METHOD AND DEVICE FOR SELECTIVE, SPECIFIC AND SIMULTANEOUS SORTING OF TARGET RARE CELLS IN A BIOLOGICAL SAMPLE

Abstract

The invention proposes a device and a method for selective, specific and simultaneous sorting of target cells in a biological sample, versatile and economical. To this end, the device according to the invention comprises: • A first row (110) of at least two fluidic chambers (111) each comprising an inlet (112) fluidically connected to at least one reservoir (1) of a biological solution, and an outlet (113); • A last row (130) of fluidic chambers (131) equal in number to the number of chambers (111) of the first row (110), each fluidic chamber (131) of the last row (130) comprising an inlet (132) fluidically connected to an outlet (113) of a chamber (111) of the first row (110), and an outlet (133); • At least one solution collection reservoir, fluidically connected to each outlet (133) of the fluidic chambers of the second row;• At least one pump suitable for circulating a sample of the biological solution in the fluidic chambers of the first row and then the last row; the fluidic chambers each comprising a surface functionalized by molecules, at least some of which are capable of forming a bond with a receptor molecule carried by the target cells.;

Description

An example of a method for detecting rare cell subpopulations includes epithelial-derived cells (CTCs), non-epithelial mesenchymal cells, cancer stem cells (CSCs), and circulating endothelial cells (CECs). The respective antibodies grafted are: anti-15 EpCAM for the epithelial-derived tumor cell (CTC) subpopulation, anti-N Cadherin for the mesenchymal cell subpopulation, anti-CD133 for the cancer stem cell (CSC) subpopulation, and anti-CD146 for the non-hematopoietic cell (CEC) subpopulation (Tables 4 and 5). Table 4: 3-level combination of particle sensors of interest with and without reservoir Floors + GPTS Chamber 1 Chamber 2 Chamber 3 Chamber 4 Rank 1- particle sensors of interest with reservoir Anti-CD45 Anti-CD45 Anti- CD45 Anti- CD45 Rank 2- particle sensors of interest without reservoir Anti- EpCAM Anti-N Cadherin Anti- CD133 Anti- CD146 Rank 3- particle sensors of interest without reservoir Anti- EpCAM Anti-N Cadherin Anti- CD133 Anti- CD146 Or Table 5: 3-level combination of particle sensors of interest with and without reservoir Floors + GPTS Chamber 1 Chamber 2 Chamber 3 Chamber 4 Rank 1- particle sensors of interest with reservoir Anti-CD45 Anti- CD45 Anti-CD45 Anti-CD45 Rank 2- particle sensors of interest without reservoir Anti- EpCAM Anti-N Cadherin Anti- CD133 Anti- CD146 Rank 3- particle sensors of interest without reservoir Anti-N Cadherin Anti- EpCAM Anti- CD146 Anti- CD133 This series assembly of particle sensors of interest with a reservoir (rank 1; see figure 3), and particle sensors of interest without a reservoir (see figure 4) allows, in a way well defined by the user, to target four subpopulations of interest in a selective, specific and simultaneous manner. The device according to the invention makes it possible to overcome the inadequacy of known detection methods using a single anti-EpCAM antibody. Indeed, a classic detection with a single anti-EpCAM antibody cannot in any case detect at the same time non-epithelial mesenchymal cells, nor cancer stem cells nor non-hematopoietic endothelial cells. EXAMPLE 2 VISUALIZATION OF CELLULAR MARKERS BY IMMUNOFLUORESCENCE Two examples of selective, specific and simultaneous detection: 1) a subpopulation of CTCs of epithelial origin, and 2) a subpopulation of non-hematopoietic cells CEC (circulating endothelial cells) by cellular protein markers in metastatic colon and prostate cancers. 1. Negative selection: leukocyte filtration The anti-CD45 leukocyte antibody targets the majority of leukocytes in the blood. Figure 8 visualizes the high leukocyte population Le. The leukocytes are labeled with the nuclear marker DAPI. 2. Positive selection A. CTC of epithelial origin EpCAM antigen is a cell adhesion molecule expressed on the surface of tumor cells of epithelial origin. Grafting the anti-EpCAM antibody onto a standard microscopy slide functionalized with GPTS (3-glycidoxypropyl-trimethoxysilane), in phosphate buffer (PBS), pH 7.4, containing 0.5% Trehalose (Sigma), allows the targeted identification of CTCs of epithelial origin. CTCs are then identified by a cell marker expressing cytokeratin, the anti-pan cytokeratin antibody, in phosphate buffer (PBS), containing 0.1% bovine serum albumin (BSA). Characterization is performed by immunofluorescence to confirm the epithelial origin of the detected cell. Figures 9 and 10 show a CTC 500, captured by the anti-EpCAM antibody (green fluorescence), from either metastatic colon cancer (Figure 9) or metastatic prostate cancer (Figure 10). The nuclear marker is DAPI (blue). B. Non-hematopoietic cells: circulating endothelial cells (CEC) MCAM is a glycoprotein expressed on the surface of the endothelial cell. Grafting the anti-MCAM antibody (anti-CD146) onto a standard microscopy slide, functionalized with GPTS (3-glycidoxypropyl-trimethoxysilane), in phosphate buffer (PBS), pH 7.4, containing 0.5% Trehalose (Sigma), allows their targeted identification and capture. CECs are then identified by the anti-CD144 antibody directed against VE-cadherin (Vascular Endothelial) in phosphate buffer (PBS), containing 0.1% bovine serum albumin (BSA). Characterization is performed by immunofluorescence to confirm the endothelial cell. Figures 11 and 12 show a 510 SCC from metastatic colon cancer (Figure 11) and a SCC from metastatic prostate cancer (Figure 12). The SCCs are characterized by immunofluorescence with the anti-CD144 antibody (purple fluorescence). The nuclear marker is DAPI (blue). A non-exhaustive list of markers that can be used to target cell subpopulations of epithelial and non-epithelial origin for their characterization and identification is given below: Cell type Marker(s) used for subpopulation isolation Epithelial EpCAM, BerEP4, MUC 1, Mesenchymal N Cadherin, Cadherin 1 1 Endothelial MCAM (CD146), VE-cadherin (CD 144) Leukocyte CD45 T lymphocyte CD3 B lymphocyte CD20 Hematopoietic stem cell CD34 Mesenchymal stem cell CD44, CD45, CD90, CD150, CD299 Tumor cell (breast cancer, prostate cancer) HER2 A list of markers that can be used to target cancer stem cell (CSC) subpopulations for their characterization and identification is given below: Tumor type Marker(s) used for CSC isolation Hematologic malignancies CD34+, CD38- Colon CD133+, CD44+, Lin- Breast CD44+, CD24- / low Melanoma ABCB5 transporter Prostate CD44+, CD133+, a2pl Pancreas CD44+, CD24+, CD133+ Glioblastoma CD133+ Liver CD133+, CD44+, CD90+ Lung CD133+ VADS CD44+ Another major and user-friendly technical advantage of the device according to the invention relates to the unique development of the characterization of the cells of interest accompanied by a series of successive molecular analyses. The molecular characterization workflow consists of a multi-step identification procedure, on the same glass slide and the same cell, 1) by means of a fluorescent protein marker (antibody), 2) by the analysis of genomic alterations (gene amplifications, deletions, and rearrangements) by fluorescence in situ hybridization (FISH) using specific DNA probes, 3) the in situ characterization of mRNAs by specific RNA probes and of microRNAs (miRNAs) by nanoprobes. EXAMPLE 3: - VISUALIZATION OF GENOMIC ALTERATIONS IN CTC SUBPOPULATIONS BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH): GENE AMPLIFICATIONS, DELETIONS, REARRANGEMENTS, USING SPECIFIC DNA PROBES. The added value of the standard microscopy slide, functionalized for the detection of CTC subpopulations of epithelial and non-epithelial origin, is its remarkable capacity to be able to undergo multiple successive treatments such as: 1) the marking of cells of interest by fluorescent cellular protein markers and its identification under a microscope, 2) a specific treatment is applied to eliminate the fluorescent background noise, then a reconditioning of the cells of interest detected by the labeling of the nuclei by nucleic probes by fluorescence in situ hybridization (FISH) to identify their genomic alterations. Figure 13 shows the 520 core of a CTC of epithelial origin from metastatic colon cancer showing gene amplification (521 dashed circles) revealed using a probe targeting the EGFR gene (Kreatech, Holland). Figure 14 shows the nucleus of a CTC 530 of epithelial origin from metastatic prostate cancer showing gene amplification (dashed circles 531 ) revealed using a probe targeting the PTEN gene (Kreatech, Holland). Figure 15 shows the nucleus of a CTC 540 of epithelial origin from metastatic prostate cancer showing gene amplification (solid circles 541, dashed circles 542 and shaded circles 543) revealed using a probe targeting the TMPRSS2-ERG fusion gene (Kreatech, Holland). The device according to the invention can also be used for two particularly interesting applications: EXAMPLE 4: LIVING CELL ANALYSIS OF mRNA AND miRNA. The capture of living and single cells by the device according to the invention and, in particular, the sensors of particles of interest with and without reservoir, is an asset for a simultaneous multiplex analysis of mRNA by means of nanoparticles functionalized by multiple nucleotide sequences and functioning as direct hybridization probes (Smart Flores, Merck-Millipore), each probe being coupled to a chromophore. On the other hand, the second advantage is its application for the analysis of miRNAs in a single living cell (Smart Flores, Merck-Millipore). MiRNAs are biomarkers of tumor progression. These are new analytical tools to complement early diagnosis, anticipate disease progression, identify high-risk patients, predict recurrence after treatment and monitor patient response to treatment. Figure 16 illustrates an example of labeling of live 560 MCF7 breast cancer cells with an endocytosis control probe for mRNA and miRNA labeling (Smart Flores, Merck-Millipore). The 560 MCF7 breast cancer cells are captured on a GPTS-functionalized microscopy slide grafted with the anti-EpCAM antibody. They are then labeled with a nanoparticle endocytosis control probe and incubated at 37°C and 5% CO2 for 12 hours. EXAMPLE 5: CELL CULTURE WITH THE TANKLESS PARTICLE OF INTEREST SENSOR For cell culture, the reservoir-free particle of interest sensor and its GPTS-functionalized slide are sterilized in absolute ethanol for 3 minutes. After drying, the floor of each chamber is grafted with the anti-EpCAM antibody for the capture of MCF7 breast cancer cells. The reservoir-free particle of interest sensor is then placed in a sterile Petri dish and incubated in an incubator at 37°C and 5% CO2 for 5 days. After 5 days, 570 clusters of MCF7 breast cancer cells have developed (Figure 17). In summary, the great advantage of the method and device according to the invention is its capacity for selective and specific, and simultaneous sorting of all heterogeneous subpopulations of circulating rare cells, whatever the stage of the disease (localized or metastatic cancer). In addition, the user-friendliness of the device according to the invention allows 1) grafting of antibodies targeting each subpopulation of cells of interest, 2) high-performance molecular analyses of the cells of interest thanks to the support of the resistant glass slide with multiple experimental manipulations, user-friendly and low cost compared to Si supports.

Claims

RFÆHDICATSOHS 1. Mesofluidic device (100) for selective, specific and simultaneous sorting of rare target cells in a biological sample at a concentration of between 1 and 10 cells per milliliter (ml) of sample, characterized in that it comprises: » A first row (110) of at least two fluidic chambers (111) with laminar flow each comprising an inlet (112) fluidically connected to a reservoir (1) of a biological solution, and an outlet (113); 10 ® A last row (130) of fluidic chambers (131) with laminar flow in number equal to the number of chambers (11 1) of the first row (1 10), each fluid chamber (131) of the last row (130) comprising: - an inlet (132) fluidly connected to an outlet (113) of a chamber (111) of the first row (110); and 15 - an exit (133); “At least one solution collection tank, fluidically connected to each outlet (133) of the fluid chambers of the second row; “At least one pump suitable for circulating a sample of the biological solution in the fluid chambers of the first row and then the last 20th row; laminar flow fluid chambers each comprising a surface functionalized by molecules, at least some of which are capable of forming a bond with a receptor molecule carried by the target cells.

2. Device according to claim 1, comprising at least one 25 intermediate row (120) of fluidic chambers (121) in number equal to the number of chambers (111) of the first row (110), each fluidic chamber (121) of an intermediate row (120) comprising an inlet (122) fluidically connected to an outlet (113) of a chamber of the first row (110), and an outlet (123).

3. Device according to any one of claims 1 or 2, in which: “the functionalized surfaces of the first row chambers are provided with molecules capable of selectively binding to one or more cell population(s) of the sample different from the target cells; » the functionalized surfaces of the chambers of the last row and, possibly of the intermediate row(s), are provided with different types of molecules capable of selectively attaching to a population(s) of target cells.

4. Device according to any one of claims 1 to 3, in which each row is constituted by a sensor of particles of interest (1100, 1200) comprising: ® a cover (1101, 1201) comprising recesses (1102, 1202) for constituting the chambers, each recess being in fluid communication with a solution inlet port (1103, 1203), and a solution outlet port (1104, 1204); ® a blade mounted in a removable and hermetic manner under the hood to constitute a bottom of the fluidic chambers, and comprising a surface, facing the hood, covered with a functionalized zone opposite each recess.

5. Device according to claim 4, in which the particle sensor of interest (1100) of the first row comprises a cover provided with a liquid supply channel, intended to be connected to the reservoir, and consisting of as many channel segments as the cover comprises recesses, the segments of the supply channel being of different sections and increasingly smaller in the direction of circulation of the liquid, each segment being in fluid communication with an inlet orifice for circulating the liquid in the chambers.

6. Device according to any one of claims 4 or 5, in which the cover is transparent.

7. Device according to any one of claims 4 to 6, in which the cover is made of cycloolefin copolymer.

8. Device according to any one of claims 1 to 7, in which the inlets of the fluid chambers of each row apart from the second row are connected to the outlets of the fluid chambers of the previous row by tubes of length less than 5 cm, preferably less than 3 cm, and an internal diameter of between 0.5 and 1.4 millimeters.

9. Device (300) according to any one of claims 1 to 3, comprising, with reference to the position of use: ® an upper cover (310) comprising a flat rim (311) and recesses (312) to constitute the chambers of the first row, each recess being in fluid communication with a solution inlet orifice (313); ® at least two functionalized blades (320-330), each comprising a surface (321-331) facing the cover covered with a functionalized zone opposite each recess, and pierced with as many outlet orifices (322-332) as there are functionalized zones; “at least one spacer (340) intended to be hermetically arranged between two functionalized blades (320-330), and comprising as many slots as the upper cover comprises recesses, each slot defining a chamber; ® a lower cover (350) comprising as many outlets (351) as the first cover (310) comprises inlets (313).

10. The device of claim 19, wherein the functionalized slides are transparent to wavelengths compatible with slide analysis instruments.

11. Device according to any one of claims 4 to 10, in which each slide comprises a visual code (220) interpretable by a computer, the code comprising information relating to the molecules of the functionalized zones of the slide.

12. Method for selective, specific and simultaneous freezing of target cells in a biological sample, characterized in that it comprises the following steps: a) functionalizing slides with molecules capable of selectively attaching to one or more cell population(s) on different areas of the slide intended to each define a bottom of a fluidic chamber; b) equipping a device according to any one of claims 1 to 11 with the blades thus functionalized; c) connecting the inlet of the first row chambers to a solution tank containing the target cells to be sorted, and the outlet of the last row chambers to a recovery tank; d) fluidly matching the outlet of the chambers of one row with the inlet of the chambers of another row until all the chambers are connected in series with each other from one row to the other, e) circulating the biological sample in the chambers, from the first row to the last row so as to fix target cells on the molecules capable of selectively fixing themselves to these cells; f) analyzing the functionalized surfaces of the slides containing the target cells fixed in step e).

13. Method according to claim 12, in which step a) consists of: al) functionalizing the areas of the first row slide with molecules capable of selectively binding to one or more cell populations, the most numerous in the sample considered and different from the target cells; a2) functionalize the areas of the blade of the following row(s) with different types of molecules capable of selectively binding with a popdaHan(s) of target cells, each functionalized zone comprising a single type of the different molecules.

Images