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    • 专利摘要:
    The present invention relates to the use of a lectin recognizing the fucose α 1-2 galactose motif as the first labeling means and possibly a second means of labeling colorectal cancer stem cells, in particular a lectin recognizing the T antigen for the implementation of a method for detecting and possibly isolating colorectal cancer stem cells, a method of detecting and possibly isolating colorectal cancer stem cells for research purposes and a method for in vitro diagnosis of the risk of cancer. recurrence of colorectal cancer and / or aggressiveness of colorectal cancer to define a prognostic value for the therapeutic adaptation of colorectal cancer, as well as a kit comprising a lectin recognizing the fucose α 1-2 galactose motif and a lectin recognizing the T antigen.
    公开号:FR3044681A1
    申请号:FR1561764
    申请日:2015-12-02
    公开日:2017-06-09
    发明作者:Aurelie Lacroix；Frederic Varnat；Ludovic Micallef；Fabrice Lalloue；Marie-Odile Jauberteau
    申请人:Universite de Limoges；
    IPC主号:
    专利说明:

    METHOD OF ISOLATING CANCER STEM CELLS
    The present invention relates to the field of cancer stem cell isolation.
    Colorectal cancer (CRC) is the third most common disease in the world. This, like any cancer, can be summed up by an abnormal cellular proliferation in a healthy tissue (here the colic mucosa) causing the appearance of a tumoral mass. One of the hypotheses put forward to explain the tumor progression as well as the mechanisms of resistance and recurrences is based on the existence of cancer stem cells. The therapeutic escape of the tumor to radio- and chemo-therapeutic treatments depends on the presence of these cells within the tumor. Therefore, the detection of these cells in the tumor tissue is a means of defining the level of aggressiveness of the tumor. The characterization of specific biomarkers of cancer stem cells is of great diagnostic and prognostic value in the treatment of cancer. However, there are currently no specific markers of cancer stem cells (CSCs) that allow their discrimination with certainty of other tumor cells. Because of their small numbers and the absence of specific markers, the major difficulties in studying CSCs lie in their isolation and characterization. The modification of glycosylated proteins is often associated with tumor progression and thus appears to be of major interest in the search for surface markers.
    There is therefore a need for a method for detecting and / or isolating colorectal cancer stem cells.
    The present invention fills this need.
    The present invention is based on the demonstration by the inventors that the fucose unit has 1-2 galactose expressed on the surface of colorectal cancer stem cells to detect and possibly isolate these cells using a lectin recognizing this pattern.
    In a first aspect, the present invention relates to the use, as a labeling means, of a lectin, for the detection and optionally the isolation of colorectal cancer stem cells.
    In a second aspect, the present invention relates to a method for detecting and possibly isolating colorectal cancer stem cells comprising labeling the colorectal cancer stem cells with a lectin. Advantageously, the present invention relates to the use of a lectin and at least one second means for labeling colorectal cancer stem cells, in particular at least one second lectin recognizing the T antigen, in particular two lectins recognizing the T antigen.
    In a third aspect, the present invention relates to a kit comprising a lectin and a second means for detecting and optionally isolating colorectal cancer cells.
    In a fourth aspect, the present invention relates to a method for diagnosing the aggressiveness and / or risk of recurrence of colorectal cancer to define a prognostic value for the therapeutic adaptation of colorectal cancer, comprising a detection step and possibly isolation of colorectal cancer cells.
    A first object of the present invention relates to the use, as the first means of labeling, of a lectin recognizing the fucose moiety at 1-2 galactose, advantageously Ulex Europaeus Agglutinin 1 (UEA-1) or Trichosanthes Japonica Agglutinin. II (TJA-II), for the implementation of a method for detecting and possibly isolating colorectal cancer stem cells.
    The present invention relates to the use, as a first labeling means, of a lectin recognizing the 1-2-galactose fucose motif, advantageously Ulex Europaeus Agglutinin 1 (UEA-1) or Trichosanthes Japonica Agglutinin II (TJA-II). for the implementation of an in vitro method for detecting and possibly isolating colorectal cancer stem cells, in particular in a colorectal biological sample.
    The present invention also relates to the use, as a first labeling means, of a lectin recognizing the 1-2-fucose galactose motif, advantageously Ulex Europaeus Agglutinin 1 (UEA-1) or Trichosanthes Japonica Agglutinin II (TJA-II). ), for the implementation of an in vivo method for detecting and possibly isolating colorectal cancer stem cells, in particular on animal models or on human models of tumors.
    For the purpose of the present invention, the term "means for labeling colorectal cancer stem cells" means a substance capable of binding specifically to a marker expressed on the surface of the colorectal cancer stem cells. The labeling means may in particular be an antibody directed against an antigenic determinant, such as a glycoprotein, a protein or a glycan. The colorectal biological sample may be in particular a tumor biopsy taken from a patient with colorectal cancer or a biopsy taken from a patient suspected of having such a cancer. The tumor biological sample may also be a colorectal cancer cell line or a tumor induced in an animal by injection of cancer cell lines, for example in mice or rats. The cell line is preferably a colorectal cancer cell line. According to this embodiment, the induced tumor contains colorectal cancer stem cells which are advantageously isolated from the other cells of the tumor in order to be studied.
    For the purposes of the present invention, the term "detection" means the identification by spectroscopic methods of the presence of colorectal cancer stem cells within a colorectal tissue.
    For the purposes of the present invention, the term "isolation" means obtaining a population of cells enriched in colorectal cancer stem cells from a colorectal tumor. For the purposes of the present invention, the term "enriched" refers to a population of cells in which the ratio of cancer stem cell number to total number of cells is at least 4, advantageously at least 6, preferably at least 6. 8 and particularly preferably at least 9 as determined by the ratio Epcam high + cells / Epcam high cells - by flow cytometry.
    Currently, the reference product for the isolation of colorectal cancer stem cells consists of magnetic beads on which are grafted anti-CD133 antibodies marketed by Miltenyi Biotec. The inventors of the present invention have demonstrated that a lectin recognizing the fucose motif at 1-2 galactose was more effective at isolating colorectal cancer stem cells than this system.
    It has also been demonstrated by the inventors that the cells could be enriched particularly efficiently by combining a lectin recognizing the fucose-1-galactose motif and a second marking means for colorectal cancer stem cells.
    The present invention therefore also relates to the use of a lectin recognizing the 1-2-fucose galactose motif and a second means for labeling colorectal cancer stem cells for the detection and, optionally, the isolation of cancer stem cells in a sample. colorectal biology.
    For the purposes of the present invention, the second labeling means is a substance that specifically recognizes one or more markers expressed on the surface of the cancer stem cells. It may especially be an antibody.
    Examples of markers of colorectal cancer stem cells include CD133, CD44, CD166 (ALCAM), CD24, CD26, CD29, EpCAM, Oct-4 and Sox-2.
    Preferably, the second means for labeling the colorectal cancer stem cells is a lectin recognizing the T antigen or a mixture of lectins recognizing the T antigen.
    Advantageously, said lectin recognizing the T antigen is selected from the group consisting of Agaricus Bisporus Agglutinin (ABA), Amaranthus Caudatus Agglutinin (ACA) and Jacaline.
    The second means of labeling the colorectal cancer stem cells can also be a mixture of lectins recognizing the T antigen, in particular chosen from the group consisting of ABA and ACA; ABA and Jacaline; and Jacaline and ACA, advantageously Jacaline and ACA.
    In one embodiment, the present invention relates to the use of a mixture of two lectins selected from the group consisting of UEA-1 and ABA; TJA-II and ABA; UEA-1 and ACA; TJA-II and ACA; UEA-1 and Jacaline; and TJA-II and Jacaline.
    In another embodiment, the present invention relates to the use of a mixture of three lectins selected from the group consisting of UEA-1, ABA and ACA; TJA-II, ABA and ACA; UEA-1, ABA and Jacaline; TJA-II, ABA and Jacaline; UEA-1, Jacaline and ACA; TJA-II, Jacaline and ACA, advantageously UEA-1, ABA and Jacaline; and UEA-1, Jacaline and ACA, preferably UEA-1, Jacaline and ACA for the implementation of a method for detecting and possibly isolating colorectal cancer stem cells in a colorectal biological sample.
    Advantageously, the mixture of three lectins consists of a lectin recognizing the fucose unit with 1-2 galactose: Jacaline: ABA or ACA, in a molar ratio of 625 to 12500: 16 to 320: 1 to 20, advantageously from 5000 to 7000: 50 to 250: 5 to 15, especially 6250: 160: 10.
    In a particular embodiment, the lectins are UEA-1, Jacaline and AC A, advantageously in a molar ratio of 625 to 12500: 16 to 320: 1 to 20, advantageously from 5000 to 7000: 50 to 250: 5 to 15, preferably 6250: 160: 10.
    In order to detect and possibly isolate the colorectal cancer stem cells, the lectin or lectins are linked directly or indirectly to a means for detecting and possibly isolating said cells.
    For the purposes of the present invention, the term "detection means and possibly isolation means" a substance or a group of substances making it possible to identify the labeling means linked to colorectal cancer stem cells. Said detection means is for example a fluorophore, a chromophore, or magnetic beads. When it is desired to isolate the colorectal cancer stem cells, a fluorophore or magnetic beads is preferably used.
    Said detection and possibly isolation means may be linked directly or indirectly to the lectin (s).
    By "directly bonded" is meant in the sense of the present invention that the detection means and optionally isolation is covalently bound to the marking means.
    For the purposes of the present invention, the term "indirectly bonded" means that the detection means is bonded to a second substance, the second substance being capable of recognizing specifically the marking means and of forming with it a bond strong enough to detect and possibly isolate colorectal cancer stem cells. The means of detection and possibly isolation can be in particular a streptavidin / biotin system.
    In one embodiment, the detection and optionally isolation means consists of biotin covalently bound to the lectin (s) and magnetic beads onto which streptavidin, avidin or an anti-biotin antibody is grafted. .
    The present invention therefore relates to the use of a biotinylated lectin recognizing the 1-2-galactose fucose motif, a first biotinylated lectin recognizing the T antigen, a second biotinylated lectin recognizing the T antigen and magnetic beads. on which are grafted streptavidin for the implementation of a method for detecting and possibly isolating colorectal cancer stem cells. Advantageously, said lectins are advantageously UEA-1, Jacaline and AB A or AC A, preferably in a ratio ranging from 625 to 12500: 16 to 320: 1 to 20, advantageously from 5000 to 7000: 50 to 250: 5 to 15 , especially 6250: 160: 10.
    In another embodiment, the means for detecting and possibly isolating is a fluorophore. The fluorophore may be covalently bound to the lectin (s) or conjugated to streptavidin, avidin or an anti-biotin antibody and the lectin (s) are biotinylated.
    The fluorophore may be any fluorophore capable of being used for flow cytometry. Such fluorophores are commercially available. It is for example Alexa fluorine 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, 750 or 790, the fluorescein isothiocyanate (FITC), Rhodamine, allophycocyanin (APC) and Phycoerythrin (PE). Advantageously, the fluorophore is Alexa fluorine 488, Alexa fluorine 594 or Alexa fluorine 633.
    The present invention thus relates to the use of a biotinylated lectin recognizing the 1-2-fucose galactose motif, a first biotinylated lectin recognizing the T antigen, a second biotinylated lectin recognizing the T antigen and a fluorophore conjugated with avidin, streptavidin or an anti-biotin antibody for the implementation of a method for detecting and possibly isolating colorectal cancer stem cells. Advantageously, said lectins are UEA-1, Jacaline and ABA or ACA, preferably in a ratio ranging from 625 to 12500: 16 to 320: 1 to 20, advantageously from 5000 to 7000: 50 to 250: 5 to 15, especially from 6250: 160: 10.
    The present invention therefore also relates to the use of a lectin recognizing the fucose-1-galactose motif, a first lectin recognizing the T antigen, a second lectin recognizing the T antigen, said lectins being conjugated to a fluorophore, for the implementation of a method for detecting and possibly isolating colorectal cancer stem cells. Advantageously, said lectins are UEA-1, Jacaline and ABA or ACA, preferably in a ratio ranging from 625 to 12500: 16 to 320: 1 to 20, advantageously from 5000 to 7000: 50 to 250: 5 to 15, especially from 6250: 160: 10. The study of cancer stem cells for research and diagnostic purposes is now a necessity, especially for the detection of new substances capable of acting against these cells. The study of these cells is also particularly useful in the context of personalized medicine.
    Advantageously, the present invention therefore relates to the use as described above, for the isolation of colorectal cancer stem cells.
    The detection of colorectal cancer stem cells, in particular their quantification, makes it possible also to evaluate the risks of tumor progression. The present invention therefore also relates to the use of a lectin recognizing the 1-2-fucose galactose motif and optionally at least one lectin recognizing the T antigen under the conditions described above, for the in vitro diagnosis of the risk. recurrence and / or aggressiveness of colorectal cancer to define a prognostic value for the therapeutic adaptation of colorectal cancer.
    A second subject of the present invention relates to an in vitro method for detecting and optionally isolating colorectal cancer stem cells (I), comprising the step of labeling the colorectal cancer stem cells of a colorectal biological sample with a lectin recognizing the 1-2-galactose fucose unit, advantageously UEA-1 or TJA-II.
    The method according to the present invention comprises, as a main step, the labeling of colorectal cancer stem cells with a lectin recognizing the fucose-1-galactose motif or the staining of colorectal cancer stem cells with a lectin recognizing the fucose motif. 1-2 galactose and at least one lectin recognizing the T antigen.
    The present invention also relates to an in vitro method for detecting and possibly isolating colorectal cancer stem cells (1a), comprising the steps of: (a) labeling colorectal cancer stem cells with a lectin recognizing the fucose motif at 1-2 galactose, to obtain a biological sample in which the colorectal cancer stem cells are labeled, (b) bringing into contact the biological sample in which the cancer stem cells are labeled with a means of detection and possibly of isolation of the cells, ( c) detection of colorectal cancer stem cells, (d) possibly isolation of colorectal cancer stem cells.
    In one embodiment, the method according to the present invention is implemented to isolate colorectal cancer stem cells. This isolation step makes it possible in particular to study the colorectal cancer stem cells detected in a colorectal tumor sample in order, for example, to discover new treatments capable of eliminating these cancerous stem cells, frequently causing recurrences and metastases. .
    The present invention therefore also relates to an in vitro method for detecting and isolating colorectal cancer stem cells (Ib), comprising the steps of: (a) labeling the colorectal cancer stem cells with a lectin recognizing the fucose motif at 1-2 galactose, to obtain a biological sample in which the colorectal cancer stem cells are labeled, (b) bringing into contact the biological sample in which the cancer stem cells are labeled with a means of detection and possibly of isolation of the cells, ( c) detection of colorectal cancer stem cells, (d) isolation of colorectal cancer stem cells. In particular, the colorectal biological sample may be a tumor biopsy previously obtained on a patient suffering from colorectal cancer. The biological sample may also be a biopsy of colorectal tissue in a patient suspected of having colorectal cancer. The tumor biological sample can also be a colorectal cancer cell line or a tumor induced in an animal by injection of cancer cell lines, for example in mice or rats, including a human cell line.
    Before staining the colorectal cancer stem cells in step (a), the cells are advantageously dissociated from each other. This dissociation of the cells can be carried out according to conventional procedures, for example by using one or more enzymes capable of separating the cells from one another without altering the glycans expressed on the surface of the cells, in particular the fucose-1-galactose unit. and the T antigen. The dissociation of the cells may for example be carried out with the Liberase® mixture marketed by Roche Diagnostic.
    The present invention therefore relates to a method (I), (Ia) or (Ib) comprising a step of dissociating the cells from each other before the step (a) of marking.
    In one embodiment, the labeling step (a) is carried out with a lectin recognizing the 1-2-galactose fucose motif as the first labeling means and a second means for labeling colorectal cancer stem cells, advantageously a lectin recognizing the T antigen or a mixture of lectins recognizing the T antigen. The lectin (s) recognizing the T antigen are advantageously chosen from the group consisting of AB A, Jacaline and AC A.
    Advantageously, the labeling step (a) is carried out with a mixture of lectins recognizing the T antigen, in particular chosen from the group consisting of AB A and ACA; ABA and Jacaline; and Jacaline and ACA, advantageously Jacaline and ACA.
    The present invention therefore relates to a method (I), (la) or (Ib) as described above, wherein the labeling step (a) is carried out with a mixture of lectins selected from the group consisting of UEA-1, ABA and ACA; TJA-II, ABA and ACA; UEA-1, ABA and Jacaline; TJA-II, ABA and Jacaline; UEA-1, Jacaline and ACA; TJA-II, Jacaline and ACA, advantageously UEA-1, ABA and Jacaline; and UEA-1, Jacaline and ACA, preferably UEA-1, Jacaline and ACA.
    Advantageously, the labeling step (a) is carried out with a lectin mixture recognizing the 1-galactose fucose motif: Jacaline: ABA or ACA in a molar ratio ranging from 625 to 12500: 16 to 320: 1 at 20, advantageously from 5000 to 7000: 50 to 250: 5 to 15, in particular from 6250: 160: 10. Preferably, the lectin mixture consists of UEA-1: Jacaline: ABA or ACA, in a varying molar ratio from 625 to 12500: 16 to 320: 1 to 20, advantageously from 5000 to 7000: 50 to 250: 5 to 15, in particular from 6250: 160: 10, in particular with a mixture of UEA-1, Jacaline and ACA in a ratio Molar 6250: 160: 10. In step (b), the biological sample is brought into contact with a means of detection and possibly isolation as defined above, that is to say magnetic beads. grafted with streptavidin, avidin or an anti-biotin antibody, the lectin (s) being biotinylated, uorophore covalently bound to the lectin, or fluorophore conjugated to streptavidin, avidin or an anti-biotin antibody, the lectin or lectins being biotinylated, the fluorophore being as defined above.
    The steps (c) of detection and optionally (d) of isolation of the cells are advantageously carried out with conventional detection and possibly cell isolation techniques such as flow cytometry or magnetic cell sorting.
    In the process as described above in which step (a) is carried out with a lectin mixture, the stem cells are advantageously simultaneously labeled with the lectin recognizing the fucose moiety at 1-2 galactose and the lectins recognizing T. antigen
    In another embodiment, the staining of the colorectal cancer stem cells in the biological sample with the lectin recognizing the fucose motif has 1-2 galactose and the lectin recognizing the T antigen or the lectin mixture recognizing the T antigen is implemented sequentially.
    In this embodiment, the method according to the present invention thus comprises the steps of: (a) labeling the colorectal cancer stem cells with a lectin recognizing the fucose-1-galactose motif, to obtain a sample in which the cancer stem cells colorectal cells are labeled with lectin recognizing the fucose-1-galactose motif, (b) detection and isolation of lectin-labeled cells recognizing the fucose-1-galactose motif, (c) labeling of isolated colorectal cancer stem cells with second labeling means, preferably a lectin recognizing the T antigen or a mixture of lectins recognizing the T antigen, (d) detecting the lectin-labeled cancer stem cells or a mixture of lectins recognizing the T antigen, (e) ) Isolation of colorectal cancer stem cells.
    In this further embodiment, the step of isolating lectin-labeled cancer stem cells recognizing the fucose-1-galactose motif (b) may be followed by a cell amplification step. Thus, after the isolation of the cells, they can be cultured in a medium which makes it possible to increase the quantity of colorectal cancer stem cells before being labeled with a second labeling means, advantageously a recognizing lectin. the T antigen or a mixture of lectins recognizing the T antigen. The step (c) can itself also be followed by a cell amplification step before the steps (d) and (e) of detection and isolation of colorectal cancer stem cells.
    When the second labeling means is a mixture of lectins, step (c) can be carried out with a first lectin recognizing the T antigen, the isolated labeled cells may optionally be subjected to a cell amplification step, and then the isolated cells can be labeled with a second lectin recognizing the T antigen.
    The inventors have demonstrated that a lectin recognizing the fucose-1-galactose motif, in particular UEA-1, optionally in the presence of a lectin recognizing the T antigen or a mixture of lectins recognizing the T antigen, makes it possible to to detect colorectal cancer stem cells in a particularly efficient way but also to isolate them.
    The present invention is particularly suitable for the study of colorectal cancer stem cells for research purposes, for example for the study of these cells in a colorectal cancer cell line or a tumor induced in an animal by injection of cancer cell lines, especially human, for example in mice or rats.
    The present invention is also particularly suitable for diagnostic application in patients with colorectal cancer, particularly in the follow-up of the disease and / or treatment.
    In fact, colorectal cancer stem cells are a particular cell population that, because of their resistance to chemotherapy treatments, lead to tumor reformation and tumor recurrence. The present invention thus makes it possible, by allowing the detection of colorectal cancer stem cells, to evaluate the risk of recurrence of colorectal cancer.
    The detection and quantification of colorectal cancer stem cells in tumor tissue also helps to determine the cancer's aggressiveness and ability to develop.
    The detection and quantification of colorectal cancer stem cells is also part of a personalized approach to medicine. In particular, the detection of colorectal cancer stem cells in the biological sample makes it possible to evaluate the prognostic value of the treatment and, if necessary, to adapt the treatment.
    A fourth subject of the present invention thus also relates to an in vitro diagnostic method for the risk of recurrence of colorectal cancer and / or the aggressiveness of colorectal cancer to define a prognostic value for the therapeutic adaptation of colorectal cancer. , comprising the step of labeling the colorectal cancer stem cells of a colorectal biological sample with a lectin recognizing the 1-2-galactose fucose motif, advantageously UEA-1 or TJA-II, or a kit comprising a lectin recognizing the 1-2-galactose fucose unit as defined above.
    The present invention more particularly relates to an in vitro diagnostic method for the risk of recurrence of colorectal cancer and / or the aggressiveness of a colorectal cancer to define a prognostic value for the therapeutic adaptation of a colorectal cancer comprising the steps of (a) labeling the colorectal cancer stem cells with a lectin recognizing the 1-2-galactose fucose motif, as the first labeling means, to obtain a biological sample in which the cancer stem cells are labeled, (b) implementing contact of the biological sample in which the cancer stem cells are labeled with a means for detecting and possibly isolating the cells, (c) detecting and possibly quantifying the colorectal cancer stem cells, (d) deducing the risk of cancer recurrence colorectal and / or aggressiveness of colorectal cancer to define a prognostic value ue for the therapeutic adaptation of colorectal cancer from the presence and possibly the quantity of colorectal cancer stem cells. Step (a) may be carried out with a lectin recognizing the fucose moiety at 1-2 galactose alone or in the presence of a second labeling means for colorectal cancer stem cells as defined above, advantageously a recognizing lectin the T antigen, in particular chosen from the group consisting of ABA, Jacaline and ACA or a mixture of lectins recognizing the T antigen, in particular chosen from the group consisting of ABA and ACA; ABA and Jacaline; and Jacaline and ACA, advantageously Jacaline and ACA.
    The present invention particularly relates to an in vitro diagnostic method for the risk of recurrence of colorectal cancer and / or the aggressiveness of colorectal cancer to define a prognostic value for the therapeutic adaptation of colorectal cancer, wherein staining step of cancer stem cells is carried out with a lectin mixture selected from the group consisting of UEA-1, AB A and AC A; TJA-II, AB A and ACA; UEA-1, Jacaline and ABA; TJA-II, Jacaline and ABA; UEA-1, Jacaline and ACA; TJA-II, Jacaline and ACA, advantageously UEA-1, Jacaline and ABA; and UEA-1, Jacaline and ACA, preferably UEA-1, Jacaline and ACA. Advantageously, the lectin molar ratio recognizing the fucose unit has 1-2 galactose: Jacaline: ABA or ACA varies from 625 to 12500: 16 to 320: 1 to 20, advantageously 5000 to 7000: 50 to 250: 5 to 15, especially in a molar ratio 6250: 160: 10.
    More advantageously, the lectins are UEA-1: Jacaline: ABA or ACA, in a molar ratio of 625 to 12500: 16 to 320: 1 to 20, advantageously 5000 to 7000: 50 to 250: 5 to 15, especially in a molar ratio 6250: 160: 10.
    In a preferred embodiment, the lectins are UEA-1, Jacaline and ACA in a molar ratio of 6250: 160: 10.
    In one embodiment, the lectin or lectins are linked to a means for detecting and possibly isolating the colorectal cancer stem cells as defined above.
    The present invention particularly relates to a method for in vitro diagnosis of the risk of recurrence of colorectal cancer and / or the aggressiveness of colorectal cancer to define a prognostic value for the therapeutic adaptation of colorectal cancer in which the medium detection and optionally isolation of the cells is selected from the group consisting of magnetic beads on which is grafted with streptavidin, Tavidine or an anti-biotin antibody, the lectin or lectins being biotinylated; a fluorophore covalently bound to the lectin (s); or a fluorophore conjugated to streptavidin, Tavidine or an anti-biotin antibody, the lectin (s) being biotinylated.
    The present diagnostic method may in particular be implemented by using for the detection step (c) and possibly cell isolation (d) flow cytometry, magnetic cell sorting or immunohistochemistry. When the method for detecting and optionally isolating the cells is flow cytometry, the fluorophore conjugated with streptavidin, Tavidine or an anti-biotin antibody is advantageously Alexa Fluor 488 or Alexa Fluor 633. When the method of detection and optionally isolated cells is immunohistochemistry, fluorophore conjugated with streptavidin, avidin or an anti-biotin antibody is preferably Alexa Fluor 488 or Alexa Fluor 594.
    A fourth subject of the present invention relates to a kit, in particular intended for the detection and possibly the isolation of colorectal cancer stem cells, comprising as a first means of labeling a lectin recognizing the fucose-1-galactose motif, advantageously UEA- 1 or TJA-II, and a second means of labeling colorectal cancer stem cells.
    Advantageously, the second means for labeling the colorectal cancer stem cells is a lectin recognizing the T antigen or a mixture of lectins recognizing the T antigen, in particular chosen from the group consisting of ABA, Jacaline and ACA.
    In one embodiment, the kit according to the invention comprises, as second labeling means, a mixture of lectins recognizing the T antigen, selected from the group consisting of ABA and ACA; ABA and Jacaline; and Jacaline and ACA, advantageously Jacaline and ACA.
    The present invention therefore advantageously relates to a kit comprising lectins selected from UEA-1, ABA and ACA; TJA-II, ABA and ACA; UEA-1, ABA and Jacaline; TJA-II, ABA and Jacaline; UEA-1, Jacaline and ACA; TJA-II, Jacaline and ACA, advantageously UEA-1, ABA and Jacaline; and UEA-1, Jacaline and ACA, preferably UEA-1, Jacaline and ACA.
    The lectins of the kit according to the invention may be in independent containers to be combined at the time of their use, or combined in a single ready-to-use container.
    When the lectins are combined in a single ready-to-use container, the lectin mixture is advantageously constituted of a lectin recognizing the fucose-1-galactose motif: Jacaline: ABA or ACA, in a molar ratio of 625 to 12500 : 16 to 320: 1 to 20, advantageously 5000 to 700: 50 to 250: 5 to 15, especially in a ratio 6250: 160: 10.
    Advantageously, the combined lectin mixture in a single ready-to-use container is advantageously constituted by UEA-1: Jacaline: ABA or ACA, in a molar ratio of 625 to 12500: 16 to 320: 1 to 20, advantageously 5000 at 700: 50 to 250: 5 to 15, especially in a molar ratio 6250: 160: 10.
    In an advantageous embodiment, the present invention relates to a kit comprising a UEA-1: Jacaline mixture: AB A or AC A, combined in a single ready-to-use container, in a molar ratio of 6250: 160: 10.
    The kit according to the present invention may advantageously contain a means for detecting and optionally isolating the cells as defined above. The detection means is therefore in particular constituted by magnetic beads on which grafted streptavidin, avidin or an anti-biotin antibody, the lectin or lectins then being biotinylated; a fluorophore covalently bound to the lectin; or a fluorophore conjugated to streptavidin, avidin or an anti-biotin antibody, the lectin (s) being then biotinylated. Advantageously, the fluorophore is any fluorophore capable of being used for flow cytometry, advantageously Alexa Fluor 488 or Alexa Fluor 633.
    The present invention therefore also relates to a kit A comprising: • a lectin recognizing the fucose-1-galactose motif, • a first lectin recognizing the T antigen, • a second lectin recognizing the T antigen, • a detection means and possibly isolating the cells. said lectins being advantageously UEA-1, Jacaline and AB A or AC A, in a molar ratio of 625 to 12500: 16 to 320: 1 to 20, advantageously 5000 to 700: 50 to 250: 5 to 15, especially in a ratio molar 6250: 160: 10.
    In a first particular embodiment, the present invention relates to a kit Al comprising: • a biotinylated lectin recognizing the fucose-1-galactose motif, • a first biotinylated lectin recognizing the T antigen, • a second biotinylated lectin recognizing the antigen T, a means for detecting and possibly isolating cells consisting of magnetic beads onto which streptavidin, avidin or an anti-biotin antibody are grafted, said lectins being advantageously UEA-1, Jacaline, AB A or AC A in a molar ratio of 625 to 12500: 16 to 320: 1 to 20, preferably 5000 to 700: 50 to 250: 5 to 15, especially in a molar ratio of 6250: 160: 10.
    Advantageously, the kit is an Al-1 kit comprising: • biotinylated UEA-1, • biotinylated ABA or biotinylated ACA, advantageously biotinylated ACA, • biotinylated jacaline, • magnetic beads on which streptavidin, avidin or an anti-biotin antibody.
    Said lectins in the Al-1 kit can be combined in a single ready-to-use container, in a biotinylated UEA-1 molar ratio: biotinylated Jacaline: biotinylated ABA or biotinylated ACA from 625 to 12500: 16 to 320: 1 to 20 preferably 5000 to 7000: 50 to 250: 5 to 15, especially in a molar ratio 6250: 160: 10.
    The present invention also relates, in a second particular embodiment, to a kit A2 comprising: • a biotinylated lectin recognizing the fucose motif at 1-2 galactose, • a first biotinylated lectin recognizing the T antigen, • a second biotinylated lectin recognizing the T antigen; a means for detecting and possibly isolating the cells consisting of a fluorophore conjugated with streptavidin, with avidin or with an anti-biotin antibody.
    The fluorophore may be any fluorophore capable of being used for flow cytometry. Such fluorophores are commercially available. It is for example Alexa fluorine 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, 750 or 790, the fluorescein isothiocyanate (FITC), Rhodamine, allophycocyanin (APC) and Phycoerythrin (PE). Advantageously, the fluorophore is Alexa fluorine 488, Alexa fluorine 594 or Alexa fluorine 633.
    Advantageously, the kit is an A2-1 kit comprising: biotinylated UEA-1, biotinylated jacaline, biotinylated ABA or biotinylated ACA, advantageously biotinylated ACA, a fluorophore conjugated to streptavidin, avidin or a anti-biotin antibody,
    Said lectins can be combined in a single ready-to-use container, in a biotinylated UEA-1 molar ratio: biotinylated Jacaline: biotinylated AB A or biotinylated ACA from 625 to 12500: 16 to 320: 1 to 20, advantageously 5000 to 7000 : 50 to 250: 5 to 15, especially in a molar ratio 6250: 160: 10.
    The kit may also be an A3 kit, comprising: a lectin recognizing the fucose-1-galactose motif, conjugated to a fluorophore, in particular as defined above, a first lectin recognizing the T antigen, conjugated to a fluorophore, especially as defined above, a second lectin recognizing the T antigen, conjugated to a fluorophore, in particular as defined above.
    Advantageously, the kit is an A3-1 kit comprising: • UEA-1 conjugated to a fluorophore, especially as defined above • Jacaline conjugated to a fluorophore, especially as defined above. • AB A or ACA conjugated to a fluorophore, especially as defined above,
    Said lectins can be combined in a single ready-to-use container, in a fluorophore-conjugated conjugate EUA-1 molar ratio: Fluorophore conjugated Jacaline: ABA conjugated with a fluorophore or ACA conjugated to a fluorophore of 625 to 12500: 16 at 320: 1 to 20, advantageously 5000 to 7000: 50 to 250: 5 to 15, especially in a molar ratio 6250: 160: 10.
    The present invention also relates to the use of a kit as defined above, for the implementation of a method for detecting and possibly isolating colorectal cancer stem cells.
    The present invention also relates to the use of a kit as defined above, for the in vitro diagnosis of the risk of recurrence and / or aggression of colorectal cancer to define a prognostic value for therapeutic adaptation. colorectal cancer. DESCRIPTION OF THE FIGURES:
    FIG. 1 shows the results of the separation of cancer stem cells on a sample of cells of the HT29 line with magnetic beads on which an anti-CD133 antibody (T-AC133) is grafted with the kit marketed by Miltenyi Biotec. (CD133 MicroBead Kit) via the glycosylated marker CD133 (prominin-1), with magnetic beads on which is grafted streptavidin and UEA-1 biotinylated (Lectin UEA-1), with the mixture UEA-1 / Jacaline / ABA ( Mix 1) and with the mixture UEA-1 / Jacaline / ACA (Mix 2).
    Figure 2 shows the Epcam High + / Epcam high ratio - after cell sorting with UEA-1 alone, two lectins, or a mixture of three UEA-1 lectins: Jacaline: ACA in equimolar proportions (Mix 2) or a UEA-mixture. 1: Jacaline: ACA, in a molar ratio 6250: 160: 10.
    Figure 3 shows the results of cell sorting with magnetic beads on different lines of colorectal cancer cells with the mixture UEA-1 / Jacaline / ABA (Mix 1 S) and with the mixture UEA-1 / Jacaline / ACA (Mix 2S) .
    Figure 4 shows the results of a clonogenicity test from unsorted (T-) cells, sorted with the Miltenyi Biotec AC133 kit (T-AC133), with a UEA-1 / Jacaline / ABA mixture (Mix 1). or a UEA-1 / Jacaline / ACA mixture (Mix2). The y-axis is expressed in pixel2.
    Figure 5 shows the results of tumor growth in vivo after transplantation of unsorted cells (T-, diamonds), cells isolated with UEA-1 / Jacaline / ACA (squares) and cells of the "negative" fraction of sorting. (non-stem cancer cells, triangles) in Nude mice. The results are expressed in volume (y-axis) as a function of time (in days). EXAMPLES
    Example 1: Isolation protocol for colorectal cancer stem cells I. Materials required Reagents and material - Biotinylated mixture specifically labeling the cancerous stem cells of colorectal cancer (prepared from individual lectins Vector Laboratories
    CELLection Kit Biotin Binder (Invitrogen)
    Magnet
    Buffers - Versene (Invitrogen)
    Buffer 1: PBS (Phosphate Buffer Saline without Ca2 + and Mg2 +) with 0.1% BSA (Bovine Serum Albumin), pH 7.4
    Buffer 2: PBS (Saline Phosphate Buffer without Ca2 + and Mg2 +) with 0.1% BSA (Bovine Serum Albumin) and 0.6% Sodium Citrate
    Buffer 3: RPMI 1640 with 1% FCS (Calf Serum Feast), ImM CaCl2 and 5mM MgCl2, pH 7.0-7.4. II. Duration of the experiment 20 min to prepare the cells 20 min to mark the cells - 20 min to incubate the cells labeled with the beads 10 min to recover the suspension not enriched in CSCs 15 min to break the bond CSCs / beads 5 min recovering the suspension enriched in CSCs of interest TOTAL: 1:30 am III, Procedure by magnetic sorting: 1. Preparation of the cells. The cells are detached from their support with Versene for 10 min at 37 ° C. 2. The cells are counted and the number of cells is adjusted to 1.10. 3. The cell suspension is centrifuged at 300 g for 10 minutes and then the supernatant is removed. 4. Blocking aspecific sites. lmL Buffer 2 is added. 5. Marking of the cells. 80 μl of Mix is added and the mixture incubated for 10 min at 4 ° C. 6. 500 μl of Buffer 2 is added to wash the cells and the suspension is centrifuged at 300g for 10 min and the supernatant is removed. 7. Balls. The cells are resuspended in 1 ml Buffer 2 and then 25 μl of previously washed beads are added and resuspended using Buffer 1. The mixture is incubated for 20 min at 4 ° C. with gentle stirring. 8. Recovery of the suspension NOT enriched in CSCs. The tube is then placed on the magnet for 2 min and the supernatant removed, keeping it in a falcon, while the tube is still in place on the magnet. 9. The tube is removed from the magnet, 1mL of Buffer 1 is added, the tube is vortexed and returned to the magnet for 2 min before retaining the supernatant in the same falcon as step 8. This step is repeated twice. 10. The cells still bound to the beads are resuspended with 200 μl of Buffer 3 preheated to 37 ° C. 4 μl of cells / beads binding buffer consisting of DNasel are added. This mixture is incubated for 15 min at room temperature with gentle stirring. 11. The suspension is stirred with a pipette vigorously 5 to 10 times in order to facilitate the release of the cells. 12. Recovery of the suspension enriched in CSCs. The tube is placed on the magnet for 2 min and the supernatant containing the cells of interest transferred into a tube containing 200 μl of buffer 3 preheated to 37 ° C. Steps 11 and 12 can be repeated again to enhance the yield.
    These experiments were performed under similar conditions with UEA-1 alone, a mixture of two lectins or with the kit from Miltenyi and four other colorectal cancer cell lines.
    The results of these different tests are presented in Figures 1 to 3.
    As shown by the results of these tests, the use of UEA-1 alone or in admixture with one or two lectins recognizing the T antigen allows the isolation of the colorectal cancer stem cells with an efficiency very much improved compared to the standard method (AC 133).
    Example 2: Clonogenicity Test
    The clonogenicity tests were performed in a 6-well plate at a density of 500 cells / cm 2 in a MEM composition medium (Gibco) supplemented with 50 units / mL of penicillin, 50 units / mL of streptomycin (Gibco) and 2.4 g / L sodium bicarbonate, 1 M HEPES buffer (Sigma Aldrich, Saint-Quentin-Fallavier, France), IX progesterone (Sigma Aldrich), IX putrescine (Sigma), 0.025 g / mL heparin (Sigma Aldrich), % (w / v) glucose (Sigma Aldrich,), IX growth supplement B27 (Invitrogen, Carisbad, CA), 20 ng / mL EGF (Sigma Aldrich), 20 ng / mL basic human FGF (Sigma Aldrich), IX supplement insulin-transferrin-sodium selenite (Roche diagnostics, Meylan, France). The evolution of the coionospheres was observed after incubation at 37 ° C in a CO2 atmosphere and quantified with ImageJ® software.
    The results are shown in Figure 4.
    The cancer stem cells isolated according to the method described in the present invention lead to the formation of larger diameter spheres than isolated cells with a kit based on the recognition of the CD133 marker.
    The method according to the present invention thus makes it possible to obtain stem cells capable of reforming tumors much more effectively than the method currently available and considered as the standard.
    Example 3: Study of the evolution of tumors in vivo
    In vivo assays adapted from the method described in Varnat F et al. EMBO Mol Med. 2009; 1 (6-7): 338-351 were made.
    For this, 3 × 10 6 cells isolated with the UEA-1 / Jacaline / ACA mixture were injected subcutaneously into the Nude mouse flank or with the negative fraction isolated with the mixture.
    Control mice were obtained by injection of cells that had not been previously sorted (cells from unsorted HT-29 lines).
    Before the tumors reached the legal limits, the animals are sacrificed and the tumors studied.
    The results of the evolution of tumor size are given in Figure 5.
    As in the in vitro experiment of Example 4, colorectal cancer stem cells lead to the formation of larger volume tumors than non-isolated cells. The isolated cells are therefore effectively enriched in colorectal stem cells, initiators of tumors.
    权利要求:
    Claims (18)
    [1" id="c-fr-0001]
    1. Use, as a first means of labeling, of a lectin recognizing the 1-2-galactose fucose motif, advantageously UEA-1 or TJA-II, for the implementation of a detection method and optionally of isolation of colorectal cancer stem cells.
    [2" id="c-fr-0002]
    2. The use according to claim 1, of a lectin recognizing the fucose-1-galactose unit and of a second marking means for colorectal cancer stem cells, advantageously chosen from a lectin recognizing the T antigen, in particular ABA, Jacaline. and ACA or a lectin mixture recognizing the T antigen, especially selected from the group consisting of ABA and ACA; ABA and Jacaline; and Jacaline and ACA, advantageously Jacaline and ACA.
    [3" id="c-fr-0003]
    3. Use according to one of claims 1 to 2, wherein the lectin or lectins are linked to a means for detecting and optionally isolating colorectal cancer stem cells, preferably consisting of magnetic beads on which streptavidin is grafted, avidin or an anti-biotin antibody, the lectins being biotinylated; a fluorophore covalently bound to the lectin; or a fluorophore conjugated to streptavidin, avidin or an anti-biotin antibody, the lectins being biotinylated.
    [4" id="c-fr-0004]
    4. Use according to one of claims 1 to 3, wherein: the lectin recognizing the fucose pattern has 1-2 galactose is biotinylated, a first lectin recognizing the T antigen is biotinylated, a second lectin recognizing the T antigen is biotinylated, the means for detecting and possibly isolating the cells consists of magnetic beads onto which streptavidin is grafted. said lectins being advantageously UEA-1, Jacaline and ABA or ACA, in a molar ratio of 625 to 12500: 16 to 320: 1 to 20, advantageously 5000 to 7000: 50 to 250: 5 to 15, especially in a 6250 molar ratio : 160: 10.
    [5" id="c-fr-0005]
    5. Kit comprising, as first labeling means, a lectin recognizing the 1-2-galactose fucose unit, advantageously UEA-1 or TJA-II, and a lectin recognizing the T antigen, advantageously chosen from ABA, ACA and Jacaline, or a lectin mixture recognizing the T antigen, preferably selected from the group consisting of ABA and ACA; ABA and Jacaline; and Jacaline and ACA, advantageously Jacaline and ACA.
    [6" id="c-fr-0006]
    Kit according to claim 5 comprising UEA-1, Jacaline and ACA in a ratio of 6250: 160: 10.
    [7" id="c-fr-0007]
    7. Kit according to one of claims 5 or 6, further comprising a means for detecting and optionally isolating the cells, preferably consisting of magnetic beads on which is grafted streptavidin, avidin or an anti-human antibody. biotin, the lectins being biotinylated, a fluorophore covalently bound to the lectin, or a fluorophore conjugated to streptavidin, avidin or an anti-biotin antibody, the lectins being biotinylated.
    [8" id="c-fr-0008]
    8. Kit according to one of claims 5 to 7, comprising: • a biotinylated lectin recognizing the fucose pattern has 1-2 galactose, • a first biotinylated lectin recognizing the T antigen, • a second biotinylated lectin recognizing the T antigen A means for detecting and possibly isolating the cells, advantageously magnetic beads on which streptavidin is grafted, said lectins being advantageously UEA-1, Jacaline, ABA or ACA in a molar ratio of 625 to 12500: 16 to 320: 1 to 20, advantageously 5000 to 7000: 50 to 250: 5 to 15, especially in a molar ratio 6250: 160: 10.
    [9" id="c-fr-0009]
    9. In vitro method for detecting and optionally isolating colorectal cancer stem cells, comprising the step of labeling the colorectal cancer stem cells of a colorectal biological sample with a lectin recognizing the fucose motif with 1-2 galactose, advantageously UEA -1 or TJA-II.
    [10" id="c-fr-0010]
    The method of claim 9 comprising the steps of: (a) labeling the colorectal cancer stem cells with a lectin recognizing the 1-2-galactose fucose motif to obtain a biological sample in which the colorectal cancer stem cells are labeled, (b) contacting the biological sample in which the cancer stem cells are labeled with a means for detecting and optionally isolating the cells, (c) detecting the colorectal cancer stem cells, (d) optionally isolating the stem cells colorectal cancer.
    [11" id="c-fr-0011]
    The method according to one of claims 9 or 10, wherein step (a) is carried out with a lectin recognizing the 1-2-fucose galactose motif as the first labeling means and a second marking means. colorectal cancer stem cells, advantageously chosen from the group consisting of ABA, Jacaline and ACA or a mixture of lectins recognizing the T antigen, in particular chosen from the group consisting of ABA and ACA; ABA and Jacaline; and Jacaline and ACA, advantageously Jacaline and ACA.
    [12" id="c-fr-0012]
    The method of claim 11, wherein the lectins are UEA-1, Jacaline and ACA in a molar ratio of 6250: 160: 10.
    [13" id="c-fr-0013]
    13. The method of claim 12, wherein the lectin or lectins are linked to a means for detecting and optionally isolating colorectal cancer stem cells, preferably consisting of magnetic beads on which streptavidin, avidin or an anti-biotin antibody, the lectins being biotinylated, a fluorophore covalently bound to the lectin, or a fluorophore conjugated to streptavidin, avidin or an anti-biotin antibody, the lectins being biotinylated.
    [14" id="c-fr-0014]
    14. In vitro diagnostic method for the risk of recurrence of colorectal cancer and / or the aggressiveness of colorectal cancer to define a prognostic value for the therapeutic adaptation of colorectal cancer, comprising a step of labeling colorectal cancer stem cells of a colorectal biological sample with a lectin recognizing the fucose motif at 1-2 galactose, advantageously UEA-1 or TJA-II.
    [15" id="c-fr-0015]
    The diagnostic method of claim 14, comprising the steps of: (a) labeling the colorectal cancer stem cells with a lectin recognizing the 1-2-galactose fucose motif, to obtain a biological sample in which the cancer stem cells are labeled (b) contacting the biological sample in which the cancer stem cells are labeled with a means for detecting and possibly isolating the cells, (c) detecting and optionally quantifying the colorectal cancer stem cells, (d) deducing the risk of recurrence of colorectal cancer and / or the aggressiveness of colorectal cancer to define a prognostic value for the therapeutic adaptation of colorectal cancer from the presence and possibly the quantity of colorectal cancer stem cells.
    [16" id="c-fr-0016]
    The diagnostic method according to claim 14 or 15, wherein step (a) is carried out with a lectin recognizing the 1-2-galactose fucose motif as the first labeling means and a second means of labeling the colorectal cancer stem cells, advantageously chosen from a lectin recognizing the T antigen, in particular AB A, Jacaline or ACA, or a mixture of lectins recognizing the T antigen, in particular ABA and ACA; ABA and Jacaline; or Jacaline and ACA, advantageously Jacaline and ACA.
    [17" id="c-fr-0017]
    The diagnostic method of claim 16, wherein the lectins are UEA-1, Jacaline and ACA in a molar ratio of 6250: 160: 10.
    [18" id="c-fr-0018]
    18. Diagnostic method according to one of claims 14 to 17, wherein the means for detecting and possibly isolating the cells is constituted by magnetic beads on which grafted streptavidin, avidin or an anti-human antibody. biotin, the lectins being biotinylated; a fluorophore covalently bound to the lectin; or a fluorophore conjugated to streptavidin, avidin or an anti-biotin antibody, the lectins being biotinylated.
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    优先权:
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    CA3005820A| CA3005820A1|2015-12-02|2016-12-02|Method for isolating cancer stem cells|
    US15/780,724| US11255858B2|2015-12-02|2016-12-02|Method for isolating cancer stem cells|
    PCT/FR2016/053197| WO2017093697A1|2015-12-02|2016-12-02|Method for isolating cancer stem cells|
    JP2018548289A| JP2018538551A|2015-12-02|2016-12-02|Methods for isolating cancer stem cells|
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