Targeted PDCD10 cancer treatment method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a PDCD10-targeted cancer treatment method and application thereof. The invention reveals for the first time that PDCD10 gene deletion can significantly improve the expression level of MHC-I on the surface of a tumor cell by inhibiting a lysosome degradation pathway of MHC-I molecules, thereby enhancing CD8 + T cell-mediated anti-tumor immune response. The deletion of PDCD10 does not influence the proliferation of tumor cells, and the effects of up-regulating MHC-I expression and enhancing anti-tumor immunity are proved in various mouse and human tumor cells, thereby providing a brand new target and strategy for overcoming the drug resistance of immune checkpoint inhibitors (ICIs), having broad spectrum and safety, and having broad application prospects. The polypeptide can be developed into a plurality of cancer treatment schemes such as PDCD10-targeted gene therapy and small-molecule inhibitors, and has extremely high clinical transformation value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cancer treatment method targeting PDCD10 and its application. Background Technology

[0002] Cancer (malignant tumors) is one of the leading causes of death worldwide. Immune checkpoint inhibitors (ICIs), as a breakthrough in cancer treatment, are currently a highly anticipated cancer immunotherapy approach, demonstrating significant efficacy in clinical applications. However, a considerable number of patients still do not respond to ICIs or experience poor efficacy. Therefore, developing new treatment strategies or enhancing the efficacy of ICIs has become a hot research topic in this field.

[0003] A key reason for the poor response or resistance of tumors to ICIs (Invasive Clinical Injectors) therapy lies in the fact that tumor cells can evade immune surveillance through various mechanisms. For example, tumor cells can genetically induce defects in antigen processing and presentation (APP), thereby evading recognition by CD8+ cytotoxic T lymphocytes (CTLs). Therefore, the current lack of efficient ICI resistance reversal strategies, broad-spectrum anti-tumor therapies, and precise methods for assessing resistance risk necessitates the development of a novel, highly targeted, and widely applicable cancer treatment technology, while also expanding its application value in drug development and clinical evaluation. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a cancer treatment method targeting PDCD10 and its application. Furthermore, it reveals for the first time the regulatory mechanism of PDCD10 (Programmed Cell Death Protein 10) on the expression of MHC-I molecules in tumor cells, providing a novel approach for cancer treatment, especially for overcoming ICI resistance.

[0005] Technical solution:

[0006] A cancer treatment application targeting PDCD10, wherein the drug enhances the expression level of MHC-I molecules on the surface of tumor cells by inhibiting the expression or activity of PDCD10 in tumor cells, thereby enhancing the anti-tumor immune response mediated by CD8+ T cells; the cancers include solid tumors such as melanoma, colon cancer, lung cancer, liver cancer, and leukemia, as well as non-solid tumors.

[0007] This invention also provides a cancer treatment method targeting PDCD10, the method comprising the following steps:

[0008] S1. Constructing a tumor cell intervention system that inhibits PDCD10: Inhibiting the expression or function of PDCD10 in tumor cells through gene editing (such as CRISPR / Cas9), small molecule inhibitors, RNA interference, and other means;

[0009] S2. Verification of intervention effect:

[0010] S21. Molecular level verification: The expression levels of MHC-I molecules on the surface of tumor cells and in total protein were detected to confirm their upregulation;

[0011] S22. Mechanism verification: It was confirmed that PDCD10 inhibition prolongs the half-life of MHC-I molecules by blocking the lysosomal pathway, without affecting the mRNA levels of MHC-I molecules and related regulatory factors;

[0012] S23. Verification of immune efficacy: The killing efficiency of CD8+ T cells against tumor cells, the amount of IFNγ secreted, and the amount of CD8+ T cell infiltration in tumor tissue were detected to confirm the enhanced anti-tumor immune response;

[0013] S24. Safety verification: Tumor cell proliferation activity was tested to confirm that PDCD10 inhibition does not affect normal cell growth;

[0014] S3. Clinical Application: The PDCD10 intervention system can be used alone or in combination with ICIs for the treatment of cancer patients, or it can be prepared into gene therapy preparations, small molecule drugs, RNAi drugs and other products.

[0015] 1.3 Application of PDCD10 as a biomarker for ICI resistance assessment

[0016] The expression level of PDCD10 in tumor tissue of cancer patients is detected. When PDCD10 is highly expressed, the patient is identified as a high-risk group for ICIs resistance; when PDCD10 is low expressed, the patient is identified as a sensitive group for ICIs, and personalized treatment plans are formulated accordingly.

[0017] 1.4 Method for constructing PDCD10 knockout tumor cell lines, comprising the following steps:

[0018] S41. Plasmid preparation: Extraction of vector control plasmid, PDCD10 sgRNA lentiviral plasmid and packaging plasmids psPAX2, pMD2.G;

[0019] S42. Lentiviral packaging: Lentiviral plasmids and packaging plasmids were co-transfected into 293T cells, and the viral supernatant was collected and concentrated after culture.

[0020] S43. Cell transfection: Infect tumor cells with a mixture of concentrated virus solution and polybrene, and repeat the transfection 2-3 times;

[0021] S44. Screening of stable cells: Puromycin was used for drug screening for 7 days, followed by culture in normal medium;

[0022] S45. Knockout Verification: PDCD10 protein expression was detected by Western Blot, confirming successful knockout.

[0023] Key Invention Points: This invention reveals for the first time that PDCD10 gene deletion can significantly increase the expression level of MHC-I on the surface of tumor cells by inhibiting the lysosomal degradation pathway of MHC-I molecules, thereby enhancing the CD8+ T cell-mediated anti-tumor immune response.

[0024] (1) Novel target mechanism: It was found that the loss of PDCD10 can upregulate the expression level of MHC-I protein in tumor cells, significantly enhance the killing of tumors by T cells and the infiltration of CD8+ T cells, and found that PDCD10 is a key target for regulating the degradation of MHC-I molecules. Its loss significantly prolongs the half-life of MHC-I by blocking the lysosomal pathway.

[0025] (2) Synergistic effect value: The mechanism by which PDCD10 deficiency upregulates the expression of MHC-I in tumor cells provides a new target for developing small molecule inhibitors against the tumor MHC-I degradation pathway, a new strategy for overcoming ICI resistance, and a new idea for developing new biomarkers to assess the risk of drug resistance in cancer or patients.

[0026] (3) Wide range of applications: The upregulation of MHC-I expression in tumor cells by PDCD10 deficiency has been verified in in vitro and in vivo experiments of several murine tumor cells such as B16-F10, MC38, and LLC, and has been preliminarily verified in in vitro experiments of several human tumor cells such as A549, Hep-3B, and THP-1, providing a new strategy for a variety of cancer therapies including solid tumors and non-solid tumors.

[0027] (4) Strong treatment safety: PDCD10 knockout does not affect cell growth and proliferation, suggesting the potential safety of targeted intervention.

[0028] Beneficial effects of the present invention

[0029] (1) Mechanism Innovation Breakthrough, Overcoming the Core Bottleneck of ICI Resistance: This invention reveals for the first time that PDCD10 negatively regulates the expression of MHC-I molecules through specific regulation of the lysosomal degradation pathway, breaking through the limitations of traditional research that only focuses on transcriptional regulation, and clearly identifying PDCD10 as a novel key target of the tumor antigen presentation pathway. By blocking PDCD10 function, the expression of MHC-I in tumor cells can be significantly enhanced at the protein degradation level, repairing antigen presentation defects, and fundamentally solving the problem of ICI non-responsiveness and drug resistance caused by immune recognition barriers, providing a new mechanism of action for tumor immunotherapy.

[0030] (2) Significant anti-tumor effects and broad-spectrum therapeutic value: In vitro experiments have confirmed that targeting PDCD10 can significantly enhance the tumor cells' sensitivity to CD8+ T cells, increase T cell IFNγ secretion, and efficiently activate the anti-tumor immune response; in in vivo animal models, it can significantly inhibit tumor growth, reduce tumor volume and weight, prolong host survival, and promote the infiltration of CD8+ T cells in tumor tissues, forming a positive feedback loop of "enhanced antigen presentation → T cell activation → tumor clearance". At the same time, this effect has been verified in six types of murine and human tumor models, including melanoma, colon cancer, lung cancer, liver cancer, and leukemia, covering both solid and non-solid tumors, and has the potential for broad-spectrum treatment across cancer types.

[0031] (3) Excellent safety profile and low risk of clinical translation: Experiments have shown that PDCD10 knockout does not affect the proliferation of tumor cells or the function of normal cells, thus avoiding the toxic damage to normal tissues caused by traditional chemotherapy drugs and some targeted drugs. Moreover, its regulatory mechanism relies on protein degradation pathways and does not involve gene transcription reprogramming, which significantly reduces the risk of off-target effects and provides a good safety guarantee for clinical application, making it highly feasible for clinical translation.

[0032] (4) Significant synergistic effects and expanded application value across multiple scenarios: Targeting PDCD10 has a natural synergistic effect with ICIs therapy. By repairing tumor immunogenicity, it can create response conditions for ICIs such as PD-1 / PD-L1 inhibitors, which is especially suitable for patients resistant to existing ICIs, significantly broadening the applicable population of ICIs. At the same time, based on the functional mechanism of PDCD10, multimodal therapeutic products such as gene therapy (such as CRISPR-mediated knockout agents), small molecule inhibitors, and RNAi drugs can be developed to adapt to different clinical treatment scenarios. In addition, PDCD10 can also serve as a biomarker for assessing the risk of ICI resistance in cancer patients, providing a scientific basis for precise treatment planning and realizing the integrated application of "treatment-assessment".

[0033] (5) Balancing academic and applied value to promote the development of the field of tumor treatment: This invention fills the research gap of PDCD10 in the field of tumor immune regulation, clarifies its core role in the antigen presentation pathway, and provides a new perspective for the study of tumor immune escape mechanisms. At the same time, the complete technical system of "target discovery-mechanism verification-efficacy confirmation-safety assessment" it constructs not only provides standardized experimental basis for the development of drugs targeting PDCD10, but also provides a reference paradigm for the discovery and application of other tumor immune targets, which is of great significance to promoting the technological progress in the field of precision immunotherapy for tumors. Attached Figure Description

[0034] Figure 1 PDCD10 knockout increases MHC-I expression on tumor cell membrane surface: Figures (A)-(G) show flow cytometry (left) and mean fluorescence intensity (MFI) statistics (right) of murine tumor cells B16-F10 (A), MC38 (B), LLC (C), human tumor cells A549 (D), Hep-3B (E), THP-1 (F), and Ramos (G), respectively.

[0035] Figure 2 PDCD10 knockout does not affect the mRNA expression levels of MHC-I genes and MHC-I regulatory genes in tumor cells: Figures (A)-(E) show the gene expression of B16-F10 (A), MC38 (B), LLC (C), A549 (D), and Hep-3B (E), respectively.

[0036] Figure 3 PDCD10 knockout increases the expression of total protein MHC-I in tumor cells: Figures (A)-(E) show the protein expression in B16-F10 (A), MC38 (B), LLC (C), A549 (D), and Hep-3B (E) cells, respectively.

[0037] Figure 4 PDCD10 mainly mediates HLA degradation through the lysosomal pathway: Western blot analysis was performed on the HLA protein expression of control and knockout cells after adding 10 μg / mL cycloheximide (CHX, A), 10 mM proteasome inhibitor MG132 (B), and 50 mM chloroquine (CQ, C) for 0, 1, 4, 8, and 18 h, respectively.

[0038] Figure 5In vitro OT-I killing experiments confirmed that PDCD10 knockout significantly enhanced the sensitivity of tumor cells to CD8+ T cell killing: Figures (A) and (C) show the tumor cell viability after 24 h of co-culture with OT1-T cells in the B16-F10 and MC38 cell control and knockout groups, respectively, with E:T representing the ratio of OT1-T cells to tumor cells; Figures (B) and (D) show the flow cytometry data (left) and cell proportion statistics (right) of T cell IFNg secretion after co-culturing OT1-T cells with B16-F10 and MC38 cell lines, respectively.

[0039] Figure 6 In vivo animal tumor model experiments confirmed that PDCD10 knockout significantly inhibited tumor growth: Figures (A)-(C) show the host survival (A), tumor growth curve (B), and tumor weight (C) of the B16-F10 cell line, respectively; Figures (D)-(F) show the host survival (D), tumor growth curve (E), and tumor weight (F) of the MC38 cell line, respectively; Figures (G)-(I) show the host survival (G), tumor growth curve (H), and tumor weight (I) of the LLC cell line, respectively; Figures (J)-(K) show tumor images of B16-F10 and LLC cells, respectively.

[0040] Figure 7 In vivo animal models validated that PDCD10 knockout increased MHC-I expression on the surface of tumor cells: Figures (A)-(C) show the flow cytometry (left) and MFI statistical plot (right) of MHC-I on the surface of B16-F10 (A), MC38 (B), and LLC (C) cell lines, respectively.

[0041] Figure 8 In vivo animal tumor model experiments confirmed that PDCD10 knockout promotes CD8+ T cell infiltration: Figures (A)-(C) show flow cytometry (left) and absolute cell count statistics (right) of CD4+ T cell and CD8+ T cell infiltration in tumor sites of B16-F10 (A), MC38 (B), and LLC (C), respectively.

[0042] Figure 9 PDCD10 knockout does not alter tumor cell proliferation activity: Figures (A)-(B) show the in vitro proliferation curves of B16-F10 (A) and MC38 (B) control and knockout cell lines, respectively.

[0043] Figure 10 PDCD10 is highly expressed in cancer tissues and is associated with the prognosis of cancer patients: Figure (A) shows the expression of PDCD10 mRNA in human tumor tissues and corresponding normal tissues in the TCGA database; Figures (B)-(G) show the Kaplan-Meier survival curve analysis of the impact of PDCD10 expression on the survival of cancer patients. Detailed Implementation

[0044] To make the above-mentioned objectives, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples. The following content is merely an example and illustration of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not deviate from the concept of the invention, they should all fall within the protection scope of the present invention.

[0045] Example 1

[0046] Construction of PDCD10 knockout cell lines:

[0047] (1) The vector control plasmid lentiCRISPR V2-PDCD10-sgNC-PuroR, the lentiviral plasmid lentiCRISPR V2-PDCD10-sgRNA-PuroR, and the two packaging plasmids psPAX2 and pMD2.G were extracted with high purity and free of endotoxin. The lentiviral plasmid and the two packaging plasmids were co-transfected into 293T cells. After 6 h, the complete culture medium was replaced. After 24 and 48 h of culture, the cell supernatant rich in lentiviral particles was collected, and virus concentrate was added, mixed, and incubated overnight. The cells were then centrifuged at 4°C for concentration and collection.

[0048] (2) Beforehand, seed tumor cells in 6-well plates. When the density reaches about 40%, remove the original culture medium and add 0.5 mL of virus concentrate + 1 mL of complete culture medium + 8 μg / mL of polybrene. Incubate at 37°C for 12-24 h for transfection. Repeat this step 2-3 times.

[0049] (3) When the cell density reaches about 70%, replace the culture medium with a complete medium containing puromycin (Puro) for drug screening. The concentration of puro depends on the cell tumor.

[0050] (4) After 7 days of Puro drug screening, the cells were cultured in normal complete culture medium for more than 24 hours to obtain different mouse and human stable cell lines with PDCD10 knockout and corresponding vector control cell lines.

[0051] (5) Protein samples were extracted from control and knockout cells respectively, and the knockout status was confirmed using PDCD10 antibody via Western blotting. The verification results are as follows: Figure 3 As shown, this indicates that the PDCD10 knockout cell line is almost completely knocked out.

[0052] Example 2

[0053] PDCD10 knockout increases the expression of MHC-I on the cell membrane surface:

[0054] (1) Control cells and PDCD10 knockout cells were seeded in 24-well plates, with 3 replicates for each group. After the adherent cells adhered, the stimulation group was stimulated with 10 ng / mL IFNg for 24 h.

[0055] (2) Digest and collect cells, wash with PBS, stain with the corresponding MHC-I flow cytometry antibody at 4°C in the dark for 30 minutes, wash with FACS buffer and resuspend cells, analyze samples using BD FACSCelesta flow cytometer, process flow cytometry data using FlowJo software, and perform statistical analysis of data using Graphpad Prism8.

[0056] (3) Experimental results are as follows Figure 1 As shown, knocking out PDCD10 on the surface of tumor cells in mouse B16-F10, MC38, LLC tumor cell lines and human A549, Hep-3B, THP1, and Ramos tumor cell lines can increase the expression of MHC-I or HLA on the surface of tumor cells.

[0057] Example 3

[0058] PDCD10 knockout does not affect changes in the mRNA levels of MHC-I and its regulatory factors in tumor cells.

[0059] (1) Control cells and PDCD10 knockout cells were seeded in 6-well plates. After the cells adhered, 10 ng / mL IFNg was added at appropriate times to stimulate them.

[0060] (2) Digest and collect cells, resuspend in 500uL Trizol, add 100uL chloroform, mix well, centrifuge at 12000rpm at 4℃ to separate layers, take 250uL of supernatant, add 250uL isopropanol, mix well, centrifuge at 12000rpm at 4℃ to obtain precipitate, wash twice with 75% ethanol prepared with DEPC water, air dry the precipitate, add 20-40uL DEPC water to dissolve, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the concentration and quality of the extracted RNA.

[0061] (3) Use reverse transcription reagent to reverse transcribe 1ug RNA sample into cDNA, dilute 10 times as template, prepare qPCR reaction mixture with SYBR Green, perform real-time fluorescence quantitative analysis using qPCR instrument, and process and analyze data using Excel and Graphpad Prism8.

[0062] (4) qRT-PCR results are as follows Figure 2As shown, PDCD10 knockout did not affect the mRNA levels of H2-Kb, H2-Db, B2M, HLA-ABC, Nlrc5, IRF1, IRF2, and ERAP1 genes in mouse B16-F10, MC38, LLC cell lines and human A549 and Hep-3B cell lines, i.e., it did not affect the mRNA levels of MHC-I genes and MHC-I regulatory genes.

[0063] Example 4

[0064] PDCD10 knockout increases the expression level of total MHC-I protein in tumor cells:

[0065] (1) Control and knockout cells were seeded into 6-well plates respectively. Due to the low MHC-I basal expression in murine tumor cells, 10 ng / mL IFNg was added for 24 h after cell adhesion. Human tumor cell lines had a higher HLA basal expression level and were not stimulated with IFNg.

[0066] (2) Digest and collect cells, wash with PBS, add 200uL RIPA lysis buffer (containing PMSF protease inhibitor) to reselect cells, vortex to mix, lyse on ice for 5min, repeat twice, centrifuge at 12000rpm at 4℃ for 10min, transfer the supernatant to a new 1.5mL centrifuge tube, add 5× loading buffer, mix well, and boil in a metal bath at 100℃ for 10min to obtain total protein sample.

[0067] (3) Western Blot detection: Prepare 10% SDS-PAGE polyacrylamide gel. After separating the protein samples by SDS-PAGE polyacrylamide gel electrophoresis, transfer them to PVDF membrane by wet transfer. After blocking, incubate with MHC-I, PDCD10 and β-Actin antibodies respectively. After overnight incubation, wash 3 times with TBST and incubate with the corresponding mouse or rabbit anti-secondary antibodies respectively. After 1 hour, wash 3 times with TBST and add developing solution for development.

[0068] (4) Western Blot results are as follows Figure 3 As shown, PDCD10 knockout increases the expression of total protein MHC-I in tumor cells.

[0069] Example 5

[0070] PDCD10 primarily negatively regulates MHC-I expression through the lysosomal degradation pathway.

[0071] (1) A549 control cells and knockout cells were seeded in 6-well plates. When the cells adhered and reached a density of 70%, 10 μg / mL cycloheximide (CHX, A), 10 mM proteasome inhibitor MG132 (B), and 50 mM chloroquine (CQ, C) were added, respectively. Samples were collected at 0, 1, 4, 8, and 18 h, and total protein was extracted. MHC-I expression was detected by Western blotting. The results of the Western blotting are shown below. Figure 4 As shown.

[0072] (2) CHX inhibits protein synthesis. Figure 4 A showed that PDCD10 knockout cells had a longer MHC-I half-life, suggesting that PDCD10 may mediate MHC-I degradation.

[0073] (3) MG132 is a proteasome inhibitor that inhibits the proteasome degradation pathway. Figure 4 B showed that MG132 treatment did not lead to the accumulation of MHC-I protein, and there was no change in the difference in MHC-I expression levels between control cells and knockout cells, suggesting that PDCD10-mediated MHC-I degradation occurs through pathways other than the proteasome.

[0074] (4) CQ is a lysosomal inhibitor. Figure 4 C showed that CQ treatment led to the accumulation of MHC-I protein, and as the inhibition time increased, the difference in MHC-I expression between control cells and knockout cells gradually narrowed or even disappeared, suggesting that PDCD10 may mediate MHC-I degradation through the lysosomal pathway.

[0075] Example 6

[0076] In vitro OT-I killing experiments confirmed that PDCD10 knockout significantly increased the sensitivity of tumor cells to CD8+ T cell killing:

[0077] (1) After euthanizing an OT-1 mouse, its spleen and lymph nodes were taken, ground in 1640 complete medium, passed through a sieve to a 15 mL centrifuge tube, and collected by centrifugation. Red blood cell lysis buffer ACK was used to lyse for 3 min, and then terminated with 1640 complete medium and washed. After resuspending, the cells were passed through a sieve to a 1 mL centrifuge tube, plated into a 24-well plate, and OVA peptide (257-264 peptide segment) was added at the same time for activation.

[0078] (2) On the day of T cell activation 48h, CFSE-stained control cells and knockout cells were seeded into 24-well plates in advance. After the cells adhered, OVA peptide was added and incubated for 2h. The activated T cells were counted. T cells were added to tumor cells at four ratios of T cells:tumor cells: 0:1, 1:1, 2:1, and 5:1. The cells were collected 24h later. One group was stained with DAPI and the tumor cell viability was detected by flow cytometry. The other group was stained with CD8 on the cell membrane surface, fixed and ruptured, stained with IFNg, and the IFNg level secreted by OT1CD8+ T cells was detected by flow cytometry.

[0079] (3) Results of the OT1 killing experiment are as follows Figure 5 As shown, Figure 5 A,5C showed that the PDCD10 knockout group had lower tumor cell survival rate and stronger T cell killing effect; Figure 5 B and 5D showed that the PDCD10 knockout group had higher levels of IFNg secretion by T cells, confirming that PDCD10 knockout significantly increased the sensitivity of tumor cells to CD8+ T cell killing.

[0080] Example 7

[0081] Animal tumor models confirmed that PDCD10 knockout significantly inhibited tumor growth:

[0082] (1) Take C57 WT mice, divide them into two groups of 6-8 mice each, and subcutaneously inoculate them with equal amounts of control cells and PDCD10 knockout cells respectively. After one week, the tumor size is measured and counted every two days.

[0083] (2) Survival statistics: The length and width of the tumor were measured, and the tumor size was expressed as the value of length × width. When the tumor size of the mouse reached 225 mm2, the mouse was euthanized, and the survival curve of the mouse was plotted. Figure 6 A, 6D, and 6G studies showed that in the B16-F10, MC38, LLC tumor model, mice inoculated with PDCD10-deficient tumors had longer survival.

[0084] (3) Tumor growth curve and weight statistics: Measure the length and width of the tumor, and use the value of tumor length × width to represent the size of the tumor, and draw the tumor growth curve accordingly. Figure 6 B, 6E, and 6H show that PDCD10 knockout inhibits tumor growth in vivo in the B16-F10, MC38, LLC tumor model. Figure 6 J, 6K is a photograph taken after the tumor was removed; Figure 6 C, 6F, and 6I represent the weighing results of the B16-F10, MC38, and LLC tumor models, respectively.

[0085] Example 8

[0086] Animal model validation shows that PDCD10 knockout increases MHC-I expression on the surface of tumor cells:

[0087] (1) Take C57 WT mice, divide them into two groups of 6-8 mice each, and subcutaneously inoculate them with equal amounts of control cells and PDCD10 knockout cells respectively.

[0088] (2) The tumor was peeled off, ground, digested with DNase and collagenase, ACK split red, washed and sieved to obtain a single tumor cell suspension. A portion was stained with MHC-I flow cytometry antibody and the expression of MHC-I on the surface of tumor cells was analyzed by flow cytometry. Figure 7 A-7C flow cytometry and MFI statistics showed that, in the B16-F10, MC38, LLC tumor models, knocking out PDCD10 increased the expression of MHC-I on the surface of tumor cells in vivo.

[0089] Example 9

[0090] Animal models confirm that PDCD10 knockout in tumors promotes CD8+ T cell infiltration:

[0091] (1) Take C57 WT mice, divide them into two groups of 6-8 mice each, and subcutaneously inoculate them with equal amounts of control cells and PDCD10 knockout cells respectively.

[0092] (2) When the tumor grows to an average size of 225 mm2, the mice are euthanized, the tumor is dissected, and after grinding, digestion with DNase and collagenase at 37°C, ACK splitting red, washing and sieving, the tumor-infiltrating immune cell single-cell suspension is obtained by enriching with lymphocyte separation fluid. A portion is stained with CD4 and CD8 flow cytometry antibodies, and the T cell infiltration in the tumor site is detected by flow cytometry.

[0093] (3) Flow cytometry analysis results are as follows Figure 8 As shown, in the B16-F10, MC38, LLC animal models, PDCD10 knockout resulted in greater CD8+ T cell infiltration at the tumor site.

[0094] Example 10

[0095] PDCD10 knockout does not alter tumor cell proliferation activity.

[0096] (1) Prepare control cells and PDCD10 knockout cell suspensions, count and seed them into 96-well plates (1500-2000 cells / well).

[0097] (2) Prepare CCK8 solution according to the ratio of culture medium: CCK8 = 10:1. Replace the original culture medium in the measurement wells with CCK8 solution, incubate at 37℃ for 2 hours, and measure the absorbance (OD) at 450nm using a microplate reader. Replace the remaining cells with fresh culture medium. Measurements were taken on days 0, 1, 2, 3, and 4.

[0098] (3) Measurement results of B16-F10 and MC38 control and knockout cell line CCK8 are as follows: Figure 9 As shown, PDCD10 knockout does not affect the in vitro proliferation and growth of tumor cells, confirming the scientific validity and rigor of the experimental results of Examples 1-9. It also suggests the potential safety of using it as a target and indicates its high potential for targeted therapy development.

[0099] Example 11

[0100] PDCD10 is highly expressed in cancer tissues and is associated with cancer patient prognosis:

[0101] The expression of PDCD10 across various cancer types was analyzed using the TCGA (Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression) databases. As shown in Figure A, the results indicate that PDCD10 mRNA expression in tumor sites is significantly higher than that in normal tissue sites across multiple cancer types.

[0102] Using the TCGA database, Kaplan-Meier survival curves were analyzed to determine the correlation between PDCD10 expression and prognosis in cancer patients. Figure BG shows that high PDCD10 expression was associated with poorer survival in various cancers, suggesting that PDCD10 may be involved in tumor progression or disease deterioration, indicating its potential as a prognostic biomarker.

Claims

1. A cancer therapeutic application targeting PDCD10, characterized in that: The application, by inhibiting the expression or activity of PDCD10 in tumor cells, enhances the expression level of MHC-I class molecules on the surface of tumor cells, and enhances the CD8+ T cell-mediated anti-tumor immune response.

2. A method of cancer treatment targeting PDCD10, characterized in that, The method comprises the following steps: S1. Inhibiting the expression or function of PDCD10 in tumor cells by gene editing, small molecule inhibitors or RNA interference; S2. Verify the PDCD10 inhibition effect, confirm that the MHC-I class molecule expression of tumor cells is up-regulated, the CD8+ T cell killing efficiency is enhanced, and the cell proliferation activity is not affected; S3. Apply the PDCD10 inhibition system alone or in combination with immune checkpoint inhibitors for cancer treatment.

3. The method of claim 2, wherein: In step S2, PDCD10 regulates the degradation of MHC-I class molecules through the lysosomal pathway, and PDCD10 inhibition can prolong the half-life of MHC-I class molecules.

4. An application of targeting PDCD10 as an ICI drug resistance evaluation biomarker.

5. Use according to claim 4, characterized in that: Detecting the expression level of PDCD10 in tumor tissues of cancer patients, high expression of PDCD10 indicates that the patient is a high-risk group for ICI drug resistance, and low expression of PDCD10 indicates that the patient is an ICI sensitive group.

6. A method of constructing a PDCD10 knock-out tumor cell line, characterized by, The method comprises the following steps: S41. Plasmid preparation: extract the vector control plasmid, PDCD10 sgRNA lentivirus plasmid and packaging plasmid psPAX2, pMD2.G; S42. Lentivirus packaging: co-transfect 293T cells with lentivirus plasmid and packaging plasmid, collect and concentrate the virus supernatant after culture; S43. Cell transfection: infect tumor cells after mixing virus concentrate with polybrene, repeat transfection 2-3 times; S44. Stable cell screening: drug screening with puromycin for 7 days, and replace with normal culture medium; S45. Knockout verification: detect PDCD10 protein expression by Western Blot to confirm successful knockout.

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