A combination of peripheral blood neutrophil phenotype markers for detecting patients with stress hormone type tumors, a kit and application thereof

Abstract

The application discloses a kind of detection pressure hormone type tumor patient's peripheral blood neutrophil phenotype marker combination, kit and application. Wherein, peripheral blood neutrophil phenotype marker combination is CD63+IL18R1, and reagent includes anti-human CD63 fluorescently labeled antibody and anti-human IL18R1 fluorescently labeled antibody.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostics, and in particular to a combination of peripheral blood neutrophil phenotypic markers, a kit, and their applications for detecting stress hormone-induced tumors. Background Technology

[0002] Recent studies have shown that cancer patients often experience changes in stress hormone levels, such as glucocorticoids, during disease progression, treatment, and psychophysiological stress. Stress hormones not only affect tumor cells themselves but also reshape the tumor immune microenvironment, particularly by influencing neutrophil activation, degranulation, and NET formation, thus promoting tumor metastasis and immune escape. Previous research has shown that chronic stress can alter neutrophil status and promote metastasis via the glucocorticoid pathway, suggesting that neutrophils are an important sensitive cell population to the effects of stress hormones.

[0003] Meanwhile, the understanding of tumor-associated neutrophil heterogeneity has been deepening both domestically and internationally. Recent reviews and studies generally agree that neutrophils are not a single homogeneous population, but rather exhibit significant plasticity in the tumor microenvironment, capable of forming different functional states such as pro-tumor, immunosuppressive, or anti-tumor; their phenotype and function are jointly regulated by tumor-derived signals, inflammatory factors, and stress-related hormones.

[0004] In existing technologies, the assessment of whether tumors are under the influence of stress hormones and the state of the tumor immune microenvironment mainly relies on tumor tissue samples, analyzed using methods such as immunohistochemistry, multiplex immunofluorescence, tissue flow cytometry, transcriptomics, or single-cell sequencing. While these methods can directly reflect the local state of the tumor, they typically suffer from drawbacks such as invasive sampling, difficulty in sample acquisition, complex testing procedures, high costs, and difficulty in dynamic and repeated monitoring. In contrast, peripheral blood samples are easy to obtain and more suitable for repeated sampling and continuous follow-up. Therefore, constructing alternative detection methods for the tumor microenvironment based on peripheral blood immune cell phenotypes has become an important research direction both domestically and internationally.

[0005] Against this backdrop, if a subset of neutrophils in peripheral blood can be identified that is sensitive to the effects of stress hormones and reflects changes in the tumor immune microenvironment, it may be possible to indirectly assess the "stress hormone-induced tumor immune microenvironment" without requiring tumor tissue resection. Based on published literature, existing research mainly focuses on the overall heterogeneity of tumor-associated neutrophils, the impact of stress hormones on neutrophil function, and the analysis of neutrophil subsets using high-dimensional omics. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies, such as reliance on tumor tissue, invasiveness, complexity, difficulty in dynamic monitoring, and lack of peripheral-specific indicators for the effects of stress hormones. It proposes a combination of phenotype markers for detecting peripheral blood neutrophils in patients with stress hormone-induced tumors, a kit, and its application.

[0007] According to a first aspect of the present invention, a reagent for detecting a combination of peripheral blood neutrophil phenotypic markers is used in the preparation of a clinical diagnostic product for identifying patients with stress hormone-induced tumors, wherein the combination of peripheral blood neutrophil phenotypic markers is CD63+IL18R1, and the reagent comprises an anti-human CD63 fluorescently labeled antibody and an anti-human IL18R1 fluorescently labeled antibody.

[0008] Preferably, the reagent further includes anti-human CD66B fluorescently labeled antibody and anti-human CD11B fluorescently labeled antibody.

[0009] Preferably, the reagents further include red blood cell lysis buffer, PBS buffer, and live / dead dye.

[0010] Preferably, the present invention uses erythrocyte lysis buffer to lyse the sample; the lysed sample is then stained with antibodies including anti-human CD63 fluorescently labeled antibody, anti-human IL18R1 fluorescently labeled antibody, anti-human CD66B fluorescently labeled antibody, anti-human CD11B fluorescently labeled antibody, and live / dead dyes; the antibody-stained sample is then placed in a flow cytometer for detection to obtain flow cytometry data including sample forward scatter (FSC), sample side scatter (SSC), and [other data]. By analyzing the flow cytometry data, it is determined whether the patient has a stress hormone-induced tumor.

[0011] Preferably, the analysis of the flow cytometry data in this invention includes: delineating a neutrophil population based on FSC and / or SSC; analyzing the proportion of CD63 and IL18R1 double-positive cells in the neutrophil population; when the proportion of CD63 and IL18R1 double-positive cells to all neutrophils in the neutrophil population is greater than a predetermined threshold, the patient is identified as a stress hormone-induced tumor patient; otherwise, the patient is identified as a non-stress hormone-induced tumor patient.

[0012] Preferably, the clinical diagnostic product is a reagent kit.

[0013] According to a second aspect of the present invention, a kit for detecting peripheral blood neutrophil phenotypic markers in patients with stress hormone-induced tumors, wherein the peripheral blood neutrophil phenotypic marker combination is CD63+IL18R1, and the kit comprises an anti-human CD63 fluorescently labeled antibody and an anti-human IL18R1 fluorescent label.

[0014] Preferably, the kit further comprises anti-human CD66B fluorescently labeled antibody and anti-human CD11B fluorescently labeled antibody.

[0015] Preferably, the kit further comprises erythrocyte lysis buffer, PBS buffer, and live / dead dye.

[0016] Preferably, the present invention uses erythrocyte lysis buffer to lyse the sample; the lysed sample is then stained with antibodies including anti-human CD63 fluorescently labeled antibody, anti-human IL18R1 fluorescently labeled antibody, anti-human CD66B fluorescently labeled antibody, anti-human CD11B fluorescently labeled antibody, and live / dead dyes; the antibody-stained sample is then placed in a flow cytometer for detection to obtain flow cytometry data including forward scattered light (FSC), side scattered light (SSC), and various fluorescence signals; the flow cytometry data is analyzed to determine whether the patient has stress hormone-induced tumors.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] Peripheral blood is used instead of tumor tissue as the test sample. No tumor tissue needs to be removed; only peripheral blood is required for testing, significantly reducing sampling trauma.

[0019] Neutrophil CD63 and IL18R1 double-positive cells were used as the core detection target. By identifying characteristic subsets of neutrophils sensitive to the effects of stress hormones, a peripheral alternative detection method for the influence of stress hormones within tumors was established.

[0020] The detection can be completed using conventional flow cytometry. Compared to single-cell sequencing or complex multi-omics methods, this invention is simpler, faster, and lower in cost, making it more conducive to standardization and clinical application.

[0021] Suitable for dynamic monitoring. Because it relies solely on peripheral blood, it is suitable for repeated testing at different time points and can be used for disease progression monitoring, efficacy observation, and risk stratification.

[0022] This invention improves the specificity and interpretability of test results. Compared to general inflammatory markers, it focuses on the specific biological state of the "stress hormone-induced tumor immune microenvironment," thus having a more clearly defined application. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the three-dimensional protein structure of the peripheral blood neutrophil surface dual markers IL18R1 (left image) and CD63 (right image) involved in this invention;

[0024] Figure 2This is a representative two-dimensional scatter plot and a representative schematic diagram of subpopulation gating for the dual-channel detection of IL18R1 and CD63 in peripheral blood neutrophils using flow cytometry, as described in this invention.

[0025] Figure 3 This invention presents a comparative statistical analysis and ROC diagnostic efficacy curve of the ratio of IL18R1 and CD63 double-positive neutrophil subsets and peripheral plasma cortisol concentration in predicting glucocorticoid-induced tumor microenvironment (TME-Steroid). Detailed Implementation

[0026] Figure 1 The diagram shows the three-dimensional spatial structure of the combined detection targets of this invention—IL18R1 (also known as IL18Rα) protein and CD63 protein—obtained from the PDB database.

[0027] Structural biological characteristics indicate that both proteins contain extracellular domains exposed on the outer side of the cell. This provides a definite material basis for the present invention to use specific antibodies labeled with different fluorescent dyes (such as PE-Cy7 and APC) to directly perform in situ dual recognition of these two targets on the surface of living cells by flow cytometry without cell perforation.

[0028] This invention aims to provide a peripheral immunoassay kit and method for detecting the stress hormone-induced immune microenvironment in tumors. By detecting CD63 and IL18R1 double-positive cells in peripheral blood neutrophils of cancer patients, it serves as a peripheral surrogate indicator reflecting the state of tumor-associated immune microenvironment influenced by stress hormones. This allows for indirect assessment of the tumor-associated immune microenvironment without requiring tumor tissue biopsy. This invention primarily addresses the problems of existing technologies, such as over-reliance on tumor tissue samples, invasive testing, complex procedures, difficulty in dynamic monitoring, and the lack of specific peripheral biomarkers for the stress hormone-induced tumor immune microenvironment. This invention can be applied to the auxiliary assessment of the stress hormone-associated immune microenvironment in cancer patients, peripheral surrogate detection, dynamic follow-up, efficacy monitoring, risk stratification, and tumor immunology research.

[0029] This invention uses flow cytometry to detect the phenotype of neutrophils in the peripheral blood of subjects. By identifying CD63 and IL18R1 double-positive cells in the neutrophil population and detecting their proportion, it serves as a peripheral surrogate indicator for assessing the state of the immune microenvironment of stress hormone-induced tumors.

[0030] In a preferred embodiment, the method includes: collecting peripheral venous blood from the subject, performing erythrocyte lysis, antibody staining, flow cytometry detection, and gating analysis to obtain the proportion of CD63 and IL18R1 double-positive cells in neutrophils. If necessary, the mean fluorescence intensity (MFI) of CD63 and / or IL18R1 in the double-positive population can be further detected to enhance the quantitative analysis capability.

[0031] Figure 2 This invention demonstrates the first-ever identification of a novel neutrophil subset (IL18R1) with microenvironmental indicative function in clinical peripheral blood samples. + CD63 + (Double positive subgroup).

[0032] In the specific detection process, the first step is to use the specific surface marker CD11B. + CD66B + The total neutrophil population in peripheral blood was delineated; then, flow cytometry was used to detect dual-channel fluorescence signals representing the target: the horizontal axis (X-axis) represents the fluorescence channel (PE-Cy7) for detecting IL18R1 expression level, and the vertical axis (Y-axis) represents the fluorescence channel (APC) for detecting CD63 expression level.

[0033] The inventors, through flow cytometry scatter plot (pseudo-color density map) analysis, discovered a distinct independent cell population drifting towards the upper right quadrant in the peripheral blood of patients acclimated to a specific tumor microenvironment. Based on this, the present invention establishes a specific polygon gate (such as...). Figure 2 (As shown in the black solid box in the middle), it is specifically used to delineate and statistically analyze cell populations that simultaneously highly express IL18R1 and CD63 (i.e., double-positive subsets). Figure 2 The comparison of representative samples on the left and right shows that the proportion of this double-positive subgroup varies greatly among different patients (the proportion in the left figure is only 3.37%, while the proportion in the right figure is as high as 30.2%). This huge difference between groups suggests that this specific subgroup has the potential to serve as an excellent clinical classification marker.

[0034] The present invention can be performed according to the order of experimental operations:

[0035] Step 1: Peripheral blood sample collection

[0036] Peripheral venous blood was collected from the subjects, preferably in EDTA anticoagulant tubes. The preferred blood volume for each subject was 2–5 mL. After collection, the samples were stored at room temperature and, preferably, tested within 4 hours.

[0037] Step 2: Sample Dispensing

[0038] Take 1 ml of anticoagulated whole blood and add it to a flow cytometry tube as a single test sample.

[0039] Step 3: Red blood cell lysis

[0040] Add 15 mL of red blood cell lysis buffer to the sample and lyse at room temperature in the dark for 10 min to remove red blood cell interference.

[0041] Step 4: Antibody staining

[0042] Fluorescently labeled antibodies CD66B and CD11B for recognizing neutrophils, fluorescently labeled antibodies for detecting CD63 and IL18R1, and inactivated / deactivated dyes were added to the whole blood sample. After adding all antibodies, the mixture was thoroughly mixed and incubated at room temperature in the dark for 15 minutes.

[0043] Step 5: Washing and Resuspension

[0044] After lysis, centrifuge at 300–500 g for 3–5 min and discard the supernatant; wash 1–2 times with PBS or flow cytometry staining buffer, and finally resuspend the cells in 200 μL buffer.

[0045] Step Six: Flow Cyclic Testing

[0046] The processed samples were placed in a flow cytometer for analysis, and forward scattered light (FSC), side scattered light (SSC), and signals from each fluorescence channel were collected. Ideally, no fewer than 10,000 cellular events should be collected from each sample.

[0047] Step 7: Gating Analysis

[0048] Perform streaming data analysis in the following order:

[0049] 1. Identify the major cell populations based on FSC / SSC (this is existing technology and will not be elaborated further).

[0050] 2. Exclude double-celled cells based on FSC-H / FSC-A or SSC-H / SSC-A (this is existing technology and will not be elaborated further);

[0051] 3. If necessary, remove dead cells based on the presence or absence of the dye (this is existing technology and will not be elaborated further).

[0052] 4. Combined with the neutrophil marker CD11B + CD66B + Delineate the neutrophil population;

[0053] 5. Analyze the proportion of CD63 and IL18R1 double-positive cells in the neutrophil population;

[0054] 6. If necessary, further read the mean fluorescence intensity (MFI) of CD63 and IL18R1.

[0055] Step 8: Result Interpretation

[0056] The proportion of CD63 and IL18R1 double-positive cells in the neutrophil population is compared with a preset threshold (e.g., 10%). If it is greater than the preset threshold, the patient is determined to be a patient with stress hormone-induced tumors; otherwise, the patient is determined not to be a patient with stress hormone-induced tumors.

[0057] Control conditions of test operation

[0058] To ensure the stability and repeatability of the test results, the following test conditions are preferred:

[0059] --Sample processing time control

[0060] Peripheral blood samples should ideally be tested within 2 to 4 hours after collection to avoid decreased cell activity or phenotypic drift due to prolonged storage.

[0061] --Anticoagulant conditions control

[0062] EDTA anticoagulant tubes are preferred for blood collection to avoid processing conditions that may affect cell activation.

[0063] --Staining conditions control

[0064] Antibody staining is preferably performed at room temperature and in the dark, with consistent incubation time to reduce nonspecific fluctuations.

[0065] --Fracturing conditions control

[0066] The type of erythrocyte lysis buffer, lysis time, and lysis volume should be kept consistent to prevent insufficient or excessive lysis from affecting cell population recognition.

[0067] --Flow Cytometry Parameter Control

[0068] The voltage, compensation settings, threshold, and number of acquisition events of the flow cytometer should be kept consistent; uniform instrument parameters should be used in the same batch of tests.

[0069] --Gating strategy control

[0070] A fixed gating strategy is used to analyze all samples to ensure that the results are comparable between different samples.

[0071] --Reference settings

[0072] Set up blank control, monostain control, and isotype control to correct background signals and help determine the double-positive detection area.

[0073] --Subject status control prioritizes excluding factors that may significantly affect neutrophil phenotype, such as recent severe infection, acute inflammation, hematological diseases, and recent high-dose hormone therapy.

[0074] Experimental results:

[0075] The inventors used the above method to test healthy control samples and tumor patient samples, and the results showed that:

[0076] The method of this invention can stably identify peripheral blood neutrophil populations and clearly detect CD63 and IL18R1 double-positive cells.

[0077] Compared with the healthy control group, the proportion of CD63 and IL18R1 double-positive neutrophils in the peripheral blood of cancer patients was significantly increased.

[0078] Under elevated levels of stress hormones or under conditions simulating stress hormone stimulation, the proportion of CD63 and IL18R1 double-positive cells in neutrophils further increases.

[0079] The increase in the proportion of double-positive cells is consistent with the trend of changes in tumor-associated immunosuppression, suggesting that this indicator can reflect changes in the immune microenvironment within the tumor after being affected by stress hormones.

[0080] Under repeated testing conditions, the proportion of double-positive cells obtained from the same sample fluctuated little, indicating that the method of the present invention has good stability and repeatability.

[0081] This invention requires only a peripheral blood sample to complete the detection, without the need to cut tumor tissue, and the detection process is simple and suitable for dynamic follow-up.

[0082] Based on the above experimental results, the following conclusions can be drawn:

[0083] CD63 and IL18R1 double-positive cells in peripheral blood neutrophils can be considered as a characteristic cell subpopulation that can reflect the state of stress hormones.

[0084] The ratio of CD63 and IL18R1 double-positive cells among neutrophils to all neutrophils can serve as a peripheral alternative indicator for assessing the state of the immune microenvironment in stress hormone-induced tumors.

[0085] The detection method described in this invention does not require obtaining tumor tissue; it can indirectly assess whether there are changes in the stress hormone-related immune microenvironment within the tumor simply by peripheral blood flow cytometry.

[0086] This invention has the advantages of simple detection, minimal invasiveness, quantifiable results, good repeatability, and suitability for dynamic monitoring. It is suitable for auxiliary detection, follow-up monitoring, and related research applications for cancer patients.

[0087] Figure 3 The paper presents a comparative statistical analysis and ROC diagnostic efficacy curve of the proportion of IL18R1 and CD63 double-positive neutrophil subsets and peripheral plasma cortisol concentration in predicting glucocorticoid-induced tumor microenvironment (TME-Steroid).

[0088] To verify the clinical diagnostic value of the newly identified "IL18R1_CD63 double-positive neutrophil subset" in predicting "glucocorticoid-induced tumor microenvironment (TME-Steroid)," the inventors conducted rigorous statistical analysis and compared it with existing conventional clinical blood biochemical indicators (cortisol) (results are shown below). Figure 3 (As shown).

[0089] like Figure 3 As shown in the left-hand box plot (difference analysis), the inventors statistically analyzed the level / concentration of this double-positive cell subset in total neutrophils and compared its differences in peripheral blood between the "Other" and "TME-Steroid" groups. The results showed a highly statistically significant difference between the two groups (p = 1.5480e-03). The data distribution indicates that the proportion of the double-positive subset was extremely low in the "Other" group (boxes concentrated below 5%), while the proportion was significantly enriched and abnormally high in the TME-Steroid group (boxes mainly concentrated between 15% and 25%). Based on this highly significant discriminative power, this invention establishes a clinically feasible cut-off value: the cut-off percentage of the double-positive cell subset can be set at approximately 10%. When the percentage exceeds this cut-off, it can be accurately determined that the patient's solid tumor is in a TME-Steroid microenvironment.

[0090] like Figure 3The right side (ROC diagnostic efficacy comparison curve) shows: To intuitively quantify and compare the diagnostic superiority of the method of this invention with existing technologies, the inventors plotted the receiver operating characteristic (ROC) curve. In existing routine clinical tests, the direct measurement of free cortisol concentration in peripheral blood is usually relied upon to indirectly assess hormone levels. However, because free hormones in the blood are easily affected by non-tumor physiological fluctuations, their predictive efficacy for the microenvironment is very limited (as shown by the blue dashed line in the figure, the AUC is only 0.77, and the specificity is poor). In contrast, the combined detection method of the IL18R1_CD63 dual biomarkers described in this invention (as shown by the red solid line in the figure) achieves a significant improvement in the area under the ROC curve (AUC), reaching 0.94. The curve exhibits a perfect upward curve in the upper left corner, indicating that the detection method possesses both extremely high sensitivity and specificity (i.e., an extremely low false positive rate).

[0091] The rigorous comparative data above fully demonstrate that the IL18R1 / CD63 double-positive neutrophil subset identified in this invention, as a functional cell readout indicator deeply "acclimated" to the microenvironment, perfectly overcomes the significant technical deficiency of traditional free biochemical markers, which are prone to fluctuation. This invention not only fully retains the non-invasive convenience of liquid biopsy but also achieves an unexpectedly high predictive accuracy (AUC=0.94), unmatched by existing technologies, providing an extremely reliable new diagnostic tool for developing personalized targeted / immunotherapy strategies in clinical practice.

[0092] Compared with existing technologies that rely on whole-transcriptome sequencing (RNA-seq) of solid tissues and blood-based biochemical assays (such as free cortisol measurement), this invention achieves the following significant advantages:

[0093] 1. Identification of a Novel Double-Positive Cell Subpopulation, Significantly Enhancing Detection Specificity: This invention is the first in the world to identify a novel neutrophil subpopulation that is "double-positive for IL18R1 and CD63". Through a dual-marker combined detection strategy, it effectively filters out "false positive" signals caused by non-specific systemic inflammation that can easily interfere with single markers, achieving more precise and highly specific targeted characterization of the solid tumor microenvironment at the level of non-invasive liquid biopsy.

[0094] 2. Targeting a stable "functional readout" indicator, achieving a qualitative leap in prediction accuracy. Traditional free cortisol detection is highly susceptible to physiological and emotional fluctuations. This invention uses specific receptors (IL18R1 / CD63) on the surface of neutrophils as the detection target, whose expression levels reflect the stable cumulative response of cells to long-term exposure to microenvironment signals. Comparative data show that this method effectively eliminates transient physiological interference, significantly improving the microenvironment prediction accuracy (AUC) from 0.77 in traditional methods to 0.94, achieving unexpected technical results.

[0095] 3. Non-invasive liquid biopsy overcomes the limitations of tissue sampling and enables dynamic monitoring throughout the entire treatment process, breaking through the bottleneck of existing RNA-seq detection which is highly dependent on invasive solid tissue biopsies. This method only requires drawing a small amount of peripheral blood to complete non-invasive assessment, which not only significantly reduces patient discomfort but also allows for high-frequency continuous blood sampling throughout the entire treatment cycle, including initial diagnosis, medication, and recurrence, enabling real-time dynamic monitoring of the tumor microenvironment evolution.

[0096] 4. Significantly reduced costs and increased efficiency, with a greatly shortened testing cycle and high clinical applicability. This invention eliminates the complex sample cold chain, nucleic acid extraction, sequencing, and bioinformatics analysis processes of RNA-seq. Direct detection using conventional flow cytometry not only significantly reduces the cost per test but also shortens the report generation time from the traditional 1-2 weeks to within hours (achieving same-day results), greatly facilitating the large-scale promotion and popularization of this testing technology in primary healthcare institutions at all levels.

[0097] The reagent kit of the present invention has the following structural components:

[0098] This invention provides a peripheral detection kit for detecting the immune microenvironment of stress hormone-induced tumors. The kit is a multi-component combined detection reagent system, comprising the following components:

[0099] -- Neutrophil recognition antibody components, used to recognize peripheral blood neutrophils, including anti-CD66B antibody and CD11B antibody;

[0100] --CD63 and IL18R1 detection antibody components are used to detect fluorescently labeled antibodies for stress-responsive neutrophil-associated states.

[0101] --Red blood cell lysis buffer components are used to remove red blood cells from peripheral blood samples to ensure the accuracy of flow cytometry analysis.

[0102] --Sample processing buffer components used for cell washing, resuspending, and maintaining cell stability.

[0103] --Instructions and interpretation standards are provided to guide the testing procedures and result interpretation.

[0104] The components of the reagent kit of the present invention are preferably packaged in individual containers and functionally combined according to the detection process:

[0105] • Antibody components (CD63, IL18R1, neutrophil markers) form the core detection module;

[0106] • Red blood cell lysis buffer and buffer solution constitute the sample processing module;

[0107] • The control group was used for quality control and result correction;

[0108] There is no mechanical connection between the components, but they form a synergistic relationship through the detection process to jointly complete the detection of specific phenotypes of peripheral blood neutrophils.

[0109] The working principle of this invention is as follows:

[0110] The activation state and expression of inflammatory receptors of neutrophils in peripheral blood change under the influence of stress hormones;

[0111] CD63 can reflect the degranulation and activation status of neutrophils;

[0112] IL18R1 is involved in inflammatory signal transduction and reflects the state of the immune response;

[0113] Under the influence of stress hormones, some neutrophils simultaneously exhibit a double-positive phenotype of CD63 and IL18R1;

[0114] The change in the proportion of this double-positive cell population can serve as a peripheral alternative indicator of the effects of intratumoral stress hormones and related changes in the immune microenvironment.

[0115] By detecting this cell population using flow cytometry, an indirect assessment of the state of the tumor immune microenvironment can be achieved.

[0116] The working process of the reagent kit of this invention is as follows:

[0117] --Sample collection, used to collect peripheral blood samples from subjects;

[0118] --Red blood cell lysis is used to remove red blood cell interference and preserve the white blood cell population;

[0119] --Antibody labeling, used to incubate samples with neutrophil recognition antibodies, CD63 antibodies, and IL18R1 antibodies;

[0120] --Flow cytometry is used to collect cell scattering and fluorescence signals using a flow cytometer;

[0121] --Gated analysis, used to sequentially delineate granulocytes → neutrophils → CD63+ IL18R1 + cell;

[0122] --Result interpretation is used to calculate the proportion of double-positive cells and compare them with the control or threshold to determine whether a stress hormone-induced tumor immune microenvironment exists.

[0123] Through the above structure and working process, the present invention achieves:

[0124] • No tumor tissue needs to be obtained; the test is completed using only peripheral blood.

[0125] • Specific phenotypic identification can be achieved using a small number of biomarkers (CD63 + IL18R1);

[0126] • Detection can be performed on a conventional flow cytometry platform, and the operation is simple;

[0127] • It can perform repeated testing at multiple time points to achieve dynamic monitoring;

[0128] • Provide a standardized and scalable peripheral detection solution.

[0129] The research results of this invention have the following characteristics: First, no tumor tissue needs to be excised; detection can be completed solely through peripheral blood. Second, the detection target is clearly defined: CD63 and IL18R1 double-positive cells in peripheral blood neutrophils. Third, the detection indicators are quantifiable, characterized by the proportion of double-positive cells and / or related average fluorescence intensity. Fourth, it is sensitive to the effects of stress hormones, reflecting the state of the immune microenvironment within the tumor after being affected by stress hormones. Fifth, the detection method is simple, with good repeatability and dynamic monitoring value. The above characteristics are preferably detected using flow cytometry. The simplified steps include peripheral blood collection, antibody staining, erythrocyte lysis, washing and resuspending, flow cytometry, gating analysis, and result interpretation. The detection results show that the proportion of CD63 and IL18R1 double-positive cells in the peripheral blood of tumor patients is significantly higher than that of healthy controls, and further increases under stress hormone stimulation, suggesting that it can serve as a peripheral indicator reflecting the state of the immune microenvironment in stress hormone-induced tumors.

[0130] Example 1: Establishment of a flow cytometry method for detecting CD63 and IL18R1 double-positive cells in peripheral blood neutrophils.

[0131] 1. Experimental Objective

[0132] A flow cytometry method based on peripheral blood samples was established to stably identify neutrophils and detect the proportion of CD63 and IL18R1 double-positive cells on their surface.

[0133] 2. Experimental Materials

[0134] 2.1 Test Sample

[0135] Two mL of peripheral venous blood was collected from the subject using EDTA anticoagulant tubes, and the samples were tested within 4 hours after collection.

[0136] The main reagents include:

[0137] Anti-human CD66B fluorescently labeled antibody, manufacturer: Elabscience, catalog number: Cat# E-AB-F1267C, working volume: 1 μL / 100 μL test;

[0138] Anti-human CD11B fluorescently labeled antibody, manufacturer: BD Biosciences, catalog number: Cat# 557754, working volume: 1 μL / 100 μL test;

[0139] Anti-human CD63 fluorescently labeled antibody, manufacturer: BioLegend, catalog number: 353007, working volume: 1 μL / 100μL test;

[0140] Anti-human IL18R1 fluorescently labeled antibody, manufacturer: BioLegend, catalog number: 313812, working volume: 1 μL / 100 μL test;

[0141] Red blood cell lysis buffer, manufacturer: Solarbio, product number: Cat# R1010;

[0142] PBS buffer, manufacturer: Vivacell;

[0143] Dead or live dye, manufacturer: BioLegend, item number: 423101.

[0144] The detection equipment includes a flow cytometer: Cytek Biosciences Full Spectrum Flow Cytometer.

[0145] Experimental Methods: 100 μL of lysed whole blood was placed in an EP tube, antibody mixture was added, and the mixture was incubated at room temperature in the dark for 15 min. After incubation, 1 mL of flow cytometry buffer was added. The sample was then centrifuged at 500 g for 5 min, the supernatant was discarded, and the sample was washed twice with 1 mL of PBS. Finally, the sample was resuspended in 100 μL of staining buffer. The sample was then analyzed by flow cytometry, and at least 10,000 cellular events were collected.

[0146] Gating strategy: First, debris is excluded based on FSC-A / SSC-A, then double TS cells are excluded based on FSC-H / FSC-A. After excluding dead cells, the granulocyte population is delineated based on the scatter plot, and the neutrophil population is determined by combining the positive signals of CD66B and CD11B. Finally, the proportion of CD63 and IL18R1 double positive cells in the neutrophil population is read, and the mean fluorescence intensity (MFI) of CD63 and IL18R1 can be further analyzed.

[0147] Experimental results: The results showed that this method can stably identify the peripheral blood neutrophil population. Neutrophils accounted for 60% ± 10% of the total number of cells detected. The background signal of CD63 and IL18R1 double positive cells was low and the clusters were clear. The gating results of repeated detections of the same sample were consistent.

[0148] This embodiment establishes a flow cytometry method for detecting CD63 and IL18R1 double-positive neutrophils in peripheral blood, which can stably detect the proportion of double-positive cells, providing a basis for subsequent applications.

[0149] This invention provides a peripheral detection kit for detecting the immune microenvironment of stress hormone-induced tumors, which has significant advantages and beneficial effects compared with the prior art.

[0150] 1. This invention uses peripheral blood as the test sample, which can assess the status of the tumor-related immune microenvironment without tumor puncture, surgical excision or other invasive tissue sampling. This significantly reduces the trauma of testing and improves patient compliance. It is especially suitable for patients who have difficulty obtaining tumor tissue samples or who need to undergo repeated testing.

[0151] 2. This invention uses CD63 and IL18R1 double-positive cells in peripheral blood neutrophils as the core detection target, with clearly defined detection indicators and quantifiable results. The proportion of these double-positive cells can be directly obtained through conventional flow cytometry, offering good intuitiveness, comparability, and reproducibility, facilitating comparative analysis between different time points and different subjects. Compared with traditional inflammatory markers or general immune cell proportions, the detection indicators used in this invention more specifically reflect the impact of stress hormones on tumor-related immune status, thus possessing stronger biological relevance and application value.

[0152] 3. This invention can be performed using a conventional flow cytometry platform, without relying on single-cell sequencing, multi-omics analysis, or complex data processing workflows. Therefore, it offers advantages such as ease of operation, rapid detection, relatively low cost, and ease of standardization, making it more suitable for widespread application in routine clinical laboratories. Furthermore, this invention indirectly reflects the state of the immune microenvironment within tumors after exposure to stress hormones through changes in the phenotype of peripheral blood neutrophils, thus overcoming the dependence of existing technologies on tumor tissue detection and providing a non-invasive alternative peripheral detection method.

[0153] 4. Since this invention only requires the collection of peripheral blood samples, it can be repeatedly tested at different stages of the patient's disease progression, before and after treatment, and changes in stress status, providing excellent dynamic monitoring capabilities. It can be used for continuous follow-up, efficacy observation, and disease assessment. Furthermore, this invention employs a standardized testing procedure, minimizing the impact of operating conditions on the test results, exhibiting good stability and repeatability, and facilitating the establishment of a standardized testing system.

[0154] 5. The CD63 and IL18R1 double-positive neutrophil phenotype detected by this invention helps identify patient groups with stress hormone-related immune abnormalities, providing a reference for patient stratification, risk assessment, and individualized intervention. Simultaneously, this invention also provides a simple and reproducible detection tool for studying the relationship between stress hormones and the tumor immune microenvironment, possessing significant research value and clinical translational potential.

[0155] In summary, this invention establishes a detection scheme for assessing the immune microenvironment of stress hormone-induced tumors without the need for tumor tissue by detecting CD63 and IL18R1 double-positive cells in peripheral blood neutrophils. It has the advantages of being minimally invasive, simple to operate, quantifiable results, highly targeted, reproducible, and suitable for dynamic monitoring, and has good application and promotion value.

[0156] Although the present invention has been described in detail above, it is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. The use of a reagent for detecting a combination of peripheral blood neutrophil phenotypic markers in the preparation of a clinical diagnostic product for identifying patients with stress hormone-induced tumors, characterized in that, The peripheral blood neutrophil phenotypic marker combination is CD63+IL18R1, and the reagents include anti-human CD63 fluorescently labeled antibody and anti-human IL18R1 fluorescently labeled antibody.

2. The application according to claim 1, characterized in that, The reagents also include anti-human CD66B fluorescently labeled antibody and anti-human CD11B fluorescently labeled antibody.

3. The application according to claim 2, characterized in that, The reagents also include red blood cell lysis buffer (PBS) and live / dead dyes.

4. The application according to claim 2, characterized in that, The samples were lysed using erythrocyte lysis buffer; the lysed samples were then stained with antibodies against human CD63, human IL18R1, human CD66B, and human CD11B, along with live and dead dyes; the antibody-stained samples were then analyzed by flow cytometry to obtain flow cytometry data, including forward scattered light (FSC) and side scattered light (SSC); the flow cytometry data were analyzed to determine whether the patient had stress hormone-induced tumors.

5. The application according to claim 4, characterized in that, Analysis of the flow cytometry data includes: delineating the major cell populations based on FSC and SSC; and analyzing the neutrophil surface marker CD66B based on the delineated cell populations. + CD11B + The expression status of CD63 and IL18R1 was used to delineate the neutrophil population, and the proportion of CD63 and IL18R1 double-positive cells in the neutrophil population was analyzed. When the proportion of CD63 and IL18R1 double-positive cells to all neutrophils in the neutrophil population was greater than a predetermined threshold, the patient was identified as a stress hormone-induced tumor patient; otherwise, the patient was identified as a non-stress hormone-induced tumor patient.

6. The application according to any one of claims 1-5, characterized in that, The clinical diagnostic product is a reagent kit.

7. A kit for detecting peripheral blood neutrophil phenotypic markers in patients with stress hormone-induced tumors, characterized in that, The peripheral blood neutrophil phenotypic marker combination is CD63+IL18R1, and the kit contains anti-human CD63 fluorescently labeled antibody and anti-human IL18R1 fluorescent label.

8. The reagent kit according to claim 7, characterized in that, The kit also contains fluorescently labeled antibodies against human CD66B and human CD11B.

9. The reagent kit according to claim 7, characterized in that, The kit also contains red blood cell lysis buffer, PBS buffer, and live / dead dye.

10. The reagent kit according to claim 9, characterized in that, The samples were lysed using erythrocyte lysis buffer; the lysed samples were then stained with antibodies against human CD63, human IL18R1, human CD66B, and human CD11B, along with live and dead dyes; the antibody-stained samples were then analyzed by flow cytometry to obtain flow cytometry data, including forward scattered light (FSC), side scattered light (SSC), and various fluorescence signals; the flow cytometry data were analyzed to determine whether the patient had stress hormone-induced tumors.

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