Use of ppfibp2 as a target in the preparation of a pharmaceutical composition for treating bladder cancer

Abstract

The application belongs to the technical field of tumor immunotherapy and molecular targeted intervention, and particularly relates to the use of PPFIBP2 as a target in the preparation of a pharmaceutical composition for treating bladder cancer. Knocking down PPFIBP2 can inhibit the malignant proliferation ability of bladder cancer cells, reduce the tumor growth rate, restore or enhance the expression of chemokines, promote the recruitment and infiltration of CD8-positive T cells, and enhance the anti-tumor immune effect. After the preparation targeting PPFIBP2 is combined with a PD-1 inhibitor, the tumor inhibition effect is more obvious, and the CD8-positive T cell infiltration and functional activation are at a higher level than any single drug group, which indicates that the combined strategy has clear transformation potential. The research results of the application suggest that PPFIBP2 is a potential therapeutic target for bladder cancer progression, and the development of a drug for inhibiting the expression level of PPFIBP2 and the combined application scheme of the drug and a PD-1 inhibitor are expected to provide a new strategy for bladder cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy and molecular targeted intervention technology, specifically relating to the use of PPFIBP2 as a target in the preparation of pharmaceutical compositions for the treatment of bladder cancer. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, exhibiting significant biological heterogeneity and varying treatment responses. In recent years, PD-1 or PD-L1-related immune checkpoint inhibitors have been increasingly used in bladder cancer treatment; however, a considerable proportion of patients still experience limited benefit, insufficient duration of response, or primary drug resistance. Relying solely on single immune checkpoint blockade is insufficient to fully reverse the immunosuppressive microenvironment created by the tumor. Therefore, there is an urgent need to identify combined intervention targets that can synergize with PD-1 inhibitors and improve the tumor's immune status.

[0003] Current research suggests that bladder cancer progression is not only related to the enhanced proliferation capacity of tumor cells themselves, but also closely related to insufficient recruitment, restricted invasion, and functional suppression of effector T cells in the tumor microenvironment. Especially when CD8-positive T cells have difficulty effectively entering the tumor parenchyma or when their effector function is insufficient after entry, PD-1 inhibitors, even if they partially relieve functional suppression, often fail to achieve ideal therapeutic effects. Therefore, if T cell entry and invasion can be improved simultaneously on the basis of PD-1 blockade, it is hoped that the efficacy of immunotherapy can be further enhanced.

[0004] PPFIBP2, PTPRF interactor protein-binding protein 2, commonly known as lipophosphoprotein β2 (Liprin-β2), is a protein-coding gene located at human 11p15.4, encoding an intracellular scaffold protein. Belonging to the Liprin family, it primarily binds to the receptor tyrosine phosphatase PTPRF, bridging the cell membrane receptor and the cytoskeleton, and regulating focal adhesion assembly and disassembly. It participates in neuronal axon growth, synaptic structure assembly and maturation, maintaining normal nerve signal transduction; and regulates cell adhesion, morphological changes, and cell migration. It is widely expressed in multiple human tissues, with higher expression levels in brain tissue, mammary gland, and testes. Current research often focuses on its mechanisms in neurodevelopmental abnormalities and tumor cell invasion and metastasis. In some livestock, this gene can also serve as a molecular breeding marker related to lactation. Currently, there are no reports of PPFIBP2 targeting bladder cancer. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide new targets for the treatment of bladder cancer and further explore the specific regulatory mechanisms of PPFIBP2 on the occurrence, development, and immune escape of bladder cancer.

[0006] The technical solution provided by this invention is the use of PPFIBP2 as a target in the preparation of a pharmaceutical composition for treating bladder cancer.

[0007] Preferably, knocking down PPFIBP2 can inhibit the malignant proliferation of bladder cancer cells and reduce tumor growth rate; at the same time, it can restore or enhance the expression of chemokines, promote the recruitment and infiltration of CD8 positive T cells, and enhance the anti-tumor immune effect.

[0008] Preferably, the pharmaceutical composition for treating bladder cancer is a formulation that targets PPFIBP2; the formulation that targets PPFIBP2 is a formulation that can reduce the expression level of PPFIBP2 or inhibit the biological function of PPFIBP2.

[0009] Preferably, the formulation targeting PPFIBP2 is shRNA.

[0010] The present invention also provides a pharmaceutical composition for treating bladder cancer, the pharmaceutical composition comprising an agent targeting PPFIBP2.

[0011] Preferably, the pharmaceutical composition further comprises a PD-1 inhibitor.

[0012] The experimental results of this invention reveal that PPFIBP2 has a dual biological role in the progression of bladder cancer: on the one hand, it promotes tumor cell proliferation, and on the other hand, it weakens CD8-positive T cell recruitment and effector function by inhibiting chemokine expression, thereby promoting tumor progression. Based on this, establishing targeted interventions around PPFIBP2 has clear technical value. Further observation in animal models shows that the combined application of PPFIBP2-targeting agents and PD-1 inhibitors exhibits more significant tumor suppression effects and higher levels of CD8-positive T cell infiltration and functional activation compared to either single-agent group, indicating that this combined strategy has clear translational potential. The results of this invention suggest that PPFIBP2 is a potential therapeutic target for bladder cancer progression, and the development of drugs that inhibit PPFIBP2 expression and their combined application with PD-1 inhibitors holds promise for providing new strategies for bladder cancer treatment. Attached Figure Description

[0013] Figure 1This study investigates the effect of PPFIBP2 on the malignant proliferation of bladder cancer. Specifically: A. The screening process for key targets in PRC subtyping; B. The importance distribution of the key target PPFIBP2 in the Qingdao and TCGA cohorts; C. The distribution difference of PPFIBP2 between tumor and NAT samples; D. The distribution difference of PPFIBP2 in single-cell data across four subtyping categories; E-F: The distribution difference of PPFIBP2 among different cell types; G. Stable knockout and overexpression of PPFIBP2 were constructed in the T24 cell line and verified by Western blotting experiments; the knockdown and overexpression models were independent experimental systems, with GAPDH and β-ACTIN used as internal controls in their respective batches; H. qPCR was used to verify the interference effect in PPFIBP2 knockdown and overexpression cell lines; I. The MTT assay was used to evaluate the changes in tumor cell growth ability after PPFIBP2 downregulation or upregulation; J. A colony formation assay was used to observe the differences in tumor cell colony formation ability after PPFIBP2 knockdown or overexpression treatment. Figure 2 This section describes the tumor-promoting ability of PPFIBP2. Figures A-B show the tumor-promoting efficacy of the PPFIBP2 gene verified through nude mouse tumorigenesis experiments. From left to right, the first image shows tumors dissected from nude mice; the second image shows changes in tumor volume at different time points; and the third image shows the difference in tumor weight between different expression level groups. A. Tumorigenesis effect after PPFIBP2 knockdown; B. Tumorigenesis effect after PPFIBP2 overexpression; C. Based on sequencing data from different tissues and omics samples, GSEA analysis was performed to explore the potential regulatory mechanism of PPFIBP2 expression differences on the microenvironment. From left to right, the first image shows the GSEA results of T24 cells overexpressing PPFIBP2 and the control cell line; the second image shows the GSEA results of the Qingdao cohort proteome sequencing analysis between PPFIBP2 high and low expression groups; the third image shows the GSEA results of the Qingdao cohort transcriptome sequencing analysis between PPFIBP2 high and low expression groups; and the fourth image shows the GSEA results of the Qingdao cohort single-cell transcriptome sequencing analysis between PPFIBP2 high and low expression groups. Figure 3 This study aimed to investigate PPFIBP2-mediated immune microenvironment remodeling in bladder cancer. Specifically, A. Validation of cytokine levels after PPFIBP2 knockdown and overexpression using high-throughput liquid microarray analysis; B. Validation of CD8+ levels after PPFIBP2 knockdown and overexpression using qPCR experiments. + C. Changes in the expression levels of T cell recruitment-related cytokines; D. Verification of the association between different PPFIBP2 expression patterns and the expression of various immune cell effector genes based on transcriptomics and proteomics; + Co-culturing T cells with cells exhibiting different PPFIBP2 expression levels validated their effect on recruiting CD8+. +E. By comparing the regulatory effects of tumor cells with different PPFIBP2 expression patterns on the anti-tumor effect of T cells through T cell-mediated tumor killing experiments; F-G. By analyzing the possible mechanisms of reduced cytotoxicity of co-cultured T cells using flow cytometry at the phenotypic and functional levels. Figure 4 To validate the immunomodulatory role of PPFIBP2 in a mouse model with a complete immune system; this part includes: A. Study design; B. Constructing a stable PPFIBP2 overexpressing cell line in the mouse MB49 cell line and validating it based on Western blotting and qPCR experiments; C. Validating the carcinogenic efficacy of PPFIBP2 in mice with complete immune function through a mouse tumorigenesis experiment; D. Differences in tumor weight among different PPFIBP2 expression groups; E. Changes in tumor volume in different PPFIBP2 expression groups over time; F. Validating the CD8+ expression in different PPFIBP2 expression groups using flow cytometry. + G. Statistical analysis of flow cytometry results; H. Typical IHC images of CD8 staining in different mouse groups; I. Verification of significantly enriched gene pathways among different PPFIBP2 expression level groups based on mouse cell line transcriptome sequencing. Figure 5 To analyze the microenvironmental differences under different PPFIBP2 expression levels based on single-cell transcriptome sequencing; including: A. UMAP maps of major cell type clusters; B. Annotation markers of different cell types; C. Differences in the proportion of different cell types between the two groups; D. UMAP maps of further subdivided T cell subpopulations; E. Differences in the proportion of different types among the subpopulations of T cells between the two groups; F. Differences in the distribution of T cell effector scores between the two groups; G. Differences in CD8 expression between the two groups in single-cell transcriptome sequencing data. + H. Differential expression of T cell recruitment factors; I. Evaluation of the correlation between NF-κB pathway and PPFIBP2 in all cancer types based on the TCGA pan-cancer dataset; II. GSEA analysis of different PPFIBP2 expressions among human tissue transcriptomes, proteomes, and human cell line transcriptomes; III. Differences in NF-κB pathway activity among different PPFIBP2 expression groups in mouse single-cell samples; IV. Changes in the expression of NF-κB-related proteins after PPFIBP2 knockout. Figure 6 To assess the efficacy of combination therapy targeting PPFIBP2; including: A. Animal experimental design diagram; B. Tumor volume changes between different treatment groups; C. Tumorigenicity between different treatment groups; D. Tumor weight differences between different treatment groups; E. Flow cytometry results between different treatment groups; F. CD8 levels between different treatment groups. +Differences in T-cell effector cell changes; G. Typical CD8 IHC staining images between different treatment groups. Detailed Implementation

[0014] To facilitate understanding of the present invention, the specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. The following examples or drawings are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0015] Examples 1 to 5 describe the relevant experimental methods, and Example 6 describes the analysis of experimental results. The T-cell-deficient BALB / C nude mice used in this invention were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd., and the fully immunocompetent C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in facilities negative for specific pathogens. All animal experiments involved in this study were conducted after approval by the institution's laboratory animal ethics committee and were strictly performed in accordance with the approved procedures.

[0016] Example 1: Screening of PRC typing-specific targets First, differential expression analysis was performed based on PRC and other subtypes. Then, the correlation between each candidate gene and LUBD score, fatty acid metabolism, and interferon-γ response was assessed based on the PRC-specific phenotype, retaining genes with consistent phenotypes in both TCGA and QD-BC (Qingdao cohort). Finally, a random forest algorithm was used to determine the candidate genes most relevant to distinguishing the PRC phenotype. The QD-BC cohort used in this embodiment included 93 bladder cancer cases with qualified H&E whole-slice images and corresponding multi-omics subtype labels. The TCGA-BLCA cohort included 375 bladder cancer cases with available H&E whole-slice images, transcriptomic data, and clinical data.

[0017] Example 2: Construction of the PPFIBP2 intervention model.

[0018] Stable knockdown (sh-PPFIBP2) and overexpression (oe-PPFIBP2) models of PPFIBP2 were constructed in representative human cell lines using small interfering RNA transfection. The successful model construction was confirmed by protein or transcriptional level assays.

[0019] 1. Overexpression plasmid amplification and extraction 1.1 Plasmid amplification The recombinant plasmid used in the overexpression experiment of this invention was constructed by Beijing Liuhe BGI Genomics Co., Ltd. The full-length mRNA information corresponding to the target gene was retrieved from the NCBI database, and the vector used was pcDNA3.1(+). Simultaneously, corresponding restriction enzyme recognition sites were designed based on the coding region sequence of the target gene. LB liquid medium was prepared according to standard methods: 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl were dissolved in deionized water and brought to a final volume of 1 L. The medium was then autoclaved for 20 min and stored at 4 ℃ for later use. During amplification, 15 mL of LB medium was added to a 50 mL centrifuge tube, and appropriate antibiotics were added based on the different vector resistances. Subsequently, 20 μL of the transformation bacterial solution provided by the company was added, and the mixture was cultured at 37 ℃ with shaking for 12–16 h. After culture, 650 μL of fresh bacterial solution was thoroughly mixed with 150 μL of 80% glycerol to prepare a bacterial culture preservation solution, which was stored at -80 ℃.

[0020] 1.2 Plasmid Extraction Plasmid isolation was performed using an endotoxin-free plasmid mini-extraction kit from Tiangen Biotech Co., Ltd., as detailed below: (1) First, add 500 μL of BL equilibration solution to the CP4 adsorption column, centrifuge at 12000 r / min for 1 min, discard the waste liquid, and then put the column back into the collection tube.

[0021] (2) Take the bacterial culture obtained overnight and place it in a centrifuge tube. Centrifuge at 12000 r / min for 1 min, remove the upper liquid, and retain the bacterial precipitate.

[0022] (3) Add 500 μL of P1 suspension premixed with RNase A to the precipitate and use vortexing to completely disperse the cells.

[0023] (4) Continue to add 500 μL of P2 lysis buffer and mix 6–8 times to ensure that the cells are fully lysed. Avoid vigorous shaking. After full lysis, the cells usually appear clear.

[0024] (5) Then add 500 μL of P4 neutralizing solution and mix 6–8 times. At this time, a white flocculent precipitate will appear. After standing at room temperature for 10 min, centrifuge at 12000 r / min for 10 min to allow the precipitate to gather at the bottom of the tube.

[0025] (6) Transfer the obtained supernatant into the CS filter column in several batches, centrifuge at 12000 r / min for 2 min, and collect the filtrate into a new 2 mL centrifuge tube.

[0026] (7) Add isopropanol to the filtrate, mix well, and then load the sample onto the CP4 adsorption column.

[0027] (8) Centrifuge at 12000 r / min for 1 min at room temperature, discard the waste liquid, and then put the adsorption column back into the collection tube.

[0028] (9) Add 500 μL of protein removal solution PD to CP4, centrifuge at 12000 r / min for 1 min, remove the waste liquid and put the column back in.

[0029] (10) Add 600 μL of PW wash buffer pre-added with anhydrous ethanol to the adsorption column, let stand for 5 min, then centrifuge at 12000 r / min for 1 min to remove waste liquid; repeat this step twice. Then continue centrifuging for 2 min, and leave at room temperature for several minutes after opening the lid to remove as much residual wash buffer as possible.

[0030] (11) Transfer CP4 to a new centrifuge tube, slowly add 100–300 μL of TB elution buffer to the center of the membrane, let stand at room temperature for 2 min, then centrifuge at 12000 r / min for 1 min. Collect the elution buffer, which is the plasmid DNA. After determining the concentration, store at -20 ℃ for later use.

[0031] 1.3 Plasmid transfection Plasmids were pre-coated according to groupings, and plasmid introduction began when the density approached 50%. Transfection system A and transfection system B were prepared separately, incubated at room temperature for 5 min, mixed thoroughly, and then incubated for another 15 min to allow DNA and liposomes to form complex particles. These were then added to the cell culture system, and after 8 h, the medium was replaced with fresh complete culture medium. Culture was continued for 24–72 h depending on the experimental objective before subsequent detection. The specific system is as follows: Table 1. Overexpression plasmid transfection system

[0032] 2. Lentiviral infection and screening Table 2 shows the siRNA sequences used.

[0033] The lentiviral vector used in this invention was constructed by Shanghai Gemma Gene Technology Co., Ltd. The target cell infection and screening process is as follows: (1) Prepare a cell suspension using complete culture medium, so that its density reaches 3–5 × 10⁻⁵. 4 Inoculate 500 μL into a 24-well plate and incubate at 37 °C for 16–24 h. Infection begins when the density reaches approximately 20%–30%.

[0034] (2) Remove the frozen virus from the low temperature environment, thaw it slowly on ice, and dilute it to the required concentration using complete culture medium according to the experimental protocol.

[0035] (3) Calculate the sample volume based on the virus titer and target MOI, remove the original culture supernatant from the well, add the corresponding volume of virus solution and infection enhancement solution, mix well and continue culturing.

[0036] (4) After about 16 hours of infection, replace with fresh complete culture medium; if the cell state declines significantly during the infection process, the medium replacement time can be advanced to 8 hours to maintain activity.

[0037] (5) Then change the culture medium as needed according to the cell growth.

[0038] (6) 72 h after infection, when the fluorescence expression is more obvious, the infection efficiency is evaluated under a microscope; cells with an infection rate of about 80% and good cell condition are selected for further passage.

[0039] (7) After the cells have grown stably, puromycin is added to the culture medium for drug screening.

[0040] (8) Observe cell morphology and proliferation status during the screening period. Uninfected cells gradually die because they cannot tolerate puromycin, while cells that successfully integrate the virus continue to survive and further expand, eventually obtaining stable expression cell lines.

[0041] 3. Real-time quantitative reverse transcription PCR experiment Total RNA extraction from cells: Total RNA from cell samples was separated using TRIzol, and the specific procedure is as follows: (1) Complete the plating according to the experimental plan and culture the cells after appropriate treatment. After removing the culture medium, wash the cells twice with pre-cooled PBS buffer, place the culture plate on ice, add 500 μL of TRIzol lysis buffer to each well, and repeatedly pipette to ensure that the cells are fully lysed. Then transfer the lysis products to 1.5 mL EP tubes.

[0042] (2) Add chloroform at 1 / 5 of the volume of the lysis solution, shake well and let stand for 10 min.

[0043] (3) Centrifuge at 12000 r / min for 15 min at 4 ℃. After centrifugation, the system is clearly divided into layers. The lower and middle layers mainly contain protein and phenolic components, while RNA is distributed in the upper aqueous phase. Carefully aspirate the top colorless and transparent liquid and transfer it to a new 1.5 mL EP tube without RNase.

[0044] (4) Add an equal volume of isopropanol to the obtained aqueous phase, mix well, place on ice for 10 min, and then centrifuge at 4 ℃ and 12000 r / min for 20 min.

[0045] (5) After discarding the supernatant, add 1 mL of 75% ethanol to wash the RNA precipitate and centrifuge at 12000 r / min for 5 min at 4 ℃. Repeat this washing step once.

[0046] (6) After removing the residual liquid, open the lid and place it at room temperature to allow the ethanol to evaporate completely.

[0047] (7) Then add 30 μL of RNase-free water to resuspend the RNA precipitate, mix well and then perform concentration detection.

[0048] RNA concentration determination: RNA concentration and purity were detected using an ultra-micro nucleic acid and protein quantification instrument. Before the assay, blank correction was performed with RNase solution, and then each sample was tested on the instrument, and the A260 / A280 ratio was recorded.

[0049] RNA reverse transcription: (1) Based on the sample RNA concentration, calculate the volume required to obtain 0.5 μg of RNA, and prepare a genomic DNA-free system with a total reaction volume of 10 μL on ice. The reaction conditions are set at 42 ℃ for 2 min, followed by incubation at 4 ℃.

[0050] (2) Prepare the reaction solution according to the preset system. The amplification program is set as follows: pre-denaturation at 95 ℃ for 30 s, for one cycle; then proceed to the amplification stage, 95 ℃ for 5 s, 60 ℃ for 30 s, for a total of 40 cycles. After amplification, record the amplification curve and the melting curve, and use 2 - Ct The method calculates the relative expression level of the target gene in each group.

[0051] Table 3 qPCR reaction system

[0052] Example 3: Verification of the effect of PPFIBP2 targeted intervention on tumor proliferation In vitro, cell viability and clonogenic assays were used to assess changes in tumor cell proliferation after PPFIBP2 downregulation. In vivo, a nude mouse xenograft model was used to verify its effect on tumor growth.

[0053] 1. MTT Experiment Bladder cancer cells in the logarithmic proliferation phase were selected, and the cell suspension was collected after trypsin treatment. The cells were centrifuged, re-mixed, and counted. Cells were then distributed at 2 × 10⁶ cells per well. 3Cells were seeded at a density of 100 μL of culture medium in 96-well plates, with a blank control well added to each well. Each treatment group had 5 parallel wells. After cell attachment stabilized, appropriate interventions were administered according to the experimental protocol, and assays were performed at designated time points. For assays, 20 μL of MTT working solution was added to each well, and incubation continued for 4 h. The liquid in the wells was then carefully removed, and 150 μL of DMSO was added to fully dissolve the generated formazan product. After vortexing and mixing, the absorbance at 490 nm was read using a microplate reader, and cell growth curves were plotted based on the results.

[0054] 2. Cloning experiment Selected PPFIBP2 overexpressing cells, PPFIBP2 knockdown cells, and their respective negative control cells in good growth condition, digested them, prepared them into suspensions, and thoroughly mixed with complete culture medium. Dispense 1 × 10⁶ cells per well. 3 One seed cell line was seeded into each well of a 6-well plate, and an appropriate amount of complete culture medium was added. The plates were gently shaken to ensure even cell distribution, and then incubated at 37 °C for approximately 2 weeks, with the medium changed periodically according to cell condition. After incubation, the culture medium was aspirated, and the cells were fixed with 4% paraformaldehyde for 30 min, followed by recrystallization with crystal violet. After staining, the cells were washed with PBS, air-dried upside down, and photographed. Colony counting was performed using ImageJ software to assess the colony-forming ability of different groups.

[0055] 3. Xenograft tumor model 1 × 10 7 Stable cell lines were subcutaneously inoculated onto the dorsal side of mice. The long diameter (L) and short diameter (W) of the tumor were measured twice weekly. Tumor volume was calculated using the formula V = 0.5 × L × W. 2 Calculation. Based on tumor growth, mice were euthanized by cervical dislocation on day 24 (overexpression group) or day 35 (knockdown group) after inoculation. Tumor tissue was dissected and photographed, and some tissue was used for subsequent molecular biological analysis.

[0056] Example 4: Validation of the effects of PPFIBP2 targeted intervention on immune recruitment and anti-tumor immunity In vitro, changes in chemokine expression were detected after PPFIBP2 intervention. Further assessments of immune response were conducted using tumor cell-immune cell co-culture assays, T-cell killing assays, and flow cytometry.

[0057] 1. Extraction of peripheral blood mononuclear cells Peripheral blood from healthy volunteers at the Affiliated Hospital of Qingdao University was used for mononuclear cell preparation. All participants fully understood the research objectives and signed written informed consent. At the start of the experiment, the collected blood samples were centrifuged at 3000 r / min for 10 min to remove the supernatant plasma. Approximately 4 mL of the blood cell fraction was then transferred to a 15 mL centrifuge tube, and PBS buffer was added and gently mixed by pipetting. Approximately 4 mL of human lymphocyte separation medium was added to a new centrifuge tube, slowly covering the blood cell suspension along the tube wall, taking care to maintain liquid separation. After centrifugation at 800 r / min for 20 min at room temperature, a mononuclear cell layer was observed at the interface. The intermediate white membrane-like cell layer was aspirated into a new centrifuge tube, and erythrocyte lysis buffer was added to remove residual erythrocytes. The cells were then washed 2–3 times with PBS. Subsequently, the cells were centrifuged at 1000 r / min for 10 min, the supernatant was discarded, and the resulting cell pellet was collected and resuspended in approximately 2 mL of culture medium for subsequent experiments.

[0058] 2. Chemotaxis experiment Chemotaxis analysis was performed in 24-well Transwell plates with 3 μm pore size. The 3 × 10⁻⁶ pores obtained above were then analyzed. 5 One T cell was dissolved in 200 μL of culture medium and added to the upper chamber. 600 μL of supernatant from different stable cell lines was added to the lower chamber. The cells were incubated at 37°C for 6 hours. Cells that migrated to the lower chamber were collected, stained with CD3 / CD8 antibody, and counted by flow cytometry.

[0059] 3. In vitro co-culture experiment of tumor cells and T cells Extracted mononuclear cells were cultured in flasks pre-coated with anti-human CD3 antibody and RetroNectin. The culture medium was supplemented with IL-2 and IFN-γ for 7 days. Human bladder cancer cells (oe-PPFIBP2, sh-PPFIBP2, and negative controls) and activated T cells were co-cultured in DMEM medium containing anti-CD3 antibody and IL-2 in 6-well plates at a 1:5 ratio for 72 hours. After treatment of the co-culture system with cell stimulation and protein transport blocking reagents, suspension T cells were collected from each well and analyzed by flow cytometry. Changes in phenotypic and functional parameters of T cells were assessed by comparing the results of direct co-culture with tumor cells expressing different PPFIBP2 states. Residual cancer cells were stained with crystal violet, dissolved in a solution containing 33% acetic acid and 67% methanol, and then the OD value was measured at 570 nm using a microplate reader to assess the viability of residual tumor cells.

[0060] 4. Flow cytometry In vitro experimental samples were collected, washed, and prepared into single-cell suspensions. In vivo experimental tissues were mechanically dissociated and filtered through a 70 μm screen. Red blood cells were removed using erythrocyte lysis buffer to obtain single-cell suspensions. For cytokine detection, cells were pre-treated with cell activators and protein transport inhibitors. In vivo experiments involved blocking cells with Fc receptor blockers after stimulation and incubation with fluorescently labeled surface antibodies in the dark. For intracellular marker detection, cells were fixed and permeabilized after surface staining. Data acquisition was performed using a NovoCyte flow cytometer for in vitro samples and an Attune NxT flow cytometer for tissue-derived samples.

[0061] 5. High-throughput liquid phase protein chip analysis (1) Sample processing: Take cells in the logarithmic growth phase and maintain them in a well plate for at least 48 h. Collect an appropriate amount of culture supernatant into an EP tube and centrifuge at 4 ℃ and 13000 r / min for 10 min to remove cell impurities. Then transfer the supernatant obtained by centrifugation into a new EP tube for subsequent detection.

[0062] (2) Reagent preparation: Before the experiment, move the reagents to room temperature and equilibrate for 30 minutes. Dilute the standard according to the instructions and set it to 7 standard + 1 Blank. Dilute the premixed beads for use and dilute the premixed detection antibody to prepare the working solution for use. Dilute the washing solution to 1x working solution and use dilution buffer to prepare PE-streptavidin to 1x working solution.

[0063] (3) Detection process per well: Add 25-50 μl each of beads, standards, quality control, and sample in sequence according to the kit instructions, and incubate at 800 rpm at room temperature for 0.5-1 h or at 4°C overnight; then, add 100-200 μl of washing buffer to rinse the beads 3 times, and then add 25-50 μl of detection antibody to incubate at 800 rpm at room temperature for 0.5-1 h; then, add 100-200 μl of washing buffer to rinse the beads 3 times, and then add 50 μl of PE-streptavidin to incubate at 800 rpm at room temperature for 10-30 min; finally, add 100-200 μl of washing buffer to rinse the beads. Repeat 3 times, then add 100-150 μl of washing solution / sheath solution and incubate at room temperature and 800 rpm for 0.5-2 min. Detect the results using a Luminex 200 platform, fit a standard curve, and finally calculate the concentration value (pg / ml).

[0064] Example 5: Validation of In vivo immune microenvironment remodeling In an immune-intact allogeneic xenograft model, the differences in the composition of immune cells in tumor tissue under different PPFIBP2 expression states were compared.

[0065] 5 × 105 A mouse-derived bladder cancer cell line (MB49-NC or MB49-oe-PPFIBP2) was subcutaneously inoculated onto the dorsal side of mice. After tumor formation, the mice were sacrificed and tumor tissue was collected for subsequent multi-omics sequencing and flow cytometry analysis.

[0066] Example 6: Preclinical Combined Treatment Model Given the low endogenous PPFIBP2 expression level in the MB49 cell line, which cannot directly simulate the immune microenvironment driven by high PPFIBP2 expression in PRC cells, this invention employs a phenotypic reproducibility strategy to construct a research model. First, PPFIBP2 is stably overexpressed in MB49 cells to construct a model mimicking PRC PPFIBP2. high A high-burden PRC-like cell model was used as the control group (NC); based on this, PPFIBP2 was further knocked down to construct a targeted intervention model (PPFIBP2). high -siPPFIBP2). In the simulation of PRC classification, PPFIBP2 high In this context, C57BL / 6J mouse models were inoculated with 5 × 10⁻⁶ mice. 5 MB49-oe-PPFIBP2 cells (feeding conditions as before). After tumor formation, mice were randomly divided into 4 groups: NC + IgG2a, NC + anti-PD-1, si-PPFIBP2 + IgG2a and si-PPFIBP2 + anti-PD-1. Treatment was performed every 3 days, including: (1) intratumoral electroporation delivery of siRNA, 15 μg si-PPFIBP2 or control NC dissolved in 30 μL sterile PBS and slowly injected into the tumor. Five minutes after injection, a square wave pulse (voltage 75-80 V, pulse width 50 ms, interval 50 ms, 3 cycles) was applied using a CUY21 EDIT II pulse generator with forceps electrodes; (2) intraperitoneal injection of antibody: 200 μg PD-1 antibody or isotype control IgG2a was administered. Tumor volume was measured twice a week during the treatment period. On day 13, mice were sacrificed according to the standard protocol and the tumors were collected for subsequent analysis.

[0067] Statistical analysis Data processing and statistical analysis in this invention were primarily performed using R v4.4.0 and GraphPad Prism 7.04. All statistical tests were two-tailed, with P < 0.05 considered statistically significant. For multiple comparisons involving high-throughput data, the Benjamini–Hochberg method was used for FDR correction; unless otherwise specified, a corrected FDR < 0.05 was used as the significance threshold. For continuous data, the analysis of differences between groups was performed using appropriate statistical methods based on the data distribution characteristics. If the data conformed to a normal distribution and had homogeneous variances after testing, Student's t-test was used for two-group comparisons, and one-way ANOVA was used for comparisons of three or more groups. If the data did not conform to a normal distribution, Wilcoxon rank-sum test was used for comparisons between two groups, and Kruskal–Wallis test was used for comparisons between multiple groups. For multiple group comparisons, pairwise comparisons were further performed according to the appropriate statistical framework when the overall difference was statistically significant. In this section, continuous variables such as cell viability, colony formation, qPCR, protein expression quantification, chemotaxis / migration assays, T-cell killing assays, flow cytometry results, and animal experimental endpoints were analyzed using Student's t-test or Wilcoxon rank-sum test, depending on the data distribution. When comparing multiple treatment groups, one-way ANOVA or Kruskal-Wallis test was used, depending on the data distribution. For repeated measurements over time, such as tumor growth curves, two-way ANOVA was used for inter-group comparisons. All cytological experiments were performed at least three times independently, and the sample size for animal experiments is indicated in the corresponding figure captions. The significance levels in the figures are as follows: ns indicates no statistical significance, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0068] Example 7, Experimental Results 1. Identification of PPFIBP2 as a key marker for PRC First, based on a four-level hierarchical system (STE, STE-like, PRC-like, PRC) based on LUBD, PRC was used as a representative of the front-end state of the continuous spectrum, and molecular features stably enriched at the PRC end were screened across cohorts. Differential expression analysis was performed between PRC and the other three groups in the QD-BC and TCGA cohorts to obtain the set of genes significantly upregulated in PRC; subsequently, the intersection of differentially expressed genes from the two cohorts was taken to obtain candidate genes stably upregulated at the PRC end. Figure 1 (A)

[0069] Secondly, to further screen candidate genes that best fit the PRC composite phenotype at the functional level, LUBD scores, fatty acid metabolism pathway activities, and interferon-γ response pathway activities were calculated for each sample in the QD-BC and TCGA cohorts, and the correlation between candidate genes and these three indicators was evaluated one by one. Only genes that simultaneously met the following conditions and showed consistent correlation directions in both cohorts were retained: significantly negatively correlated with LUBD scores, significantly positively correlated with fatty acid metabolism pathway activities, and significantly negatively correlated with IFN-γ response pathway activities. A set of candidate genes highly consistent with the PRC-defined phenotype of low dedifferentiation, high lipid metabolism, and relative immunosuppression was obtained.

[0070] Finally, to prioritize the identification of key molecules that contribute most to PRC and have potential target value within the candidate set, random forest classification models were constructed in two cohorts, with PRC / PRC-like and other subtypes as outcome variables, and candidate genes were ranked based on variable importance indices. Figure 1 (Middle B). The results consistently showed that the classic luminal transcription factor PPARG consistently ranked high in variable importance, further validating the luminal differentiation lineage characteristics corresponding to PRC. In addition to known lineage regulators such as PPARG, PPFIBP2 also consistently ranked high in variable importance in both cohorts and was persistently highly expressed in PRC tumors. Figure 1 Based on its specific high expression pattern in PRC / PRC-like cells and the known functions of the Liprin-α family in cytoskeleton remodeling and vesicle transport, PPFIBP2 was ultimately identified as a core candidate effector molecule linking the PRC complex phenotype and will be the focus of subsequent mechanism and function studies.

[0071] 2. PPFIBP2 promotes malignant proliferation of bladder cancer. qRT-PCR and Western Blot showed that PPFIBP2 was effectively downregulated or upregulated in the corresponding cells. Figure 1 (G, H). Functional experiments showed that PPFIBP2 significantly promoted tumor cell growth: MTT assay showed that knockdown of PPFIBP2 significantly reduced cell proliferation rate, while overexpression significantly promoted cell growth (G, H). Figure 1 (I); Plate colony formation assays further confirmed that downregulation of PPFIBP2 significantly weakened colony formation ability, while overexpression of PPFIBP2 significantly increased the number of colonies formed. Figure 1 (J).

[0072] 3. PPFIBP2 has a significant cancer-promoting ability. Tumor growth was significantly slowed in mice inoculated with PPFIBP2 knockdown cells, with a significant decrease in both tumor volume and weight at the endpoint; conversely, xenografts overexpressing PPFIBP2 showed significantly faster growth. Figure 2 (A, B). To further analyze the biological characteristics of PPFIBP2 from the perspective of transcriptional program, we integrated data from four levels: PPFIBP2 overexpressing cell lines, QD-BC transcriptome and proteome, and PRC single-cell sequencing, and performed gene set enrichment analysis on PPFIBP2 high and low expression groups ( Figure 2 (C) Results showed that EMT-related gene sets were consistently negatively enriched in all datasets, suggesting that high PPFIBP2 expression is closely related to EMT inhibition and epithelial phenotype maintenance, providing molecular evidence for the high-proliferation but relatively limited growth characteristics of PRCs. Notably, GSEA results also showed that multiple pathways closely related to cytokines and immune-related signal transduction (such as cytokine–cytokine receptor interaction, cytokine activity, etc.) consistently showed significant negative enrichment in the PPFIBP2 high-expression group, suggesting that PPFIBP2 may be accompanied by systemic inhibition of immune-related signaling networks while promoting tumor cell proliferation. Based on this clue, we further systematically evaluated the role of PPFIBP2 at the level of the immune microenvironment.

[0073] 4. PPFIBP2-mediated immune microenvironment remodeling in bladder cancer This invention first systematically evaluates the effect of PPFIBP2 on tumor secretion at the protein level. A liquid-phase cytokine antibody microarray was used to detect the culture supernatant of PPFIBP2 knockdown and overexpression bladder cancer cell lines. The results showed that PPFIBP2 overexpression significantly downregulated multiple pro-inflammatory chemokines, particularly CCL3 / MIP-1α, CCL4 / MIP-1β, and CCL5 / RANTES; conversely, in the supernatant of PPFIBP2 knockdown cells, the secretion of these chemokines was significantly increased. Figure 3 (A). qRT-PCR verified the above changes at the transcriptional level, confirming that PPFIBP2 has a stable negative regulatory effect on chemokines such as CCL3 / 4 / 5. Figure 3 (B). Correlation analysis combining clinical multi-omics data showed that PPFIBP2 expression abundance was correlated with CD8. + The expression of T cell effector molecules (GZMA, GZMB, PRF1, CD8A) was significantly negatively correlated. Figure 3 The results (C) suggest that upregulation of PPFIBP2 is closely related to the weakening of cytotoxic T cell recruitment and activation signals.

[0074] To clarify the functional consequences of the aforementioned chemokine alterations on T cell behavior, we constructed a tumor cell-T cell co-culture system and conducted functional experiments. In the Transwell migration assay, PPFIBP2 overexpression significantly reduced the number of T cells migrating towards the tumor cell supernatant, while PPFIBP2 knockdown partially restored or even enhanced the chemotactic migration ability of T cells. Figure 3 (D) indicates that PPFIBP2-mediated downregulation of CCL3 / 4 / 5 can directly weaken tumor cell recruitment of T cells. In T cell-mediated killing experiments, the sensitivity of tumor cells to T cell cytotoxicity was significantly reduced in the PPFIBP2 overexpression group, while PPFIBP2 knockdown significantly enhanced the susceptibility of tumor cells to T cell killing. Figure 3 (E). Further flow cytometry analysis showed that after co-culturing with PPFIBP2-overexpressing tumor cells, CD8... + IFN-γ in T cells + and TNF-α + The proportion of functional subgroups decreased significantly; conversely, knocking down PPFIBP2 resulted in the above-mentioned functional CD8 subgroups. + Increased proportion of T cells ( Figure 3 (F, G).

[0075] In a comprehensive experiment involving liquid-phase chips and co-culture, PPFIBP2 weakens CD8 by downregulating chemokines such as CCL3 / 4 / 5. + T cell recruitment and effector function lead to an immunosuppressive phenotype in PRC tumors.

[0076] 5. In vivo functional validation of PPFIBP2 To validate the PPFIBP2-related phenotype and elucidate its immune regulatory mechanism in mice with a complete immune system, we constructed an allogeneic xenograft model in C57BL / 6 mice using MB49 bladder cancer cells. Figure 4 (A). After qRT-PCR and Western Blot confirmed stable overexpression of PPFIBP2 ( Figure 4 (B) Continuous monitoring showed that, compared with the control group, PPFIBP2 overexpression significantly accelerated tumor growth, with both tumor volume and weight significantly increased at the endpoint (P < 0.001). Figure 4 (CE). This result further confirms the pro-proliferative and tumorigenic effects of PPFIBP2 in immune intact hosts, consistent with the high-proliferative clinical phenotype associated with PRC.

[0077] Based on this, flow cytometry results showed that CD8+ was present in tumors of the PPFIBP2 overexpression group. + The proportion of T cell infiltration was significantly lower than that in the control group, and IFN-γ +TNF-α + Functional CD8 + The absolute number of T cells decreased synchronously. Figure 4 (F, G). IHC staining further confirmed that in tumor regions with high PPFIBP2 expression, CD8... + T cell density was significantly reduced. Figure 4 (H). Enrichment analysis of bulk RNA-seq in mouse tumor tissues showed that cytokine-related pathways such as Cytokine–cytokine receptor interaction were significantly downregulated in the PPFIBP2 overexpression group. Figure 4 (I) This result is highly consistent with the cytokine pathway inhibition observed in the aforementioned human cell lines and clinical cohorts, suggesting that cytokine-mediated immune blockade is one of the key mechanisms of PPFIBP2-related immunosuppression.

[0078] To analyze the microenvironment remodeling process at single-cell resolution, we performed single-cell transcriptome analysis on tumors in the control group and the PPFIBP2 overexpression group (n = 3 / group). Figure 5 (A). Cell component analysis showed that the proportion of tumor epithelial cells was significantly increased in the PPFIBP2 overexpression group, while the proportions of T cells and myeloid cells were significantly decreased. Figure 5 (B and C). Further subgroup analysis showed that CD8 has antitumor activity. + Effector memory T cells (CD8) + Tem) and Th1 cells were significantly reduced or even nearly absent in the PPFIBP2 overexpression group. Figure 5 (D, E). Notably, based on T cell toxicity scores, there was no significant difference between the two groups ( Figure 5 The results (F) suggest that PPFIBP2-related immunosuppression can lead to T cell dysfunction. Consistent with this, in tumor cell clusters, the expression of key chemokines such as CCL3 / 4 / 5 was significantly downregulated in the PPFIBP2 overexpression group (F). Figure 5 (G), further supporting the view that low chemokine axis activity leads to limited recruitment of effector T cells.

[0079] At the mechanistic level, considering that NF-κB is a classic transcriptional activator of chemokines such as CCL3 / 4 / 5, we focused on evaluating the relationship between PPFIBP2 and NF-κB signaling. Pan-cancer analysis showed that PPFIBP2 expression was generally negatively correlated with NF-κB pathway activity scores in various tumor types, including bladder cancer, with the most significant correlation observed in bladder cancer. Figure 5(H). Further analysis using clinical proteomics, transcriptomics, and human cell line data revealed a persistent negative enrichment of NF-κB-related genes in the PPFIBP2-overexpressing group. At the single-cell level, NF-κB pathway activity scores were significantly decreased in both human PRC tumor cell subsets and mouse tumor cell clusters. Figure 5 (I, J). Western blot confirmed that knockdown of PPFIBP2 enhanced p65 phosphorylation levels, suggesting increased NF-κB activity. Figure 5 (Middle K). The above multiple pieces of evidence collectively point to a strong association between PPFIBP2 upregulation and NF-κB signal suppression.

[0080] In summary, PPFIBP2 plays a dual role in the tumor microenvironment as both a proliferative driver and an immune rejection regulator within tumor cells: on the one hand, its high expression continuously enhances the proliferation capacity of tumor epithelial cells and accelerates the increase in tumor burden; on the other hand, it may inhibit NF-κB signaling and downregulate key chemokines such as CCL3 / 4 / 5, thereby weakening cytokine-receptor interactions and chemokine signaling, and limiting CD8 at its source. + T cells enter the tumor parenchyma. This complex phenotype of high proliferation and immunosuppression is highly consistent with the characteristics defined in the aforementioned analysis.

[0081] 6. Design and evaluation of combination therapy strategies based on PPFIBP2 targeting In the C57BL / 6 allogeneic xenograft model, the present invention administered anti-PD-1 monoclonal antibody treatment or corresponding control treatment to the NC group and the experimental group, respectively. Figure 6 (Category A). Results showed that in the NC group with PRC characteristics, anti-PD-1 therapy alone did not significantly inhibit tumor growth; tumor volume and endpoint tumor weight were similar to the IgG control group, suggesting that PD-1 monotherapy is unlikely to exert a significant tumor-suppressive effect in the immunosuppressive microenvironment driven by high PPFIBP2 expression. Reducing PPFIBP2 expression alone significantly slowed tumor growth, indicating that relieving PPFIBP2 itself has a clear anti-tumor effect. Notably, tumor burden control was most significant in the combination therapy group, with tumor volume and weight significantly lower than either monotherapy group (Category A). Figure 6 (BD), suggesting that targeting PPFIBP2 can significantly enhance the anti-tumor effect of PD-1 blockade.

[0082] To clarify the relationship between combined therapeutic effects and anti-tumor immune remodeling, this invention further analyzes intratumoral CD8 in each group. + T cell infiltration and functional status. Flow cytometry showed that IFN-γ in the combination therapy group... + and TNF-α + Functional CD8 +The proportion of T cells was significantly higher in the PD-1 monotherapy group and the control group, suggesting that PPFIBP2 downregulation first restored CD8. + T cell recruitment to the tumor, and PD-1 blockade enhances the effector function of already infiltrated T cells, with both working synergistically to achieve the transition from immunosuppression to immunosensitivity. Figure 6 (E, F). IHC further confirmed at the in situ level that CD8+ was present in the tumor region of the combined treatment group. + T cell infiltration density was significantly increased, consistent with flow cytometry analysis. Figure 6 (G).

[0083] This invention provides a novel approach to bladder cancer treatment, combining a PPFIBP2-targeting agent with a PD-1 inhibitor, which can further enhance anti-tumor efficacy on top of existing immune checkpoint blockade. The combined application described in this invention reduces tumor volume and endpoint tumor weight more effectively than either agent alone, demonstrating a more definitive tumor burden control capability. This combined application can increase intratumoral CD8-positive T cell infiltration levels and enhance the proportion of functional CD8-positive T cells such as IFN-gamma-positive and TNF-alpha-positive cells, thereby improving the immune microenvironment of bladder cancer. This invention constructs a combined regimen around the key target PPFIBP2, maintaining a clear technical route and providing a translational basis for subsequent optimized treatment of patients with low immune response.

Claims

1. Use of PPFIBP2 as a target in the preparation of pharmaceutical compositions for the treatment of bladder cancer.

2. The use according to claim 1, characterized in that: Knocking down PPFIBP2 can inhibit the malignant proliferation of bladder cancer cells and reduce tumor growth rate; at the same time, it can restore or enhance the expression of chemokines, promote the recruitment and infiltration of CD8 positive T cells, and enhance the anti-tumor immune effect.

3. The use according to claim 1, characterized in that: The pharmaceutical composition for treating bladder cancer is a formulation that targets PPFIBP2; the formulation that targets PPFIBP2 is a formulation that can reduce the expression level of PPFIBP2 or inhibit the biological function of PPFIBP2.

4. The use according to claim 3, characterized in that: The formulation targeting PPFIBP2 is shRNA.

5. A pharmaceutical composition for treating bladder cancer, characterized in that: The pharmaceutical composition contains a formulation that targets PPFIBP2.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition also contains a PD-1 inhibitor.

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