METHOD FOR SELECTING, MANIPULATING AND ISOLATING CIRCULATING TUMOR CELLS IN BODY FLUIDS BY LASER-ASSISTED TRANSFER

Abstract

A method and system for detecting, manipulating and isolating circulating tumor cells found in organic fluids comprising a simplified laser assisted transfer process. A liquid specimen of the organic fluid containing an enriched population of tumor cells and a population of non-tumor cells is spread onto a donor substrate with an interlayer comprising a semi-transparent polymeric adhesive tape. A fluorescent staining of at least one of the cell populations present in the liquid specimen is required to identify-using optical and fluorescence microscopy, the location of the cells to be manipulated and/or isolated. The laser beam energy is absorbed by the polymeric adhesive tape giving place to the formation of a blister that mechanically interacts with the liquid specimen and such interaction can induce the expulsion of a portion of the liquid specimen comprising the selected tumor cells or non-tumor cells, which can be a single cell or a cell cluster, towards the receiving substrate.

Claims

1. A method for detecting and manipulating tumor cells found in organic fluids comprising the steps of: a) providing a liquid specimen of the organic fluid containing an enriched population of tumor cells and a population of non-tumor cells; b) fluorescently staining at least one of the two cell populations wherein a different fluorescence dye is used for each population when both populations are stained; c) providing a donor substrate comprising a transparent support and a semi-transparent polymeric adhesive tape adhered to the transparent support; d) dispensing and spreading the liquid specimen onto the semi-transparent polymeric adhesive tape; e) providing a receiving substrate; f) placing the receiving substrate facing the surface of the donor substrate where the liquid specimen is spread maintaining a separation distance between them; g) visualizing the same portion of the liquid specimen by means of optical and fluorescence microscopy; h) comparing the optically and fluorescently obtained images to identify the location of tumor cells or non-tumor cells; i) providing a laser beam focused on the interface between the transparent support and the semi-transparent polymeric adhesive tape of the donor substrate; j) directing the laser beam to the identified location of at least one tumor cell or at least one non-tumor cell; so that the laser beam energy is absorbed by the semi-transparent polymeric adhesive tape at the identified location; giving place to the formation of a blister on the polymeric adhesive tape that generates a mechanical perturbation in the liquid specimen leading to the movement of at least one cell or cluster of cells.

2. The method according to claim 1, wherein the blister mechanically propels a portion of the liquid specimen towards the receiving substrate, the propelled liquid comprising at least one tumor cell without any non-tumor cells.

3. The method according to claim 1, wherein the blister mechanically propels a portion of the liquid specimen towards the receiving substrate, the propelled liquid comprising at least one non-tumor cell without any tumor cells.

4. The method according to claim 2, wherein the portion of the liquid specimen propelled by the laser beam comprises a single cell.

5. The method according to claim 2, wherein the portion of the liquid specimen propelled by the laser beam comprises a cluster of cells.

6. The method according to any of the claim 2, wherein only the tumor cells or only the non-tumor cells are stained.

7. The method according to claim 6, wherein the portion of the liquid specimen propelled by the laser beam comprises at least one non stained cell.

8. The method according to claim 6, wherein the portion of the liquid specimen propelled by the laser beam comprises at least one stained cell.

9. The method according to claim 1, wherein the generated blister leads to the disaggregation of a cluster of cells.

10. The method according to claim 1, wherein tumor cells comprise at least one of circulating tumor cells and disseminated tumor cells.

11. The method according to claim 1, wherein the non-tumor cells comprise at least one of hematopoietic cells and white blood cells.

12. A system for detecting, manipulating and isolating tumor cells from an organic fluid comprising: a donor substrate comprising a transparent support and a semi-transparent polymeric adhesive tape adapted to be adhered on one side of the transparent support and further adapted to receive, on the side opposite to the transparent support, a liquid sample of the organic fluid containing an enriched population of tumor cells and a population of non-tumor cells wherein at least one of the two cell populations are fluorescently stained, a receiving substrate arranged at a distance from the donor substrate while facing the liquid sample; a visualization system adapted to provide a visualization of the same portion of the liquid sample by means of optical and fluorescence microscopy and further adapted to compare the optically and fluorescently obtained images, a laser system adapted to focus a laser beam on the interface between the transparent support and the semi-transparent polymeric adhesive tape of the donor substrate and further adapted to direct the laser beam to the location of at least one tumor cell or at least one non-tumor cell so that the laser beam energy is absorbed by the semi-transparent polymeric adhesive tape giving place to the formation of a blister on the polymeric adhesive tape that generates a mechanical perturbation in the liquid specimen leading to the movement of at least one cell or cluster of cells.

13. A system for detecting, manipulating and isolating tumor cells from an organic fluid according to claim 12, wherein the semi-transparent polymeric adhesive tape comprises at least one thermoplastic polyimide sheet.

14. A system for detecting, manipulating and isolating tumor cells from an organic fluid according to claim 12, wherein the semi-transparent polymeric adhesive tape comprises at least one layer of a transparent adhesive silicone.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a schematic illustration of the transparent support of the donor substrate;

[0015] FIG. 2 schematically illustrates the semi-transparent polymeric adhesive tape comprising of the semi-transparent thermoplastic polymer sheet and the transparent adhesive polymer layer;

[0016] FIG. 3 shows a schematic illustration of the semi-transparent polymeric adhesive tape adhered to the transparent support wherein both elements comprise the donor substrate;

[0017] FIG. 4 schematically illustrates the arrangement of the liquid specimen containing the two cell populations with respect to all the components of the donor substrate;

[0018] FIG. 5 schematically illustrates a preferred arrangement of the donor substrate, the liquid specimen, the receiving substrate and the two pieces employed to separate the liquid specimen from the receiving substrate;

[0019] FIG. 6 shows a schematic illustration of the arrangement of the liquid specimen with respect to the objective lens of the optical and fluorescence microscopy system;

[0020] FIG. 7 schematically illustrates the arrangement of the focalized laser beam with respect to the donor substrate, the liquid specimen and the receiving substrate;

[0021] FIG. 8 shows a schematic illustration of the liquid specimen with the two populations of cells, wherein one of them is fluorescent stained and the other one is not stained, with respect to the donor and receiving substrate and the objective lens of the microscopy system;

[0022] FIG. 9 shows an illustration of the so called laser negative selection wherein the laser beam induced a blister that propels a droplet containing a single non-stained tumor cell.

[0023] FIG. 10 shows an illustration of the so called laser negative selection wherein the laser beam induced a blister that propels a droplet containing a cluster of non-stained tumor cells.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In the present disclosure, the expression organic fluid refers to liquids with a high probability to find circulating tumor cells, CTCs, or disseminated tumor cells, DTCs, such as for example, peripheral blood flow and bone marrow. Additionally, the expression tumor cells refers to CTCs and DTCs. Likewise, the expression non-tumor cells refers to different cell populations not being CTCs and DTCs that are present in the liquid specimen after the enrichment process. In the case that the liquid specimen is obtained from the peripheral blood flow or bone marrow, hematopoietic cells constitute the largest population even after the enrichment and therefore, are considered non-tumor cells in this context.

[0025] According to the previous glossary of terms, the liquid specimen contains two differentiated cell populations called tumor and non-tumor cells where the tumor population is necessarily enriched for applying this method. In addition, a fluorescent staining of at least one of the populations is also required to identify the cells to be isolated by means of the simplified laser-assisted transfer process. The staining process is performed using specific fluorescent antibodies to the cell populations and can be carried out according to one of three alternatives: the first one, where the stained population corresponds to the tumor cells; the second one, where the stained population corresponds to non-tumor cells and the third one, where both populations are stained with different florescence dyes. The fluorescent staining of CTCs and DTCs involves the use of the epithelial cell adhesion molecule, EpCAM, antigens. In the case of non-tumor cells, the fluorescent staining involves the use of CD45, the leukocytes common antigen, LCA, which is expressed on hematopoietic cells. In a preferred embodiment of this method, the liquid specimen is enriched using the method of cellular isolation by density gradient centrifugation (Ficoll) and mechanical filters while fluorescent staining is performed with EpCAM-FITC for tumor cells and CD45 PE for non-tumor cells. After the enrichment process or as consequence of it, the liquid specimen containing the processed cells results in a suspension of the cells in a biocompatible liquid medium or cell culture liquid medium. Different options for biocompatible media such as the RPMI-1640 medium supplemented with fetal bovine serum (FBS), L glutamine, Beta Mercaptoethanol and antibiotics (penicillin and streptomycin); 2% (w/v) sodium alginate and 0.5-1.5% (w/w) methylcellulose can be used for this method.

[0026] A transparent support is required as a base for the different elements of the donor substrate. The transparency requirement is due to two reasons: first, the necessity of inspection of the liquid specimen by optical and fluorescence microscopy for selecting the location where the laser beam is focused for propelling the liquid specimen and second, for minimizing the absorption of part of the beam radiation by the transparent support which has effects during the blister generation. In a preferred embodiment of this method, the transparent support is a thin flat piece of glass with similar optical and morphological properties in both surfaces of the piece. In a more preferred embodiment of this method, the transparent support is a 1-mm thick soda-lime glass. FIG. 1 shows a schematic illustration of the transparent support 100.

[0027] The donor substrate is also comprised by a component that absorbs the laser energy. A semi-transparent polymeric adhesive tape has the role of laser energy-absorbent and at the same time allows the visual inspection of the liquid specimen due to its semi-transparent condition to the visible light. The semi-transparent polymeric adhesive tape has at least a sheet of a semi-transparent thermoplastic polymer and a transparent layer of an adhesive polymer. In a preferred embodiment of this method, the semi-transparent thermoplastic material is a semi-transparent thermoplastic polyimide with a range of thickness between 10 and 100 m and the transparent adhesive polymer corresponds to a transparent adhesive silicone of a thickness ranging from 10 to 100 m. FIG. 2 shows a schematic illustration of the structure of the polymeric adhesive tape 200 wherein the sheet of the semi-transparent thermoplastic polymer 210 and the layer of the transparent adhesive polymer 220 are represented. In order to prepare the donor substrate 300, the semi-transparent polymeric adhesive tape 200 is adhered to the transparent support 100 as shown in FIG. 3, with the adhesive surface of the tape 220 in contact with the transparent support 100.

[0028] A volume in the range of tens of microliters of the liquid specimen 400 is dispensed and spread onto the semi-transparent thermoplastic polymer surface 210. In a preferred embodiment of this method when using a thermoplastic polyimide adhesive tape, the polyimide surface is not enough hydrophilic, which causes that the dispensed liquid acquires a semispherical shape even after several trials of spreading. This behavior is not convenient for subsequent steps of the method. Consequently, a previous functionalization of the semi-transparent polyimide surface is needed for making this surface more hydrophilic. The polyimide functionalization is accomplished introducing the donor substrate into a vacuum sample desiccator to extract as much as possible the oxygen content by means of a mechanical pump. Subsequently, the desiccator containing the donor substrate is submitted to a microwave-oven-generated plasma using 1000 W during 5 s. After this process the polyimide surface is ready to receive the liquid specimen.

[0029] Different liquid dispensers can be used to dispense the liquid specimen onto the donor substrate. Some examples of dispensers are pipettes, tips, needles and syringes. In a preferred embodiment of this method, a micropipette is employed to dispense between 5 to 15 L of the liquid specimen 400 containing the enriched population of CTCs or DTCs. The spreading of the liquid specimen can be carried out using specific tools to spread liquids onto flat surfaces such as blades and rolls. Nevertheless, some inconveniences related to recovering the remaining liquid in the tool are unavoidable. To minimize this difficulty, the liquid specimen can be spread with the tip of the same micropipette used to dispense the liquid specimen. In another preferred embodiment of this method, vibrations can be generated in the dispensed liquid by means of for example ultrasounds to provoke the spreading of the liquid specimen until to cover an area sufficiently extended. In a preferred embodiment of this method an area ranging between 10 to 200 mm.sup.2 is covered forming a liquid film with a thickness ranging between 30 to 70 m. FIG. 4 shows a schematic illustration of the donor substrate with liquid specimen 400 spread on the semi-transparent polymeric adhesive tape 200.

[0030] A wide variety of reservoirs, plates, dishes, slides, sheets, liquid reservoirs such multi-well plates and tubes can be employed as receiving substrates in this method. The only restriction involving the receiving substrate arrangement is related to the separation distance needed between this substrate and the liquid specimen. Such separation distance can range between 0.1 and 20 mm. In accordance with following developments of the method, the receiving substrate is located in front of the donor substrate, in such a way that the part of the liquid propelled, as a consequence of the laser impact, can be deposited onto the receiving substrate. In a preferred embodiment of the method, the donor substrate 300 is arranged with the liquid specimen facing down respect to the receiving substrate as shown in FIG. 5. The separation distance between the liquid specimen and the receiving substrate can be accomplished by holding both substrates separately and controlling the separation between them by means of stage controllers. Other strategy consists on employing the same holding for both receiving and donor substrates and inserting two pieces of a flat material, necessarily thicker than the liquid specimen, between both substrates. These pieces are placed on the extremes of both substrates avoiding contact with the liquid specimen. In a preferred embodiment of this method, two pieces of crystalline silicon of about 550 m of thickness are used as separators between the substrates donor and receiving. In another preferred embodiment of this method, two pieces of polymeric adhesive tape are used as separator. Therefore, in this method, the separation distance can ranged depending on the thickness of the material used as separator (between 0.1 and 2 mm preferably). FIG. 5 illustrates the location of the two separation pieces 600 interposed between the donor 300 and receiving 500 substrates.

[0031] A microscopy system allowing the simultaneous inspection of the liquid specimen in the optical and fluorescence fields is employed. In a preferred embodiment of this method, the same objective lens 700 with a sub-micrometric precision is used for the optical and fluorescence inspection, in such a way that two simultaneous images of the same field view, one optical and the other fluorescent, can be obtained from the liquid specimen. FIG. 6 shows the arrangement of the liquid specimen with respect to the objective lens of the optical and fluorescence microscopy system. A linear displacement system comprising two separate translation controllers is used to independently move the donor substrate 300 containing the liquid specimen spread on it and the receiving substrate 500 with respect to the objective lens 700. In a preferred embodiment of this method, two motorized linear stages with micrometric precision are used to independently translate the donor substrate 300 containing the liquid specimen 400 and the receiving substrate 500 so that the liquid specimen can be inspected through a fixed objective lens.

[0032] A laser beam 810 is focalized on the interface between the transparent support 100 and the semi-transparent thermoplastic polymeric adhesive tape 200. The laser radiation is absorbed by the semi-transparent thermoplastic polymeric adhesive tape generating a vapor pocket whose pressure produces the debonding of the polymeric adhesive tape from the transparent support and the deformation in the thermoplastic polymer, called blister, which can mechanically propels a droplet of liquid, depending on the laser energy, onto the receiving substrate. Each propelled droplet contains a single cell or a cluster of cells, so the laser-assisted transfer process induces the isolation of the selected cell from the liquid specimen. FIG. 7 illustrates the arrangement of the laser source 800 and the focalized laser beam 810 with respect to the donor 300 and receiving substrates 500. In a preferred embodiment of this method, the laser source is an ultraviolet multi-frequency pulsed laser operating in the nanosecond regime. A 355 nm laser focalized to obtain a range of energy density between 0.3 and 50 J/cm.sup.2 is used in a preferred embodiment of this method. The tuning of the laser energy density gives place to different work regimes in terms of the effects caused by the mechanical impulse generated in the blister formation on the selected cell or cell clusters. In first place, a threshold value of energy density leading to droplet transfer and consequently to cell transfer is determined. Values of energy density very above this threshold are considered inefficient, since they would induce cellular damage and contamination due cell direct exposition to the laser irradiation and possible polymer releasing. Values close to the threshold are considered the optimal work regimen for the cell isolation. Finally, values close to but below the threshold can be used to generate certain perturbation in the liquid not involving drop transfer. In this case, the generated blister lead to a movement of cells within the liquid specimen. If a cluster of cells is perturbed under these specific energy conditions, the generated blister can induce a disaggregation of the selected clusters of cells, which is convenient for a subsequent single cell isolation.

[0033] It is important to mention that in the optimal regimen, the laser energy can be tuned in order to transfer higher volume of liquid containing cell aggregations or clusters so not only single cell but also cell clusters can be transferred. In a preferred embodiment of this method, when working with a 355 nm 10 ns pulsed laser and a 25 m semitransparent polymeric adhesive tape, the optimal energy density range is found between 1.0 to 1.8 J/cm.sup.2. Energy densities between 0.3 and 1.0 J/cm.sup.2 would induce a separation of clusters into different single cells without propelling any cell to the receiving substrate. Finally, energy densities higher than 1.8 J/cm.sup.2 could induce thermal damage of liquid specimen.

[0034] In this method, there are two options of laser-assisted transfer according to the cell populations that can be tumor cells and non-tumor cells. In the first option the tumor cells are transferred whereas in the second option the non-tumor cells are transferred. In either option, two further alternatives exist depending on the fluorescence condition of the cells. If the cells are fluorescently stained their identification is straightforward from a fluorescence microscopy inspection. On the other hand, when transferring non-stained cells, a comparison between simultaneous optical and fluorescence images of the same field view is required in order to identify the location of the cell to be transferred.

[0035] FIG. 8 shows among other elements a schematic illustration of the two populations of cells when one of them is fluorescently stained. In this case, non-tumor cells are represented as fluorescently stained 410 while tumor cells are represented as non-stained 420. In order to isolate the most interesting population which is the tumor cells (CTCs and DTCs) the previous mentioned optical and fluorescence comparison is needed to identify the locations of such cells. In this particular case, the tumor cells have kept unaltered after the enrichment and staining processes. The laser-assisted transfer isolation of a tumor cell in this particular condition is equivalent to the well-known negative selection process of CTCs and DTCs and therefore, it is called laser negative selection in this disclosure. FIG. 9 shows among other elements a schematic illustration of the liquid specimen 400 where the non-tumor cells are represented as fluorescently stained cells 410 while the tumor cells 420 are represented as non-stained. Thus, the laser impinging on a specific location of a non-stained tumor cell 420 gives place to a blister 230 in the semi-transparent polymeric adhesive tape that propels a droplet of the liquid specimen 430 containing a single tumor cell in such a way that, the laser negative selection is accomplished. In a preferred embodiment of this method, a single pulse of an UV laser system working in the nanosecond regime leads to the ejection of a single droplet with a volume ranging from 1 to 500 pL and containing a single non-stained CTC or DTC. When the tumor cells are fluorescently stained, the process of selection and isolation is equivalent to the well-known positive selection process for CTCs and DTCs. Thus, when the laser-assisted transfer is applied to isolate fluorescently stained tumor cells, the term laser positive selection is adopted to describe this process. In addition, this method also allows transferring the non-tumor cells, leaving the tumor cells in the donor substrate. Due to the fact that, a simple energy tuning enables the method to transfer higher volumes of liquid, the previous description is also applicable to clusters of cells. FIG. 10 shows a schematic illustration of the laser negative selection of a cluster of non-stained tumor cells 440. In this case, laser energy must be adjusted to allow transferring enough amount of liquid comprising the aggregation of two or more tumor cells.