Use of ferritin heavy chain 1 (FTH1) inhibitor in preparation of anti-tumor drugs

Abstract

This invention discloses the application of ferritin heavy chain 1 (FTH1) inhibitors in the preparation of antitumor drugs. This invention collects pre-treatment FFPE tumor tissue from cancer patients receiving anti-PD-1 / L1 therapy. By integrating transcriptomic and proteomic data from baseline samples, FTH1 is screened as a key candidate molecule for immunotherapy resistance. Experiments demonstrate that FTH1 knockout significantly improves the sensitivity of cancer patients to anti-PD-1 / L1 immunotherapy and effectively inhibits tumor growth; conversely, FTH1 overexpression leads to resistance to anti-PD-1 / L1 immunotherapy. This invention can be applied to the development or preparation of antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of ferritin heavy chain 1 (FTH1) inhibitors in the preparation of antitumor drugs. Background Technology

[0002] Lung cancer is the most common malignant tumor worldwide, ranking first in both incidence and mortality among all cancers. Lung cancer is mainly divided into two categories: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), with NSCLC primarily consisting of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). In recent years, advancements in surgical treatment, radiotherapy, and targeted therapy have significantly extended the survival time of NSCLC patients; however, a large number of patients still do not benefit from these treatments. This is especially true for patients with advanced NSCLC, who often face limited treatment options and poor clinical prognosis.

[0003] Currently, with the rapid development of immunotherapy, immune checkpoint inhibitors targeting programmed death receptor-1 (PD-1) and its ligand (PD-L1) have become one of the most promising treatments for NSCLC. However, primary and secondary resistance are significant problems, with the objective response rate for patients with advanced NSCLC receiving PD-1 / L1 inhibitor monotherapy being only 26%–44.8%. Although immunotherapy combined with chemotherapy can improve the response rate to some extent (47.6%–63.5%), some patients still cannot achieve true clinical benefit due to intolerance to the side effects of chemotherapy. Therefore, finding new immunotherapy combination regimens to overcome immunotherapy resistance is a pressing clinical challenge that needs to be explored and resolved.

[0004] The efficacy of cancer immunotherapy depends on the dynamic interaction between tumor cells and the tumor immune microenvironment (TIME)—a complex system that regulates anti-tumor immune responses and therapeutic responses. TIME is composed of both immunostimulatory and immunosuppressive components, which interact dynamically through a complex signaling network, ultimately determining the efficacy of immunotherapy. Therefore, identifying key mechanisms that can reprogram the immunosuppressive TIME into an immune-active state has become a core strategy for developing novel combination therapies and improving therapeutic efficacy. Summary of the Invention

[0005] The technical problems to be solved by this invention are how to develop or prepare combination drugs of PD-1 / L1 inhibitors for cancer patients and / or how to develop or prepare molecular inhibitors of immunotherapy resistance in cancer patients and / or how to develop or prepare drugs to alleviate, treat or adjuvant therapies for cancer and / or how to develop immunotherapy resistance targets for cancer patients.

[0006] To address the aforementioned technical problems, this invention first provides the use of substances that inhibit or knock out FTH1 (ferritin heavy chain 1) protein or inhibit the function, activity, or expression of its mRNA or its encoding gene in the development or preparation of products having any of the following functions:

[0007] A1) Treatment of tumors; A2) Inhibit the occurrence and development of tumors; A3) Inhibits tumor metastasis; A4) Improve the prognosis of cancer patients; The FTH1 protein may be one of the following proteins: B1) The amino acid sequence is that of the protein in sequence 1 of the sequence listing; B2) The amino acid sequence is that of sequence 3 in the sequence listing; B3) A protein derived from B1) or B2) or having the same function as the protein shown in B1) or B2) by substitution and / or deletion and / or addition of one or more amino acid residues. B4) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1), B2) or B3).

[0008] To address the aforementioned technical problems, this invention also provides the application of the FTH1 protein described above as a therapeutic target in the preparation or screening of products having any of the following functions: C1) Drugs for treating tumors; C2) Drugs that inhibit the occurrence and development of tumors; C3) Drugs that inhibit tumor metastasis; C4) Drugs that improve the prognosis of tumors.

[0009] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0012] In the aforementioned proteins, the 80% or more identity can be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0013] In the above applications, the substance that regulates the activity or content of the protein may be a substance that knocks out the coding gene of the protein and / or a substance that regulates the expression of the coding gene of the protein.

[0014] In the above applications, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the gene transcription level; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0015] In the above applications, the regulation of gene expression can be achieved by inhibiting or reducing gene expression, which can be achieved by gene knockout or gene silencing.

[0016] Gene knockout refers to the inactivation of a specific target gene through changes in its DNA sequence.

[0017] Gene silencing refers to the phenomenon of reducing gene expression without altering the genomic DNA sequence. Gene silencing includes transcriptional silencing (such as DNA methylation and heterochromatinization) and posttranscriptional silencing (such as RNA interference and microRNA-mediated regulation).

[0018] In the above applications, the substance regulating gene expression can be a reagent that inhibits or reduces the expression of the gene. The reagent that inhibits or reduces the expression of the gene can be a gene knockout reagent, such as a reagent that knocks out the gene via CRISPR-Cas9, or a reagent that knocks out the gene via homologous recombination. The reagent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0019] In the above applications, the tumor may be lung cancer. In various specific embodiments of the present invention, the tumor is non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC).

[0020] The products described above also fall within the scope of protection of this invention.

[0021] The product may be a drug. The FTH1 protein may be derived from humans or mice.

[0022] To address the aforementioned technical problems, this invention also provides the use of substances that inhibit or knock out FTH1 protein or inhibit the function, activity, or expression of its mRNA or its encoding gene in the development or preparation of tumor immunotherapy products.

[0023] To address the aforementioned technical problems, the present invention also provides a composition that may contain an immunotherapeutic antibody and a substance that inhibits the function, activity, or expression of FTH1 protein or its mRNA or its encoding gene.

[0024] In one specific embodiment of the present invention, the substance that inhibits the function, activity or expression of FTH1 protein or its mRNA or its encoding gene is baicalin; the immunotherapy antibody is an anti-PD-1 antibody or / and an anti-PD-L1 antibody.

[0025] To address the aforementioned technical problems, the present invention also provides the application of the composition described above in the development or preparation of products having any of the following functions: A1) Treatment of tumors; A2) Inhibit the occurrence and development of tumors; A3) Inhibits tumor metastasis; A4) Improve the prognosis of cancer patients.

[0026] To address the aforementioned technical problems, the present invention also provides a method for constructing a tumor drug screening model, the method comprising constructing the model by inhibiting or reducing the activity of the proteins described above or the expression of the genes encoding the proteins described above in the model species.

[0027] In the above method, the inhibition or reduction of the activity of the protein described above in the target species, target species cell line, or target species tissue, and / or the expression level of the gene encoding the protein described above, can be achieved by knocking out the gene encoding the protein described above in the model species.

[0028] In the applications described above, the tumor may be a solid tumor or a metastatic tumor. The product described above may be a drug.

[0029] The tumors mentioned above can be solid tumors or metastatic tumors.

[0030] The above-mentioned inhibition or reduction of the expression level of the protein-coding gene in the target species, target species cell line or target species tissue can be achieved by any means in the prior art, so as to induce deletion mutation, insertion mutation or base change mutation in the gene, thereby reducing or losing gene function, specifically chemical mutagenesis, physical mutagenesis, RNAi, genome editing or homologous recombination, etc.

[0031] Among the aforementioned site-specific genome editing methods, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, clustered regularly interspaced short palindromic repeats / CRISPR-associated (CRISPR / Cas9 system) technology, and other technologies capable of site-specific genome editing can be employed. Regardless of the method used, the entire coding gene of the aforementioned protein can be targeted, or individual elements regulating the expression of the coding gene can be targeted, as long as gene function loss or reduction is achieved. For example, exons of the coding gene of the aforementioned protein can be used as targets.

[0032] The inhibition or reduction of the activity of the protein described above in the target species, target species cell line, or target species tissue, and / or the expression level of the gene encoding the protein described above, can be achieved by knocking out the gene encoding the protein described above in the target species, target species cell line, or target species tissue.

[0033] The method described above may include introducing a substance into the target species that inhibits or reduces the expression of the protein-coding genes described above; the substance that inhibits or reduces the expression of the protein-coding genes described above may be any of the following substances: c1) Nucleic acid molecules that inhibit or reduce the expression of the protein-coding genes mentioned above; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3).

[0034] The nucleic acid molecule mentioned above may be a DNA molecule that expresses gRNA targeting the protein-coding genes of B1), B2) or B3), or gRNA targeting the protein-coding genes of B1), B2) or B3).

[0035] The applications or methods described above are not for disease diagnosis. They are not intended to directly obtain disease diagnoses or health status results from living humans or animals.

[0036] The above applications or methods are for non-disease treatment purposes. They are not intended to restore or restore health or reduce suffering in living human or animal bodies.

[0037] This invention aims to identify key drug resistance molecules and related mechanisms in NSCLC immunotherapy. Primarily, it uses transcriptomic and proteomic data from baseline formalin-fixed paraffin-embedded (FFPE) samples of NSCLC patients receiving PD-1 / L1 blockade therapy to screen for key molecules of immunotherapy resistance. Combining clinical samples, functional experiments, co-immunoprecipitation (Co-IP), dual-luciferase reporter gene assays, and mouse models, it explores the molecular mechanisms of immunotherapy resistance. Inhibitors of these resistance molecules are identified through virtual screening based on molecular docking, and their efficacy and safety, both alone and in combination with PD-1 / L1 inhibitors, are investigated.

[0038] This invention explores the potential mechanisms of immunotherapy resistance in NSCLC: by collecting baseline tissue samples from NSCLC patients receiving PD-1 / L1 immunotherapy before treatment, transcriptomic and proteomic analyses were performed to screen for the key resistance molecule FTH1; and the close association between FTH1 and PD-1 / L1 immunotherapy resistance was verified using clinical tissue samples. Simultaneously, this invention also investigates the relevant mechanisms of the candidate molecule FTH1 in NSCLC immunotherapy resistance and explores the feasibility of combining FTH1 inhibitors (baicalin) with PD-1 inhibitors as a potential treatment regimen in a mouse model, aiming to provide ideas and clues for novel clinical treatments of NSCLC.

[0039] This invention found that NSCLC patients with higher FTH1 expression levels had poorer response to PD-1 / L1 blockade therapy. Transcriptomic data, immunohistochemical staining, and functional experiments showed that FTH1 was positively correlated with M2 macrophage infiltration and CD8 expression. + T cell infiltration was negatively correlated. Mechanistically, FTH1 promotes CXCL16-mediated M2 macrophage polarization and chemotaxis by activating the PPAR-γ signaling pathway; and FTH1 can bind to GATAD2B and act on the CXCL16 promoter, regulating CXCL16 transcription. Furthermore, the FTH1 inhibitor baicalin, when used in combination with PD-1 inhibitors in an NSCLC mouse model, effectively inhibited tumor growth and demonstrated reliable safety.

[0040] This invention clarifies that FTH1 is a key drug resistance molecule in NSCLC immunotherapy and elucidates its resistance mechanism. Baicalin overcomes resistance to anti-PD-1 / L1 immunotherapy in non-small cell lung cancer by reversing FTH1-mediated M2 macrophage immunosuppression. Importantly, the study observed that baicalin can enhance the efficacy of PD-1 blockers in NSCLC patients by inhibiting FTH1 expression without inducing serious adverse reactions. This finding provides new ideas and clues for developing NSCLC treatment regimens.

[0041] The beneficial effects of this invention are: Immunotherapy has revolutionized the treatment landscape of NSCLC, bringing significant survival benefits to some patients. However, primary and secondary drug resistance limits its efficacy; therefore, elucidating the resistance mechanisms and identifying potential combination therapy targets is urgent. This invention is the first to discover that FTH1 is a novel driver molecule for PD-1 / L1 immunotherapy resistance in NSCLC: FTH1 promotes CXCL16-mediated M2 macrophage polarization and recruitment, and reduces CD8+ through ferroptosis-dependent inhibition of HMGB1 release. +T cell infiltration constructs an immunosuppressive tumor immune microenvironment (TIME), thereby mediating resistance to anti-PD-1 / L1 immunotherapy. Furthermore, mouse models have demonstrated that the FTH1 inhibitor baicalin combined with a PD-1 inhibitor has significant efficacy in NSCLC, comparable to chemotherapy combined with a PD-1 inhibitor regimen, without significant organ toxicity.

[0042] This invention collected pre-treatment FFPE tumor tissue from NSCLC patients receiving anti-PD-1 / L1 immunotherapy. By integrating transcriptomic and proteomic data from baseline samples, FTH1 was screened as a key candidate molecule for immunotherapy resistance. Importantly, the gene regulation of FTH1 expression has a significant impact on the outcome of anti-PD-1 / L1 immunotherapy: FTH1 knockout significantly improves treatment sensitivity and effectively inhibits tumor growth in both LUAD and LUSC models; conversely, FTH1 overexpression leads to resistance to anti-PD-1 / L1 immunotherapy. These results collectively indicate that FTH1 is a key regulator of immunotherapy response in NSCLC, and inhibiting FTH1 may be an effective strategy to overcome immunotherapy resistance.

[0043] This invention further explores the mechanism by which FTH1 mediates immunotherapy resistance. Time-dependent immunotherapy (TIME) is widely considered a key determinant of immunotherapy efficacy, and its heterogeneity is a significant reason for the differences in clinical outcomes of anti-PD-1 / L1 immunotherapy. This invention is the first to discover that FTH1 remodels immunosuppressive TIME through two independent pathways: Firstly, nuclear-localized FTH1 binds to GATAD2B and acts on the CXCL16 promoter, regulating CXCL16 transcription and secretion (CXCL16 is a member of the α-chemokine subfamily, known to recruit and polarize M2 macrophages); M2 macrophages possess anti-inflammatory and immunosuppressive phenotypes and can exert pro-tumorigenic effects through various mechanisms. Secondly, FTH1 inhibits ferroptosis-dependent HMGB1 release, and HMGB1 is a key member of damage-associated molecular patterns (DAMPs). Ferropreservation can trigger immunogenic cell death by releasing HMGB1 and other DAMPs. These two pathways work synergistically to construct an immunosuppressive microenvironment, ultimately mediating anti-PD-1 / L1 immunotherapy resistance in NSCLC.

[0044] This invention is the first to discover that FTH1-mediated M2 macrophage immunosuppression is a novel mechanism of anti-PD-1 / L1 immunosuppression resistance in NSCLC; baicalin can effectively reverse this immunosuppressive microenvironment by targeting FTH1. These findings suggest that FTH1 holds promise as a predictive biomarker for immunotherapy response and a potential therapeutic target; baicalin combined with anti-PD-1 / L1 immunotherapy may be a novel strategy to overcome immunotherapy resistance in NSCLC patients. Attached Figure Description

[0045] Figure 1 To investigate the relationship between FTH1 expression and the efficacy of anti-PD-1 / L1 therapy in NSCLC patients. (A) Proteomic volcano plot showing differentially expressed proteins between PR and PD patients; (B) Transcriptomic volcano plot showing differentially expressed genes between PR and PD patients; (C) Venn diagram of proteomic and transcriptomic results; (D) Immunohistochemical analysis of FTH1 protein levels in PD and non-PD patients; (E) ROC curve analysis based on FTH1 protein levels in archived tissues (assessing progression-free survival (PFS) of anti-PD-1 / L1 therapy in NSCLC patients); (F) Kaplan-Meier survival analysis based on FTH1 protein levels in archived tissues (PFS of anti-PD-1 / L1 therapy in NSCLC patients); (G) ROC curve analysis based on FTH1 transcriptional levels in archived tissues (assessing PFS of anti-PD-1 / L1 therapy in NSCLC patients); (H) Kaplan-Meier survival analysis based on FTH1 transcriptional levels in archived tissues (PFS of anti-PD-1 / L1 therapy in NSCLC patients).

[0046] Figure 2 To reduce the antitumor efficacy of PD-1 inhibitors in immunocompetent mice. (A) Western blot detection of FTH1 expression in KLN-205 and LLC cells; (B) Schematic diagram of NSCLC mouse model establishment and administration regimen; (C) Tumor nodules in C57BL / 6 mice after inoculation with FTH1-NC, FTH1-KO, FTH1-VEC, and FTH1-OE LLC tumors; (D) Tumor volume changes in C57BL / 6 mice after inoculation with the above LLC tumors and treatment with anti-PD-1; (E) Tumor nodules in DBA-2J mice after inoculation with FTH1-NC, FTH1-KO, FTH1-VEC, and FTH1-OE KLN205 tumors; (F) Tumor volume changes in DBA-2J mice after inoculation with the above KLN205 tumors and treatment with anti-PD-1.

[0047] Figure 3FTH1 is a key molecule in tumor immune resistance. (A) CCK-8 assay to detect growth curves of LLC and KLN-205 cells (FTH1-NC, FTH1-KO, FTH1-VEC, FTH1-OE); (B) Changes in tumor volume in nude mice after inoculation with LLC and KLN-205 cells (FTH1-NC, FTH1-KO, FTH1-VEC, FTH1-OE); (C) Western blot to detect FTH1 expression in H358, H520, and A549 cells; (D) CCK-8 assay to detect growth curves of H358, H520, and A549 cells (FTH1-NC, FTH1-KO, FTH1-VEC, FTH1-OE); (E) Changes in tumor volume in nude mice after inoculation with H358, H520, and A549 cells (FTH1-NC, FTH1-KO, FTH1-VEC, FTH1-OE).

[0048] Figure 4 To investigate the role of FTH1 in promoting tumor-associated macrophage infiltration and M2 macrophage polarization. (A-B) Correlation analysis of FTH1 and CD163 expression in LUSC (A) and LUAD (B) from the TCGA cohort; (C-D) Correlation analysis of FTH1 expression and M2 macrophage infiltration in LUSC (C) and LUAD (D) from the TCGA cohort; (E-F) Correlation analysis of FTH1 expression and M2 macrophage infiltration in LUSC (E) and LUAD (F) from the CICAMS cohort; (G) Representative immunohistochemical images of FTH1 and M2 macrophages in LUAD and LUSC from the CICAMS cohort; (I-K) Transwell chemotaxis assay to detect the chemotactic effect of H358(I), H520(J), and A549(K) cells (FTH1-NC, FTH1-KO, FTH1-VEC, FTH1-OE) on PMA-activated macrophages; (L) Schematic diagram of the co-culture system of tumor cells and PMA-activated THP-1 macrophages; (M) RT-qPCR detection of M2 marker expression in PMA-activated THP-1 macrophages co-cultured with H358 and H520 cells (FTH1-NC, FTH1-KO, FTH1-VEC, FTH1-OE).

[0049] Figure 5FTH1 promotes tumor-associated macrophage chemotaxis and M2 polarization by regulating CXCL16 expression. (A-B) Correlation analysis of CXCL16 expression and M2 macrophage infiltration in LUSC (A) and LUAD (B) from the TCGA cohort; (C-D) Correlation analysis of CXCL16 expression and M2 macrophage infiltration in LUSC (C) and LUAD (D) from the CICAMS cohort; (E) Representative immunohistochemical images of CXCL16 in NSCLC from the CICAMS cohort; (F-G) Western spectral analysis. blot analysis was performed to detect the expression of CXCL16 and FTH1 in LLC, KLN-205, A549, H358, and H520 cells (FTH1-NC, FTH1-KO, FTH1-VEC, and FTH1-OE); (H~J) ELISA was used to detect the CXCL16 level in the supernatant of H358(H), H520(I), and A549(J) cells (FTH1-NC, FTH1-KO, FTH1-VEC, and FTH1-OE); (K~M) Transwell chemotaxis assay was used to detect the expression of H3... Chemotaxis of 58 (K), H520 (L), and A549 (M) cells (FTH1-OE and CXCL16 knockdown of FTH1-OE) on PMA-activated macrophages; (N) Chemotaxis of different concentrations of exogenous CXCL16 on PMA-activated macrophages; (O~Q) RT-qPCR detection of M2 marker expression in PMA-activated THP-1 macrophages co-cultured with A549 (O), H358 (P), and H520 (Q) cells (FTH1-OE and CXCL16 knockdown of FTH1-OE).

[0050] Figure 6CXCL16, derived from tumor cells, promotes M2 macrophage polarization by regulating the PPAR-γ signaling pathway. (A) Bubble diagram of differentially enriched signaling pathway genes in PMA-activated THP-1 macrophages after CXCL16 stimulation; (B) RT-qPCR detection of M2 marker expression in PMA-activated THP-1 macrophages induced by IL-4, IL-13, and CXCL16; (C) RT-qPCR detection of M2 marker expression in PMA-activated THP-1 macrophages stimulated by CXCL16 after CD36 inhibitor intervention; (D) RT-qPCR detection of PPAR-γ pathway molecule expression in PMA-activated THP-1 macrophages induced by IL-4, IL-13, and CXCL16; (E) Western blot detection of CD36 expression in PMA-activated THP-1 macrophages induced by IL-4, IL-13, and CXCL16; (F) Western blot detection of CD36 expression in PMA-activated THP-1 macrophages induced by IL-4, IL-13, and CXCL16; blot analysis was performed to detect the interaction between GATAD2B and FTH1 in the nuclei of H358, H520, and A549 cells (FTH1-OE); (G) Luciferase activity was detected by dual-luciferase reporter gene assay 24 hours after transfection of H358, H520, and A549 cells with FTH1 and GATAD2B overexpression plasmids and control plasmids; (H) RT-qPCR was performed to detect CXCL16 expression in the transfected cells.

[0051] Figure 7 To demonstrate the synergistic anti-tumor immune response of baicalin combined with anti-PD-1 immunotherapy in a mouse model of NSCLC. (A) Molecular docking binding energy diagram of baicalin and FTH1 protein; (B) Details of NSCLC mouse model establishment and treatment administration regimen; (C) Tumor nodules in C57BL / 6 mice inoculated with FTH1-VEC and FTH1-OE LLC tumors and treated with baicalin, baicalin combined with PD-1 antibody, and PD-1 antibody combined with chemotherapy; (D) Tumor volume changes in the above C57BL / 6 mice; (E) Tumor nodules in DBA-2J mice inoculated with FTH1-VEC and FTH1-OE KLN205 tumors and treated with the above three treatments; (F) Tumor volume changes in the above DBA-2J mice.

[0052] Figure 8Analysis of tumor immune microenvironment infiltration in a mouse model of LLC lung adenocarcinoma immunotherapy. (A) Typical schematic diagram of M2 macrophage and CD8+ T lymphocyte infiltration before and after drug intervention in FTH1 knockout (LLC-KO) and control (LLC-NC) lung adenocarcinoma mouse models; (B) Infiltration levels of M2 macrophage and CD8+ T lymphocytes before and after drug intervention in FTH1 knockout / control lung adenocarcinoma mouse models; (C) Typical schematic diagram of M2 macrophage and CD8+ T lymphocyte infiltration before and after drug intervention in FTH1 overexpression / control lung adenocarcinoma mouse models; (D) Infiltration levels of M2 macrophage and CD8+ T lymphocytes before and after drug intervention in FTH1 overexpression / control lung adenocarcinoma mouse models.

[0053] Figure 9 This study analyzes the infiltration of the tumor immune microenvironment in a mouse model of KLN205 squamous cell carcinoma of the lung. (A) Typical schematic diagrams of M2 macrophage and CD8+ T lymphocyte infiltration before and after drug intervention in FTH1 knockout (KLN205-KO) and control (KLN205-NC) squamous cell carcinoma mouse models; (B) Infiltration levels of M2 macrophage and CD8+ T lymphocytes before and after drug intervention in FTH1 knockout / control squamous cell carcinoma mouse models; (C) Typical schematic diagrams of M2 macrophage and CD8+ T lymphocyte infiltration before and after drug intervention in FTH1 overexpression / control squamous cell carcinoma mouse models; (D) Infiltration levels of M2 macrophage and CD8+ T lymphocytes before and after drug intervention in FTH1 overexpression / control squamous cell carcinoma mouse models.

[0054] Figure 10 To investigate the effect of FTH1 expression on the chemotactic capacity of CD8+ T lymphocytes in NSCLC cells. (A) Effect of FTH1 knockout / control group on the chemotactic capacity of CD8+ T lymphocytes in A549, H358 and H520 cell lines; (B) ELISA detection of HMGB1 protein levels in FTH1 knockout / control group in A549, H358 and H520 cell lines.

[0055] Figure 11 Scutellaria baicalensis can effectively inhibit FTH1 expression in non-small cell lung cancer. (A) Western blot experiment to detect the effect of scutellaria baicalensis stimulation on FTH1 and CXCL16 expression in LLC, KLN-205, H358 and H520 wild-type cells.

[0056] Figure 12To analyze the infiltration of the tumor immune microenvironment in a FTH1-overexpressing mouse lung adenocarcinoma model. (A) FTH1 expression and M2 macrophage infiltration levels in subcutaneous tumor tissues of FTH1-overexpressing mice with lung adenocarcinoma after treatment with baicalin monotherapy, baicalin combined with anti-PD-1 (baicalin+PD-1 mAb), and anti-PD-1 combined with chemotherapy (PD-1 mAb+CT); (B) FTH1 expression and M2 macrophage infiltration levels in subcutaneous tumor tissues of FTH1-overexpressing mice with adenocarcinoma after treatment with baicalin monotherapy, baicalin combined with anti-PD-1, and anti-PD-1 combined with chemotherapy.

[0057] Figure 13 To analyze the tumor immune microenvironment infiltration in a FTH1-overexpressing squamous cell carcinoma mouse model. (A) FTH1 expression and M2 macrophage infiltration levels in subcutaneous tumor tissues of FTH1-overexpressing squamous cell carcinoma mice after treatment with baicalin monotherapy, baicalin combined with anti-PD-1 (baicalin+PD-1 mAb), and anti-PD-1 combined with chemotherapy (PD-1 mAb+CT); (B) FTH1 expression and M2 macrophage infiltration levels in subcutaneous tumor tissues of FTH1-overexpressing squamous cell carcinoma mice after treatment with baicalin monotherapy, baicalin combined with anti-PD-1, and anti-PD-1 combined with chemotherapy.

[0058] Figure 14 No significant serious adverse reactions were observed with the combined use of baicalin and anti-PD-1 monoclonal antibody. (A) In the LLC lung adenocarcinoma treatment model, the combined use of baicalin and anti-PD-1 monoclonal antibody did not cause significant tissue damage to important organs such as the heart, liver, spleen, lungs, and kidneys; (B) In the KLN205-lung squamous cell carcinoma treatment model, the combined use of baicalin and anti-PD-1 monoclonal antibody did not cause significant tissue damage to important organs such as the heart, liver, spleen, lungs, and kidneys. Detailed Implementation

[0059] Animal viruses: The public may obtain the biological material from the applicant in accordance with the relevant national biosafety regulations. The biological material is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0060] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0062] This invention included 12 NSCLC patients from the Cancer Hospital of the Chinese Academy of Medical Sciences (CICAMS, Beijing) who received PD-1 / L1 inhibitor monotherapy. Pretreatment baseline formalin-fixed paraffin-embedded (FFPE) specimens were collected to screen for key resistance molecules. Patient-related clinical characteristics are shown in Table 1. To validate the clinical value of the key resistance molecule FTH1, archived FFPE samples from 26 NSCLC patients from CICAMS who received PD-1 / L1 inhibitor monotherapy were also collected. Relevant clinical information is shown in Table 2. The treatment response was assessed using the Criteria for Evaluating the Efficacy of Treatment in Solid Tumors (Version 1.1). In addition, clinical cohort samples from 189 surgically resected NSCLC patients were collected from the CICAMS biobank for immunohistochemical exploration and analysis. Tumor and normal tissues were collected from all patients for analysis, and none had received radiotherapy or chemotherapy prior to enrollment. This invention has been approved by the CICAMS Ethics Committee, and all enrolled patients signed written informed consent forms.

[0063] Table 1. Clinical information of 12 patients included in the study who underwent transcriptome and proteome sequencing.

[0064] Table 2. Clinical information of NSCLC patients who received anti-PD-1 / L1 monoclonal antibody immunotherapy at CICAMS Hospital

[0065] RNA sequencing and data-independent acquisition (DIA) analysis: Twelve baseline FFPE samples were collected from 12 NSCLC patients who received PD-1 / L1 inhibitor monotherapy for transcriptomic and proteomic analysis. Transcriptomic sequencing was performed using Illumina Truseq technology, with mRNA enrichment via oligonucleotide dT magnetic beads. Proteomic analysis was conducted using data-independent acquisition (DIA) technology on a ThermoScientific Q Exactive HF high-resolution mass spectrometer. By integrating multi-omics data, biomarkers of resistance to anti-PD-1 / L1 therapy were screened.

[0066] The experimental methods used in the embodiments of this invention are as follows: Cell culture: This invention uses human NSCLC cell lines (A549, H358, H520) and mouse NSCLC cell lines (LLC, KLN-205). The A549, H358, and H520 cell lines were cultured in RPMI-1640 medium (Corning, catalog number 10-040-CV) containing 10% fetal bovine serum (FBS) and 1% streptomycin antibiotic; the LLC and KLN-205 cell lines were cultured in DMEM medium (Corning, catalog number 10-013-CVR) containing 10% FBS and 1% streptomycin antibiotic. All NSCLC cell lines were obtained from the American Type Culture Collection (ATCC).

[0067] Human CD8 + T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and used to extract CD8+ cells. + T-cell isolation kit (Miltenyi Biotec, catalog number 130-096-495); isolated CD8 cells + T cells were cultured in a medium containing CD3-28 activating magnetic beads (Miltenyi Biotec, catalog number 130-128-758) and 100 ng / mL interleukin-2 (IL-2). All cells were cultured in a 37°C, 5% CO2 incubator.

[0068] Lentiviral-mediated stable overexpression of FTH1: Using the synthesized gene fragment as a template, the human FTH1 coding gene (Sequence 2 in the sequence listing) and the mouse FTH1 coding gene (Sequence 4 in the sequence listing) were amplified by PCR and cloned into the lentiviral vectors pPS-BVC-LW334 (Beijing Hesheng Gene Co., Ltd.) and GL107 (Shanghai OBio Technology Co., Ltd.), respectively.

[0069] Sequence 2 (5'-3'): ATGACGACCGCGTCCACCTCGCAGGTGCGCCAGAACTACCACCAGGACTCAGAGGCCGCCATCAACCGCCAGATCAACCTGGAGCTCTACGCCTCCTACGTTTACCTGTCCATGTCTTACTACTTTGACCGCGATGATGTGGCTTTGAAGAACTTTGCCAAATACTTTCTTCACCAATCTCATGAGGAGAGGGAACATGCTGAGAAACTGATGAAGCTGCAGAACCAACGAGGTGGCCGAATCTTCCTTCAGGATATCAAGAAACCAGACTGTGATGACTGGGAGAGCGGGCTGAATGCAATGGAGTGTGCATTACATTTGGAAAAAAATGTGAATCAGTCACTACTGGAACTGCACAAACTGGCCACTGACAAAAATGACCCCCATTTGTGTGACTTCATTGAGACACATTACCTGAATGAGCAGGTGAAAGCCATCAAAGAATTGGGTGACCACGTGACCAACTTGCGCAAGATGGGAGCGCCCGAATCTGGCTTGGCGGAATATCTCTTTGACAAGCACACCCTGGGAGACAGTGATAATGAAAGC。

[0070] Sequence 4 (5'-3'): .

[0071] Lentivirals carrying the human FTH1 gene and the mouse FTH1 gene were prepared by co-transfecting HEK-293T cells with recombinant vectors and packaging systems (pLP1, pLP2, pLP / VSVG) using Lipofectamine™ 3000 (Invitrogen). Subsequently, human NSCLC cell lines (A549, H358, and H520) and mouse NSCLC cell lines (LLC and KLN205) derived from ATCC were infected with the lentiviruses in medium containing 5 μg / mL polybrene (Sigma-Aldrich, catalog number P4505). After screening with puromycin (Thermo Fisher Scientific, catalog number A1113803), the expression level of FTH1 in infected cells was verified by Western blot and real-time quantitative polymerase chain reaction (RT-qPCR). Finally, overexpressing cell lines (FTH1-OE) of human NSCLC cell lines (A549, H358 and H520) and overexpressing cell lines (LLC-FTH1-OE and KLN205-FTH1-OE) of mouse NSCLC cell lines were obtained.

[0072] FTH1 knockout cell construction: FTH1 knockout cell lines were constructed using the CRISPR-Cas9 system. Single guide RNAs (sgRNAs) targeting human FTH1 (sgRNA sequence: 5'-CATGGACAGGTAAACGTAGG-3') and mouse FTH1 (sgRNA sequence: 5'-GTAGTTCTGGCGCACTTGC-3') were cloned into the lentiviral vectors lentiCRISPR V2_U6 and pLV-hU6 (Beijing Hesheng Gene Co., Ltd.), respectively, and co-transfected with packaging plasmids into HEK-293T cells using Lipofectamine™ 3000 (Invitrogen). Viral supernatant was collected after 48 hours and stored at -80°C. Human NSCLC cell lines (A549, H358, and H520) and mouse NSCLC cell lines (LLC and KLN205) infected with the virus were selected with puromycin for 72 hours, and then isolated as single clones using limiting dilution. By performing Sanger sequencing and Western blot on the CRISPR-targeted genomic regions, the success of FTH1 knockout was verified. Ultimately, knockout cell lines (FTH1-KO) of human NSCLC cell lines (A549, H358, and H520) and mouse NSCLC cell lines (LLC-FTH1-KO and KLN205-FTH1-KO) were obtained.

[0073] Small interfering RNA (siRNA) transfection: Human CXCL16 protein knockdown was achieved by transfecting siRNA with Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific). Specific steps included: transfecting human NSCLC cell lines (H358, H520, and A549) at a concentration of 5 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 1 cell / well and transfected after reaching 70%–80% confluence. Following the optimized kit protocol, 1 μg of siRNA targeting CXCL16 (sequence: 5'-GGAAGUUGUUAUUGUGGUA-3') was transfected into the cells. Functional analysis was performed 24 hours after transfection.

[0074] Cell proliferation experiment: Cell proliferation capacity in vitro was assessed using the Cell Counting Kit-8 (CCK-8). A CCK-8 kit (Dojindo, catalog number CK04) was added to each well, and absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Varioskan LUX) at 0, 24, 48, 72, and 96 hours.

[0075] In vivo mouse model experiments: All animal experiments were conducted in accordance with guidelines approved by the CICAMS Animal Care and Use Committee. C57BL / 6J mice were obtained from Beijing Huafukang Biotechnology Co., Ltd. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 5 Tumor cells from several mouse NSCLC cell lines (LLC-FTH1-VEC, LLC-FTH1-OE, LLC-FTH1-NC, LLC-FTH1-KO, KLN205-FTH1-VEC, KLN205-FTH1-OE, KLN205-FTH1-NC, and KLN205-FTH1-KO cells) were suspended in 100 μL of phosphate-buffered saline (PBS) and subcutaneously injected into the right flank of mice to establish mouse tumor xenograft models (subcutaneous tumor models of LUAD's FTH1-VEC, FTH1-OE, FTH1-NC, and FTH1-KO cells, and mouse subcutaneous tumor models of LUSC's FTH1-VEC, FTH1-OE, FTH1-NC, and FTH1-KO cells). Tumor volume was measured every two days using calipers and calculated using the formula: volume = 0.5 × length × width², with the maximum tumor diameter controlled to be within 2.0 cm. LLC-FTH1-VEC and KLN205-FTH1-VEC are LLC and KLN205 transgenic empty vector cell lines, respectively; LLC-FTH1-NC and KLN205-FTH1-NC are LLC and KLN205 control cell lines, respectively.

[0076] Drug intervention was initiated when the tumor volume reached 100±25 mm³ (mean ± standard deviation). All drugs were administered via intraperitoneal injection at a dose of 10 mg / kg, as detailed below: - PD-1 antibody (BioXCell, catalog number BP0273): Dosage on days 1, 4, 7, and 10 (10 mg / kg / dose); - Baicalin (Selleck, catalog number S2269): Administer on days 1, 3, 5, 7, 9, and 11 (10 mg / kg / dose); - Paclitaxel (Selleck, catalog number S1150): Administered on days 1, 5, and 9 (10 mg / kg / dose); - Cisplatin (Selleck, catalog number S1166): Administer on days 1 and 8 (10 mg / kg / dose); - Pemetrexed (Selleck, catalog number S5971): Administer on days 1, 4, 8, and 11 (2 mg / kg / dose).

[0077] The control group received 100 uL of PBS at the same administration time and volume.

[0078] Enzyme-linked immunosorbent assay (ELISA): The secretion levels of CXCL6 and HMGB1 in cell supernatant were detected using an HMGB1 (MultiSciences, catalog number JL13693) and CXCL16 (MultiSciences, catalog number JL11662) ELISA kit. The absorbance of each well at 450 nm was measured using a microplate reader (ThermoFisher Scientific, Varioskan LUX).

[0079] Real-time quantitative polymerase chain reaction (RT-qPCR) analysis: Total RNA was extracted from various cell types using a rapid RNA purification kit (Esunbio, catalog number ES-RN001); cDNA was synthesized using a FastKing reverse transcription kit (TIANGEN, catalog number KR116); real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using an ABI 7900HT real-time PCR instrument with SYBR Green mixture (QIAGEN, catalog number 330502). GAPDH was used as an internal control gene, and 2... - The ΔCt method was used to calculate the relative expression level of the target gene.

[0080] Western blot: Total protein was extracted using RIPA lysis buffer (Applygen, C1053) containing protease and phosphatase inhibitors (Applygen, P1265). Protein concentration was determined using a BCA kit (Thermo Scientific, 23227). Extracted protein samples were loaded onto 10% SDS-PAGE gels for electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH07850). The transferred membranes were blocked with 5% skim milk and incubated overnight at 4°C with primary antibody.

[0081] The primary antibodies used in this invention include: FTH1 antibody (Cell Signaling Technology, catalog number 4393), CXCL16 antibody (Proteintech, catalog number 60123-1-Ig), CD163 antibody (Abcam, catalog number ab156769), microtubule antibody (Tubulin, Abcam, catalog number ab6046), GAPDH antibody (Cell Signaling Technology, catalog number 2118), and actin antibody (Actin, Abcam, catalog number ab179467). Horseradish peroxidase (HRP)-labeled secondary antibody (Abcam, catalog number 6721) was added, and after incubation at room temperature for 2 hours, images were acquired using an imaging system (General Electric, AI600UV).

[0082] Co-immunoprecipitation (Co-IP): Nuclear extracts were prepared using the NE-PER nucleocytoplasmic extraction kit (Thermo Fisher Scientific, catalog number 78835) according to the manufacturer's instructions. The pretreated lysis buffer was incubated overnight at 4°C with anti-DYKDDDDK magnetic beads (Thermo Fisher Scientific, catalog number A36797) by rotation. The magnetic beads were washed five times with TBS-T buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween-20) on ice, followed by elution with 2×Laemmli buffer and boiling at 95°C for 5 minutes to remove the bound protein. GATAD2B (Proteintech, catalog number 25679-1-AP) in the eluent was analyzed by Western blotting.

[0083] Dual-luciferase reporter gene assay: A549 and H358 cells (5 × 10⁶ cells / well) were seeded in 24-well plates. 5Transfection was performed 24 hours after culture. The following plasmids were co-transfected using Lipofectamine™ 3000 (Invitrogen): (1) 500 ng CXCL16 promoter-driven firefly luciferase reporter plasmid (Beijing Hesheng Gene Co., Ltd.); (2) 10 ng pRL-Renilla luciferase plasmid (Beijing Hesheng Gene Co., Ltd.); (3) 500 ng FTH1 overexpression plasmid (the lentiviral overexpression vector prepared above) or GATAD2B overexpression plasmid (Beijing Hesheng Gene Co., Ltd.) (with an empty vector as a control). 24 hours after transfection, luciferase activity was measured using a dual-luciferase reporter gene detection system (Promega) according to the manufacturer's instructions. The activity of *Rhizopus rubrum* luciferase in the same sample was used as an internal control to calibrate firefly luciferase activity and control transfection efficiency.

[0084] Immunohistochemistry (IHC): Immunohistochemistry was used to detect the expression levels of FTH1, CD8, CD163, and CXCL16 in FFPE tissue specimens from 189 patients and in a mouse model (mouse tumor xenograft model). Primary antibodies used for immunohistochemical staining included: FTH1 antibody (Cell Signaling Technology, catalog number 4393), CD8 antibody (Abcam, catalog number ab17147), CD163 antibody (Abcam, catalog number ab156769), and CXCL16 antibody (Proteintech, catalog number 60123-1-Ig).

[0085] The staining scores of FTH1 and CXCL16 in each tissue sample were calculated using the following formula: Immunohistochemical score = staining intensity × percentage of positive tumor cells × 100; CD8 + T cells and CD163 + The proportion of macrophages = CD8 in the stromal region + Lymphocytes or CD163 + Macrophage count / total nucleated cell count in the stromal region. All slides were independently evaluated by two pathologists unaware of the clinical parameters.

[0086] Immunofluorescence: Immunofluorescence staining was performed using rabbit anti-FTH1 primary antibody (Abcam, catalog number ab75973), followed by incubation with Alexa Fluor® 555-labeled anti-rabbit secondary antibody (CST, catalog number 4413). Cell nuclei were counterstained using DAPI-containing mounting medium (Invitrogen, catalog number P36935), and fluorescence signals were observed using a confocal laser scanning microscope.

[0087] CD8 +T cell and macrophage chemotaxis assay: CD8 + T cell and macrophage chemotaxis assays were performed using 24-well Transwell chambers (Corning, catalog number 3415). Tumor cells were seeded in the lower chamber and cultured until 80% confluence was achieved, then subjected to starvation treatment with medium containing 1% FBS; CD8+ cells were then... + T cells and M0 polarized macrophages were suspended in serum-free culture medium and seeded into the upper chamber.

[0088] -CD8 + T cell count: After 24 hours of co-culture, migrating cells in the lower chamber were collected and analyzed by flow cytometry; - Macrophage migration: After incubation for 72 hours, fix the upper chamber with 4% paraformaldehyde for 15 minutes, wash twice with PBS, and remove unmigrated cells from the surface of the Transwell membrane with cotton swabs; stained the migrated cells with Coomassie Brilliant Blue for 15 minutes, washed twice with PBS, and observed under a microscope after drying.

[0089] Macrophage polarization: THP-1 cells were cultured in medium containing 100 ng / mL phorbol ester (PMA) for 48 hours to induce differentiation into M0 macrophages. Subsequently, they were cultured in medium containing 100 ng / mL PMA, 20 ng / mL IL-4, and 20 ng / mL IL-13 for 72–96 hours to induce differentiation into M2 macrophages. Macrophage polarization assays were performed using 24-well Transwell chambers (Corning, catalog number 3412). The mRNA expression levels of CD163 and CD206, characteristic markers of M2 macrophages, were detected by RT-qPCR to assess macrophage polarization.

[0090] Statistical analysis: Statistical analysis was performed using R (version 3.6.0), SPSS (version 21.0), and GraphPad Prism (version 9.0). Differences between groups for continuous data were analyzed using the Kruskal-Wallis H test or the Mann-Whitney U test; categorical data were analyzed using Fisher's exact test; Pearson correlation analysis was used to calculate correlation coefficients; and two-way ANOVA was used to analyze tumor growth curves in the two groups of mice. All statistical tests were two-tailed, and P < 0.05 was considered statistically significant.

[0091] Example 1. The FTH1 inhibitor baicalin combined with anti-PD-1 / L1 immunotherapy has significant efficacy in NSCLC. This invention involves collecting baseline tissue samples from NSCLC patients receiving PD-1 / L1 immunotherapy before treatment, performing transcriptomic and proteomic analyses, and screening for the key drug resistance molecule FTH1. The amino acid sequence of human FTH1 protein is sequence 1 in the sequence listing, and the amino acid sequence of mouse FTH1 protein is sequence 3 in the sequence listing.

[0092] Sequence 1: MSTARTVFRQAFKSEYAGNLNWERQMHYLSLKEEWEKILEESQAALGDKTVTWNYSDKKSIYFYTAEVHKKADEVIEELFYKYYLKDKNDPVDVKYLLKMKDSYKDKEDLIQHLDDIKAKNNDGDYFVLDSFDKDPNKLFQ EYAKNLGKNESLIKIMESAKKAGFQILNERLTLTKDYKSLTENFKNLFEKIKELGLKDAQQLYQEYLQKANKENKSEYLDDLKKRLNEYTKDIQQKLKDYQAQIDKFVSQLFRMFKKHGKDSYVTGLSTRMMVKKGEEET.

[0093] Sequence 3: MSTASVFRQAFKSEYQGNINWERQMHYLSLKEEWEKILEESQAALGDKTITWNYSDKKSIYFYTAEVHKKADEVIEELFYKYYLKDKNDPVDVKYLLKMKDSYKDKEDLIQHLDDIKAKNNDGDYFVLDDFDKDPNKLF QEYAKNLGKNESLIKIMESAKKAGFQILNERLTLTKDYKSLTENFKNLFEKIKELGLKDAQQLYQEYLQKANKENKSEYLDDLKKKLNEYTKEIQQKLKDYQAQIDKFVSQLFRMFKKHGKDKYVTGLSTRMMVRKGE.

[0094] 1. FTH1 expression is associated with primary resistance to PD-1 / L1 inhibitor therapy in NSCLC patients. 1.1 Screening for key molecules in PD-1 / L1 antibody resistance in NSCLC This study included 12 NSCLC patients who received PD-1 / L1 inhibitor monotherapy, of whom 8 (66.7%) were pathologically diagnosed with lung adenocarcinoma (LUAD) and 4 (33.3%) with lung squamous cell carcinoma (LUSC); 6 patients achieved partial response (PR) and 6 patients experienced disease progression (PD). The clinical information of the enrolled patients is shown in Table 1.

[0095] To screen key molecules for PD-1 / L1 inhibitor resistance in NSCLC, proteomic and transcriptomic analyses were performed on baseline tumor samples from 12 patients. Figure 1 (A and B), analyzed differentially expressed molecules between the treatment-sensitive group (PR group, indicating partial remission) and the treatment-resistant group (PD group, indicating disease progression), and performed Venn analysis based on proteomic and transcriptomic data ( Figure 1 (C) Results showed that FAH, LAMC2, SPRR3, CALML3, EPB41L4B, CD8A, FTH1, MMP28, KRT6B, and GLTP proteins may be associated with the response of NSCLC patients to PD-1 / L1 antibodies. Among them, elevated FTH1 expression was associated with reduced clinical benefit from immunotherapy, suggesting that it may play a role in primary resistance to PD-1 / L1 inhibitors. The FTH1 gene encodes the heavy chain subunit of ferritin, the main intracellular iron storage protein; FTH1 is mainly located in the cytoplasm, but can also be present in the nucleus or secreted extracellularly. It has ferrooxidase activity and can react with hydroxyl radicals to release unstable Fe²⁺. + Transformed into stable Fe³ + This reduces the oxidative stress caused by the Fenton reaction due to excessive iron and inhibits ferroptosis.

[0096] 1.2 Verifying the association between FTH1 and prognosis in an independent queue In an independent cohort containing 26 archived FFPE tissue samples, the clinical value of FTH1 in immunotherapy was further validated by immunohistochemistry and RT-qPCR. Figure 1 (D). The results consistently showed that patients in the PD group had higher FTH1 expression levels, while patients with low FTH1 expression benefited more from immunotherapy and had better survival prognosis. Figure 1 ROC curve analysis further confirmed the predictive value of FTH1 for progression-free survival (PFS), with AUC values ​​of 0.861 and 0.969 at the protein and transcriptional levels, respectively. Figure 1 (E and G in the middle).

[0097] 1.3 Evaluation of the relationship between FTH1 levels and the efficacy of PD-1 inhibitors using a mouse model Using CRISPR-Cas9 technology and lentivirus-mediated FTH1 transfection, FTH1 knockout (KO) and overexpression (OE) cell lines of mouse NSCLC LLC and KLN-205 cells were constructed, respectively. Figure 2(A); The method for constructing FTH1 knockout (KO) cell lines of mouse NSCLC cells LLC and KLN-205 cells is as follows: A single guide RNA (sgRNA) targeting mouse FTH1 (sgRNA sequence: 5'-GTAGTTCTGGCGCACTTGC-3') was cloned into the lentiviral vector pLV-hU6 (Beijing Hesheng Gene Co., Ltd.), and co-transfected with packaging plasmids using Lipofectamine™ 3000 (Invitrogen) into LLC and KLN-205 cells respectively to obtain FTH1 knockout (KO) cell lines of LLC and KLN-205 cells.

[0098] The method for constructing overexpression (OE) cell lines of mouse NSCLC LLC and KLN-205 cells is as follows: The mouse FTH1 encoding gene (sequence 4 in the sequence listing) was cloned into the lentiviral vector GL107. Lentiviral cells carrying the mouse FTH1 gene were then co-transfected with recombinant vectors and packaging systems (pLP1, pLP2, pLP / VSVG) using Lipofectamine™ 3000 (Invitrogen). Subsequently, lentiviral mouse NSCLC cell lines LLC and KLN-205 cells were cultured in a medium containing 5 μg / mL polybrene (Sigma-Aldrich, catalog number P4505). After selection with puromycin, the expression level of FTH1 in infected cells was verified by Western blot and real-time quantitative polymerase chain reaction (RT-qPCR).

[0099] FTH1 negative control (NC), FTH1-KO, FTH1 empty vector (FTH1-VEC), FTH1-OE LLC and KLN205 tumor cells were subcutaneously transplanted into C57BL / 6J, DBA-2J mice and Nude mice (all from Huafukang Biotechnology Co., Ltd.) to establish tumor models. Figure 2 (B)

[0100] Consistent with clinical cohort results: FTH1-OE mice treated with PD-1 inhibitors showed significantly weaker tumor growth inhibition than the control group (VEC), while FTH1-KO mice treated with PD-1 inhibitors showed significantly better results than the control group (NC). Figure 2 (C~F).

[0101] In summary, high FTH1 expression is closely associated with resistance to PD-1 inhibitor therapy in NSCLC patients. Low FTH1 expression, on the other hand, promotes the efficacy of anti-PD-1 immunotherapy.

[0102] 2. Analysis of the mechanism of FTH1 level and the efficacy of PD-1 blockers 2.1 FTH1 mediates resistance to PD-1 / L1 inhibitors by constructing an immunosuppressive tumor immune microenvironment (TIME) To further explore how FTH1 affects the efficacy of PD-1 inhibitors in NSCLC, the present invention first analyzed the effect of regulating FTH1 expression on tumor growth: in vitro experiments showed that FTH1 knockout could effectively inhibit tumor cell proliferation, while under immunodeficient conditions, overexpression of FTH1 had no significant effect on cell proliferation ( Figure 3 in A); however, in immunocompetent mice, the growth rate of FTH1-OE tumors was accelerated, suggesting that FTH1 may affect tumor growth through an immune-dependent mechanism ( Figure 3 in B).

[0103] In addition, the present invention constructed NSCLC cell lines (A549, H358, H520) with human FTH1 knockout and overexpression ( Figure 3 in C), and in vitro and in vivo experiments both confirmed that under immunodeficient conditions achieved using Nude mice, overexpression of FTH1 did not affect tumor growth ( Figure 3 in D~3E). Therefore, it is speculated that FTH1 may affect tumor growth by regulating TIME and act synergistically with immune cells in TIME.

[0104] 2.2 Based on the TCGA database, analyze the correlation between FTH1 and infiltration of various immune cells in NSCLC Analysis was performed on the lung cancer transcriptome data TCGA-LUAD (https: / / portal.gdc.cancer.gov / projects / TCGA-LUAD) and TCGA-LUSC (https: / / portal.gdc.cancer.gov / projects / TCGA-LUSC) in the TCGA database, and the results showed that in both LUSC and LUAD, FTH1 was significantly positively correlated with infiltration of M2 macrophages ( Figure 4 in A~D). The above findings were further verified by immunohistochemical detection of the expression of CD163 (a classic molecular marker of M2 macrophages) in NSCLC surgical specimens ( Figure 4 in E~H). A chemotaxis experiment was constructed using the Transwell chamber system to explore the effect of changes in FTH1 expression on the chemotaxis of macrophages (THP-1 cells activated by PMA), and the results showed that FTH1 could effectively promote macrophage chemotaxis ( Figure 4 in I~K).

[0105] Co-culture tumor cells with macrophages (see the schematic diagram of the co-culture model in Figure 4The results showed that compared with the control group (FTH1-NC or FTH1-VEC), the expression levels of M2 macrophage markers (CD163, CD206) were decreased in the FTH1-KO group, while the expression levels of these markers were significantly increased in the FTH1-OE group. Figure 4 (M). The above results suggest that FTH1 may also promote the polarization of M2 macrophages.

[0106] To further clarify the specific mechanism by which FTH1 mediates resistance to anti-PD-1 immunotherapy through regulation of TIME, immunohistochemical analysis was performed on LLC and KLN205 tumors with FTH1-NC, FTH1-KO, FTH1-VEC, and FTH1-OE types to detect differences in their TIME. Figure 8-9 The results showed that in both LUAD and LUSC, the infiltration of M2 macrophages in the FTH1-OE group was higher than that in the FTH1-VEC group; while the FTH1-KO group showed greater immunogenicity and less M2 macrophage infiltration. Furthermore, CD8+ levels varied among the groups. + T-cell infiltration also differed: in KLN-205 and LLC tumors, the FTH1-OE group had higher CD8 counts. + T cell infiltration was less in the FTH1-VEC group, while CD8 in the FTH1-KO group was less. + Increased T cell infiltration. Chemotaxis assays also confirmed that CD8+ was present in the FTH1-KO group. + T cell infiltration was significantly higher than in the control group. Figure 10 (A). ELISA experiments showed that the HMGB1 level in the FTH1-KO group was higher than that in the FTH1-NC group ( Figure 10 (B) Considering that FTH1 is a key ferroptosis inhibitor, it is speculated that FTH1 knockdown may increase ferroptosis, leading to the release of more HMGB1, and thus recruiting more CD8+. + T cells.

[0107] 2.3 FTH1 drives M2 macrophage polarization and infiltration by upregulating CXCL16 To further investigate the mechanism by which FTH1 promotes M2 macrophage polarization, supernatants from FTH1-overexpressing and control cell lines (A549, H358, and H520) were collected for proteomic analysis. NSCLC data from the TCGA database were then analyzed using the CIBERSORT algorithm. Correlating the results of the two analyses revealed that CXCL16 may be a key mediator linking FTH1 to M2 macrophage infiltration in NSCLC. Figure 5 (A~B)

[0108] Immunohistochemical staining was performed on tumor tissues from 189 NSCLC patients in the CICAMS cohort. The results showed that CXCL16 expression was significantly positively correlated with M2 macrophage infiltration in both LUSC and LUAD. Figure 5 C~E). Western blot ( Figure 5 FTH1 overexpression and RT-qPCR experiments showed that both CXCL16 protein and transcriptional expression levels increased; ELISA further confirmed that the secretion level of CXCL16 in the tumor cell supernatant of the FTH1-OE group was higher than that of the FTH1-VEC group. Figure 5 (H~J).

[0109] Furthermore, the study also examined the changes in the ability of FTH1-overexpressing tumor cells to recruit macrophages after transient knockdown of CXCL16. The results showed that the recruitment effect of FTH1 overexpression on macrophages was significantly weakened after CXCL16 expression was reduced. Figure 5 (M-K). Co-culturing CXCL16-knockdown FTH1-OE cells with macrophages showed a weakened M2-type polarization effect in macrophages. Figure 5 (O~Q).

[0110] The effect of exogenous CXCL16 recombinant protein on macrophage recruitment was investigated. The results showed that the recruitment effect on macrophages was enhanced with increasing exogenous CXCL16 concentration gradient. Figure 5 (N); and the recombinant CXCL16 protein can effectively upregulate the expression of IL-4 and IL-13-induced M2 macrophage markers, suggesting that CXCL16 can promote M2 macrophage polarization (N); Figure 6 (Middle B). In summary, it is speculated that FTH1 may promote macrophage infiltration and M2 polarization by regulating CXCL16 expression.

[0111] 2.4 Tumor-derived CXCL16 promotes M2 macrophage polarization through the PPAR-γ pathway. To investigate the specific molecular mechanism by which CXCL16 regulates M2 polarization in macrophages, transcriptome analysis was performed on macrophages from the control group and the CXCL16-stimulated group to analyze differentially expressed genes. Enrichment analysis showed that differentially expressed genes were mainly enriched in the PPAR pathway (…). Figure 6 (A). Previous studies have confirmed that the PPAR-γ pathway plays a key role in macrophage M2 polarization, and PPAR-γ-dependent lipid metabolism and uptake regulation can drive the M2 polarization process.

[0112] RT-qPCR validation showed that CXCL16 stimulation significantly increased the transcriptional levels of key genes in the PPAR-γ pathway (CD36, LPL, FABP5). Figure 6(D); Western blot experiments showed that after CXCL16 intervention, the expression level of CD36 protein in macrophages was significantly increased ( Figure 6 (E); and CD36 inhibitors can effectively inhibit CXCL16-induced macrophage M2 polarization ( Figure 6 (C). The above results suggest that tumor cell-derived CXCL16 can effectively activate the macrophage PPAR-γ pathway, thereby promoting macrophage M2 polarization, with CD36 possibly being a key molecule in this process.

[0113] 3. FTH1 regulates CXCL16 transcription by binding to the CXCL16 promoter via GATAD2B. To clarify the specific mechanism by which FTH1 regulates CXCL16 expression, based on existing literature reports (FTH1 can be located in the cytoplasm and nucleus) and immunofluorescence results (FTH1 is expressed in the nucleus of lung cancer cells), it is speculated that FTH1 in the nucleus may be involved in the transcriptional regulation of CXCL16.

[0114] First, the nuclei and cytoplasm of FTH1-overexpressing lung cancer cells were isolated. FTH1-binding proteins were screened using CoIP, and identified by protein mass spectrometry. Simultaneously, transcription factors that might bind to the CXCL16 promoter region were searched using the GeneCards website. Based on the combined results, it was inferred that GATAD2B may be a key transcription factor in FTH1 regulation of CXCL16 transcription. Western blot further confirmed the interaction between FTH1 and GATAD2B. Figure 6 (F); Dual-luciferase reporter gene assays showed that nuclear-localized FTH1 can bind to GATAD2B and regulate CXCL16 expression through transcriptional activation. Figure 6 (G~H).

[0115] 4. Baicalin enhances the efficacy of PD-1 blockers in NSCLC patients by inhibiting FTH1 expression. 4.1 Virtual Screening of FTH1 Inhibitors Based on Molecular Docking In summary, FTH1 plays a crucial role in the remodeling of the lung cancer tumor microenvironment and may be a key target for resistance to immunotherapy in NSCLC. To screen for effective inhibitors of FTH1 protein, the 3D structure of FTH1 protein (ID: P02794) was downloaded from the Uniprot website, and the protein structure with the highest precision (PDB ID: 2CIH) was selected for virtual screening. Autodock Vina software was used for molecular docking-based virtual screening, and combined with previous literature reports, it is speculated that baicalin (CAS: 21967-41-9) may be an FTH1 inhibitor. Figure 7(A). Western blot analysis showed that with increasing baicalin concentration gradient, the expression levels of FTH1 and CXCL16 in A549, H358, and H520 cells decreased accordingly. Figure 11 ).

[0116] 4.2 Analysis of the combination of FTH1 inhibitor baicalin and PD-1 monoclonal antibody in the treatment of NSCLC To preliminarily explore the value of the FTH1 inhibitor baicalin combined with PD-1 monoclonal antibody in the treatment of NSCLC, three treatment regimens were set up in the FTH1-OE mouse subcutaneous tumor models of LUAD and LUSC: baicalin alone, baicalin combined with PD-1 monoclonal antibody, and PD-1 monoclonal antibody combined with chemotherapy (LUSC: cisplatin combined with paclitaxel; LUAD: cisplatin combined with pemetrexed). Specific dosing regimens are detailed below. Figure 7 B.

[0117] The results showed that in LUAD and LUSC mouse models, baicalin combined with PD-1 monoclonal antibody in the FTH1-OE group effectively inhibited tumor growth, and the efficacy was comparable to that of PD-1 monoclonal antibody combined with chemotherapy. Figure 7 (C~F).

[0118] To further investigate the changes in time intervals (TIME) in different groups of mice, immunohistochemical staining of FTH1 and CD163 was performed on tumor tissues from mice receiving different treatment regimens. The results showed that baicalin effectively reduced the expression level of FTH1 in tumors in vivo; simultaneously, baicalin combined with PD-1 monoclonal antibody effectively reduced the proportion of M2 macrophage infiltration to a certain extent. Figure 12-13 It is speculated that the combination of the two may activate TIME as a "hot tumor" by reshaping it, thereby promoting the anti-tumor immune response.

[0119] 4.3 Analysis of whether baicalin combined with PD-1 monoclonal antibody causes serious damage to vital organs in mice To investigate whether baicalin combined with PD-1 monoclonal antibody causes severe damage to vital organs in mice, tissues from the heart, spleen, lungs, liver, and kidneys of mice treated with baicalin combined with PD-1 monoclonal antibody were collected and stained with hematoxylin and eosin (HE) to assess the degree of damage in different organ tissues. The results showed that no significant histological damage or severe inflammation was observed in the heart, spleen, lungs, liver, or kidneys of mice treated with baicalin combined with PD-1 monoclonal antibody. Figure 14 In summary, baicalin combined with PD-1 monoclonal antibody is well tolerated in vivo and did not induce significant adverse reactions.

[0120] This invention discovers that inhibiting FTH1 expression can improve the response rate of NSCLC patients to PD-1 / L1 immunotherapy by alleviating immunosuppression, thereby overcoming treatment resistance. Previous studies have confirmed that baicalin can effectively downregulate FTH1 expression and inhibit tumor progression by triggering tumor cell ferroptosis. This invention further confirms through molecular docking analysis that baicalin has a strong binding affinity to FTH1; and verifies in human and mouse NSCLC cells that baicalin can significantly reduce FTH1 levels. Furthermore, this study is the first to explore the synergistic anti-tumor efficacy of baicalin combined with anti-PD-1 immunotherapy in LUAD and LUSC mouse models. The results show that the tumor-suppressive effect of this combination regimen is comparable to that of anti-PD-1 immunotherapy combined with chemotherapy, with no significant damage to vital organs. These results indicate that the baicalin combined with anti-PD-1 immunotherapy regimen has good safety and feasibility, and may provide a new treatment option for NSCLC patients who cannot tolerate chemotherapy-related adverse reactions.

[0121] In summary, this invention is the first to discover that FTH1-mediated M2 macrophage immunosuppression is a novel mechanism of resistance to anti-PD-1 / L1 immunotherapy in NSCLC; baicalin can effectively reverse this immunosuppressive microenvironment by targeting FTH1. These findings suggest that FTH1 holds promise as a predictive biomarker for immunotherapy response and a potential therapeutic target; baicalin combined with anti-PD-1 / L1 immunotherapy may be a novel strategy for overcoming immunotherapy resistance in NSCLC patients.

[0122] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of substances that inhibit, knock out, or silence ferritin heavy chain 1 protein or its mRNA or its encoding gene in the development or preparation of products having any of the following functions: A1) Treatment of tumors; A2) Inhibit the occurrence and development of tumors; A3) Inhibits tumor metastasis; A4) Improve the prognosis of cancer patients; The ferritin heavy chain 1 protein is the following protein: B1) The amino acid sequence is that of the protein in sequence 1 of the sequence listing; B2) The amino acid sequence is that of sequence 3 in the sequence listing; B3) A protein derived from B1) or B2) or having the same function as the protein shown in B1) or B2) by substitution and / or deletion and / or addition of one or more amino acid residues. B4) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1), B2) or B3).

2. The use of the ferritin heavy chain 1 protein as a therapeutic target in the preparation or screening of products having any of the following functions: C1) Drugs for treating tumors; C2) Drugs that inhibit the occurrence and development of tumors; C3) Drugs that inhibit tumor metastasis; C4) Drugs that improve the prognosis of tumors.

3. The application according to claim 1 or 2, characterized in that: The tumor is lung cancer.

4. The product as described in any one of claims 1-3.

5. The use of substances that inhibit, knock out, or silence ferritin heavy chain 1 protein or inhibit the function, activity, or expression of its mRNA or its encoding gene in the development or preparation of tumor immunotherapy products.

6. A composition, characterized in that: The composition contains an immunotherapy antibody and a substance that inhibits the function, activity, or expression of ferritin heavy chain 1 protein or its mRNA or its encoding gene.

7. The composition according to claim 6, characterized in that: The substance that inhibits the function, activity, or expression of ferritin heavy chain 1 protein or its mRNA or its encoding gene is baicalin; the immunotherapy antibody is an anti-PD-1 antibody and / or an anti-PD-L1 antibody.

8. The use of the composition of claim 6 or 7 in the development or preparation of products having any of the following functions: A1) Treatment of tumors; A2) Inhibit the occurrence and development of tumors; A3) Inhibits tumor metastasis; A4) Improve the prognosis of cancer patients.

9. A method for constructing a tumor drug screening model, the method comprising constructing the model by inhibiting or reducing the activity of the protein of claim 1 or the expression of the gene encoding the protein of claim 1 in a model species.

10. The method according to claim 9, characterized in that: The inhibition or reduction of the activity of the protein of claim 1 or / and the expression level of the gene encoding the protein of claim 1 in the target species, target species cell line or target species tissue is achieved by knocking out the gene encoding the protein of claim 1 in the model species.

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