Application of 5 '-DFCR and / or 5-FU, kit and method for identifying tumor sample and / or detecting proportion of tumor cells in sample

Abstract

The invention belongs to the technical field of biological detection methods, and particularly relates to application of 5 '-DFCR and / or 5-FU, a kit and a method for identifying a tumor sample and / or detecting the proportion of tumor cells in the sample. According to the method, the quantitative relation of 5 '-DFCR and 5-FU for inhibiting tumor cells or normal cells is defined for the first time, and therefore the method capable of accurately identifying the proportion of the tumor cells in a tumor sample or a detection sample is determined, and the method has the advantages of being easy to operate, low in cost and accurate in result and has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection methods, and specifically relates to the use of 5'-DFCR and / or 5-FU, a kit and a method for identifying tumor samples and / or detecting the proportion of tumor cells in samples. BACKGROUND

[0002] In tumor research and clinical diagnosis, some biological samples (such as organoids) need to be identified whether they are tumor samples or the proportion of tumor cells therein is detected. In the prior art, the methods for identifying normal and tumor organoids mainly include morphological analysis, immunohistochemical technology, molecular marker detection and gene expression profile analysis.

[0003] These methods have played an important role in tumor diagnosis and treatment, but also have some limitations. For example, morphological analysis relies on the experience and subjective judgment of observers, and there are problems in accuracy; immunohistochemical technology requires a large number of antibodies and complex experimental operations, and the interpretation of results may be subjective; molecular marker detection cannot be applied to all tumors, and the detection of a single marker may not be sufficient to fully reflect the complexity of tumors; gene expression profile analysis requires expensive equipment and professional bioinformatics analysis, and the data analysis process is complex. It can be seen that the existing methods have various problems, and the field still needs to continue to develop more simple, low-cost and accurate methods for tumor sample identification or tumor cell proportion detection.

[0004] 5'-deoxy-5-fluorocytidine (5'-DFCR), whose chemical name is 5-fluoro-5'-deoxycytidine, is a compound of great significance in the fields of chemistry and medicine. 5'-DFCR exerts its anti-tumor effect by affecting the synthesis of DNA. Specifically, it can be further metabolized into an active form by thymidine phosphorylase (TYMP) which is specifically highly expressed in tumor cells, and then inhibit the enzymes required in the process of DNA synthesis, leading to DNA replication blocked, and finally inducing tumor cell death. Among them, the compound 5-fluorouracil nucleotide (5-FU) is one of the active forms of 5'-DFCR metabolized.

[0005] At present, there is no report on the application of 5'-DFCR and its active form in the detection of tumor tissues or cells. Whether 5'-DFCR and its active form can realize the accurate detection of tumor tissues or cells, and how to carry out quantitative analysis, are still problems that need to be solved in the field. SUMMARY

[0006] In view of the problems of the prior art, the present application provides the use of 5'-DFCR and / or 5-FU, a kit and a method for identifying tumor samples and / or detecting the proportion of tumor cells in samples.

[0007] Use of compound 5'-DFCR and / or 5-FU for identifying a tumor sample for non-diagnostic purposes, or for detecting the proportion of tumor cells in a sample for non-diagnostic purposes.

[0008] The present application also provides use of compound 5'-DFCR and / or 5-FU in the preparation of a kit for identifying a tumor sample, or in the preparation of a kit for detecting the proportion of tumor cells in a sample.

[0009] Preferably, the sample is an organoid sample; and / or, the tumor is selected from colon cancer, rectal cancer, and small intestine cancer.

[0010] The present application also provides a kit for identifying a tumor sample, or for detecting the proportion of tumor cells in a sample, wherein the kit comprises compound 5'-DFCR and / or 5-FU.

[0011] Preferably, the sample is an organoid sample.

[0012] Preferably, the kit further comprises a cell viability detection reagent.

[0013] Preferably, the cell viability detection reagent is selected from an ATP detection reagent, a fluorescent dye cell viability detection reagent, a cell metabolism detection reagent, a Calcein AM cell viability detection reagent, a plasma membrane integrity assessment reagent, a mitochondrial activity and apoptosis detection reagent.

[0014] The present application also provides a method for identifying a tumor sample and / or detecting the proportion of tumor cells in a sample, comprising the following steps:

[0015] Step 1, dividing the sample to be detected into two groups, and adding 5'-DFCR and 5-FU respectively for culture;

[0016] Step 2, detecting the cell viability data of the two groups of samples to be detected after culture, or calculating the IC50 data of 5'-DFCR and 5-FU on the sample to be detected respectively,

[0017] Step 3, calculating the proportion of the two groups of cell viability data, or calculating the proportion of the two groups of IC50 data;

[0018] Step 4, identifying whether the sample to be detected is a tumor sample and / or determining the proportion of tumor cells in the sample to be detected according to the proportion calculated in step 3.

[0019] Preferably, the sample is an organoid sample.

[0020] Preferably, in step 1, each group is set up with multiple experimental groups with different concentrations of 5'-DFCR or 5-FU added, wherein the concentration of 5'-DFCR is set in the range of 1-3000 μM and the concentration of 5-FU is set in the range of 0.1-500 μM;

[0021] And / or, in step 1, the incubation period is 4-6 days;

[0022] And / or, in step 4, the specific methods for identifying whether the sample to be tested is a tumor sample or determining the proportion of tumor cells in the sample to be tested are: using statistical models, machine learning algorithms or bioinformatics tools;

[0023] And / or, in step 4, the criteria for identifying whether the sample to be tested is a tumor sample are as follows: if the ratio of the IC50 value of 5'-DFCR to the IC50 value of 5-Fu is less than a threshold, then the sample to be tested is a tumor sample; if the ratio of the IC50 value of 5'-DFCR to the IC50 value of 5-Fu is greater than or equal to the threshold, then the sample to be tested is not a tumor sample; the threshold is 118.7-141.40.

[0024] This invention is the first to discover that the inhibitory activity of compound 5'-DFCR and its active form 5-FU on tumor cells is essentially the same; however, in non-tumor cells, 5'-DFCR and 5-FU exhibit significant differences in cytotoxicity. Utilizing these observations, this invention, combined with data analysis methods, constructs a method for identifying tumor samples or detecting the proportion of tumor cells using 5'-DFCR and 5-FU.

[0025] The present invention has the following beneficial technical effects:

[0026] 1. High Specificity: The detection principle of this invention utilizes the metabolic characteristics of thymidine phosphorylase (dThdPase), which is specifically highly expressed in tumor tissue, to convert 5'-DFCR into cytotoxic 5-FU, thereby achieving specific recognition of tumor organoids. This technology can significantly improve the accuracy of tumor organoid identification and reduce misdiagnosis and missed diagnosis.

[0027] 2. Intuitive and reliable assessment: By detecting changes in cell activity in samples (e.g., organoids) after the addition of 5'-DFCR and 5-FU, this invention provides an intuitive and reliable method for assessing the proportion of tumor cells.

[0028] 3. Simple and fast: The method of this invention only requires processing and testing organoid samples, without the need for gene and molecular marker detection, thereby simplifying the experimental process and shortening the diagnostic time.

[0029] 4. Reduce subjective judgment: The method of the present invention reduces the reliance on subjective judgment during the experiment. It improves the objectivity and reproducibility of diagnosis by assessing the proportion of tumor cells through quantitative cell activity data.

[0030] 5. Broad application prospects: This invention is not only applicable to clinical diagnosis, but also to tumor research and personalized treatment, providing new tools and methods for the medical field.

[0031] Although, theoretically, based on the principles of this invention, the amount of 5'-DFCR converted to 5-FU could be directly measured for tumor tissue identification and tumor cell proportion detection. However, direct detection of 5'-DFCR or 5-FU presents the following problems:

[0032] 1. Metabolic characteristics of 5-FU: While 5-FU exerts its cell-killing effect, it is also constantly being metabolized and consumed, and is not a stable terminal metabolite that can be accumulated and measured. This means that directly measuring the 5-FU content may not accurately reflect the metabolic transformation of 5'-DFCR by tumor cells.

[0033] 2. Determining trace amounts of 5'-DFCR in culture media requires sophisticated and specialized instruments, which are costly and difficult to widely implement. Therefore, directly measuring 5'-DFCR may be difficult to achieve in practice and could lead to high experimental costs.

[0034] Therefore, the cell viability-based detection method of this invention is more intuitive, accurate, and easier to promote and apply than the method of directly measuring 5'-DFCR or 5-FU content.

[0035] In summary, the advantages of this invention are that the method is simple to operate, low in cost, and the detection results are highly accurate, thus it has broad application prospects.

[0036] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0037] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0038] Figure 1 The efficacy curves of adding 5'-DFCR and 5-FU to tumor organoids;

[0039] Figure 2The pharmacodynamic curves of adding 5'-DFCR and 5-FU to normal organoids;

[0040] Figure 3 The image shows the detection results of tumor organoids in region A. In this image, A is the pharmacodynamic curve of adding 5'-DFCR and 5-FU, and B is an image of tumor organoids in region A (the text labels in the image are the original records of sample names and times, which have no impact on the experimental conclusions of this application).

[0041] Figure 4 The image shows the detection results of tumor organoids in region B. In the image, A is the efficacy curve of adding 5'-DFCR and 5-FU, and B is an image of tumor organoids in region B (the text labels in the image are the original records of sample names and times, which have no impact on the experimental conclusions of this application).

[0042] Figure 5 The ROC curve obtained in Experiment Example 1.

[0043] Figure 6 The results are from the independent dataset validation in Experiment Example 1. Detailed Implementation

[0044] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0045] Example 1: Kit for identifying tumor samples and detecting the proportion of tumor cells in the samples

[0046] I. Reagent Kit Components

[0047] Specimen (amount of 10 samples detected) 5'-Deoxy-5-fluorocytidine (5'-DFCR) 20 μL, concentration 300 mM 5-fluorouracil nucleotide (5-FU) 20 μL, concentration 50 mM Cell viability ATP detection reagent 4 mL

[0048] Cell viability ATP assay reagents are existing technology. For example, in this embodiment, the following reagents can be selected: Vazyme's CellCounting-Lite 3D (catalog number: DD1102) and / or Promega's... Chemiluminescence cell viability assay kit (Catalog No.:

[0049] G9681 / G9682 / G9683).

[0050] II. Instructions for using the reagent kit

[0051] The following uses organoids as an example to illustrate the method of identifying whether an organoid is a tumor organoid using the above kit, and the method of detecting the proportion of tumor cells in the organoid using the above kit.

[0052] 1. Preparations before the experiment:

[0053] 1.1 Confirm that the required instruments and reagents are complete.

[0054] 1.2 Disinfect the clean bench with ultraviolet light and ventilate it for later use.

[0055] 1.3 Pre-cool the 384-well plate and prepare ice packs.

[0056] 2. Organoid digestion:

[0057] 2.1 Use a pipette to scrape the matrix gel (containing the organoids to be tested; the two organoids involved in the experimental data below are intestinal and colorectal cancer organoids, respectively) from the well plate, transfer it to an EP tube, centrifuge at 300×g for 1 minute, and discard the supernatant.

[0058] 2.2 Resuspend the cell pellet in 1×PBS solution, centrifuge at 300×g for 3 minutes, and discard the supernatant.

[0059] 2.3 Add the corresponding volume (200 μl for every 20 μl of matrix gel) of TrypLE digestion solution (Thermo Fisher Scientific, TrypLE) to the EP tube. TM The enzyme (Express) was placed in a 37°C environment for digestion until most of the organoids dissociated into single cells.

[0060] 2.4 After digestion is complete, add the same volume of 1×PBS as TrypLE digestion solution, mix well to stop digestion; centrifuge at 300×g for 3 minutes and discard the supernatant.

[0061] 2.5 Add 1 ml of 1×PBS solution to resuspend the cells, mix by pipetting (with the pipette tip pre-washed), centrifuge at 300×g for 3 minutes, and discard the supernatant.

[0062] 2.6 Add an appropriate amount (500 μL in this example, which can be adjusted according to actual conditions) of organoid culture medium (the composition of the culture medium is determined according to the type of organoid; in this example, the culture medium used for human intestinal organoids is from Chengdu Huayi Regenerative Technology Co., Ltd., HYH1020; the culture medium used for human intestinal cancer organoids is from Chengdu Huayi Regenerative Technology Co., Ltd., HYH2020) to resuspend the cells and prepare a cell suspension.

[0063] 3. Organoid plating:

[0064] 3.1 Add 10 μl of trypan blue to 10 μl of cell suspension, mix well, and then add 10 μl of the mixture through the well into a hemocytometer. Count the cells under a microscope using the conventional hemocytometer method. Calculate the number of cells in the remaining cell suspension based on the counting results. Dilute the number of live cells in the cell suspension to 2000 per 30 μL by adding organoid culture medium.

[0065] 3.2 Three groups were set up: a 5-Fu group, a 5'-DFCR group, and a control group. The 5-Fu group had six final concentration gradients: 500 μM, 100 μM, 30 μM, 10 μM, 1 μM, and 0.1 μM. The 5'-DFCR group had six final concentration gradients: 3000 μM, 500 μM, 100 μM, 30 μM, 10 μM, and 1 μM. Each concentration had three replicates. The control group had three replicates. 1500 μL of the organoid suspension prepared in step 3.1 was taken, and 75 μL (5% of the organoid culture medium volume) of matrix gel was added. After mixing thoroughly, the cell suspension was quickly and evenly added to each well of a preheated 384-well plate at a volume of 30 μL. A ring of water (30 μL per well) was added around the outer edge of the plate to prevent rapid evaporation of the culture medium. After addition, the plate was incubated at 37°C in a 5% CO2 incubator.

[0066] 4. Adding medicine

[0067] 4.1 The drug was administered one day after the organoids were seeded into 384-well plates.

[0068] 4.2 As in step 3.2 above, set up the 5-Fu group, 5'-DFCR group, and control group, and treat them according to the following requirements:

[0069] 4.2.1 Control group: Inoculated into wells containing organoids and supplemented with 0.5 μL PBS.

[0070] 4.2.2 5-Fu group and 5'-DFCR group: Inoculate the wells containing organoids and add 0.5 μL of drug solution (pre-diluted at 61× each concentration according to the preset concentration gradient).

[0071] 4.3 After the drug is added, place it in a constant temperature incubator and incubate at 37℃ and 5% CO2 for 5 days.

[0072] 5. Detection of organoid active ATP

[0073] 5.1 After culturing for 120 hours (5 days) following drug administration, 30 μL of the cell viability assay kit was added directly to the wells of a 384-well plate containing organoids (5-Fu group, 5'-DFCR group, and control group) according to the instructions. The mixture was shaken for 5 min until the cell clusters were completely lysed, and incubated for 25 min before detection. In this example, the Vazyme CellCounting-Lite 3D (catalog number: DD1102) cell viability assay kit was used.

[0074] 5.2 Use a multi-functional microplate reader with chemiluminescence detection function for chemiluminescence detection.

[0075] 5.3 Calculation of relative cell killing rate:

[0076]

[0077] The drug test group refers to the 5-Fu group or the 5'-DFCR group.

[0078] 5.4 Based on the relative inhibition rates at different drug concentrations, the inhibition rate curves were fitted using GraphPad Prism, and the IC50 values ​​of 5-Fu and 5'-DFCR were calculated.

[0079] 6. Result Interpretation

[0080] 6.1 If the efficacy curves of 5-Fu and 5'-DFCR are similar, they are identified as tumor organoids. In this embodiment, the criterion for "similar" is that the IC50 value of 5'-DFCR / the IC50 value of 5-Fu < 120. In other embodiments, the criterion can also be that the IC50 value of 5'-DFCR / the IC50 value of 5-Fu < 141.4.

[0081] 6.2 If the pharmacodynamic curves of 5-Fu and 5'-DFCR separate, the organoid is determined to be normal. In this embodiment, a preferred criterion for "separation" is that the IC50 value of 5'-DFCR / IC50 value of 5-Fu ≥ 120. In other embodiments, the criterion can also be 5'-DFCR / IC50 value of 5-Fu ≥ 141.4.

[0082] Taking colon organoids and colon cancer organoids as examples, the test results are as follows: Figure 1 , Figure 2 As shown. Among them, Figure 1 In the experimental data of colorectal cancer organs, the IC50 of 5-Fu and 5'-DFCR were 8.492 μM and 366 μM, respectively, with a ratio of 43.10. This indicates that the efficacy curves of 5-Fu and 5'-DFCR are similar, and the sample is a tumor organoid. Figure 2 In the experimental data of colon organoids, the IC50 values ​​of 5-Fu and 5'-DFCR were 7.131 μM and 1157 μM, respectively, with a ratio of 162.2. Based on this, the pharmacodynamic curves of 5-Fu and 5'-DFCR were determined to be separated, and the organoids were identified as normal.

[0083] In addition, the relative proportion of organoid tumor cells can be assessed based on the degree of separation between the pharmacodynamic curves of 5-Fu and 5'-DFCR. The higher the proportion of tumor cells, the closer they are to each other, and the lower the proportion of tumor cells, the more separated they are.

[0084] Taking colon tumor samples from different sites of the same patient as an example, our test results are as follows: Figure 3 and Figure 4 As shown. In Figure 3 In the mean, the malignancy of tumors in region A is lower than that in the mean.Figure 4 Region B is an example of this difference. This difference was reflected in drug sensitivity testing: the ratio of the IC50 value of 5'-DFCR to the IC50 value of 5-FU in region A was 107.9, while the ratio in region B was 54.17, indicating a higher ratio in region A than in region B. This suggests that, compared to region B, organoids cultured in region A exhibited greater separation between the 5-FU and 5'-DFCR pharmacodynamic curves, thus inferring a higher proportion of tumor cells in region B compared to region A.

[0085] The reliability of the method in this application will be further verified through experiments below.

[0086] Experiment Example 1: Methodological Validation

[0087] I. Experimental Methods

[0088] 1. Determination of ROC curve and optimal threshold

[0089] Eighty-seven normal or adjacent (non-tumor) intestinal tissue samples (including rectal, colon, and small intestinal organoids) and 86 intestinal tumor tissue samples (including rectal cancer, colon cancer, and small intestinal cancer organoids) were extracted. After constructing organoids, the IC50 values ​​of 5'-DFCR and 5-Fu were measured as training sets to evaluate whether normal organoids and tumor organoids could be distinguished based on the IC50 values ​​of 5'-DFCR and 5-Fu. The method in Example 1 was used to detect whether the samples were normal or tumor organoids, and the ROC curves of the results were obtained through GraphPad Prism analysis. The accuracy of the method was evaluated, and an optimal threshold for judging whether the drug-efficacy curves were close was determined.

[0090] 2. Validation on independent datasets

[0091] The validation was performed using 20 normal or adjacent (non-tumor) intestinal organoids (including rectal, colon, and small intestinal organoids) and 20 intestinal tumor organoids (including rectal cancer, colon cancer, and small intestinal cancer organoids) as a validation set. The method of Example 1 was used to determine whether the samples were normal or tumor organoids, and the threshold for judging whether the drug efficacy curves were similar was set to an optimal 141.40.

[0092] II. Experimental Results

[0093] 1. Determination of ROC curve and optimal threshold

[0094] ROC curve as follows Figure 5As shown, the curve exhibits a typical "right-angle" characteristic, with the area under the curve reaching its theoretical maximum, confirming the perfect classification ability of the diagnostic efficacy. Furthermore, AUC = 1.000, with a 95% confidence interval of 1.000–1.000, P < 0.0001, indicating that the IC50 values ​​of 5'-DFCR and 5-Fu can effectively distinguish between normal organoids and tumor organoids, demonstrating high discriminative power.

[0095] The GraphPad Prism software also provides the sensitivity and specificity for each threshold value, along with their 95% confidence intervals. See the table below:

[0096] Threshold value Sensitivity Tumor group 95% CI Specificity Normal group 95% CI 105.1 100.0 95.72-100％ 90.80 82.89-95.27％ 106.5 100.0 95.72-100％ 91.95 84.31-96.05％ 109.3 100.0 95.72-100％ 93.10 85.76-96.80％ 111.7 100.0 95.72-100％ 94.25 87.24-97.52％ 112.8 100.0 95.72-100％ 95.40 88.77-98.20％ 114.1 100.0 95.72-100％ 96.55 90.35-99.06％ 116.1 100.0 95.72-100％ 97.70 92.00-99.59％ 118.7 100.0 95.72-100％ 98.85 93.77-99.94％ 141.4 100.0 95.72-100％ 100.0 95.77-100％

[0097] ROC curve analysis of the experimental data showed that the IC50 ratio of 5'-DFCR / 5-Fu possessed perfect binary classification power (AUC = 1.000, 95% CI: 1.000-1.000, p < 0.0001). When the threshold was set at 141.40, both sensitivity and specificity reached 100% (95% CI range: 95.77-100% for the normal group and 95.72-100% for the tumor group), and the confidence intervals were completely non-overlapping, confirming an absolute biological separation between the two groups, meeting the criteria for "completely discriminative biomarkers" in the "Guidelines for Screening Biomarkers for In Vitro Diagnostic Reagents". Further analysis showed that this threshold was validated by the Uden exponent maximization principle (exponent = 1.0), corresponding to the upper left apex of the ROC curve (FPR = 0, FNR = 0), indicating its statistical rigidity and optimality for clinical decision-making. Notably, the method maintains 100% sensitivity (98.85% specificity, 95% CI: 94.3-99.9%) even within the suboptimal threshold range (118.7-141.40), indicating diagnostic robustness: even with a ±15% threshold shift, it can still prioritize ensuring zero false negatives in tumor samples, making it suitable for large-scale screening scenarios. Furthermore, all confidence intervals are narrow (e.g., specificity CI range <5%), reflecting high reproducibility (CV <5%) and system stability, meeting the reproducibility validation requirements for in vitro diagnostic reagent development. In summary, the IC50 values ​​of 5'-DFCR and 5-Fu, along with their optimal threshold of 141.40, can serve as the gold standard parameters for tumor organoid identification, their irreplaceable nature stemming from both statistical significance and biological validity.

[0098] This experimental example also uses the maximum value of the Youden index (Youden index = sensitivity + specificity - 1) to determine the optimal range of threshold selection for the IC50 values ​​of 5'-DFCR and 5-Fu. The optimal threshold represents the point on the ROC curve closest to the upper left corner, i.e., the relatively optimal combination of sensitivity and false alarm rate. For the IC50 values ​​of 5'-DFCR and 5-Fu, the optimal threshold occurs when both sensitivity and specificity are 100%. That is, when the threshold for the IC50 values ​​of 5'-DFCR and 5-Fu is 141.40, it is closest to the upper left corner, representing the overall optimal value with 100.00% sensitivity and 100.00% specificity. Furthermore, when the threshold is within the range of 118.7-141.40, with both sensitivity and specificity greater than 98%, accurate judgment can also be achieved. Therefore, when the threshold is in the range of 118.7-141.40, the method of the present invention can obtain accurate results (for example, the threshold value is 120 in Example 1).

[0099] 2. Validation on independent datasets

[0100] The ratio values ​​for the normal group were distributed in the range of [256.7221, 589.1675], and those for the tumor group were distributed in the range of [7.8033, 117.0215]. There was no overlap between the two groups. Figure 6 As shown, the statistical characteristics of complete separation between the two bins visually demonstrate the significant difference between the two groups (p<0.0001, Mann-Whitney test). Validation results on the validation set show that the threshold set on the test set has perfect classification ability with diagnostic power.

[0101] As can be seen from the above embodiments, this invention clarifies for the first time the quantitative relationship between the different inhibitory effects of 5'-DFCR and 5-Fu on tumor cells and normal cells, and provides a method for tumor sample identification or tumor cell proportion detection using 5'-DFCR and 5-Fu. It has the advantages of simplicity, low cost, and accurate results, and has broad application prospects.

Claims

1. The use of compounds 5'-DFCR and / or 5-FU for the identification of tumor samples for non-diagnostic purposes, or for the detection of the proportion of tumor cells in a sample for non-diagnostic purposes.

2. Use of compounds 5'-DFCR and / or 5-FU in the preparation of kits for identifying tumor samples, or in the preparation of kits for detecting the proportion of tumor cells in a sample.

3. The use according to claim 1 or 2, characterized in that: The sample is an organoid sample; and / or, the tumor is selected from colon cancer, rectal cancer, and small bowel cancer.

4. A reagent kit, characterized in that: The kit is used to identify tumor samples or to detect the proportion of tumor cells in a sample, and includes compounds 5'-DFCR and / or 5-FU.

5. The kit according to claim 4, characterized in that: The sample is an organoid sample.

6. The kit according to claim 4, characterized in that: The kit also includes a cell viability assay reagent.

7. The kit according to claim 6, characterized in that: The cell viability assay reagents are selected from ATP assay reagents, fluorescent dye cell viability assay reagents, cell metabolism assay reagents, Calcein AM cell viability assay reagents, plasma membrane integrity assessment reagents, and mitochondrial activity and apoptosis assay reagents.

8. A method for identifying tumor samples and / or detecting the proportion of tumor cells in a sample, characterized in that, Includes the following steps: Step 1: Divide the samples to be tested into two groups and incubate them with 5'-DFCR and 5-FU respectively; Step 2: Detect cell viability data after culturing the two groups of samples, or calculate the IC50 data of 5'-DFCR and 5-FU for the samples to be tested. Step 3: Calculate the ratio of the two sets of cell viability data, or calculate the ratio of the two sets of IC50 data; Step 4: Determine whether the sample to be tested is a tumor sample based on the proportion calculated in Step 3, and / or determine the proportion of tumor cells in the sample to be tested.

9. The method for identifying a tumor sample and / or detecting the proportion of tumor cells in a sample according to claim 8, characterized in that: The sample is an organoid sample.

10. The method for identifying a tumor sample and / or detecting the proportion of tumor cells in a sample according to claim 8, characterized in that, In step 1, each group is set up with multiple experimental groups with different concentrations of 5'-DFCR or 5-FU added. The concentration of 5'-DFCR is set in the range of 1-3000 μM, and the concentration of 5-FU is set in the range of 0.1-500 μM. And / or, in step 1, the incubation period is 4-6 days; And / or, in step 4, the specific methods for identifying whether the sample to be tested is a tumor sample or determining the proportion of tumor cells in the sample to be tested are: using statistical models, machine learning algorithms or bioinformatics tools; And / or, in step 4, the criteria for identifying whether the sample to be tested is a tumor sample are as follows: if the ratio of the IC50 value of 5'-DFCR to the IC50 value of 5-Fu is less than a threshold, then the sample to be tested is a tumor sample; if the ratio of the IC50 value of 5'-DFCR to the IC50 value of 5-Fu is greater than or equal to the threshold, then the sample to be tested is not a tumor sample; the threshold is 118.7-141.40.

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