CN113567675B - Liquid biopsy companion diagnosis and kit for hexokinase 2 inhibitors of tumors - Google Patents

Abstract

The application provides a concomitant diagnosis method and a kit of a hexokinase 2 inhibitor for tumors, and also provides application of a hexokinase 1 antibody and a hexokinase 2 antibody in the diagnosis and the kit. Through the detection of body fluid samples containing tumor cells of tumor patients by utilizing hexokinase 1 antibody substances and hexokinase 2 antibody substances, patients sensitive to hexokinase 2 inhibitors are found, so that accurate treatment is performed. Since body fluid is more available than tumor tissue, this detection mode can eliminate the damage to the patient caused by tumor tissue biopsy.

Description

Liquid biopsy companion diagnosis and kit for hexokinase 2 inhibitors of tumors

Technical Field

The present application relates to the field of medical detection. In particular to the concomitant diagnosis of hexokinase 2 inhibitors on cancer patients by means of liquid biopsy to achieve the aim of accurate medical treatment.

Background

An abnormality in energy metabolism is one of the fundamental characteristics of malignant tumor cells. Tumor cells tend to adopt the metabolic mode of glycolysis even under aerobic conditions, a phenomenon known as the Warburg effect. Aiming at the characteristic of aerobic glycolysis of tumor cells, the key enzyme is used as a drug target point, so that the utilization rate of glucose is reduced, the growth of the tumor cells is hindered, and the method is one of ideas of cancer treatment and drug development.

Hexokinase (HK) is a key and rate-limiting enzyme in the first step of cellular glucose metabolism, catalyzing the phosphorylation of glucose to glucose-6-phosphate. Phosphorylated glucose becomes unstable and cannot cross the cell membrane, facilitating its further metabolism within the cell. In humans, there are 5 isozymes of hexokinase, HK1, HK2, HK3, HK4 and HKDC1 (Hexokinase Domain Containing 1), respectively, of which HK1 and HK2 are the most important. HK1 is expressed in most tissues and is considered a "housekeeping gene" enzyme. HK2 is expressed in fetal tissue as well as in a few adult tissues, including bone, cardiac muscle and adipose tissue. HK3 is expressed at low levels in almost all tissues and is inhibited by physiological concentrations of glucose. HK4, also known as glucokinase, is expressed primarily in the pancreas and liver for maintaining blood glucose constant. HKDC1 is arranged adjacent to HK1 on the same chromosome and is associated with gestational diabetes. Of the five hexokinases above, HK2 has the greatest tumor-related effect. HK2 has higher catalytic activity than HK1 and has high affinity to glucose. HK2 is combined with Voltage-dependent anion channel protein VDAC (Voltage-Dependent Anion Channel) on mitochondria, so that ATP generated by mitochondria can be preferentially obtained, and the ATP can meet the requirement of rapid growth of tumors. The research at present finds that HK2 is highly expressed in a plurality of tumor tissues such as lung cancer, breast cancer, prostate cancer, osteosarcoma and the like. The major subtype of normal brain tissue hexokinase is HK1, whereas gliomas express large amounts of HK2. Likewise, normal liver tissue expresses almost only HK4, and in highly malignant liver cancer, HK4 is silenced, instead HK2 and a small amount of HK1.

Aiming at the characteristic of high expression of HK2 in malignant tumors, various HK2 inhibitors have been developed at present, but are not yet approved to enter clinic. HK2 inhibitors are mainly of two types, the first being the direct use of short hairpin RNAs to reduce or silence HK2; the second is a small molecule inhibitor of HK2, such as 3-bromopyruvate (3-Bromopyruvic Acid), which can separate HK2 from VDAC on mitochondria, inhibit HK2 activity, and ultimately lead to cell death. Previous studies found that the inhibitory effect of HK2 inhibitors on tumors depends on whether the tumor simultaneously expressed HK1 (e.g., xu and Herschman, A Tumor Agnostic Therapeutic Strategy for Hexokinase 1-Null/Hexokunase 2-Positive Cancer, cancer Research,2019, DOI:10.1158/0008-5472. CAN-19-1789). If the tumor simultaneously expresses higher HK1, the tumor can survive by metabolizing glucose through HK1 even if HK2 is completely inhibited. Whereas HK1 negative or very low tumors were significantly more sensitive to gene silencing and inhibitors of HK2 than HK1 positive tumors. Thus, HK2 inhibitors are believed to be primarily useful for HK1 negative/very low tumors. From this study result, it is possible to judge whether or not the tumor tissue belongs to a HK 1-negative/low and HK 2-positive tumor by simultaneously detecting the amounts of HK1 and HK2 in the tumor tissue (by RNA or protein), and judge whether or not the tumor is sensitive to the HK2 inhibitor, so as to predict the therapeutic effect of the HK2 inhibitor on the tumor.

In clinical practice, however, it is often the case that no tumor tissue is available in the patient, and liquid biopsies provide an alternative method. The process of outward spreading of tumor cells into various body fluids, such as blood, pleural effusion, cerebrospinal fluid, urine, etc., and the detection of tumor cells in these fluid samples is known as fluid biopsy. Liquid biopsies are more convenient and less damaging to patients than tumor tissue biopsies, and are therefore increasingly accepted clinically. Unlike tissue samples, the proportion of tumor cells in body fluid samples tends to be low, and there are a large number of benign cells that interfere with detection, especially those that tend to highly express HK1 and lowly express HK2. Therefore, we cannot directly and indiscriminately detect the values of the cells HK1 and HK2 in a body fluid sample like tumor tissue to judge the values of the HK1 and HK2 of the tumor cells of a patient, but should identify the tumor cells in the body fluid sample and detect the values of the HK1 and HK2 for the tumor cells. Meanwhile, since the tumor cell fraction in the body fluid has already died or is undergoing apoptosis, resulting in protein degradation, it is preferable to identify active tumor cells in the body fluid sample and to detect the values of HK1 and HK2 thereof.

Disclosure of Invention

The application aims to provide a method and a reagent for carrying out the concomitant diagnosis of a hexokinase 2 inhibitor by using a body fluid sample of a tumor patient.

The present application first provides a concomitant diagnostic method of a hexokinase 2 inhibitor against a tumor, comprising: forming complexes with each of the HK1 and HK2 proteins in the patient body fluid sample cells with a labeled hexokinase 1 (HK 1) antibody material and a labeled hexokinase 2 (HK 2) antibody material; and detecting a marker signal of the complex in the cell to indicate the tumor cell contained in the body fluid sample and the respective expression levels of HK1 and HK2 in the tumor cell.

In a specific embodiment of the present application accompanying a diagnostic method, the body fluid sample is blood, cerebrospinal fluid, pleural effusion or urine of a tumor patient, more particularly urine of a bladder cancer patient.

In a specific embodiment of the present application that accompanies the diagnostic method, the forming of the complex comprises staining the body fluid sample cells with a fluorescently labeled HK1 antibody material and a fluorescently labeled HK2 antibody material; and the detection comprises obtaining a fluorescent signal of the stained cells, preferably further comprising staining the cells with a labeled, in particular fluorescently labeled, antibody substance for the depletion of leukocytes.

The application also provides a kit for concomitant diagnosis of a hexokinase 2 inhibitor against a tumor, or for a concomitant diagnostic method of the application, comprising: hexokinase 1 (HK 1) and hexokinase 2 (HK 2) antibody substances, preferably, the HK1 and HK2 antibody substances are labeled HK1 and labeled HK2 antibody substances. The kit of the application also optionally comprises labeled antibody substances for the depletion of leukocytes, preferably antibody substances directed against the leukocyte antigen CD45 or antibody substances directed against cytokeratin.

In a specific embodiment of the kit of the application, the HK1 antibody and HK2 antibody are a labeled HK1 antibody and a labeled HK2 antibody, respectively, or a combination of HK1 antibody and a labeled HK1 secondary antibody and a combination of HK2 antibody and a labeled HK2 secondary antibody. The antibody substance for eliminating leukocytes is an antibody against leukocyte antigen CD45 or an antibody against cytokeratin. The label is preferably a fluorescent label.

The application also relates to the use of a combination of hexokinase 1 (HK 1) and hexokinase 2 (HK 2) antibody substances, comprising: use in concomitant diagnosis of an HK2 inhibitor against a tumor; the application in preparing detection reagent for HK2 inhibitor accompanying diagnosis of tumor; or in the preparation of the kit of the application.

In the concomitant diagnosis to which the present application relates, the level of HK1 expression of tumor cells lower than that of the bladder cancer cell line UMUC-3 is determined as HK1 low expression.

According to the application, by combining the HK1 antibody substance and the HK2 antibody substance, the sensitivity of a tumor patient to the HK2 inhibitor can be judged according to the expression level of the HK1 of the tumor cells while detecting the tumor cells in the patient sample based on the expression level of the HK2 of the sample cells, so that the accurate treatment can be realized.

Drawings

FIG. 1 shows the results of detection of HK1 and HK2 in three bladder cancer cell lines RT4, 5637 and UMUC-3.

FIG. 2 shows the HK2 inhibition curve of the low grade bladder cancer cell line RT4 versus the high grade bladder cancer cell line UMUC-3, wherein the HK2 inhibitor is 3-bromopyruvate.

FIG. 3 shows the results of HK1, HK2 measurements of tumor tissue from 42 patients with bladder cancer, wherein the abscissa represents HK1 fluorescence values and the ordinate represents HK2 fluorescence values, both mean and standard deviation are used to represent data. These patients included 25 high-grade tumor patients (dark grey), 16 low-grade tumor patients (grey) and 1 benign bladder tumor patient (light grey).

Fig. 4 shows the detection results of HK1 and HK2 of the urine sample and paired tumor tissue sample of 4 patients with high-grade bladder cancer, wherein the upper part of the figure shows the detection results of tumor cells in urine, and the lower part of the figure shows the detection results of tissue samples.

FIG. 5 shows the percentage decrease in high glycolytic activity tumor cells after 4 high grade bladder cancer patients urine samples were treated with the HK2 inhibitor 3-bromopyruvate (1 mM concentration) for 4 hours.

Detailed Description

Glycolysis is a commonly used energy metabolism mode for tumors, and Positron Emission Tomography (PET) technology developed based on the characteristic has been used for clinically diagnosing tumors and finding metastatic lesions thereof. In addition to diagnosis, the development of specific inhibitors against the basic tumor feature of glycolysis has been one of the targets in the field of drug development, and HK2 highly expressed in various tumors is one of the most promising targets. Although some HK2 inhibitors have been reported, there are several problems with their use in therapy: 1) Although HK2 is inhibited, tumor cells remain viable through HK1 compensation, thus requiring typing of tumors, HK1 negative or low expressing tumors are sensitive to HK2 inhibitors; 2) Because HK2 is still expressed in a small number of tissues of a human body, the administration of the HK2 system can cause certain side effects, but a patient with bladder cancer can act on the local part of the bladder by a perfusion mode, so that the side effects can be reduced. Combining the above two points, tumors negative or under-expressed for HK1 in bladder cancer patients may be sensitive to HK2 inhibitors. However, the previous literature only studied tumor cell lines (non-bladder cancer cell lines) and animal models constructed based on these tumor cell lines, and therefore all involved pure tumor cells, whereas many benign cells were present in actual tumors, and were not completely composed of tumor cells, and therefore it was not possible to directly measure the amount of HK1 to determine.

Since HK2 itself is closely related to the Warburg effect of the tumor, HK2 can be combined with other markers to detect tumor cells with glycolytic activity in urine samples from patients with bladder cancer, providing a basis for further detection of HK1 values.

The concomitant diagnostic method of the application involves the detection of tumor cells in a liquid sample from a patient. The principle, method, judgment criteria for identifying rare tumor cells from human body fluid using tumor marker HK2, and advantages thereof, which are applicable to the identification of tumor cells involved in the present application, are described in detail in chinese patent application (CN 2019102852422, "detection method and detection kit for rare tumor cells", assigned by the present inventors) filed on 4/10/2019. The disclosure of this patent application is incorporated by reference in its entirety. According to the method described in the application, the cells with high HK2 expression/white blood cell marker negative/cell nucleus positive staining in the human body fluid sample are identified as tumor cells, for example, the cells with high HK2 expression/CD 45 negative/DAPI positive staining in the human body fluid sample are identified as rare tumor cells; and further identifying tumor cells positive for epithelial cell markers in the human fluid sample as epithelial-mesenchymal-transformed tumor cells or tumor cells of mesenchymal origin. The HK2 highly expressed cells may refer to cells in which the fluorescence signal value reaches a predetermined threshold after HK2 fluorescent staining treatment, for example, the average value of HK2 fluorescence values of white blood cells (such as CD45 positive cells) at the time of fluorescence detection is increased by five times the standard deviation as the threshold for judging HK2 highly expressed. These methods are also suitable for the present application.

The present application provides HK2 inhibitors for cancer (e.g., bladder cancer) along with diagnostic reagents and kits that can detect tumor tissue from a sample of fluid (e.g., urine) containing tumor cells, and screen samples sensitive to HK2 inhibitors by detection of the HK1/HK2 ratio. For example, the application can screen the sample sensitive to the HK2 inhibitor by taking the ratio of HK1/HK2 lower than 0.4 as a threshold value, and has the advantage that the ratio is not changed by the condition of the sample and the difference of the fixed dyeing condition unlike the simple detection of the fluorescence value of HK1. In addition, since the proportion of tumor cells in urine samples is generally low, it is necessary to identify tumor cells therein by using a marker and then detect the fluorescence values of HK1 and HK2 and calculate the proportion. Specifically, the present application provides a urine sample-based HK2 inhibitor companion diagnostic kit in which tumor cells in urine are identified by combining HK2 (or optionally with another marker (CD 45 or CK)) and then the HK1/HK2 ratio is calculated based on the HK1 to HK2 fluorescence values of these tumor cells, this liquid biopsy-based test result being consistent with the results of a tumor tissue biopsy.

Alternatively, the level of HK1 expression in a tumor cell line that has been shown to be sensitive to an inhibitor may be selected as a threshold for determining low expression of HK1 in the tumor cell to be tested. In the examples that follow, the level of HK1 expression of the bladder cancer cell line UMUC-3 can be used in a concomitant diagnosis as a reference for determining low expression levels in test tumor cells, particularly bladder cancer cells. For example, cells in urine are stained and imaged under the same conditions as UMUC-3 cells, the median of UMUC-3 fluorescence is the criterion of low HK1, and if the median of HK1 of tumor cells (HK 2 is high and non-white blood cells) in urine is lower than the median of UMUC-3, it is the low expression of HK1.

Specific embodiments of hexokinase 2 inhibitors of the present application along with diagnostic reagents and kits comprise: a fluorescent-labeled hexokinase 1 (HK 1) antibody substance, a fluorescent-labeled hexokinase 2 (HK 2) antibody substance, and a fluorescent-labeled antibody substance for leukocyte depletion; after the above materials stain the cells in urine, the HK1 fluorescence value of the non-leucocyte and HK2 high expression cells is counted; if the median of these cellular HK1 fluorescence values is low, the patient is sensitive to the HK2 inhibitor. The fluorescently labeled antibody species is a fluorescently labeled antibody, or a combination of an antibody and a fluorescently labeled secondary antibody.

However, the present application is not limited to fluorescein labeling, and other labels used in the art for proteins may be used as long as efficient, quantitative signal detection is achieved.

The antibody substance used for the exclusion of leukocytes may be an antibody substance against leukocyte antigen CD45 or an antibody substance against Cytokeratin (CK), i.e. CD45 negative cells are non-leukocytes or CK positive cells are non-leukocytes. The kit of the application may also include a nuclear dye to further exclude interference with tumor cell detection in the sample.

The application mainly relates to the detection of the sensitivity of tumor cells to hexokinase 2 inhibitors in a body fluid sample for accurate treatment, and has obvious advantages compared with tumor tissue biopsy in the concomitant diagnosis based on body fluid, such as easy implementation, reduction of damage or no damage of patients, and the like. However, as mentioned above, and as shown in the examples below, the methods of the application can also be used for the concomitant diagnosis of glucokinase 2 inhibitors based on tumor tissue samples.

In the embodiment of the application, the values of HK1 and HK2 of tumor cells in a urine sample of a patient with bladder cancer are used as a liquid biopsy means to judge whether the patient is sensitive to the HK2 inhibitor. The kit provided by the application can detect the values of HK1 and HK2 of active tumor cells in urine of a patient with bladder cancer.

Although the following examples refer only to urine samples from patients with bladder cancer, it will be readily understood by those skilled in the art that the present application is not so limited and that the principles, methods and reagents of the present application are applicable to liquid samples from other oncology patients, such as blood, cerebrospinal fluid, hydrothorax, etc.

The following examples use 3-bromopyruvate as an HK2 inhibitor for testing the sensitivity of tumor cells to HK2 inhibitors, but those skilled in the art will readily appreciate that the accompanying diagnostic methods and reagents of the present application are applicable to other HK2 inhibitors in the art.

The application will be further illustrated with reference to specific examples. It should be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Modifications and adaptations of the application will occur to those skilled in the art and are intended to be within the scope of the application in practice.

The technical means used in the following examples are conventional technical means well known to those skilled in the art, and the raw materials and reagents used are commercially available products, unless otherwise specified.

Example 1: HK2 inhibitors inhibit bladder cancer tumor cell lines

A. Detection of HK1, HK2 of bladder cancer tumor cell lines

1) Preparing 0.5 ml of cell (RT 4, 5637, UMUC-3 and white blood cells isolated from healthy human blood) suspension (100 tens of thousands of cells/ml);

2) Cell fixation: 50 microliters of DSP (Dithiobis (succinimidyl propionate,50 mM) was mixed with 50 microliters of SPDP (N-succinimidyl 3- (2-pyridysith) propionate,50 mM) and 400 microliters of phosphate buffer was added, and this formulated 500 microliter fixative was quickly mixed with 500 microliters of cell suspension in the previous step, added to a tabletting warehouse and left to stand at room temperature for 40 minutes to fix cells;

3) And (3) tabletting: centrifuging (100 g,5 min) with a tablet-throwing machine, discarding the supernatant, adding 100 microliters of Tris-HCl (100 mM), and reacting for 10min at room temperature; washing three times with PBS, and then taking out the glass slide from the slide preparation bin;

4) Cell membrane: 100 microliters of 0.5% Triton X-100 phosphate buffer solution was added and allowed to permeate for 15 minutes at room temperature;

5) Cell closure: 100 microliters of phosphate buffer solution containing 10% sheep serum and 3% BSA was added and blocked for 1 hour at room temperature;

6) Incubation resistance: 100 microliters of primary antibody mixture, containing 2% of primary HK1 antibody (murine: abnova H00003098-B01P) and 1% of primary HK2 antibody (rabbit, abcam ab 209847), was added, and washed overnight at 4℃with phosphate buffer;

7) Secondary antibody incubation: adding 100 microliters of secondary antibody mixed solution which comprises 0.25%Alexa Fluor 555 labeled goat anti-mouse secondary antibody and 0.25%Alexa Fluor488 labeled goat anti-rabbit secondary antibody, incubating for 1 hour at room temperature, and washing with phosphate buffer solution;

8) Nuclear staining: DAPI is dyed for 10min at room temperature, and is washed by phosphate buffer solution;

9) Imaging by a high-speed fluorescence imaging device;

10 Statistics of cellsHK1 and HK2Is a fluorescent value of (a).

Results: as shown in FIG. 1, the bladder cancer cell line highly expressed HK2 compared to leukocytes, while the high-grade bladder cancer cell line UMUC-3 had low expression of HK1.

B. HK2 inhibitors inhibit bladder cancer tumor cell lines

1) Preparing a mother solution with the concentration of 100mM of the HK2 inhibitor 3-bromopyruvate by using DMSO, subpackaging, and preserving at-20 ℃;

2) After bladder cancer cell lines (RT 4 and UMUC-3) are digested by pancreatin, the bladder cancer cell lines are resuspended by a complete culture medium (the cell lines correspond to basic culture medium and 10% fetal bovine serum) and then dripped on a micropore array chip, the pore diameter of the micropore on the chip is 30 mu m, 2% matrigel is pre-paved, and the bladder cancer cell lines are stood for 5 minutes to enable cells to enter micropores, so that the cells in the micropores can be prevented from being lost in subsequent operation;

3) Preparing working solutions with different concentrations from 3-bromopyruvate by using a complete culture medium, replacing the culture medium on a chip to treat cells, incubating for 24 hours in an incubator at 37 ℃, and adding DMSO (dimethyl sulfoxide) with the same volume into the culture medium by using a control group under the same conditions as a dosing group;

4) Cell viability staining solution was prepared before the end of incubation, i.e. 0.5. Mu.L calcein AM (viable cell dye) and 2. Mu. L ethidium homodimer-1 (dead cell dye) were added to 1 ml Dulbecco's phosphate buffer;

5) Washing the original culture medium with Dulbecco's phosphate buffer solution after the incubation is finished, adding the cell activity staining solution, incubating for 30 minutes at room temperature, and then washing with the phosphate buffer solution for imaging;

6) The percent of viable cells at different drug concentrations was calculated for different cell lines to plot drug inhibition curves.

Results: as shown in FIG. 2, UMUC-3 cells with low expression of HK1 were sensitive to the HK2 inhibitor at a half inhibitory concentration (IC 50) of about 240. Mu.M relative to RT4 cells with high expression of HK1. This experiment demonstrates that a bladder cancer cell line with low expression of HK1 is sensitive to an HK2 inhibitor, while the amount of HK1 of UMUC-3 can be used as a criterion for judging sensitivity to an HK2 inhibitor.

Example 2: detection of bladder cancer tissue samples

1) Baking slices: placing the tissue slices into a 63 ℃ oven for wax drying for one hour;

2) Dewaxing: after the baking of the flakes is finished, taking out and putting the flakes into a xylene No. 1 jar for soaking for 15 minutes, putting the flakes into a xylene No. 2 jar for soaking for 15 minutes, putting the flakes into an absolute alcohol No. 1 jar for soaking for 5 minutes, putting the flakes into an absolute alcohol No. 2 jar for soaking for 5 minutes, putting the flakes into a 90% alcohol jar for soaking for 5 minutes, putting the flakes into a 80% alcohol jar for soaking for 5 minutes, putting the flakes into a 70% alcohol jar for soaking for 5 minutes, putting the flakes into a deionized water jar for soaking for 5 minutes, and putting the flakes into a phosphate buffer solution jar for soaking for 5 minutes;

3) Antigen retrieval: placing the dewaxed slice into EDTA (MVS-0099 of Ministry of biological technology development Co., fuzhou, inc.) repairing liquid at 97 ℃ for soaking for 20 minutes, naturally cooling the EDTA repairing liquid cylinder containing the slice to room temperature, taking out the slice, placing the slice into a deionized water cylinder for soaking for 5 minutes, placing the slice into a phosphate buffer liquid cylinder for soaking for 5 minutes, and finally taking out the slice for airing;

4) Penetrating: 0.5% Triton X-100 was permeabilized for 15 minutes;

5) Closing: phosphate buffer solution containing 10% sheep serum and 3% BSA was blocked for 1 hour at room temperature;

6) Incubation resistance: the antibody (HK 1 murine primary antibody: abnova H00003098-B01P; HK2 rabbit primary antibody: abcam ab 209847) was diluted 1:100 and then added to the sections overnight, washed with phosphate buffer solution;

7) Secondary antibody incubation: diluting an antibody (Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody; alexa Fluor 555-conjugated goat anti-mouse secondary antibody) according to a ratio of 1:400, adding the diluted antibody to a slice, and dyeing the slice for 1 hour at room temperature, and washing with a phosphate buffer solution;

8) Nuclear staining: DAPI is dyed for 10min at room temperature, and is washed by phosphate buffer solution;

9) The high-speed fluorescence imaging device images.

Results: as shown in FIG. 3, high-grade patients had high HK2 expression and low HK1 expression, while low-grade patients and benign tumor patients were reversed. HK1 expression is low in some patients, which are mostly high-grade patients, meaning that some high-grade patients may be sensitive to HK2 inhibitors.

Example 3: detection of urine from patients with bladder cancer

1) Pretreatment of urine: the urine samples of four bladder cancer patients are respectively filtered by a 350-mesh nylon net, centrifuged (410 g,5 minutes) to remove the supernatant, and 0.5 ml of phosphate buffer solution is added for resuspension; if hematuria exists, the erythrocytes are firstly lysed and then the subsequent step of testing is carried out;

2) Cell fixation: 50 microliters of DSP (Dithiobis (succinimidyl propionate,50 mM) was mixed with 50 microliters of SPDP (N-succinimidyl 3- (2-pyridysith) propionate,50 mM) and 400 microliters of phosphate buffer was added, and this formulated 500 microliter fixative was quickly mixed with 500 microliters of cell suspension in the previous step, added to a tabletting warehouse and left to stand at room temperature for 40 minutes to fix cells;

3) And (3) tabletting: centrifuging (100 g,5 min) with a tablet-throwing machine, discarding the supernatant, adding 100 microliters of Tris-HCl (100 mM), and reacting for 10min at room temperature; washing three times with PBS, and then taking out the glass slide from the slide preparation bin;

4) Cell permeabilization: 100 microliters of 0.5% Triton X-100 phosphate buffer solution was added and allowed to permeate for 15 minutes at room temperature;

5) Cell closure: 100 microliters of phosphate buffer solution containing 10% sheep serum and 3% BSA was added and blocked for 1 hour at room temperature;

6) Incubation resistance: 100 microliters of primary antibody mixture, containing 2% of primary HK1 antibody (murine: abnova H00003098-B01P) and 1% of primary HK2 antibody (rabbit, abcam ab 209847), was added, and washed overnight at 4℃with phosphate buffer;

7) Secondary antibody incubation: adding 100 microliters of secondary antibody mixed solution which comprises 0.25%Alexa Fluor 555 labeled goat anti-mouse secondary antibody and 0.25%Alexa Fluor488 labeled goat anti-rabbit secondary antibody, incubating for 1 hour at room temperature, and washing with phosphate buffer solution;

8) Cytokeratin staining: 100 microliters of an antibody dilution containing 2% of the murine primary antibody HK1 was added dropwise to the slide glass to block the Alexa Fluor 555-labeled goat anti-mouse secondary antibody, incubated at 37℃for 1 hour, washed with a phosphate buffer solution, 100 microliters of an antibody dilution containing 0.5% of the APC-labeled pan-CK was added, and washed with a phosphate buffer solution overnight at 4 ℃;

9) Nuclear staining: DAPI is dyed for 10min at room temperature, and is washed by phosphate buffer solution;

10 High-speed fluorescence imaging device;

11 Number of pan-CK positive and HK2 high-expression cells and their HK1 fluorescence values were counted.

Results: 180, 226, 694 and 632 pan-CK positive, HK2 high-expression high glycolytic activity tumor cells were identified in urine samples of these 4 patients with high-grade bladder cancer, and the fluorescence values of HK1 (dark grey) and HK2 (light grey) are shown in the upper graph of FIG. 4 by violin curves and quartiles, respectively. Fluorescence values of HK1, HK2 of paired tumor tissue samples of these 4 patients are shown in the following plots, expressed as mean and standard deviation, respectively. The figure shows that HK1 values of highly glycolytic active tumor cells in urine are consistent with the trend of tumor tissue.

Example 4: HK2 inhibitors inhibit bladder cancer tumor cell lines

1) Cells in urine of the bladder cancer patient of example 3 were divided into two parts, one part was incubated with 1mM of the HK2 inhibitor 3-bromopyruvate in an incubator at 37℃for 4 hours, and the other part was used as a control group, and the same volume of DMSO was added to the incubator at 37℃for 4 hours;

2) Cell viability staining solution was prepared before the end of incubation, i.e. 0.5. Mu.L calcein AM (viable cell dye) and 2. Mu. L ethidium homodimer-1 (dead cell dye) were added to 1 ml Dulbecco's phosphate buffer;

3) Washing the original culture medium with Dulbecco's phosphate buffer solution after the incubation is finished, adding the cell activity staining solution, incubating for 30 minutes at room temperature, and then washing with the phosphate buffer solution for imaging;

results: as shown in fig. 5, the greatest decrease in high glycolytic activity tumor cells in urine from T2, T3 patients showed that it was very sensitive to HK2 inhibitors, consistent with low expression of high glycolytic activity tumor cells HK1 in both samples, among 4 patients.

Thus, urine detection kits based on HK2 antibodies and HK1 antibodies can be used to detect low HK1 expression bladder cancer and test for sensitivity to HK2 inhibitors.

Claims (7)

1. Use of a combination of hexokinase 1 (HK 1) and hexokinase 2 (HK 2) antibody substances, comprising:

the application of the kit in preparing a detection kit for detecting tumor cells in a body fluid sample of a patient through liquid biopsy and carrying out diagnosis of HK2 inhibitor aiming at the tumor cells,

wherein: the HK1 antibody material and HK2 antibody material are contacted with sample cells of the patient's body fluid to detect the levels of HK1 and HK2 expression in the cells, respectively, and to determine that the cells in which HK2 is highly expressed are tumor cells, and that low or negative HK1 expression in the tumor cells indicates that the tumor cells or patient are sensitive to the HK2 inhibitor.

2. The use of claim 1, wherein the HK1 and HK2 antibody substances are labeled HK1 and labeled HK2 antibody substances.

3. The use according to claim 1, wherein the kit further comprises a labeled antibody material for the depletion of leukocytes.

4. The use according to claim 3, wherein the antibody substance for the depletion of leukocytes is an antibody substance against the leukocyte antigen CD45 or an antibody substance against cytokeratin.

5. The use of any one of claims 1-4, wherein the patient fluid sample is blood, pleural effusion, cerebrospinal fluid, or urine.

6. The use of any one of claims 1-4, wherein the patient is a bladder cancer patient and the body fluid sample is urine.

7. The use according to claim 6, wherein the level of HK1 expression of the tumor cells is determined to be HK1 low expression, as compared to the level of HK1 expression of the bladder cancer cell line UMUC-3.