Kit for evaluating tumor microenvironment uridine metabolic activity and prognosis of lung cancer patient

Abstract

The invention discloses a kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment of a lung cancer patient, the kit comprises a UPP1 antibody, an EPCAM antibody, a CD68 antibody and a CD66b antibody which are labeled by different fluorescence, the UPP1 antibody is combined with a green fluorescent dye Alexa Fluor 488, the EPCAM antibody is combined with a red fluorescent dye Alexa Fluor 594, the CD68 antibody is combined with a blue fluorescent dye Alexa Fluor 650, and the CD66b antibody is combined with a yellow fluorescent dye Alexa Fluor 555. According to the kit, the expression level of UPP1 in tumor cells and immune cells is detected, cell specific markers EPCAM, CD68 and CD66b are combined for quantitatively evaluating the metabolic activity of uridine in the tumor microenvironment and prognostic metabolic activity, and a scientific basis is provided for early diagnosis and prognostic evaluation of lung cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment in lung cancer patients. Background Art

[0002] Currently, in the prognosis assessment of lung cancer patients, common technical means include imaging examinations (such as CT scans), tumor marker detections (such as CEA, CYFRA21-1, etc.), and pathological analyses (such as tissue section staining, immunohistochemistry, etc.). These methods provide certain reference values for clinical practice to a certain extent, but there are also some key deficiencies, mainly including: (1) Imaging examinations: Although imaging means such as CT scans have advantages in tumor localization and monitoring tumor size, they cannot comprehensively evaluate biological characteristics such as metabolic activities, immune responses, and cell functions in the tumor microenvironment. Imaging methods are difficult to effectively reflect the immune escape mechanism in the tumor microenvironment, especially unable to quantitatively analyze the interaction between tumor cells and immune cells. (2) Tumor marker detections: Conventional tumor markers such as CEA, CYFRA21-1, etc. play a certain role in the early screening and diagnosis of lung cancer, but their specificity and sensitivity are relatively low, and they usually lack the ability to directly reflect the metabolic characteristics of the tumor microenvironment, especially there are obvious limitations in the accuracy of evaluating immune system responses and metabolic activities. (3) Pathological analyses: Although traditional tissue section staining and immunohistochemistry methods can evaluate the phenotypic characteristics of tumor cells and the expression of molecular markers, there are great limitations in the analysis of complex metabolic activities in the tumor microenvironment. Most of the existing technologies can only detect single markers, lacking a comprehensive evaluation of the metabolic activity interaction between tumor cells and immune cells, and it is difficult to comprehensively reveal the dynamic changes of the tumor microenvironment.

[0003] Therefore, the existing technologies are relatively limited in the ability to evaluate the tumor microenvironment and immune responses in multiple aspects. Especially in the comprehensive analysis of tumor metabolic characteristics and immune escape mechanisms, there is a lack of effective tools for comprehensive and quantitative evaluation.

[0004] In the prior art, kits have been applied to tumor metabolism research, especially immunohistochemical staining kits for metabolic-related proteins such as lactate dehydrogenase and glucose transporters. However, most of them focus on the detection of tumor cell metabolite markers and fail to comprehensively evaluate the metabolic interaction between immune cells and tumor cells in the tumor microenvironment. There are still the following defects and deficiencies: (a) Single protein labeling: Currently, most kits can only detect a single metabolic marker, which limits the overall analysis of the complex metabolic activities in the tumor microenvironment; (b) Lack of cell-specific analysis: Existing kits usually only focus on the metabolic activities of a single type of cell and cannot distinguish the metabolic differences between tumor cells and immune cells, thus unable to fully reflect the metabolic dynamics and interactions between different cell types in the tumor microenvironment; (c) Inability to quantitatively evaluate: Current kits mainly rely on antibody staining or ELISA detection and fail to achieve multiplex quantitative analysis of uridine metabolism-related proteins, resulting in the inability to accurately evaluate the changes in metabolic activities in different cell populations. Especially in the tumor immune microenvironment, quantitative evaluation of the metabolic state is crucial.

[0005] Research has found that uridine metabolism plays a crucial role in the functions and survival of tumor cells and immune cells. Tumor cells adapt to metabolic stress and promote their own proliferation by altering the uridine metabolism pathway. At the same time, they regulate the functions of immune cells through uridine metabolism to help tumors evade immune surveillance. Uridine metabolism-related protein (UPP1) has become a key target in the analysis of the tumor microenvironment. Although there are already some antibodies for UPP1 applied to cell metabolism research on the market, the application of these antibodies in clinical prognosis evaluation is still limited, mainly having the following problems: (i) Lack of cell marker combination: Although existing antibodies can detect UPP1, most antibodies can only be used alone and cannot simultaneously bind cell-specific markers such as EPCAM, CD68, and CD66b, thus unable to comprehensively evaluate the metabolic activities in tumor cells and immune cells and limiting the quantitative analysis of the metabolic state in different cell populations. (ii) Lack of immune cell metabolism labeling: In the current antibody combinations, there are few labels for uridine metabolism-related proteins in immune cells such as macrophages and neutrophils, and the role of immune cells in tumor immune escape has not been fully explored. (iii) Insufficient detection accuracy: Existing antibodies are mostly detected based on traditional immunohistochemical methods and lack a high-sensitivity, multiplex quantitative detection platform. Due to the inability to accurately evaluate the complex metabolic activities in the tumor microenvironment, the comprehensive evaluation of tumor metabolic characteristics faces challenges. Summary of the Invention

[0006] In view of the above deficiencies in the prior art, the purpose of the present invention is to provide a kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment in lung cancer patients. By detecting the expression levels of UPP1 in tumor cells and immune cells and combining with cell-specific markers EPCAM, CD68, and CD66b, this kit comprehensively and quantitatively analyzes the uridine metabolic activity, is used to quantitatively evaluate the metabolic activities between tumor cells and immune cells in the tumor microenvironment, and provides a scientific basis for the early diagnosis, prognosis evaluation, judgment of immunotherapy effect, and personalized treatment of lung cancer.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides the application of a uridine metabolic marker in the preparation of a product for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment in lung cancer patients, and the uridine metabolic marker is UPP1.

[0009] Preferably, the product includes reagents for detecting UPP1 and cell-specific markers, and the cell-specific markers include one or more of EPCAM, CD68, and CD66b.

[0010] More preferably, the product includes reagents for detecting UPP1 and cell-specific markers, and the cell-specific marker is EPCAM.

[0011] More preferably, the product includes reagents for detecting UPP1 and cell-specific markers, and the cell-specific marker is CD68.

[0012] More preferably, the product includes reagents for detecting UPP1 and cell-specific markers, and the cell-specific marker is CD66b.

[0013] More preferably, the product includes reagents for detecting the combination of UPP1, EPCAM, CD68, and CD66b.

[0014] The present invention also provides the application of reagents for detecting uridine metabolic markers in the preparation of a product for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment in lung cancer patients, and the uridine metabolic marker is UPP1.

[0015] Preferably, the product includes reagents for detecting UPP1 and cell-specific markers, and the cell-specific markers include one or more of EPCAM, CD68, and CD66b.

[0016] More preferably, the product includes reagents for detecting the combination of UPP1, EPCAM, CD68, and CD66b.

[0017] The present invention also provides a kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment in lung cancer patients, comprising UPP1 antibodies, EPCAM antibodies, CD68 antibodies and CD66b antibodies labeled with different fluorescent dyes.

[0018] Preferably, the kit further comprises a PBS buffer solution, a BSA blocking agent and a PBST washing solution.

[0019] Preferably, the kit is an immunohistochemical staining kit comprising UPP1 antibodies, EPCAM antibodies, CD68 antibodies and CD66b antibodies labeled with different fluorescent dyes.

[0020] More preferably, the UPP1 antibody is conjugated with the green fluorescent dye Alexa Fluor 488; the intensity of the green fluorescent signal generated reflects the level of uridine metabolic activity and is used to rapidly identify the distribution and expression of UPP1 in cells;

[0021] and / or the EPCAM antibody is conjugated with the red fluorescent dye Alexa Fluor 594; after staining, the tumor cells emit red fluorescent signals, which are used to distinguish tumor cells from other cell populations;

[0022] and / or the CD68 antibody is conjugated with the blue fluorescent dye Alexa Fluor 650; the generated blue fluorescent signal is used to distinguish macrophages from other cell populations and show the distribution of macrophages and their role in the tumor microenvironment;

[0023] and / or the CD66b antibody is conjugated with the yellow fluorescent dye Alexa Fluor 555; it can make neutrophils present yellow fluorescent signals, define the immune cell population, and facilitate the quantitative analysis of their role in the tumor microenvironment.

[0024] Compared with the prior art, the present invention provides a kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment in lung cancer patients, which has the following beneficial effects:

[0025] (1) Multiplex immunofluorescence labeling system: Multiplex immunofluorescence staining technology can simultaneously detect uridine metabolism markers in multiple cell types such as tumor cells, macrophages, and neutrophils in the tumor microenvironment by using different fluorescently labeled antibodies, providing a comprehensive analysis of the metabolic activities and immune responses in the tumor microenvironment. Compared with traditional single-labeling methods, multiplex labeling can better reflect the interactions between different cell types, thus improving the diagnostic accuracy and prognostic evaluation ability. In the present invention, four specific antibodies, UPP1, EPCAM, CD68, and CD66b, are labeled with four different fluorescent dyes, enabling the simultaneous detection of uridine metabolism markers in different cell populations and performing multiplex quantitative analysis on the same tissue section, efficiently and more accurately reflecting the metabolic activities and immune responses in the tumor microenvironment, and significantly improving the sensitivity and accuracy.

[0026] (2) Precise quantification ability: Through the analysis of the intensity of quantitative fluorescence signals, it is possible to accurately evaluate the changes in uridine metabolism activities in tumor cells and immune cells. Combining multiplex immunofluorescence labeling with quantitative image analysis provides strong support for the early diagnosis of lung cancer, the prognostic evaluation of patients, and the monitoring of the efficacy of immunotherapy.

[0027] (3) High sensitivity and high specificity: Each antibody has been conjugated with high-quality fluorescent dyes, having a high fluorescence signal intensity and low background noise, ensuring high-sensitivity and high-specificity analysis results.

[0028] (4) Simplified operation process: It is simply designed and the operation process is straightforward. The antibodies have been pre-labeled with fluorescent dyes, reducing the complexity of the staining steps. The staining effect is stable, saving time, and ensuring the consistency and repeatability of the results.

[0029] (5) Wide application prospects: This kit can be widely used in the analysis of metabolic activities of lung cancer and other types of cancers, and is particularly suitable for the research of the tumor immune microenvironment, the prognostic evaluation of patients, and the monitoring of the efficacy of immunotherapy.

[0030] (6) Strong applicability: This kit is applicable to tissue samples of lung cancer patients in clinical and research settings, and can effectively evaluate the uridine metabolism status of different cell populations in the tumor microenvironment, thereby inferring the biological characteristics of the tumor and the status of the immune microenvironment, providing support for the formulation of personalized treatment plans, early diagnosis, and the evaluation of the efficacy of immunotherapy. Brief Description of the Drawings

[0031] Figure 1 It is a schematic diagram of the detection principle and multiplex immunofluorescence labeling of the kit used in the examples for evaluating the uridine metabolism activity and prognosis of the tumor microenvironment of lung cancer patients.

[0032] Figure 2In the examples, the sensitivity and specificity ROC curves of UPP1, EPCAM, CD68, and CD66b as markers for the prognosis of lung cancer patients were verified by the pairwise combination of UPP1+CD68+, UPP1+CD66b+, and UPP1+EPCAM+.

[0033] Figure 3 It is a representative immunofluorescence picture in the examples. Detailed implementation manners

[0034] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below in conjunction with the accompanying drawings and specific examples. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. It should be noted that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all fall within the protection scope of the present invention.

[0035] The following examples provide a kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment of lung cancer patients, accurately evaluating the uridine metabolic activity in the tumor microenvironment of lung cancer patients and its relationship with prognosis through immunofluorescence staining technology. The composition includes four highly specific antibodies, which are respectively combined with four different fluorescent dyes for multiplex immunofluorescence labeling, specifically as follows:

[0036] (1) UPP1 antibody: An antibody against the key enzyme UPP1 in uridine metabolism, which can efficiently bind to UPP1 in tumor cells and immune cells, and is combined with the green fluorescent dye Alexa Fluor 488 to produce a bright green fluorescent signal. The signal intensity reflects the level of uridine metabolic activity, and the green fluorescent signal is clearly visible, facilitating the rapid identification of the distribution and expression of UPP1 in cells.

[0037] (2) EPCAM antibody: As a tumor cell marker, the EPCAM antibody can specifically recognize lung cancer cells. This antibody has been combined with the red fluorescent dye Alexa Fluor 594, and after staining, the tumor cells emit a significant red fluorescent signal, facilitating the distinction between tumor cells and other cell populations.

[0038] (3) CD68 antibody: Used to label macrophages in the tumor microenvironment. The CD68 antibody is combined with the blue fluorescent dye Alexa Fluor 650, which can accurately show the distribution of macrophages and their role in the tumor microenvironment. The blue fluorescent signal makes the distinction between macrophages and other cell populations more obvious.

[0039] (4) CD66b antibody: As a marker for neutrophils, the CD66b antibody can specifically recognize neutrophils and bind to the yellow fluorescent dye Alexa Fluor 555, causing neutrophils to exhibit a yellow fluorescent signal. This signal clearly demarcates the immune cell population, facilitating the quantitative analysis of their role in the tumor microenvironment.

[0040] The following examples present an immunofluorescence staining method for tissue sections or cell specimens of lung cancer patients, including the following steps:

[0041] (1) Sample preparation: Collect tissue sections or cell specimens from lung cancer patients. After being fixed with formalin, embedded in paraffin, and sectioned, they are used for immunofluorescence staining. The section thickness is 4 - 5 microns to ensure sufficient antibody penetration and signal intensity.

[0042] (2) Blocking: Use 5% BSA or 10% normal goat serum to block the sections, prevent non-specific binding, and reduce background signal interference.

[0043] (3) Antibody incubation: According to the kit instructions, incubate the four fluorescently labeled antibodies with the sample at room temperature for 1 hour. During the incubation process, the antibodies specifically bind to the target proteins (UPP1, EPCAM, CD68, CD66b) to ensure clear labeling of each cell population (tumor cells, macrophages, and neutrophils).

[0044] (4) Washing: Thoroughly wash the sample with PBS buffer to remove unbound antibodies and ensure clear and accurate final images.

[0045] (5) Microscopic observation: Observe the sample with a fluorescence microscope, set appropriate fluorescence channels, and observe the green (Alexa Fluor 488), red (Alexa Fluor 594), blue (Alexa Fluor 650), and yellow (Alexa Fluor 555) fluorescent signals respectively. Clearly distinguish the distribution of tumor cells, macrophages, and neutrophils through different color fluorescence channels, and simultaneously quantitatively analyze the uridine metabolic activity in these cell populations.

[0046] (6) Quantitative analysis and image processing: Through image analysis software, quantitatively analyze the immunofluorescence images to ensure quantitative measurement of the UPP1 expression level in different cell populations.

[0047] (7) Fluorescent signal intensity determination: Evaluate the expression level of UPP1 in cells through the fluorescent signal intensity. Stronger green fluorescence (Alexa Fluor 488) indicates high expression of UPP1, suggesting higher uridine metabolic activity.

[0048] (8)Cell population segmentation and classification: Use EPCAM, CD68, and CD66b antibodies to label and distinguish tumor cells, macrophages, and neutrophils. Analyze the expression of UPP1 in each cell population through the intensity and distribution of fluorescence signals, and then evaluate the uridine metabolic activity of each cell population.

[0049] (9)Result output: Output the fluorescence signal intensity data of each cell population, and conduct comprehensive analysis in combination with the clinical information (prognosis data) of the patient. The results help to provide a basis for the prognosis assessment and treatment plan formulation of lung cancer patients.

[0050] Example 1

[0051] This example presents a kit, which synthesizes or purchases four specific antibodies as follows:

[0052] UPP1 antibody, targeting the key enzyme UPP1 of uridine metabolism, purified and labeled with Alexa Fluor 488;

[0053] EPCAM antibody, targeting the tumor cell surface marker EPCAM, purified and labeled with Alexa Fluor594;

[0054] CD68 antibody, targeting the macrophage marker CD68, purified and labeled with Alexa Fluor 650;

[0055] CD66b antibody, targeting the neutrophil marker CD66b, purified and labeled with Alexa Fluor 555.

[0056] Fluorescent dye labeling: Use the standard fluorescent dye labeling method to bind each antibody to the corresponding fluorescent dye, and use a crosslinking agent (NHS ester) to ensure the stable binding of the fluorescent dye to the antibody.

[0057] Kit assembly: Package the labeled antibodies together with blocking agents, washing solutions, antibody diluents, etc. in the kit, including 4 fluorescently labeled antibodies, PBS buffer, BSA blocking agent, and PBST washing solution.

[0058] The operating procedure (SOP) of the kit is as follows:

[0059] Sample preparation: Select lung cancer patient tissue sections or tumor cells, fix, paraffin embed, and section them. The section thickness is 4 - 5 microns to ensure antibody permeability.

[0060] Antibody Incubation: Operate according to the kit instructions. First, block the tissue sections to prevent non-specific binding using a blocking agent. Then, incubate the diluted UPP1, EPCAM, CD68, and CD66b antibodies on the sections for 1 hour respectively to ensure specific binding of the antibodies to the target proteins. After incubation, wash with PBS buffer to remove unbound antibodies.

[0061] Microscopic Observation: Observe the samples under a fluorescence microscope and observe the fluorescence signals of each cell population under the corresponding fluorescence channels, and record the fluorescence signal intensities of different cell populations (tumor cells, macrophages, neutrophils).

[0062] Data Analysis: Use image analysis software to process the immunofluorescence images, quantify the intensities of green, red, blue, and yellow fluorescence signals, so as to evaluate the expression level of UPP1 in each cell type and further analyze the uridine metabolic activity in the tumor immune microenvironment. Finally, through the analysis of the intensity and distribution of fluorescence signals, comprehensively evaluate the metabolic state and immune response in the tumor microenvironment, providing a basis for the prognosis evaluation and treatment plan formulation of patients.

[0063] As Figure 1 shown, the four fluorescently labeled antibodies and their applications in tissue samples are as follows:

[0064] UPP1 Antibody: Conjugated with the green fluorescent dye Alexa Fluor 488, used to label the expression location of the uridine metabolism-related protein UPP1, and the green fluorescence signal reflects the expression intensity of UPP1 in the tissue.

[0065] EPCAM Antibody: Conjugated with the red fluorescent dye Alexa Fluor 594, used to specifically identify tumor cells, and the red fluorescence shows the distribution of tumor cells.

[0066] CD68 Antibody: Conjugated with the blue fluorescent dye Alexa Fluor 650, used to identify macrophages in the tumor microenvironment, and the blue fluorescence signal distinguishes macrophage populations;

[0067] CD66b Antibody: Conjugated with the yellow fluorescent dye Alexa Fluor 555, used to identify neutrophils, and the yellow fluorescence label shows the distribution and enrichment areas of neutrophils.

[0068] Figure 1The region of the fluorescence microscopy image shows the typical immunofluorescence images formed after the tumor tissue is stained with the above four antibodies. It is found that the expression characteristics of various cell populations and the metabolic enzyme UPP1 in the tumor microenvironment can be clearly distinguished; the multiple fluorescence labeling in the figure reflects the quantitative analysis of the uridine metabolic activity in the tumor microenvironment by jointly detecting the expression of UPP1 in tumor cells, macrophages, and neutrophils, providing technical support for the early diagnosis, prognosis evaluation, and immunotherapy monitoring of lung cancer patients.

[0069] To verify the sensitivity and specificity of UPP1, EPCAM, CD68, and CD66b as markers for judging the prognosis of lung cancer patients, the above kits were combined in pairs, namely UPP1+CD68+, UPP1+CD66b+, and UPP1+EPCAM+, and then the sensitivity and specificity of the prognosis of lung cancer patients were determined. The results are as Figure 2 shown.

[0070] In summary, the present invention combines the uridine metabolic marker UPP1 and markers of different cell types EPCAM, CD68, and CD66b, and uses the multiple immunofluorescence staining technique to comprehensively and accurately evaluate the state of metabolic activities and immune responses in the tumor microenvironment of lung cancer patients, providing a more accurate basis for early diagnosis, prognosis evaluation, and personalized treatment.

[0071] Representative immunofluorescence images of UPP1, EPCAM, CD68, and CD66b antibodies are as Figure 3 shown.

[0072] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a uridine metabolism biomarker in the preparation of a product for evaluating the uridine metabolism activity and prognosis of the tumor microenvironment in lung cancer patients, wherein the uridine metabolism biomarker is UPP1.

2. The use according to claim 1, wherein the product comprises reagents for detecting UPP1 and cell-specific markers; the cell-specific markers include one or more of EPCAM, CD68, and CD66b.

3. The use according to claim 2, wherein the product comprises reagents for detecting UPP1 and a cell-specific marker, and the cell-specific marker is EPCAM; or the product comprises reagents for detecting UPP1 and a cell-specific marker, and the cell-specific marker is CD68; or the product comprises reagents for detecting UPP1 and a cell-specific marker, and the cell-specific marker is CD66b; or the product comprises reagents for detecting the combination of UPP1, EPCAM, CD68, and CD66b.

4. Use of a reagent for detecting a uridine metabolism biomarker in the preparation of a product for evaluating the uridine metabolism activity and prognosis of the tumor microenvironment in lung cancer patients, wherein the uridine metabolism biomarker is UPP1.

5. The use according to claim 4, wherein the product comprises reagents for detecting UPP1 and cell-specific markers; the cell-specific markers include one or more of EPCAM, CD68, and CD66b.

6. The application according to claim 5, characterized in that, the product comprises reagents for detecting UPP1 and a cell-specific marker, and the cell-specific marker is EPCAM; or the product comprises reagents for detecting UPP1 and a cell-specific marker, and the cell-specific marker is CD68; or the product comprises reagents for detecting UPP1 and a cell-specific marker, and the cell-specific marker is CD66b; or the product comprises reagents for detecting the combination of UPP1, EPCAM, CD68, and CD66b.

7. A kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment in lung cancer patients, characterized in that, It includes UPP1 antibodies, EPCAM antibodies, CD68 antibodies, and CD66b antibodies with different fluorescent labels.

8. The kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment of lung cancer patients according to claim 7, characterized in that, The kit further comprises a PBS buffer, a BSA blocking agent, and a PBST washing solution.

9. The kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment of lung cancer patients according to claim 7, characterized in that The kit is an immunohistochemical staining kit comprising UPP1 antibodies, EPCAM antibodies, CD68 antibodies, and CD66b antibodies with different fluorescent labels.

10. The kit for evaluating the uridine metabolic activity and prognosis of the tumor microenvironment of lung cancer patients according to claim 7, characterized in that, The UPP1 antibody is conjugated with the green fluorescent dye Alexa Fluor 488; and / or the EPCAM antibody is conjugated with the red fluorescent dye Alexa Fluor 594; and / or the CD68 antibody is conjugated with the blue fluorescent dye Alexa Fluor 650; and / or the CD66b antibody is conjugated with the yellow fluorescent dye Alexa Fluor 555.

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