Application of CXCR2-CLEC4N + neutrophil in product for detecting immunotherapy curative effect of bladder urinary tract epithelial cancer

Abstract

The invention discloses an application of CXCR2-CLEC4N + neutrophil as a marker in a product for detecting the immunotherapy curative effect of bladder urinary tract epithelial carcinoma. The product comprises a reagent or a medicine for detecting the infiltration degree of the CXCR2-CLEC4N + neutrophil in the immunotherapy curative effect of the bladder urinary tract epithelium cancer. The relation between neutrophil cells and immunotherapy is screened through an scRNA-seq technology, CXCR2-CLEC4N + granulocytes are used as the biomarker for predicting the immunotherapy effect of bladder urothelium carcinoma, and the marker can improve the accuracy of selecting patients suitable for ICBs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a CXCR2 - CLEC4N + Application of neutrophils as a biomarker in products for detecting the efficacy of immunotherapy for bladder urothelial carcinoma. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, with urothelial carcinoma of the bladder (UCB) being the most prevalent pathological type. Approximately 70% of bladder cancer patients have non-muscle-invasive bladder cancer (NMIBC), while 30% have muscle-invasive bladder cancer (MIBC). In recent years, immune checkpoint inhibitors (ICBs) have shown some efficacy in the treatment of advanced metastatic urothelial carcinoma of the bladder. However, the application of ICBs in clinical practice still faces several challenges: on the one hand, the objective response rate of ICBs in patients with advanced bladder cancer is only 14.8%-21%, indicating that approximately 80% of patients have primary resistance to ICB treatment; on the other hand, some patients initially respond well to ICBs, but acquire resistance still occurs as treatment progresses. Therefore, in-depth research into the primary and acquired resistance mechanisms to immunotherapy is of great clinical significance for improving the efficacy of immunotherapy for bladder cancer.

[0003] Recent studies have shown that neutrophils play multiple roles in tumor development, progression, and metastasis: on the one hand, neutrophils can kill tumor cells through various mechanisms; on the other hand, tumor cells can also promote tumor immune escape and generate immunotherapy resistance by dissociating cytokines or interacting with other cells in the tumor microenvironment (TME). Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, the main objective of this invention is to provide a CXCR2... - CLEC4N + Neutrophils are used as biomarkers to detect the efficacy of immunotherapy for bladder urothelial carcinoma.

[0005] The objective of this invention is achieved through the following technical solution: CXCR2 - CLEC4N+ Application of neutrophils as a biomarker in products for detecting the efficacy of immunotherapy for bladder urothelial carcinoma.

[0006] In some specific embodiments, the product used to detect the efficacy of immunotherapy for bladder urothelial carcinoma includes CXCR2 as a marker of the efficacy of immunotherapy for bladder urothelial carcinoma. - CLEC4N + Reagents or drugs for assessing the degree of neutrophil infiltration.

[0007] The CXCR2 - CLEC4N + The degree of neutrophil infiltration can be calculated using multiplex immunofluorescence. In some specific embodiments, the CXCR2 - CLEC4N + Application of neutrophils in the preparation of products for the prevention or treatment of bladder urothelial carcinoma.

[0008] In some specific embodiments, the CXCR2 - CLEC4N + The application of neutrophils in combination with ICBs in the preparation of products for the prevention or treatment of bladder urothelial carcinoma can enhance the therapeutic effect against bladder urothelial carcinoma.

[0009] Regarding the definition: The term "detection" is used in its broadest sense herein to include both qualitative and quantitative measurements of target molecules. Detection includes simply identifying the presence of a target molecule in a sample as well as determining whether the target molecule is present in the sample at a detectable level.

[0010] As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, such as predictive, diagnostic, and / or prognostic indicators. Biomarkers can serve as indicators of a specific subtype of disease or condition (e.g., cancer) characterized by certain molecular, pathological, histological, and / or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide copy number alterations (e.g., DNA copy number), peptides, peptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipid-based molecular markers.

[0011] As used herein, the term “sample” refers to a composition obtained or derived from a subject and / or individual of interest that contains cells and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” or variations thereof refers to any sample obtained from a subject of interest that is expected to or is known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, and tissue culture fluids, tissue extracts such as homogenized tissue, cell extracts, and combinations thereof.

[0012] As used herein, “treatment” refers to a clinical intervention that attempts to alter the natural course of the disease in the individual being treated, and may be implemented during the course of clinicopathological development. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and eliminating or improving prognosis.

[0013] Predicting prognosis refers to predicting the process or outcome of a patient's condition, but does not mean that the process or outcome can be predicted with 100% accuracy. Predicting prognosis means determining whether the likelihood of certain processes or outcomes has increased, not determining the likelihood of certain processes or outcomes occurring by comparing them to a situation where they do not occur.

[0014] Compared with the prior art, the present invention has at least the following advantages: The application disclosed in this invention uses scRNA-seq technology to screen the relationship between neutrophil cells and immunotherapy, and discloses CXCR2. - CLEC4N + Neutrophils serve as a biomarker to predict the efficacy of immunotherapy for bladder urothelial carcinoma, and this biomarker can improve the accuracy of selecting suitable patients for ICBs. Attached Figure Description

[0015] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0016] Figure 1This study demonstrates the heterogeneity of neutrophil subsets in mouse bladder cancer BD single-cell sequencing and the relationship between different subsets and anti-PD-1 therapy in mouse bladder cancer. Figure A presents a dimensionality reduction plot of neutrophil subsets in mouse bladder cancer single-cell sequencing analysis; Figure B uses maturity scores to determine the maturity of different neutrophil subsets; Figure C preliminarily classifies neutrophil subsets into three groups based on surface markers; Figure D verifies the sorting of mouse bladder cancer granulocytes using flow cytometry; and Figure E observes the relationship between different neutrophil subsets and immunotherapy by adoptive infusion of different neutrophil subsets combined with anti-PD-1 therapy. Figure 2 Revealed CXCR2 - CLEC4N + The relationship between neutrophils and immunotherapy in bladder cancer patients; Figure 3 The functions, maturation states, and differentiation trajectories of different neutrophil subsets are shown; Figure A reveals the differentiation trajectory of neutrophils through pseudo-time series analysis; Figures B and C score the functions of different neutrophil subsets. Figure 4 CXCR2 was displayed - CLEC4N + Neutrophil antitumor function depends on the production of peroxides; Figure A compares the amount of hydrogen peroxide produced by different neutrophil subsets under PMA stimulation and non-stimulation; Figure B shows the ability of neutrophils to kill tumor cells by co-culturing different neutrophil subsets with tumor cells and detecting surviving tumor cells. Detailed Implementation

[0017] The CXCR2 will be discussed below with reference to the accompanying drawings. - CLEC4N + The use of neutrophils as a biomarker to predict the efficacy of immunotherapy for bladder urothelial carcinoma is described in detail. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0018] I. CXCR2 - CLEC4N + Neutrophil counts are associated with good outcomes from immunotherapy for bladder urothelial carcinoma. This application utilizes single-cell sequencing analysis, flow cytometry analysis, and adoptive infusion models to compare the relationship between different neutrophil subsets and immunotherapy.

[0019] Single-cell sequencing analysis of neutrophil subsets in mouse bladder cancer. Single-cell sequencing analysis method: Single-cell RNA-seq libraries were generated using a 10X Genomics Chromium Controller instrument and the Chromium Single Cell 3′ V3 kit (10X Genomics, Pleasanton, CA). Briefly, cells were concentrated to approximately 1000 cells / μL and loaded into each channel to generate single-cell gel bead emulsions (GEMs). Following a reverse transcription step, GEMs were cleaved, and barcoded cDNA was purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed, ligated with adapters, and subjected to indexed PCR amplification. The final libraries were quantified using the Qubit high-sensitivity DNA kit (Thermo Fisher Scientific), and the size distribution of the libraries was determined using a high-sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). All libraries were sequenced at 150 bp paired ends using an Illumina sequencer (Illumina, San Diego, CA). Raw data was processed using CellRanger (version V7.2.0) to generate a raw unique molecular identifier (UMI) count matrix, which was then converted into a Seurat object using the R package Seurat (version V4.1.1). Cells with UMI counts less than 500 or UMI counts exceeding 15% mitochondrial origin were identified as low-quality cells and removed. Double-packets were removed from each sample using DoubleFinder. Batch effects between samples were removed using Harmony, and the top 30 PCA analyses were performed. Seurat's FindNeighbors function was used to construct a shared nearest neighbor graph, upon which unsupervised clustering analysis was performed using the FindClusters function in Seurat with the parameter set to "resolution = 0.1". The graph was visualized using a UMAP plot and further clustered into subgroups. From the graph, it can be seen that ( Figure 1 (A) Neutrophil subsets can be divided into five subgroups: N0, N1, N2, N3, and N4. Maturity scoring of these subgroups revealed that N0 and N1 have lower maturity and are classified as immature neutrophils; while N3 and N4 have higher maturity and are classified as mature neutrophils; N2 is intermediate between mature and immature, warranting further investigation. Figure 1 B). To further classify the N2 subpopulation, this application screened key biomarkers for this subpopulation and found that two biomarkers, Cxcr2 and Clec4N, can classify neutrophils into three major categories: Cxcr2 + Clec4N - These belong to mature neutrophils N3 and N4, Cxcr2- Clec4N - These belong to immature neutrophils N0 and N1, Cxcr2 - Clec4N + Belongs to subgroup N2 ( Figure 1 C).

[0020] Flow cytometry was used to separate neutrophil subsets to verify their impact on immunotherapy response. Flow cytometry analysis: Mouse tumor tissues were processed as follows: each tumor was digested at 37°C with 5 mL of digestion buffer containing 2.5 mg DNase I and 5 mg collagenase for 30 minutes with shaking. The resulting suspension was filtered through a 70 μm sieve and resuspended in PBS containing 5% FBS. For surface labeling, cells were incubated with fluorescein-labeled antibodies at 4°C for 20 minutes. For intracellular cytokine staining, cells were fixed and permeabilized with flow cytometry buffer (00-5523-00, ThermoFisher) for 30 minutes, followed by antibody incubation at room temperature in the dark. Flow cytometry data were collected using a BD Fortessa analyzer and analyzed using FlowJo software. From the figure, it can be seen that ( Figure 1 D), the flow cytometry method used in this application can effectively separate the three major neutrophil subsets required in this application, Cxcr2. + Clec4N - (N3 and N4), Cxcr2 - Clec4N - (N0 and N1) and Cxcr2 - Clec4N + (N2). To further investigate the impact of different subgroups on the responsiveness of bladder cancer to immunotherapy, this application constructed an animal model. Regarding the treatment method, this application specifies the subgroups with tumor volumes reaching 100-200 mm². 3 Around day 7, tumor-bearing mice were randomly divided into four groups: Group 1 served as the control group, receiving 100 μl PBS intraperitoneally and 50 μl PBS intratumorally; Group 2 received anti-PD-1 treatment, receiving 100 μl PBS intraperitoneally and 50 μl PBS intratumorally; Group 3 received 100 μl PBS intraperitoneally and received different neutrophil subsets (5 × 10⁻⁶ cells per day). 6 The first group received treatment with 50 μL of PBS intratumorally; the fourth group received anti-PD-1 treatment via intraperitoneal injection of 100 μL, and simultaneously received different neutrophil subsets (5 × 10⁶ cells / mL). 6Mice were treated with 50 μL of PBS via intratumoral injection. Each mouse received 200 μg of anti-PD-1 every 3 days for a total of 5 treatments. The tumors were derived from homologous mouse subcutaneous heterotopic xenografts (tumor volume greater than 400 mm²). 3 Purification time: 5 × 10⁻⁶ 6 Neutrophils were injected intratumorally once every 3 days for a total of 5 times.

[0021] From the figure, we can see that ( Figure 1 E), reinfusion of mature Cxcr2 + Clec4N - (N3 and N4) and immature Cxcr2 - Clec4N - Neither N0 nor N1 neutrophils effectively sensitized the therapeutic effect of ICBs in bladder cancer; only the infusion of Cxcr2... - Clec4N + Only neutrophils of the (N2) type can effectively sensitize the therapeutic effect of ICBs in bladder cancer. These results confirm that the novel neutrophil subset discovered in this application is closely related to the responsiveness to ICBs in bladder cancer treatment.

[0022] II. CXCR2 - CLEC4N + The relationship between neutrophils and immunotherapy in bladder cancer patients This application compares CXCR2 using a computational scoring method. - CLEC4N + The relationship between neutrophils and the efficacy of immunotherapy in bladder cancer patients.

[0023] For CXCR2 - CLEC4N + Mapping of neutrophils in a transcriptome sequencing cohort of bladder cancer samples was performed using bulk RNA-seq samples from different tumors obtained from the literature. CIBERSORTx was used for deconvolution to obtain relative cell type frequency estimates for 10 different immune cell subsets (LM10 feature matrix). Subsequently, representative cell type-specific expression profiles and per-gene variance were estimated at each aggregated group level (non-responders [NR] vs. responders [R]) using CIBERSORTx [mode: Group]. To obtain robust statistical measures, the data were then analyzed using bootstrap simulations based on a negative binomial (NB) model—where the NB dispersion estimate per gene was calculated as (CVGeneA^2)⁻¹ / m(GeneA).

[0024] Each simulated sample was analyzed using Gene Set Variation Analysis (GSVA) ​​[^v1.47.3^]. As can be seen from the figure ( Figure 2Whether in bladder cancer (BLCA), or in glioma (GBM), skin melanoma (SKCM), gastric adenocarcinoma (STAD), etc., CXCR2 - CLEC4N + A higher number of neutrophils is closely associated with better responsiveness to ICBs therapy.

[0025] III. Quasi-time series analysis reveals CXCR2 - CLEC4N + Neutrophils are in an intermediate transitional state between immaturity and maturity, and exhibit a strong oxidative stress capacity, capable of efficiently producing reactive oxygen species (ROS). This application reveals the secrets of CXCR2 through pseudo-temporal methods, functional enrichment methods, and gene scoring methods. - CLEC4N + Characteristics and functions of granulocytes.

[0026] For CXCR2 - CLEC4N + Regarding the origin of neutrophils, this application analyzed their differentiation trajectory using a pseudo-time series approach. The results showed that the mature subpopulations N3 and N4 were located at the end of differentiation, while the immature subpopulations N0 and N1 were located at the beginning of differentiation; the N2 subpopulation was located on a separate branch. This suggests that the N2 subpopulation may have differentiated from N0 and N1 via a different pathway, rather than from immature differentiation. Figure 3 A). Therefore, this application names the N2 subgroup as the respiratory burst period.

[0027] To understand CXCR2 - CLEC4N + The functional characteristics of granulocytes were assessed using GO enrichment scores and GSVA scores. As shown in the figure ( Figure 3 B), GO enrichment hint CXCR2 - CLEC4N + The reactive oxygen species (ROS) pathway and NRF2 pathway in neutrophils were significantly enriched; further functional analysis of GO further confirmed that ROS production, pentose phosphate, and antioxidant protection pathways were significantly activated in the N2 subset. Figure 3 C).

[0028] IV. CXCR2 - CLEC4N + Neutrophils can directly kill tumors by producing peroxides. In in vitro experiments, this application revealed the differences in the ability of different neutrophil subsets to produce peroxides, and elucidated the antitumor immune functions of different neutrophil subsets through a co-culture system.

[0029] To compare the ability of different neutrophil subsets to produce peroxides, this application sorts out different subsets of neutrophils within tumor-bearing mice, cultures them, and tests their peroxide production capacity.

[0030] The specific testing method is as follows: tumor volume is calculated by multiplying the longest diameter (L) by the square of the vertical diameter (W). 2 Then the product is divided by 2 to calculate (V = L × W). 2 / 2); Regarding treatment methods and grouping, this application applies to tumors with a volume of 100-200 mm. 3 Around day 7, tumor-bearing mice were randomly divided into four groups: Group 1 was the control group, receiving 100 μl PBS intraperitoneally and 50 μl PBS intratumorally; Group 2 received anti-PD-1 treatment, receiving 100 μl PBS intraperitoneally and 50 μl PBS intratumorally; Group 3 received 100 μl PBS intraperitoneally and received different neutrophil subsets (5 × 10⁻⁶ cells per day). 6 The first group received treatment with 50 μL of PBS intratumorally; the fourth group received anti-PD-1 treatment via intraperitoneal injection of 100 μL, and simultaneously received different neutrophil subsets (5 × 10⁶ cells / mL). 6 Mice were treated with 50 μL of PBS via intratumoral injection. Each mouse received 200 μg of anti-PD-1 every 3 days for a total of 5 treatments. The tumors were derived from homologous mouse subcutaneous heterotopic xenografts (tumor volume greater than 400 mm²). 3 Purification time: 5 × 10⁻⁶ 6 Neutrophils were injected intratumorally once every 3 days for a total of 5 times.

[0031] From the figure, we can see that ( Figure 4 (A) The N2 subpopulation showed a significantly stronger ability to produce hydrogen peroxide than the N0, N1, N3, and N4 subpopulations, especially under PMA stimulation, where the N2 subpopulation demonstrated even greater hydrogen peroxide production. This result further suggests that the N2 subpopulation may function by producing ROS such as hydrogen peroxide.

[0032] For evaluating CXCR2 - CLEC4N + The tumor-killing ability of neutrophils is dependent on peroxides. This application co-cultures three major neutrophil subsets with tumor cells, then detects surviving tumor cells to observe their tumor-killing ability. As can be seen from the figure (…). Figure 4 B) The N2 subset has a stronger killing ability against tumor cells.

[0033] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. CXCR2 - CLEC4N + Use of neutrophils as a marker in products for detecting the efficacy of immunotherapy in bladder urothelial cancer.

2. Use according to claim 1, characterized in that, Products for detecting bladder urothelial carcinoma immunotherapy efficacy include detecting bladder urothelial carcinoma immunotherapy efficacy in CXCR2 - CLEC4N + Agents or drugs for detecting the degree of neutrophil infiltration.

3. Use according to claim 1, characterized in that, The CXCR2 - CLEC4N + Use of neutrophils in the manufacture of a product for the prevention or treatment of bladder urothelial cancer.

4. Use according to claim 3, characterized in that, The CXCR2 - CLEC4N + Use of neutrophils in combination with ICBs in the manufacture of a product for the prevention or treatment of bladder urothelial cancer.

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