixture of MAH-II and GSL-II lectins, and/or the mixture of EEL and PTL-I lectins, and/or the mixture of EEL and GSL-II lectins, and/or the mixture of PTL-I and GSL-II lectins, and/or the mixture MAH-II, EEL and PTL-I, and/or the mixture of MAH-II, EEL and GSL-II lectins, and/or the mixture of MAH-II, PTL-I and GSL-II lectins and/or the mixture of EEL, PTL-I and GSL-II lectins and/or the mixture of MAH-II, EEL, PTL-I lectins and GSL-II, said lectins being conjugated with a fluorophore, and optionally the mixture of MAH-II and EEL lectins, and/or the mixture of MAH-II and PTL-I lectins, and/or the mixture of MAH-II and GSL-II lectins, and/or the mixture of EEL and PTL-lectins I, and/or the mixture of EEL and GSL-II lectin, and/or the mixture of PTL-I and GSL-II lectins, and/or the mixture of MAH-II, EEL and PTL-I and/or the mixture of MAH-II, EEL and GSL-II lectins, and/or the mixture of MAH-II, PTL-I and GSL-II lectins and/or the mixture of EEL, PTL-I and GSL-II lectins and/or the mixture of MAH-II, EEL, PTL-I and GSL-II lectins, said lectins being conjugated to biotin, a radioisotope, an enzyme or gold beads.

Description

DESCRIPTION OF THE FIGURES

[0284] FIG. 1 shows the results of the separation of cancer stem cells of hormone-dependent cancers on a sample of cells from the MCF-7 line, a line of cancer cells of breast origin.

[0285] Cancer stem cells are examined from a sorting of cells from the MCF-7 line, using the Epcam High+/Epcam High ratio, with the use of magnetic beads onto which streptavidin is grafted and the use of the following biotinylated lectins: Biotinylated MAH-II (Lectin MAH-II), Biotinylated EEL (Lectin EEL), Biotinylated PTL-I (Lectin PTL-I), Biotinylated GSL-II (Lectin GSL-II), a mixture in equal quantity (weight ratio 1:1) of biotinylated lectins MAH-II/EEL (Mixture 1) or mixture in unequal quantity (weight ratio 2:1) of biotinylated lectins MAH-II/EEL (Mixture 2).

[0286] FIG. 2 shows sections of breast tissue samples taken from biopsies of healthy (FIG. 2A) or breast cancer (FIG. 2B) patients:

[0287] FIG. 2A: Treatment of healthy tissue by mixing an equal quantity of the two MAH-II/EEL lectins (1:1 ratio) demonstrates the absence of labeling of the cells, demonstrating the absence of breast cancer stem cells in healthy tissue.

[0288] FIG. 2B: Treatment of tumor tissue by mixing an equal amount of the two MAH-II/EEL lectins (1:1 ratio) demonstrates the ability of this mixture of lectins to selectively label cancer stem cells of hormone-dependent cancer target organs, in this case to selectively label breast cancer stem cells (dark area).

[0289] FIG. 3 shows sections of ovarian tissue samples taken from biopsies of healthy (FIG. 3A) or ovarian cancer (FIG. 3B) patients:

[0290] FIG. 3A: Treatment of healthy tissue by mixing an equal amount of the two MAH-II/EEL lectins (1:1 ratio) demonstrates the absence of labeling of the cells, demonstrating the absence of ovarian cancer stem cells in healthy tissue.

[0291] FIG. 3B: Treatment of tumor tissue by mixing an equal amount of the two MAH-II/EEL lectins (1:1 ratio) demonstrates the ability of this mixture of lectins to selectively label cancer stem cells of hormone-dependent cancer target organs, in this case to selectively mark ovarian cancer stem cells (dark area).

[0292] FIG. 4 shows sections of uterine tissue samples taken from biopsies of healthy (FIG. 4A) or uterine cancer (FIG. 4B) patients:

[0293] FIG. 4A: Treatment of healthy tissue by mixing an equal amount of the two MAH-II/EEL lectins (1:1 ratio) demonstrates the absence of cell labeling, demonstrating the absence of uterine cancer stem cells in healthy tissue.

[0294] FIG. 4B: Treatment of tumor tissue by mixing an equal amount of the two MAH-II/EEL lectins (1:1 ratio) demonstrates the ability of this mixture of lectins to selectively label cancer stem cells of hormone-dependent cancer target organs, in this case to selectively mark cancer stem cells in the uterus (dark area).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1: Protocol for the Isolation of Cancer Stem Cells from Breast Cancer

[0295] I. Materials Required

[0296] Reagents and Materials [0297] Biotinylated individual lectin or mixture of biotinylated lectins specifically marking Cancer Stem Cells of hormone-dependent cancers, here breast cancer (prepared from individual lectins from Vector Laboratories and Emelca Biosciences) [0298] CELLection Biotin Binder kit (Invitrogen) containing magnetic beads coupled to streptavidin by a DNA bond [0299] Magnet

[0300] Buffers [0301] Versene (Invitrogen) comprising phosphate buffered saline (PBS) and EDTA [0302] Buffer 1: PBS (Phosphate buffered saline without Ca.sup.2+ and Mg.sup.2+) with 0.1% BSA (Bovine serum albumin), pH 7.4 [0303] Buffer 2: PBS (Phosphate Buffer Saline without Ca.sup.2+ and Mg.sup.2+) with 0.1% BSA (Bovine Serum Albumin) and 0.6% sodium citrate [0304] Buffer 3: RPMI 1640 with 1% FCS (fetal calf serum), 1 mM CaCl.sub.2) and 5 mM MgCl.sub.2, pH 7.0-7.4.

[0305] II. Duration of the Experiment [0306] 20 min to prepare cells [0307] 20 min to label cells [0308] 20 min to incubate labeled cells with the beads [0309] 10 min to recover the suspension not enriched in CSCs [0310] 15 min to break the CSCs/beads bond [0311] 5 min to recover the suspension enriched in CSCs of interest [0312] TOTAL: Ih30

[0313] III. Magnetic Sorting Procedure:

[0314] 1. Preparation of cells. The cells of the MCF-7 line (mammary cancer cell line) are detached from their support with Versene for 10 min at 37 C.

[0315] 2. The cells are counted and the number of cells is adjusted to 1.10.sup.7 in the sample.

[0316] 3. The cell suspension is centrifuged at 300 g for 10 min then the supernatant is eliminated.

[0317] 4. Blocking of non-specific sites. 1 mL of Buffer 2 is added.

[0318] 5. Cell labeling. A total amount of lectins of 10 g is added, so that when there is a plurality of lectins, the amounts of each are the same.

[0319] Thus, are added: [0320] 10 pg of an individual biotinylated lectin chosen from: chosen from MAH lectins

[0321] II, EEL, PTL-I and GSL-II, or [0322] 5 pg of each lectin for mix 1 (MAH-II/GSL-II) [0323] 6.66 pg of MAH-II lectin and 3.33 pg of GSL-II lectin for mixture 2.

[0324] The mixture then obtained is incubated for 10 min at 4 C.

[0325] 6. 500 pL of Buffer 2 is added in order to wash the cells and the suspension is centrifuged at 300 g for 10 min and the supernatant is removed.

[0326] 7. Addition of beads. The cells are resuspended in 1 mL of Buffer 2 and then 25 L of magnetic beads coupled to streptavidin washed beforehand are added and resuspended using Buffer 1. The mixture is incubated for 20 min at 4 C. with gentle stirring.

[0327] 8. Recovery of the suspension NOT enriched in CSCs. The tube is then placed on the magnet for 2 min. Cells labeled with biotinylated lectins and bound to magnetic beads coupled to streptavidin, precipitate in the direction of the magnet (magnetic cell sorting) and are then separated from unlabeled cells. The supernatant containing unlabeled cells is then removed, keeping the tube placed on the magnet, and stored in a Falcon tube.

[0328] 9. The tube containing the labeled cancer stem cells is then removed from the magnet. 1 mL of Buffer 1 is added. The tube is vortexed and replaced on the magnet for 2 min, then the supernatant is again removed and stored in the same falcon as in step 8. This step is repeated twice.

[0329] 10. The labeled cancer stem cells, still bound to the magnetic beads, are resuspended using 200 m1 of Buffer 3 preheated to 37 C. 4 l of cell/bead binding cleavage buffer consisting of DNasel are added. This mixture is incubated for 15 min at room temperature with gentle stirring.

[0330] 11. The suspension is stirred with a pipette vigorously 5 to 10 times in order to facilitate the release of the cells.

[0331] 12. Recovery of the suspension enriched in CSCs. The tube is placed on the magnet for 2 min. The magnetic beads are then separated from the labeled cancer stem cells and the supernatant containing the labeled cancer stem cells is transferred to a tube containing 200 L of buffer 3 preheated to 37 C. Steps 11 and 12 can be repeated again to enrich the yield.

[0332] These experiments were performed under similar conditions with each of the lectins individually (MAH-II, EEL, PTL-I, GSL-II), a mixture in equal quantity (weight ratio 1:1) of two lectins (Mixture 1: MAH-II/EEL) or a mixture in unequal quantity (weight ratio 2:1) of two lectins (Mixture 2:2 MAH-II/EEL).

[0333] The results of these different tests are presented in FIG. 1.

[0334] As shown by the results of these tests, the use of mixture 1, namely MAH-II lectins and EEL in an equimolar mixture, allows the isolation of cancer stem cells of hormone-dependent cancer target organs, in this case breast cancer, and this predominantly compared to other trials carried out in parallel.

[0335] These results also make it possible to demonstrate that the cancer stem cells of breast cancer present on their surface predominantly O-linked glycans and in particular the disialyl-T group [NeuAc 2-3Gal 1-3 (NeuAc 2-6) GalNAc] and galactosylated glycans, in particular the Fuc1-2Gal 1-3GlcNAc group of the H antigen.

[0336] Indeed, MAH-II and EEL lectins reveal a particular efficiency in isolating and detecting this type of cancer stem cells.

Example 2: Clonogenicity Test

[0337] The objective of a clonogenicity test is to observe the capacity of cells to reform spheres (corresponding in the patient to the reform of a tumor mass) and therefore their proliferative capacity.

[0338] The clonogenicity test is in this example used to confirm the presence of cancer stem cells of hormone-dependent cancers and to quantify said cells in a sample after isolation of cancer stem cells of hormone-dependent cancers by the isolation method described herein. invention. It thus makes it possible to demonstrate the effectiveness of the isolation method according to the invention compared to a control sample not subjected to this method (unsorted cells).

[0339] The clonogenicity tests were carried out in a 6-well plate at a density of 500 cells/cm.sup.2 in a medium of RPMI composition (Gibco) supplemented with 50 units/ml of penicillin, 50 units/ml of streptomycin (Gibco) and 2.4 g/L of sodium bicarbonate, 1 M of HEPES buffer (Sigma Aldrich, Saint-Quentin-Fallavier, France), 1 progesterone (Sigma Aldrich), 1 putrescine (Sigma), 0.025 g/ml heparin (Sigma Aldrich), 30% (m/v) glucose (Sigma Aldrich), 1 Growth Supplement B27 (Invitrogen, Carlsbad, Calif.), 20 ng/mL EGL (Sigma Aldrich), 20 ng/mL Human Basic LGL (Sigma Aldrich), 1 insulin-transferrin-sodium selenite supplement (Roche diagnostics, Meylan, France).

[0340] The evolution of colonies was observed after incubation at 37 C. in a CO.sub.2 atmosphere for three weeks and quantified with ImageJ software.

[0341] Cancer stem cells of hormone-dependent cancer target organs isolated using the isolation method described in the present invention lead to the formation of spheres unlike the control. This is a clonogenicity test having unsorted cells as control (T-), against cells sorted positively by biotinylated MAH-II (MAH-II Lectin), biotinylated EEL (Lectin EEL), biotinylated PTL-I (Lectin PTL-I), biotinylated GSL-II (Lectin GSL-II), mixing in equal quantity (weight ratio 1:1) of biotinylated lectins MAH-II/EEL (Mixture 1), mixing in unequal quantity (2:1 weight ratio) of biotinylated lectins 2 MAH-II/EEL (Mixture 2). The method according to the present invention therefore makes it possible to obtain stem cells capable of reforming tumors (results not shown).

Example 3: Visible Labeling of Lectins on a Paraffinized Histological Section (FIG. 2: Breast)

[0342] Equipment used: Paraffin blocks, Ice, Microtome, Superfrost slides, Bond Max automaton (Leica Microsystems) with computer, Leica consumables (alcohol, washing buffer, ER1 buffer, dewax buffer, labels, coverslips, tubes), PBS-buffer 5% BSA, Biotinylated Lectins (MAH-II and GSL-II (Vector Lab), EEL and PTL-I (Emelca Biosciences)), Bond Intense R detection kit (Leica), Leica mounting medium, coverslips and microscope.

[0343] The paraffin blocks containing the breast cancer samples from each of the patients identified by their number (given by the pathological anatomy department) were placed in ice for about 1 hour in order to be cooled, in order to facilitate their microtome cut to a thickness of 4 m.

[0344] So-called superfrost blades, this for maximum adhesion of the cut tissue have been identified by the same numbers as those on the blocks. A drop of water was placed in the center of each of these slides.

[0345] The sections were made with a microtome and placed on the drop of water previously deposited. The slides were then placed on a hotplate at 37 C. to facilitate their adhesion and the excess water was removed. All of the slides produced were placed in an oven at 37 C. in order to dry them.

[0346] The rest of the manipulation involved the Bond Max automaton from Leica connected to a computer with software controlling the automaton. While the slides are in the oven, all of the immunohistochemical labeling manipulation was prepared, starting by checking the level on the automatic device of each of the products necessary for carrying out the manipulation, then identification of the blades with their same number on the software controlling the PLC. Labels allowing a standardized protocol were generated. The dilution of lectins and their quantity were calculated and the necessary kit prepared. It should be noted that each of the products used had to be scanned and the level reset to zero before each of the experiments carried out.

[0347] The labels were then stuck on their corresponding blades at the outlet of the oven and the coverslips, plastic elements placed on the cup allowing a homogeneous distribution of the product over the entire surface of the blade during handling thanks to the contact properties, were placed on each of the slides.

[0348] The slide rack was placed in the machine and after recognition by the reader of each of the elements and slides identified by their bar codes present on the labels, manipulation was initiated. It began with heat dewaxing using the Dewax product from Leica, which subsequently made the antibodies accessible. This step as well as all the others was followed by washes, thanks to the 10 Bond Wash previously diluted, this on three occasions.

[0349] This step was followed by a pretreatment for 5 min with ER1 buffer from Leica, corresponding to a citrate buffer at pH=6, which makes it possible to unmask the antigens, that is to say to make them accessible.

[0350] Biotinylated lectin or a mixture of two biotinylated lectins chosen from MAH-II, EEL, PTL-I and GSL-II, diluted in a solution of PBS with 5% BSA (in order to prevent non-specific attachments due to its saturating power), was placed on the cup for 20 min. The Bond Intense R detection kit (Leica) thanks to the intervention of a streptavidin-HRP playing the role of secondary antibody made it possible, through its properties, to reveal the biotinylated lectin (s) in brown thanks to the properties of DAB, a substrate HRP (horseradish peroxidase) enzyme, which reveals the biotin/streptavidin-HRP complex. A step of bluish counterstaining thanks to the presence of hematoxylin was then carried out for 7 min in order to make the entire sample identifiable.

[0351] The slides have been removed from the machine. The sections were then rehydrated by dipping the slides manually in an alcohol bath twice for 5 min. This rehydration step was continued with a toluene bath for 5 min as well. The slides could therefore be mounted by adding a drop of mounting medium (Leica).

[0352] The slides were finally observed under a microscope.

[0353] The labeling of healthy tissue, obtained from a breast biopsy, by mixing an equal amount of the two MAH-II/EEL lectins (ratio 1:1) showed the absence of labeling of the cells. It can therefore be deduced from this that there are no breast cancer stem cells in this tissue (FIG. 2A).

[0354] Several cell types coexist in tumor tissue: non-tumor stem cells, non-tumor stem cells, tumor stem cells and non-tumor non-stem cells. The labeling of a tumor tissue by the mixture in equal quantity of MAH-II/EEL lectins (ratio 1:1) demonstrates the ability of this mixture of lectins to selectively label cancer stem cells of hormone-dependent cancer target organs, in this case to selectively label breast cancer stem cells (FIG. 2B).

Example 4: Visible Labeling of Lectins on a Paraffinized Histological Section (FIG. 3: Ovary)

[0355] The protocol for this manipulation is identical to that of Example 3 above. However, the samples contained in the paraffin blocks necessary for carrying out this present example are obtained from ovarian cancers originating from patients. The labeling of healthy tissue, obtained from an ovarian biopsy, by mixing an equal amount of the two MAH-II/EEL lectins (ratio 1:1) showed the absence of labeling of the cells. It can therefore be deduced from this that there are no ovarian cancer stem cells in this tissue (FIG. 3A).

[0356] In tumor tissue, several cell types coexist: tumor non-stem cells, non-tumor stem cells, tumor stem cells and non-tumor non-stem cells. The labeling of a tumor tissue by the mixture in equal quantity of MAH-II/EEL lectins (ratio 1:1) demonstrates the ability of this mixture of lectins to selectively label cancer stem cells of hormone-dependent cancer target organs, in this case to selectively mark ovarian cancer stem cells (FIG. 3B).

Example 5: Visible Labeling of Lectins on Paraffinized Histological Section (FIG. 4: Uterus)

[0357] The protocol for this manipulation is identical to those of Example 3 and of Example 4 above. However, the samples contained in the paraffin blocks necessary for carrying out this present example are obtained from cancers of the uterus originating from patients.

[0358] The labeling of healthy tissue, obtained from a uterine biopsy, by mixing an equal amount of the two MAH-II/EEL lectins (ratio 1:1) showed the absence of labeling of the cells. It can therefore be deduced from this the absence of uterine cancer stem cells in this tissue (FIG. 4A).

[0359] In tumor tissue, several cell types coexist: tumor non-stem cells, non-tumor stem cells, tumor stem cells and non-tumor non-stem cells. The labeling of a tumor tissue by the mixture in equal quantity of MAH-II/EEL lectins (ratio 1:1) demonstrates the ability of this mixture of lectins to selectively label cancer stem cells of hormone-dependent cancer target organs, in this case to selectively label uterine cancer stem cells (FIG. 4B).