Method for detecting and typing rare tumor cells in body fluid sample and kit therefor

Abstract

A method for detecting and typing rare tumor cells having high metabolic activity in a body fluid sample, comprising the following steps: incubating nucleated cells in a body fluid sample with a first metabolic marker and a second metabolic marker capable of producing fluorescence signals; detecting, by means of high-throughput imaging, uptake of all the fluorescence signals of the metabolic markers by cells, so as to determine the energy metabolism mode and intensity of the cells; and identifying and typing, according to the fluorescence signals of the metabolic marker combination, tumor cells having high metabolic activity in the body fluid sample. Further provided is a kit used for the detecting and typing method, comprising a microwell array chip, a first metabolic marker and a second metabolic marker capable of producing fluorescence signals, and fluorescence-labeled antibodies specific to leukocyte common antigen. The method and the kit identify and type energy metabolism modes of rare tumor cells in a body fluid sample based on fluorescence signal characteristics, and the operation is simple and fast, reducing the possibility of losing rare tumor cells.

Claims

1. A method for detecting and typing tumor cells with high metabolic activity in a body fluid sample, comprising: step A, incubating karyotes from the body fluid sample with a fluorescein-labeled first metabolic marker, a fluorescein-labeled second metabolic marker, and a fluorescein-labeled anti-CD45 antibody, wherein the first metabolic marker is 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), and the second metabolic marker is resazurin; step B, detecting fluorescence signals generated by the fluorescein labels associated with the karyotes by high-throughput imaging so as to determine energy metabolism; and step C, identifying tumor cells with high metabolic activity in the body fluid sample which are the karyotes having fluorescence signals of both of the metabolic markers stronger than corresponding positive cut-off values of 2-NBDG and resazurin and not having fluorescence signal of the anti-CD45 antibody.

2. The method according to claim 1, wherein the body fluid sample is selected from the group consisting of blood, pleural effusion, peritoneal effusion and cerebrospinal fluid, and wherein the incubation is for a time of from 2 minutes to 2 hours.

3. The method according to claim 1, wherein the cut-off value of 2-NBDG is a mean value of 2-NBDG fluorescence signal values of all anti-CD45 positive cells plus 5 folds of standard deviation, and the cut-off value of resazurin is a mean value of resazurin fluorescence signal values of all the anti-CD45 positive cells plus 3 folds of standard deviation.

4. The method according to claim 1, wherein step A further comprises adding the fluorescein-labeled karyotes from the body fluid sample into a microwell array chip, the microwell array chip including multiple addressable microwells for accommodating cells.

5. The method according to claim 1, wherein step A further comprises enriching tumor cells in the body fluid sample.

6. A kit for detecting and typing tumor cells in a body fluid sample, wherein the kit comprising: (a) a microwell array chip, including multiple addressable microwells for accommodating cells; (b) a fluorescein-labeled first metabolic marker capable of producing a fluorescence signal; (c) a fluorescein-labeled second metabolic marker capable of producing a fluorescence signal; and (d) a fluorescein-labeled anti-CD45 antibody; wherein the first metabolic marker is 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), and the second metabolic marker is resazurin.

7. The kit according to claim 6, wherein the number of the microwells on the microwell array chip is from 5 thousand to 500 thousand, and the bottom of the microwells on the microwell array chip is closed or has one or more micropores with a diameter of from 5 μm to 10 μm.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a microphotograph of a certain numbered area on one microwell array chip, the purpose of the numbering is to conveniently locate the cells on the chip. The microwell array chip has 400 to 2500 groups of such numbered areas, in total 100000 to 500000 micropores.

(2) FIG. 2 shows a scatter diagram of uptake fluorescence values of 2-NBDG and resazurin of all the anti-CD45 negative cells on one chip according to examples of the present invention, in this figure two straight lines respectively represent the cut-off values of 2-NBDG and resazurin high uptake, wherein the method for determining the cut-off value of 2-NBDG is the mean value of the 2-NBDG uptake values of the anti-CD45 positive cells plus 5 folds of a standard deviation, the method for determining the cut-off value of resazurin is the mean value of the resazurin uptake values of anti-CD45 positive cells plus 3 folds of the standard deviation.

DETAILED DESCRIPTION

(3) The inventors, through extensive and deep researches, firstly develop a method for detecting the rare tumor cells in the patient's body fluid sample based on the energy metabolism mode and conducting functional typing to them, whereas the tumor cells having different energy metabolism modes have different malignant degrees and metastatic potentials.

(4) Researches indicate that the feature of the tumor cell metabolism is to replace oxidative phosphorylation of normal tissue cells with high level of aerobic glycolysis. Because of low efficiency of glycolysis, the tumor cells need to uptake a large number of glucose. Further researches find that besides glucose, the tumor cells may also have other energy substance source, such as glutamine, a large number of reductive enzymes are accumulated in the cells. In the present invention, the rare tumor cells in the body fluid sample are identified according to a biological principle namely the capacity of uptaking glucose or the capacity of reducing resazurin by the tumor cells is much higher than the normal cells. In order to increase specificity of the identification, in the present invention of the leukocyte is further excluded by using the leukocyte surface marker anti-CD45. Because 2-NBDG and resazurin can both produce the fluorescence signal, a very large number of cells can be identified within a short time with the help of a high-speed fluorescence imaging equipment, thus the invention is not required to firstly conduct a complicated enrichment process on the rare tumor cells in the body fluid sample like the reported method. For the body fluid sample such as pleural effusion and cerebrospinal fluid, because of its low total cell number, the detection can be directly conducted without enriching the tumor cells. For a blood sample, the number of the cells can be reduced by a magnetic sphere negative selection of a simple labeled CD45 antibody after lysis and removal of erythrocytes, then the detection is conducted. If the blood sample is less or the CTC number is more, the detection can be directly conducted without the negative selection.

(5) The method described in the present invention has similarities with a tumor imaging detection method for detecting glucose uptake by the tissue with a radioactive glucose analog (.sup.18F-FDG, 2-fluorine-18-fluoro-2-deoxy-D-glucose) which has been used in clinic. .sup.18F-FDG is transported into the cells by a glucose transporter, and phosphorylated under the action of hexokinase, to generate 6-PO.sub.4-.sup.18F-FDG and accumulate in the cells and can be detected by Positron Emission computed Tomography (PET). Therefore, the PET imaging based on the radioactive glucose analog .sup.18F-FDG can be used to show site, morphology, size, number of the tumor and radioactive distribution within the tumor, in clinic it is mainly used in diagnosis of a malignant tumor and differential diagnosis of benign or malignancy, clinical staging, evaluating efficacy and monitoring recurrence and the like. The vast majority of the benign focuses do not uptake or slightly uptake .sup.18F-FDG. In clinic, uptake amount of .sup.18F-FDG by the focus is measured by using a half dose therapy of SUV (standard uptake value), and benign and malignancy of the tissue are identified, generally SUV>2.5 is considered as a malignant tumor, SUV<2.0 can be considered as a benign lesion.

(6) For the tumor cells, they have various different phenotypes, genetic characteristics and metabolism modes. In the present invention, two or more metabolic markers are used to collectively detect the characteristic metabolism mode of the tumor cells, these metabolism modes are closely related to the malignant degree and the metastatic potential; compared with other molecular characteristics, it is able to more simply and reasonably reflect the malignant degree and the metastatic potential of the tumor cells.

Terminology

(7) As used herein, “2-NBDG” refers to 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose, which is a glucose analog with a fluorescent label, and can be used in detecting uptake of glucose by the cells.

(8) “Resazurin” refers to 7-hydroxy-3H-phenoxazin-3-one 10-oxide, its English name is Resazurin, it is a blue dye having only a weak fluoresce, but it can be irreversibly reduced to a resorufin having a strong red fluorescence, and can be used in detecting the reductive enzyme within the cells.

(9) “2-NBDG high uptake” means that the amount of 2-NBDG uptaken by the cells is higher than the mean value of the amounts of 2-NBDG uptaken by the leukocyte in the sample plus five folds of the standard deviation.

(10) “Resazurin high uptake” means that the amount at which the cells reduce resazurin uptaken by them into a fluorescent form is higher than the mean value of the amounts of resazurin uptaken and reduced by the leukocytes plus three folds of the standard deviation.

(11) The preferred examples of the invention are given below in conjunction of the accompanying drawings, in order to illustrate the technical solution of the invention in detail. For the experiment methods in which the specific conditions are not indicted in the following examples, they generally follow the conventional conditions, for example the conditions described in Sambrook et al., Molecular cloning: laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or follow the conditions recommend by the manufacturer. Unless otherwise specified, percentage and part are weight percentage and weight part. In the examples, all culture mediums, cell lines, and antibodies are all commercially available.

EXAMPLES

(12) Detection of 2-NBDG and resazurin uptaken by rare tumor cells in a lung cancer patient's pleural effusion sample

(13) One micorporouse array PDMS chip is provided, its structure is as shown in FIG. 1, it includes 400 numbered areas, each area includes about 350 micropores with a diameter of 25 μm, in total 14 thousand micropores, and they can accommodate about 600 thousand karyotes.

(14) In this example, the method comprises the following steps:

(15) (1) 5 ml of lung cancer patient's pleural effusion was centrifuged (500 g, 5 minutes) and the cells were isolated, the erythrocyte was remove by using an erythrocyte lysate of BD Company, the cells were resuspended with a Hank's balanced salt solution (HBSS) and washed, finally resuspended in 500 ml of HBSS;

(16) (2) 2 ml of Allophycocyanin (APC) labeled anti-CD45 antibody was added into 500 ml of cell suspension (about 1 million cells) which is then turned over on a turnover instrument and incubated for 1 hour;

(17) (3) the 500 ml of cell suspension is centrifuged, a supernatant was discarded, then the cells were diluted with HBSS, and a cell suspension was dripped onto two microwell array chips, standing for 10 minutes, a microscopic bright field picture of the chip is as shown in FIG. 1, the cells were substantially in the micropores, but because some of the leukocytes were small in size, there were more than one cells in some of the micropores;

(18) (4) the solution on the chip surface was removed, and fluorescein labeled glucose analog 2-NBDG (400 μM) and resazurin (1 μM) were added onto each chip, standing in an incubator of 37° C. for 15 minutes;

(19) (5) after completion of the incubation, the chip was washed with an ice PBS for 8 times, and imaged with a high-speed fluorescent imaging equipment, and fluorescence values of 2-NBDG, resazurin and anti-CD45 of each cell were recorded.

(20) Cut-off values of 2-NBDG and resazurin can be calculated according to the fluorescence values of 2-NBDG and resazurin in anti-CD45 positive leukocyte, if higher than this cut-off value the cells are cells with high metabolic activity. FIG. 2 shows a scatter diagram of the uptake values of 2-NBDG and resazurin in all anti-CD45 negative cells, the anti-CD45 negative cells can be divided into 4 subgroups according to the cut-off values, namely a cell subgroup of anti-CD45 negative/resazurin being high, a cell subgroup of anti-CD45 negative/2-NBDG being high, a cell subgroup of anti-CD45 negative/resazurin, 2-NBDG being both high, a cell subgroup of anti-CD45 negative/resazurin, 2-NBDG being both low. Wherein, a definition of resazurin and 2-NBDG high uptake has been illustrated in the “terminology”. The cells in the first three subgroups (the cell subgroup of anti-CD45 negative/resazurin high, the cell subgroup of anti-CD45 negative/2-NBDG high, the cell subgroup of anti-CD45 negative/resazurin, 2-NBDG being both high of) were taken out one by one to conduct a single cell sequencing and conduct an in situ tissue comparison, it was found that most of these cell had EGFR 19del mutation consistent with the in situ tissue, wherein 7 cells among 10 cells of anti-CD45 negative/resazurin high had the EGFR 19del mutation, 16 cells among 20 cells of anti-CD45 negative/2-NBDG being high had the EGFR 19del mutation, 3 cells among 4 cells of anti-CD45 negative/resazurin, 2-NBDG being both high had the EGFR 19del mutation. These cells having the EGFR 19 del mutation were all the tumor cells, whereas 8 cells in which the EGFR19del mutation was not detected were found having a large fragment of gene amplification and deletion by means of genome copy number variation detection, conforming to the characteristics of the tumor cells. In the cells of anti-CD45 negative/resazurin, NBDG being both low, a large proportion of them are not the tumor cells, and a large number of cells are unable to successfully conduct the single cell genome sequencing, this may be related to the fact of the cells being in an apoptosis state.

(21) The cells with two different metabolism modes were found in multiple lung cancer patient samples, their number and relative proportion were not identical, a further genome copy number variation analysis on the tumor cells with two kinds of metabolism modes found that these two types of tumor cells were different in terms of copy number variation, specifically manifested in that the cells with glucose high uptake generally had amplification of PIK3CA gene and deletion of PTEN gene, whereas the cells with resazurin high uptake tended to have amplification of MYC gene. Such a difference in the genome aspect reflect the difference between the two types cells in signaling pathway activating mode and the differences in terms of metastatic potential and drug resistance and the like.

(22) At present, in clinic, by using a radioactive substance which can be phagocytized or swallowed by the tumor cells, by applying a PET imaging of the radioactive substance in showing site, morphology, size, number of the tumors and radioactive distribution within the tumor, in clinic the invention are mainly used in diagnosis of malignant tumor and differential diagnosis of benign and malignancy, clinical staging, evaluating efficacy and monitoring the recurrence and the like.

(23) The present invention quantitatively detects the glucose uptake capacity of the rare tumor cells by a labeled glucose analog, such as fluorophore labeled D-glucose analog 2-NBDG which has a metabolic pathway similar to D-glucose. The fluorophore labeled D-glucose analog 2-NBDG enters into the cells via a glucose transporter (GLUT), then C-6 position of which is phosphorylated by a hexokinase. Researches have shown that, compared with a benign cell, 2-NBDG can be rapidly uptaken by a malignant tumor cell, thereby becoming an optical marker for detecting the malignant tumor cell.

(24) Some of the tumor cells also have other source of energy substance, but glycolytic process with high intensity makes a large number of reductive enzymes to accumulate within the tumor cells, these enzymes can rapidly reduce a resazurin without fluorescence signal to an oxidized form with fluorescence signal, so as to quantitatively characterize the amount of the reductive enzymes within the cell, thereby to detect the intensity of glycolysis.

(25) The invention, based on the glucose analog having fluorescence signal and a glycolysis-related reductive enzyme detection method and kit, detects energy metabolism mode and intensity of the very rare tumor cells in the tumor patient's body fluid sample, so as to conduct a typing of energy metabolism function on these tumor cells, thereby to effectively select the tumor cells with high malignant degree and great metastatic potential.

(26) It should be understood that, the above-mentioned examples are merely used to illustrate the invention but not to limit the protection scope of the invention, one skilled in the art can make various variations or modifications in the invention based on the principle disclosed by the invention and the disclosed content, these equivalent forms likewise fall into the protection scope of the appended claims.