A biomarker for predicting sensitivity / prognosis of platinum-containing chemotherapy drug systemic treatment of non-small cell lung cancer and application thereof

Abstract

This invention discloses a biomarker for predicting the sensitivity / prognosis of non-small cell lung cancer (NSCLC) treated with platinum-based chemotherapy drugs, and its application, belonging to the field of tumor biology technology. The biomarker is MT-ND6. MT-ND6 is an indicator of NSCLC's sensitivity to platinum-based chemotherapy, helping to avoid unnecessary treatment, reduce ineffective treatment, and minimize unnecessary chemotherapy side effects. The biomarker MT-ND6 can also serve as an independent marker distinguishing NSCLC tumor tissue from adjacent normal tissue; the expression level of MT-ND6 protein in tumor tissue is significantly higher than in normal tissue, demonstrating diagnostic value. This invention also discloses a detection reagent, a method for screening the biomarker, and a method for predicting / evaluating the sensitivity / prognosis of NSCLC treated with platinum-based chemotherapy drugs. The biomarker of this invention can be used to prepare diagnostic products for NSCLC, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of tumor biology technology, specifically relating to a biomarker for predicting the sensitivity / prognosis of platinum-based chemotherapy systemic therapy for non-small cell lung cancer and its application. Background Technology

[0002] Lung cancer can be broadly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with the latter accounting for approximately 85% of all lung cancers. NSCLC mainly includes three subtypes: lung adenocarcinoma (LUAD), squamous cell carcinoma (LUSC) (also known as lung squamous cell carcinoma), and large cell carcinoma (LCC). Each subtype has different biological characteristics and treatment responses. Lung adenocarcinoma accounts for approximately 40%-50% of NSCLC cases, usually originating in the peripheral lung regions, and is more common in non-smokers and women; squamous cell carcinoma accounts for approximately 25%-30% of NSCLC cases, is more common in smokers, and usually occurs in the central lung region; large cell carcinoma is relatively rare, characterized by high malignancy, poor cell differentiation, and a poor prognosis. [3] .

[0003] Adjuvant therapy for non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer, after surgery; and platinum-based systemic therapy for advanced, unresectable NSCLC: treatment strategies for lung cancer depend on the type and stage of the cancer, the patient's overall health, and individual differences. Surgery is the preferred treatment for early-stage NSCLC, but selecting appropriate patients, managing postoperative recurrence and complications, and effectively combining it with other treatment modalities remain major challenges. In the past decade, the discovery of a series of driver mutations, such as EGFR mutations and their corresponding targeted therapies, has greatly advanced the treatment of NSCLC, especially the NSCLC subtype of lung adenocarcinoma. In contrast, for squamous cell lung cancer patients with PTEN mutations, DDR2 mutations, and FGFR1 amplification, there are still no successful targeted therapies. [4] Currently, there are no targeted drugs, treatment options, or clinically valuable biomarkers for squamous cell lung cancer. In recent years, although tumor immunotherapy has significantly prolonged the survival of some NSCLC patients, current clinical tumor immunotherapy targeting PD-1 / PD-L1 is not specific to NSCLC, and PD-L1 is not a perfect predictor of pan-cancer immunotherapy. [5] Therefore, for lung cancer patients with advanced lung cancer who are eligible for platinum-based adjuvant therapy, developing more diverse, more effective, and safer treatment options to achieve long-term survival for NSCLC patients, as well as developing a batch of biomarkers with clinical guiding significance and scientifically guiding combination therapy, is of great research significance and is urgently needed in clinical practice.

[0004] The current challenges of neoadjuvant therapy for lung cancer using platinum-based chemotherapy and immunotherapy include: low response rates, with the highest response rate not exceeding 30%; risks associated with platinum-based neoadjuvant therapy, with 5% of patients experiencing tumor progression after chemotherapy, missing the surgical window and becoming inoperable; and uncertain neoadjuvant therapy duration. Therefore, developing effective biomarkers to guide the screening and treatment of patients receiving neoadjuvant therapy with platinum-based chemotherapy (including platinum-based + immunotherapy) is of great significance.

[0005] Currently, there are no molecular markers in clinical practice for non-small cell lung cancer (NSCLC) that can accurately predict the efficacy of platinum-based (including cisplatin and carboplatin) combination therapy before chemotherapy, particularly for squamous cell lung cancer. The treatment window includes both neoadjuvant therapy before surgery and adjuvant therapy after lung cancer surgery. Chinese patent CN 108107134 B, which describes a biomarker for predicting the sensitivity of platinum-based therapy for small cell lung cancer, only targets the adenocarcinoma subtype of NSCLC.

[0006] Currently, there is no single, effective, sensitive predictive biomarker in clinical practice for predicting platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides a biomarker for predicting the sensitivity / prognosis of non-small cell lung cancer patients to platinum-based chemotherapy (including platinum-based drugs used in combination with other chemotherapy drugs, or with immunotherapy, or with radiotherapy, or in combination thereof).

[0008] Previous research by the inventors suggested that the mitochondrial genome is a target of platinum-based chemotherapy drugs. This invention proposes the hypothesis that "mitochondrial gene expression may be involved in the sensitivity of platinum-based chemotherapy drugs." [6,7] Therefore, the applicant, for the first time, utilized public databases to obtain the half-maximal inhibitory concentration (IC50) of platinum-based drugs (including cisplatin and carboplatin) in NSCLC, as well as the expression of 13 mitochondrial genome-encoded protein genes, and examined their correlation. A series of in vitro and functional experiments were conducted to confirm that MT-ND6 regulates the sensitivity to platinum-based chemotherapy. Clinical sample studies further confirmed that MT-ND6 expression alone has an independent predictive effect on the prognosis of NSCLC patients, and its efficacy in predicting the sensitivity of NSCLC patients to platinum-based systemic therapy.

[0009] The purpose of this invention is to successfully develop MT-ND6, a protein encoded by the mitochondrial gene, and to use conventional IHC (immunohistochemistry) to detect tumor samples from patients.

[0010] In patient cohort 1: 2 patients with non-small cell lung cancer (squamous cell lung cancer) who underwent neoadjuvant systemic chemotherapy with platinum-based chemotherapy; IHC (immunohistochemistry) analysis of biopsy samples (patients to be treated with neoadjuvant systemic chemotherapy with platinum-based chemotherapy) showed that detecting MT-ND6 protein expression in tumor tissue alone could effectively indicate the sensitivity of non-small cell lung cancer patients to the efficacy of systemic chemotherapy with platinum-based chemotherapy. Imaging evaluation indicated that non-small cell lung cancer patients with high MT-ND6 expression showed initial disease remission.

[0011] Patient group 2: 33 non-small cell lung cancer (NSCLC) patients who had not received systemic platinum-based chemotherapy and were undergoing adjuvant systemic platinum-based chemotherapy after surgery. Immunohistochemistry (IHC) was used to detect MT-ND6 expression in postoperative samples (patients scheduled for adjuvant systemic platinum-based chemotherapy). Patients with high MT-ND6 expression had significantly better prognoses than those with low expression. High expression of MT-ND6 in tumor tissue can effectively indicate the sensitivity of NSCLC patients to systemic platinum-based chemotherapy for NSCLC.

[0012] Patient group 3: 87 cases of non-small cell lung cancer (squamous cell lung cancer). Patients with high expression of MT-ND6 protein in tumor tissue detected by IHC had a significantly better prognosis than patients with low expression. High expression of MT-ND6, an independent biomarker, is a good prognostic biomarker for patients with non-small cell lung cancer.

[0013] Patient cohort 4: IHC analysis of paired non-small cell lung cancer tumors and adjacent normal tissues showed that the MT-ND6 protein level in tumor tissues was significantly higher than that in adjacent normal tissues.

[0014] This invention points out that the expression level of MT-ND6 protein in tumor tissues of NSCLC patients can not only be used alone as an indicator to predict the sensitivity of non-small cell lung cancer to platinum-based chemotherapy, but also as an independent biomarker to distinguish tumor tissue from adjacent normal tissue: the expression of MT-ND6 protein in tumor tissue is significantly higher than that in normal tissue, thus having diagnostic value. Furthermore, high expression of MT-ND6 protein can effectively predict the prognostic survival of NSCLC patients (especially those with squamous cell lung cancer), with patients exhibiting high expression generally having a better prognosis.

[0015] This invention, based on multiple NSCLC cell lines in the laboratory, nonspecifically inhibited mitochondrial gene expression, significantly antagonizing cisplatin-induced drug sensitivity in NSCLC. Furthermore, this invention successfully constructed monoclonal and polyclonal NSCLC cells with mitochondrial organelle-specific high expression of MT-ND6 protein, confirming that MT-ND6-overexpressing transgenic lung cancer cells alone are highly sensitive to cisplatin. Simultaneously, this invention provides a method for quantitatively analyzing DNA damage at the single-cell level and confirms the molecular biological basis for the high sensitivity of MT-ND6-overexpressing lung cancer cells to cisplatin: it exacerbates cisplatin-induced increased nuclear DNA damage. Based on the screening of mitochondrial genes involved in platinum drug sensitivity, the use of mitochondrial gene expression inhibitors as tools for regulating mitochondrial genes, and the biological basis of constructing MT-ND6-overexpressing transgenic cells and developing a method for detecting cisplatin-induced DNA damage, combined with clinical and imaging data of NSCLC patients, this invention confirms that the mitochondrial gene protein MT-ND6 alone has the efficacy in predicting the drug sensitivity of NSCLC patients treated with platinum-based chemotherapy systems, as well as in assessing patient survival prognosis.

[0016] This invention provides a biomarker for predicting the sensitivity / prognosis of platinum-based chemotherapy systemic therapy for non-small cell lung cancer. The biomarker is MT-ND6, and its amino acid sequence is shown in SEQ ID NO.1.

[0017] SEQ ID NO.1 MT-ND6-WT amino acid sequence (174)

[0018] MMYALFLLSVGLVMGFVGFSSKPPSPIYGGLVLIVSGVVGCVIILNFGGGYMGLMVFLIYLGGMMV VFGYTTAMAIEEYPEAWGSGVEVLVSVLVGLAMEVGLVLWVKEYDGVVVVVNFNSVGSWMIYEGEG SGLIREDPIGAGALYDYGRWLVVVTGWTLFVGVYIVIEIARGN*

[0019] The expression level of MT-ND6 protein in tumor tissue of NSCLC patients can serve as an independent marker to distinguish tumor tissue from adjacent normal tissue. The expression of MT-ND6 protein in tumor tissue is significantly higher than that in normal tissue, which has diagnostic value.

[0020] Specifically, the MT-ND6 indicator predicts the sensitivity of non-small cell lung cancer (NSCLC) patients to systemic platinum-based chemotherapy, avoiding unnecessary treatment: For NSCLC patients receiving neoadjuvant systemic platinum-based chemotherapy (including combination with immunotherapy), the MT-ND6 indicator guides the screening of sensitive NSCLC patients receiving systemic platinum-based chemotherapy, increasing the response rate of these patients to neoadjuvant systemic platinum-based chemotherapy and reducing the risk of missing the surgical window; and / or,

[0021] For adjuvant therapy with platinum-based chemotherapy after radical resection of non-small cell lung cancer (NSCLC) patients, the biomarker MT-ND6 is used to guide the screening of NSCLC patients sensitive to adjuvant therapy with platinum-based chemotherapy, thereby improving the treatment prognosis of these patients and avoiding unnecessary treatment and toxic side effects; and / or,

[0022] For patients with advanced non-small cell lung cancer (NSCLC) who have poor physical condition and face significant toxicity from platinum-based chemotherapy drugs, the biomarker MT-ND6 is used to guide the screening of patients with advanced NSCLC who may be insensitive to systemic treatment with platinum-based chemotherapy drugs, thereby avoiding exacerbation of disease progression; and / or,

[0023] For patients who have never received platinum-based chemotherapy, the biomarker MT-ND6, as a single biomarker, is highly expressed in patients and can effectively indicate that NSCLC patients have a good survival prognosis. It is a potentially good prognostic biomarker for the diagnosis and treatment of NSCLC patients.

[0024] Specifically, the non-small cell lung cancer includes lung adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, among others.

[0025] Specifically, the platinum is selected from one or more of cisplatin, carboplatin, nedaplatin, lobaplatin, etc.

[0026] Specifically, the systemic treatment with platinum-based chemotherapy drugs includes combination therapy with platinum-based drugs and other chemotherapy drugs, combination therapy with platinum-based drugs and immunotherapy, combination therapy with platinum-based drugs and radiotherapy, or combinations thereof.

[0027] The present invention also provides a detection reagent / kit that can be used to detect the expression of the biomarkers described above.

[0028] Specifically, the detection reagent / kit is used to prepare products for the diagnosis of non-small cell lung cancer;

[0029] Specifically, the detection reagent / kit is used to prepare products for the detection of prognostic biomarkers in non-small cell lung cancer; and / or,

[0030] Specifically, the detection reagent / kit is used to prepare products for predicting the sensitivity / prognosis of platinum-based chemotherapy systemic therapy for non-small cell lung cancer; and / or,

[0031] Specifically, the detection reagent / kit is used to prepare a product for the treatment of non-small cell lung cancer by targeting and regulating MT-ND6 expression.

[0032] Specifically, the non-small cell lung cancer includes lung adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, among others.

[0033] This invention also provides a method for screening biomarkers as described above, namely a data mining method, which can be widely applied to tumor drug therapy research, especially in proposing mitochondrial biomarkers and predicting the sensitivity of platinum-based drugs. This method is not only applicable to studying the correlation between platinum-based drugs such as cisplatin and carboplatin and mitochondrial gene expression in non-small cell lung cancer, but can also be extended to other solid tumors and hematological malignancies to explore the relationship between the therapeutic effect of platinum-based drugs and the sensitivity of mitochondrial gene and protein expression, and to develop biomarkers for predicting the sensitivity of platinum-based drug therapy.

[0034] The method described first utilizes the public database #1 (The Genomics of Drug Sensitivity in Cancer Project). http: / / www.cancerrxgene.org / The study obtained the IC50 (half-maximal inhibitory concentration) of cisplatin and carboplatin in all tumor cells. Then, using the Cancer Cell Line Encyclopedia database (http: / / www.broadinstitute.org / ccle / home), the expression levels of 13 genes encoding target mitochondrial genome proteins in target tumor cells were obtained. Pearson correlation coefficients were used to preliminarily screen for mitochondrial gene-encoded biomarkers that could potentially predict the sensitivity of target tumor cells to platinum-based chemotherapy systemic treatment. Further validation was performed using laboratory cell data, and a functional laboratory was designed to investigate the biological effects of these key biomarkers and their role in elucidating drug sensitivity mechanisms. Finally, the efficacy of these key biomarkers was investigated based on clinical samples.

[0035] The method specifically includes the following steps:

[0036] The first step involved obtaining data on the half-maximal inhibitory rate (IC50) of platinum-based drugs against non-small cell lung cancer (NSCLC) cells, as well as the expression levels of mitochondrial genes in NSCLC cells, from databases. Preliminary screening was conducted to identify mitochondrial gene-encoded biomarkers that could potentially predict the sensitivity of NSCLC to platinum-based chemotherapy systems. A hypothesis was proposed that genes encoding mitochondrial proteins could indicate the sensitivity of NSCLC to platinum-based chemotherapy systems.

[0037] The second step involved detecting the sensitivity of non-small cell lung cancer (NSCLC) cell lines to systemic treatment with platinum-based chemotherapy drugs. RT-qPCR was used to quantitatively analyze the gene expression levels of relevant biomarkers. The experiment confirmed that the gene expression level of the mitochondrial protein-encoded MT-ND6 was negatively correlated with the half-maximal inhibitory concentration (50% inhibition) of NSCLC patients treated with platinum-based chemotherapy drugs. Western blotting (WB) was then used to quantitatively analyze the protein expression level of MT-ND6 in NSCLC cell lines, further confirming the negative correlation between the protein expression level of the mitochondrial protein-encoded MT-ND6 and the 50% inhibition rate of NSCLC patients treated with platinum-based chemotherapy drugs.

[0038] The third step is to verify the biological effects. Through functional experiments of inhibiting and overexpressing the biomarker MT-ND6, it is confirmed that the level of the biomarker MT-ND6 is directly related to the degree of nuclear DNA damage induced by platinum-based chemotherapy drugs in non-small cell lung cancer, and the cellular and molecular mechanisms by which the biomarker MT-ND6 regulates the sensitivity of non-small cell lung cancer to systemic treatment with platinum-based chemotherapy drugs are revealed.

[0039] The fourth step involved proposing clinical diagnostic biomarkers. Immunohistochemical detection of the biomarker MT-ND6 revealed that MT-ND6 levels in tumor samples from non-small cell lung cancer (NSCLC) patients were significantly higher than in adjacent normal tissues. High expression of MT-ND6 in tumor tissues of NSCLC patients indicated better prognosis, with patients exhibiting high MT-ND6 expression having significantly longer survival than those with low expression. The optimal expression cutoff point for the MT-ND6 H-Score in tumor tissues was determined using the surv-cutpoint function, and patients were divided into high-expression and low-expression groups based on this cutoff value. The overall survival (OS) of the high-expression group was significantly longer than that of the low-expression group (P = 0.044).

[0040] The fifth step is to verify the biomarkers for predicting clinical efficacy. In patients with non-small cell lung cancer who receive systemic therapy with platinum-based drugs after surgery, the survival of patients with high expression of the biomarker MT-ND6 is significantly higher than that of patients with low expression. High expression of the biomarker MT-ND6 can effectively indicate better sensitivity of non-small cell lung cancer to systemic therapy with platinum-based drugs after surgery.

[0041] Step 6: Further validation of clinical efficacy prediction biomarkers. For non-small cell lung cancer patients who received platinum-based systemic therapy (platinum-based chemotherapy combined with immunotherapy) before surgery, high expression of the biomarker MT-ND6 after imaging evaluation can effectively indicate better sensitivity of non-small cell lung cancer patients who received platinum-based systemic therapy before surgery.

[0042] This invention also provides a method for predicting / evaluating the sensitivity / prognosis of platinum-based chemotherapy systemic therapy (including gemcitabine + cisplatin, paclitaxel + cisplatin, paclitaxel + nedaplatin, paclitaxel + cisplatin + radiotherapy, docetaxel + cisplatin + radiotherapy, etc.) to non-small cell lung cancer. The method predicts / evaluates the sensitivity / prognosis of platinum-based chemotherapy systemic therapy to non-small cell lung cancer by detecting the expression of the biomarkers described above.

[0043] This invention also provides the application of the biomarker MT-ND6 as a drug target in the preparation of drugs for the prevention / delay / treatment / diagnosis of non-small cell lung cancer.

[0044] The drug is used to delay or prevent the occurrence of non-small cell lung cancer.

[0045] Target Function: MT-ND6 is the only light chain protein gene encoded by mitochondrial DNA. The MT-ND6 protein is a core subunit of mitochondrial respiratory chain complex I, crucial for the stability and function of the electron transport chain. Its abnormally high expression plays an important role in promoting tumorigenesis and progression, as well as contributing to drug resistance.

[0046] Diagnostic applications: Based on changes in MT-ND6 protein expression levels, it can serve as an early diagnostic biomarker for non-small cell lung cancer. Through tissue biopsy detection, it can determine the patient's risk of developing cancer or the progression of the disease.

[0047] Preventive use: Develop interventional drugs targeting MT-ND6 for high-risk populations to delay or prevent the onset of non-small cell lung cancer.

[0048] Drug development strategy: Develop inhibitors or activators of the MT-ND6 protein to regulate mitochondrial dysfunction, improve the metabolic microenvironment of cancer cells, and inhibit tumor cell proliferation and migration. Combine gene editing technology to target and regulate the expression level of the MT-ND6 gene for precision medicine.

[0049] Detection technologies: Develop specific antibodies or probes based on the MT-ND6 protein, and detect its expression level in tissue, blood, secretions, and metabolite samples using immunohistochemistry, ELISA, Western blot, and non-invasive detection methods. Utilize CRISPR or RNA interference technology to regulate MT-ND6 gene expression for research on MT-ND6-related molecular mechanisms and drug screening.

[0050] Clinical advantages: Detection of MT-ND6 protein levels enables early diagnosis of non-small cell lung cancer, improving the accuracy of treatment timing; therapeutic drugs developed targeting MT-ND6 protein have high targeting and selectivity, and can reduce toxic side effects on normal tissues.

[0051] Future Prospects: The application of this biomarker, combined with the concept of precision medicine, can provide a new solution for personalized treatment of non-small cell lung cancer, while also promoting innovative development in the fields of tumor molecular diagnostics and drug development.

[0052] The present invention also provides a modulator / inhibitor / antagonist, wherein the modulator / inhibitor / antagonist includes IMT1, etc.

[0053] The present invention also provides a drug / drug composition comprising, as described above, a modulator / inhibitor / antagonist, and / or a pharmaceutically acceptable carrier.

[0054] Preferably, the pharmaceutically acceptable carrier refers to a carrier that, when properly administered to animals or humans, does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0055] Preferably, the drug / drug composition may further contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0056] Specifically, the drug / drug composition is administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0057] Preferably, the dosage form of the drug / drug composition includes injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories, etc. All of the above dosage forms of the drug / drug composition can be prepared according to conventional methods in the pharmaceutical field.

[0058] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0059] Preferably, the route of administration of the drug / drug composition is parenteral, injection, or oral. Injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous injection. The drug / drug composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, gaseous, or liquid form, i.e., aqueous, non-aqueous, or suspension, more preferably tablets, capsules, granules, injections, or infusions. More preferably, it is administered intravascularly, subcutaneously, intraperitoneally, or intramuscularly. Preferably, the drug / drug composition can also be administered as an aerosol or spray, i.e., nasal administration; or intrathecal, intramedullary, or intraventricular administration. More preferably, the drug / drug composition can also be administered transdermally, topically, intraenterally, intravaginally, sublingually, or rectally.

[0060] The drug / pharmaceutical composition of the present invention can be formulated into various dosage forms as needed, and the dosage beneficial to the patient can be determined by a physician based on factors such as patient type, age, weight, general disease condition, and route of administration. The route of administration may include, for example, injection or other treatment methods. The dosage level of the drug / pharmaceutical composition of the present invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen may be a single injection or multiple injections, or may be adjusted. The selected dosage level and regimen are rationally adjusted based on various factors including the activity and stability (i.e., half-life) of the cellular drug / pharmaceutical composition, formulation, route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0061] The therapeutically effective dose of the drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the useful dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0062] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology, etc.

[0063] Furthermore, the drug / drug composition may be used alone and / or in combination with other drugs.

[0064] This invention also provides a method for detecting and analyzing the expression of the biomarker MT-ND6 protein as described above at the lung tissue level. The method is an immunohistochemical method, comprising the following steps:

[0065] Step 1: Detection Method (Immunohistochemical Staining Procedure)

[0066] (1) Dewaxing: The slide is placed in xylene 3-5 times for 10 minutes, anhydrous ethanol 1-2 times for 5-10 minutes, 95% ethanol, 85% ethanol and 75% ethanol for 5-10 minutes each, washed with tap water for 5-10 minutes, and distilled for 1-2 minutes; preferably, the slide is placed in xylene 3 times for 10 minutes, anhydrous ethanol 2 times for 10 minutes, 95% ethanol, 85% ethanol and 75% ethanol for 5 minutes each, washed with tap water for 5 minutes, and distilled for 1 minute.

[0067] (2) Antigen retrieval: Boiling antigen retrieval solution, treated in a water bath at 96-98℃ for 15-20 minutes, and then cooled naturally; preferably, treated in a water bath at 98℃ for 15 minutes;

[0068] (3) Rinse: Rinse with distilled water 1-2 times for 1 minute each time, and with PBS 2-3 times for 5 minutes each time; preferably, rinse with distilled water 1 time for 1 minute each time, and with PBS 3 times for 5 minutes each time.

[0069] (4) Blocking endogenous peroxidase activity: 3% hydrogen peroxide solution was directly added to the tissue sample and left to stand at room temperature for 20 minutes;

[0070] (5) Rinse: 2-3 times with PBS for 5 minutes each time; preferably, 3 times with PBS for 5 minutes each time.

[0071] (6) Primary antibody: Dilute the primary antibody at a ratio of (1:(800-1000)) and drop it onto the tissue, incubate overnight at 4°C, and PBS 2-3 times for 5 minutes each time; preferably, dilute the primary antibody at a ratio of (1:1000) and drop it onto the tissue, incubate overnight at 4°C, and PBS 3 times for 5 minutes each time.

[0072] (7) EnVisionplus assay kit: Staining with LeicaBond staining machine; This kit is used from blocking endogenous peroxidase activity to the primary antibody and until the completion of DAB staining.

[0073] (8) Dehydration: After rinsing the counterstained slides in water, immerse the slides in 75% ethanol for 1 minute, then transfer them to 85% ethanol for 1 minute, then immerse the slides in 95% ethanol for 1 minute, and repeat this step once (2 times, 1 minute each time); finally, immerse the slides in anhydrous ethanol for 1 minute, and repeat this step once (2 times, 1 minute each time).

[0074] (9) Mounting: After mounting with neutral quick-drying adhesive, scan the entire slide and observe the staining results.

[0075] Step 2, Analytical Methods (Scoring Method for Immunohistochemical Results):

[0076] If the staining result obtained in step one is brownish-yellow, this indicates a positive signal.

[0077] The scoring method for interpreting immunohistochemical results includes: for specific criteria of MT-ND6, a positive signal should be clearly located in the cytoplasm. Three to five tumor cells from 40x high-power fields are randomly selected, and DAB-stained blocks are segmented using ImageJ-IHC-profiler software. The percentage of areas with varying staining grayscale values ​​is automatically calculated: the percentage of strongly positive areas, positive areas, low-positive areas, and negative areas. The score is calculated based on the immunohistochemical interpretation criteria (H-Score) using the staining intensity and the percentage of positive cells. Preferably, five tumor cells from 40x high-power fields are randomly selected.

[0078] The calculation formula is H = ∑(I × PC), where I represents staining intensity, typically rated as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining); PC represents the percentage of positive cells for each intensity; and H represents the H-Score. The H-Score ranges from 0 to 300, where 0 represents no positive cell staining and 300 represents 100% strong staining.

[0079] In a specific implementation, the results of the analysis method are as follows: Figure 12 As shown.

[0080] The reagents used in the analytical method include:

[0081] The antigen retrieval solution is a citric acid antigen retrieval solution, directly purchased from Biosharp (China), catalog number BL151A;

[0082] Primary antibody: Rabbit anti-human MT-ND6 polyclonal antibody, directly purchased from Abclonal (China), catalog number: A17991;

[0083] The main components of the secondary antibody and chromogenic system are directly purchased from Leica (Germany) and are suitable for the Leica automated immunohistochemistry staining machine, catalog number: DS9800.

[0084] Peroxidase blocking agent: (3-4)% v / v hydrogen peroxide.

[0085] Primary antibody preparation reagents: rabbit anti-mouse IgG (<10 M μg / mL) contained in 10% v / v animal serum, Tris buffer (pH 7.4), and 0.09% ProClin. TM 950;

[0086] Polymer: Anti-rabbit poly-HRP-IgG (<25 μg / mL), containing 10% v / v animal serum, Tris buffer (pH 7.4), and 0.09% ProClin. TM 950;

[0087] DAB Part 1: 66Mm 3,3′ diaminobenzidine hydrochloride solution;

[0088] DAB Part B: <= 0.1% (v / v) hydrogen peroxide solution, containing stabilizer;

[0089] Hematoxylin: <0.1% hematoxylin solution

[0090] PBS powder: purchased directly from Biosharp (China), catalog number BL601.

[0091] EnVisionplus test kits are purchased directly from Leica. See the attached document for descriptions of all relevant reagents. Figure 14 .

[0092] Immunohistochemistry (IHC) is a method for detecting the expression of specific proteins in tissue sections through antigen-antibody reactions. This invention uses a fully automated immunohistochemistry analyzer to complete the entire detection process. This differs from existing manual immunohistochemistry (manual IHC) methods in terms of operation, efficiency, and accuracy, as detailed below:

[0093] The operating methods differ: In this invention, the entire detection process (such as tissue section processing, antibody incubation, staining, and rinsing) is completed by machine, making the operation highly automated. Existing technologies involve manual detection, where each step is performed manually, including antibody preparation, incubation time control, and staining reaction observation, making the operation rather cumbersome.

[0094] The efficiency difference lies in the fact that this invention automates the entire process, typically handling large numbers of samples and allowing for multiple tests to be performed simultaneously, significantly improving work efficiency and making it particularly suitable for batch testing needs. Existing manual testing techniques are suitable for small batches of samples, but are less efficient and prone to disruptions in continuity and accuracy due to human error.

[0095] Consistency differs from repeatability: The automated equipment of this invention provides higher consistency and repeatability, reduces human error, and ensures consistent processing conditions for each sample. Existing manual testing techniques may suffer from differences between different operators, resulting in poor repeatability and consistency of results.

[0096] Precision differs from result quality: The automated equipment of this invention can precisely control the conditions of each step, such as antibody incubation time, staining reaction time and temperature, ensuring uniform staining and accurate results. Existing manual operations struggle to guarantee precise control of each step and are easily affected by human factors (such as operation time and temperature control), potentially leading to uneven staining or deviations in experimental results.

[0097] The operational difficulty differs: This invention requires only simple machine setup and sample insertion, with a low operational threshold and minimal professional requirements for technicians. Existing manual testing techniques, on the other hand, require a high level of technical skill and experience, are complex and time-consuming, and may pose a significant challenge to novice or inexperienced technicians.

[0098] The economic costs differ: This invention requires a higher initial investment, including equipment purchase and maintenance costs, but in the long run, it can save labor costs and improve work efficiency, making it suitable for high-throughput testing needs. Existing manual testing technologies have lower initial investment, mainly in consumables and labor costs, but labor costs and operation time are higher, and it may be uneconomical for high-throughput testing.

[0099] The present invention also provides a method for screening candidate drugs for treating non-small cell lung cancer, alleviating or preventing non-small cell lung cancer metastasis and / or improving the prognosis of non-small cell lung cancer. The method includes detecting the effect of the candidate drug on the level of anti-MT-ND6 autoantibody in a subject or a sample obtained from the subject. A decrease in the level of anti-MT-ND6 autoantibody after using the candidate drug indicates that the candidate drug has the effect of treating non-small cell lung cancer, alleviating or preventing non-small cell lung cancer metastasis and / or improving the prognosis of non-small cell lung cancer.

[0100] The present invention also provides a method for preventing / delaying / treating non-small cell lung cancer, the method comprising administering to the individual an effective amount of the modulator as described above, or the drug / drug composition as described above.

[0101] The present invention also provides an application, which includes the use of the biomarkers as described above, or the detection reagents / kits as described above, the modulators / inhibitors / antagonists as described above, or the drugs / drug compositions as described above, or the methods as described above in the preparation of drugs for the prevention / delay / treatment / diagnosis of non-small cell lung cancer, products for the diagnosis of non-small cell lung cancer, products for the sensitivity prediction / prognosis of platinum-based chemotherapy drug system therapy for non-small cell lung cancer, products for the treatment of non-small cell lung cancer by targeting MT-ND6 regulated expression, etc.

[0102] In this invention, the biomarkers, or the detection reagents / kits, or the modulators / inhibitors / antagonists, or the drugs / drug compositions, or the methods, or the applications described above, as described above, include non-small cell lung cancer such as lung adenocarcinoma, squamous cell carcinoma, and large cell carcinoma; the platinum-based chemotherapy includes combination therapy with platinum-based drugs and other chemotherapy drugs, combination therapy with platinum-based drugs and immunotherapy, combination therapy with platinum-based drugs and radiotherapy, or combinations thereof; the amino acid sequence of MT-ND6 is shown in SEQ ID NO.1.

[0103] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0104] Currently, there are no clinically significant biomarkers for predicting the efficacy of platinum-based neoadjuvant therapy, postoperative adjuvant platinum-based therapy, and platinum-based therapy for lung cancer with driver gene occult squamous cell carcinoma. Furthermore, for patients with squamous cell carcinoma receiving systemic platinum-based chemotherapy (combination therapy / systemic therapy), current clinically tested indicators including Ki67, P63, p40, CK5&6, NUT, CK7, TIF-1 (SPT24), NapsinA, and Syn are not specific biomarkers for guiding systemic platinum-based chemotherapy and lack predictive power. This invention is based on the MT-ND6 protein, developed with a solid biological research foundation. Its single indicator strongly indicates the efficacy of platinum-based chemotherapy and can effectively indicate the treatment effect of platinum-based chemotherapy (including platinum-based drugs combined with other chemotherapeutic agents, platinum-based drugs combined with immunotherapy, platinum-based drugs combined with radiotherapy, etc., or combinations thereof) in patients with squamous cell carcinoma. This invention relates to a method for detecting the biomarker MT-ND6 protein. Compared to existing technologies that develop biomarkers based on multiple transcripts or metabolites to indicate sensitivity to platinum-based chemotherapy, this invention utilizes IHC detection and analysis based on a single pathological biomarker from tumor tissue. This method is recognized as simple, easy to preserve samples, easy to operate, and yields easily reproducible results, making it widely applicable in general hospitals. It is simple to operate and widely available. Compared to other biomarker combinations, this invention has significant advantages. Using commercially available antibodies and based on traditional IHC detection, a single biomarker has the efficacy of sensitive predictive power for systemic therapy with platinum-based chemotherapy drugs (combined with immunotherapy, radiotherapy, etc.). It does not require complex models or diverse detection methods.

[0105] This invention is based on the traditional immunohistochemical (IHC) detection method in pathology, focusing on the expression analysis of MT-ND6, a single biomarker encoded by the mitochondrial gene, in tumor tissue. The IHC method has several recognized advantages, including simplicity of operation, ease of sample preservation, convenient detection procedures, high reproducibility of experimental results, and broad applicability to existing testing platforms in general medical institutions. These characteristics make the IHC method highly accessible and practical in clinical practice.

[0106] Compared to other detection methods that require multiple biomarker combinations or complex models, this invention fully utilizes commercially available antibodies, using the single biomarker MT-ND6 as the core, and achieves the prediction of sensitivity to platinum-based chemotherapy (including combined immunotherapy, radiotherapy, and other systemic therapies) and the prognosis of survival in patients who have not received platinum-based treatment based on traditional IHC detection. The significant advantages of this method include: Highly efficient prediction: A single biomarker can effectively indicate the sensitivity of NSCLC tumors to platinum-based chemotherapy regimens, avoiding reliance on complex models; Cost advantage: No need to introduce expensive or difficult-to-obtain reagents, sequencing instruments, or analysis; the detection process is economical and efficient; Ease of operation: Traditional IHC detection methods are widely known and easy to master, requiring no special equipment or training; It can be used in conjunction with existing high-throughput IHC detection instruments, facilitating high-throughput patient testing; Stable results: The high repeatability and reliability of the detection method provide a solid foundation for clinical application; Wide applicability: It is compatible with existing testing conditions in general hospitals, requiring no additional technical upgrades. Attached Figure Description

[0107] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0108] Figure 1a , Figure 1b The association between the transcriptional expression levels of mitochondrial protein-encoding genes in lung cancer and sensitivity to platinum-based chemotherapy drugs was investigated in the CCLE (Cancer Cell Line Encyclopedia) database: 1A. Correlation analysis results of the transcriptional levels of 13 mitochondrial protein-encoding genes (MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-COⅠ, MT-COⅡ, MT-COⅢ, MT-ATP6, MT-ATP8, and MT-CYB and the CIS-IC50 (cisplatin half-maximal inhibitory rate) of 13 lung squamous cell carcinoma (LUSC) cell lines; 1B. Correlation analysis results of the transcriptional levels of 13 mitochondrial genes and the CBP-IC50 of 5 lung squamous cell carcinoma cell lines; 1C. Ranking results of the correlation between the transcriptional levels of 13 mitochondrial genes and the CIS-IC50 of lung squamous cell carcinoma cells from high to low; 1D. Ranking results of the correlation between the transcriptional levels of 13 mitochondrial genes and the CBP (carboplatin)-IC50 of lung squamous cell carcinoma cells from high to low.

[0109] Figure 2a , Figure 2b: Screening of platinum-based drug sensitivity for 7 NSCLC cell lines (H226, LK-2, H520, Y-90, H1581, H1703, SK-MES-1): 2A. Laboratory self-testing of IC50 of 7 NSCLC cell lines CIS (cisplatin); 2B. Laboratory self-testing of IC50 of 7 NSCLC cell lines CBP (carboplatin).

[0110] Figure 3 The study investigated the association between the transcriptional expression levels of mitochondrial genes in seven NSCLC cell lines and their sensitivity to platinum-based chemotherapy drugs. The study included: 3A. Transcriptional levels of MT-ND6, MT-ND5, MT-COⅠ, and MT-ND4 in seven lung squamous cell carcinoma cell lines tested in the laboratory; 3B. Correlation analysis between the transcriptional levels of MT-ND6, MT-ND5, MT-COⅠ, and MT-ND4 and CIS-IC50 in seven lung squamous cell carcinoma cell lines tested in the laboratory; 3C. Correlation analysis between the transcriptional levels of MT-ND6, MT-ND5, MT-COⅠ, and MT-ND4 and CBP-IC50 in seven lung squamous cell carcinoma cell lines tested in the laboratory; 3D. A ranking of the correlation between the transcriptional levels of four mitochondrial genes tested in the laboratory and the CIS-IC50 and CBP-IC50 of lung squamous cell carcinoma cells, from highest to lowest. Note that a Pearson correlation coefficient (r) close to 1 indicates a positive correlation between transcriptional levels and platinum-based drugs; a r close to -1 indicates a negative correlation.

[0111] Figure 4 The association between the protein expression level of the mitochondrial gene MT-ND6 in seven NSCLC cell lines and the sensitivity to platinum-based chemotherapy drugs was investigated: 4A. MT-ND6 protein levels in seven lung squamous cell carcinoma cell lines tested in the laboratory, with beta-tubulin as an internal control protein; 4B. Left figure: Correlation analysis between MT-ND6 protein levels and cisplatin IC50 in seven lung squamous cell carcinoma cell lines tested in the laboratory; Right figure: Correlation analysis between MT-ND6 protein levels and carboplatin IC50 in seven lung squamous cell carcinoma cell lines tested in the laboratory. Note: When the Pearson correlation coefficient r is close to 1, the transcriptional level is positively correlated with platinum-based drugs; when r is close to -1, the transcriptional level is negatively correlated with platinum-based drugs.

[0112] Figure 5a , Figure 5b In vitro experiments – Investigation of the sensitivity of mitochondrial gene expression to platinum-based drugs: 5A. Effects of cisplatin (CIS) vs. cisplatin combined with the mitochondrial transcription inhibitor (IMT1) on the proliferation of four lung squamous cell carcinoma cell lines; 5B. Effects of carboplatin (CBP) vs. carboplatin combined with the mitochondrial transcription inhibitor (IMT1) on the proliferation of two lung squamous cell carcinoma cell lines. Where: ns: P>0.05, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001.

[0113] Figure 6 : Confirmation of MT-ND6 protein overexpression and mitochondrial localization in NSCLC cells H226: 5A. Validation results of MT-ND6 protein overexpression level in H226 cells, where NC: empty vector group cells, OE: overexpression group cells; 5B. Localization results of the overexpression marker MT-ND6 in mitochondria of H226 cells: MT-ND6 (marker): red, MT-COⅠ (protein encoded by the mitochondrial gene itself, located in mitochondria): green, DAPI (4',6-diamidinyl-2-phenylindole, located in the nucleus): blue; scale bar: 25 μm.

[0114] Figure 7 : CIS significantly increased the toxicity of MT-ND6 overexpressing lung squamous cell carcinoma cells: 7A: Effect of CIS on the proliferation of cells in the MT-ND6 protein overexpression (OE) group and NC group (control); 7B: Effect of CIS on the proliferation of cells in the H226-monoclonal MT-ND6 overexpression (OE) group and NC group.

[0115] Figure 8 Investigation of DNA damage induced by different MT-ND6 expression levels in cells treated with cisplatin: The left image shows immunofluorescence staining, with blue representing the cell nucleus and green representing the DNA damage marker Gamma-H2AX; the right image shows the quantitative analysis of nuclear DNA damage at the single-cell level. Note: ****: P < 0.0001, two-tailed t-test showed significant difference.

[0116] Figure 9: Differences in MT-ND6 protein expression in tumor tissue and adjacent normal tissue samples of lung squamous cell carcinoma patients and Kaplan-Meier survival analysis: 9A. Panoramic scan and 40x objective: Results of MT-ND6 protein expression levels in tumor tissue and paired adjacent normal tissue of the same lung squamous cell carcinoma patient (Note: Blue staining represents cell nuclei, and brownish-yellow cytoplasm represents MT-ND6 antibody staining); 9B. OS survival curves between the high and low MT-ND6 protein expression groups in tumor tissue of 87 patients. Where: P < 0.05 is considered statistically significant; scale bar: 2.5 mm (1.7 ×), 50 μm (40 ×).

[0117] Figure 10 OS curves between high and low expression of MT-ND6 protein in tumor tissues of 33 patients with NSCLC (squamous cell lung carcinoma) who had not received neoadjuvant therapy and received platinum-based chemotherapy after surgery. P < 0.05 was considered statistically significant.

[0118] Figure 11Imaging assessment of MT-ND6 protein expression and the efficacy of platinum-based chemotherapy combined with immunotherapy in patients with squamous cell carcinoma of the lung: 11A. Under 40x objective lens: Results of MT-ND6 protein expression in paraffin-embedded tumor tissue samples from two patients with squamous cell carcinoma of the lung who received two cycles of platinum-based chemotherapy (cisplatin + paclitaxel) combined with immunotherapy (tislelizumab) (blue staining represents cell nuclei, and brownish-yellow cytoplasm represents MT-ND6 antibody staining); 11B. Enhanced CT images of the lungs before and after treatment in two patients with squamous cell carcinoma of the lung who received two cycles of platinum-based chemotherapy (cisplatin + paclitaxel) combined with immunotherapy (tislelizumab). Scale bar: 50μm (40×).

[0119] Figure 12 The scoring method for judging immunohistochemical results.

[0120] Figure 13 Design a pattern for the amplification of the target gene.

[0121] Figure 14 This is the reagent instruction manual. Detailed Implementation

[0122] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0123] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0124] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0125] This invention discloses a biomarker for predicting the sensitivity / prognosis of non-small cell lung cancer (NSCLC) treated with platinum-based chemotherapy drugs, and its application, belonging to the field of tumor biology technology. The biomarker is MT-ND6. MT-ND6 is used to predict the sensitivity of NSCLC to platinum-based chemotherapy, avoiding unnecessary treatment and reducing ineffective treatment and the resulting unnecessary toxic side effects of chemotherapy. The biomarker MT-ND6 can also serve as an independent marker to distinguish NSCLC tumor tissue from adjacent normal tissue; the expression level of MT-ND6 protein in tumor tissue is significantly higher than in normal tissue, thus having diagnostic value. This invention also discloses a detection reagent, a method for screening the biomarker, and a method for predicting / evaluating the sensitivity / prognosis of NSCLC treated with platinum-based chemotherapy drugs. The biomarker of this invention can be used in the preparation of products for the diagnosis of NSCLC, products for predicting the sensitivity of NSCLC to platinum-based (systemic) therapy, products for detecting prognostic biomarkers for NSCLC patients, and products for the treatment of NSCLC, showing broad application prospects.

[0126] Unless otherwise specified, the experimental materials used in the examples are all conventional biochemical reagents.

[0127] Example 1: Hypothesis proposed based on correlation.

[0128] This invention, using the CCLE database, proposes a significant negative correlation between the IC50 value of platinum-based chemotherapy (including cisplatin CIS and carboplatin CBP) and MT-ND6 gene expression in NSCLC (non-small cell lung cancer) tumor cells, and hypothesizes that MT-ND6 expression level can indicate the sensitivity of NSCLC lung squamous cell carcinoma cells to platinum-based drugs.

[0129] To explore the relationship between mitochondrial gene transcription levels and sensitivity to platinum-based chemotherapy drugs in lung cancer, this invention obtained relevant data from various lung cancer cell lines from the CCLE database. Specifically, this included: expression data of mitochondrial protein-coding genes (MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-COⅠ, MT-COⅡ, MT-COⅢ, MT-ATP6, MT-ATP8, and MT-CYB) from 13 lung squamous cell carcinoma (LUSC) cell lines; and IC50 values ​​of cisplatin (CIS) in 13 LUSC cell lines and carboplatin (CBP) in 5 LUSC cell lines.

[0130] Pearson correlation analysis revealed that the transcriptional levels of these 13 mitochondrial respiratory chain protein-coding genes were generally negatively correlated with the IC50 value of CIS in lung squamous cell carcinoma cell lines, with MT-ND6 showing the strongest negative correlation (see Figure 1, A and B). Furthermore, gene transcriptional levels in lung squamous cell carcinoma cell lines also showed a negative correlation with the IC50 value of CBP, although the negative correlation of MT-ND6 was not the highest, it was still significant (see Figure 1, B). Figure 1b (C and D in the middle).

[0131] The results of this invention indicate that the expression of multiple mitochondrial genes in lung squamous cell carcinoma cell lines is significantly negatively correlated with sensitivity to platinum-based drugs (CIS and CBP), especially the MT-ND6 gene. Therefore, it is hypothesized that MT-ND6 expression levels can indicate the sensitivity of NSCLC lung squamous cell carcinoma cells to platinum-based drugs.

[0132] Example 2: Laboratory Validation of Correlation

[0133] This invention uses laboratory measurements to detect the IC50 values ​​of cisplatin (CIS) and carboplatin (CBP) in seven lung squamous cell carcinoma cell lines, as well as the expression levels of mitochondrial protein-coding genes. Consistent with the analysis results from the CCLE database, the expression levels of mitochondrial protein-coding genes and the IC50 value of cisplatin were generally negatively correlated, with MT-ND6 showing the strongest negative correlation.

[0134] To verify the negative correlation between mitochondrial gene transcriptional expression levels in the CCLE database and sensitivity to platinum-based chemotherapy drugs, this invention first used a CCK-8 assay kit (supplier: MCE, trade number: HY-K0301) to determine the survival rates of seven lung squamous cell carcinoma cell lines in the laboratory after treatment with proportionally diluted CBP for 96 hours. The survival data were then analyzed using nonlinear regression analysis with GraphPadPrism 9 software to calculate the IC50 values ​​of these seven lung squamous cell carcinoma cell lines for CIS and CBP. The IC50 values ​​after CIS treatment are shown below. Figure 2a The IC50 values ​​after CBP processing are shown in the figure. Figure 2b B. (e.g.) Figure 2a , Figure 2b As shown, lung squamous cell carcinoma cells are more sensitive to CIS than CBP.

[0135] This invention uses the mitochondrial genes MT-ND6, MT-ND5, MT-COⅠ, and MT-ND4, which have high correlations with the CCLE database, as representative genes for evaluating the transcriptional levels of seven lung squamous cell carcinoma cell lines. Figure 3A). This invention designed specific primers for these genes and performed quantitative analysis using conventional RT-qPCR. To more accurately assess the expression levels of these genes in lung squamous cell carcinoma cells, normal lung tissue was used as a control group for analysis.

[0136] Primer name sequence (5' to 3')

[0137] Internal reference gene Actin Fw TCACCCACACTGTGCCCATCTACGA (SEQ ID NO.6)

[0138] Internal reference gene Actin Rv CAGCGGAACCGCTCATTGCCAATGG (SEQ ID NO.7)

[0139] MT-ND4 FwTCTGTGCTAGTAACCACGTTC(SEQ ID NO.8)

[0140] MT-ND4 Rv AAAACCCGGTAATGATGTCG(SEQ ID NO.9)

[0141] MT-ND5 Fw TCATCGCTACCTCCCTGACA(SEQ ID NO.10)

[0142] MT-ND5 Rv ATCCTGCGAATAGGCTTCCG(SEQ ID NO.11)

[0143] MT-ND6 Fw ATATACTACAGCGATGGCTA(SEQ ID NO.12)

[0144] MT-ND6 Rv AATCCTACCTCCATCGCTA(SEQ ID NO.13)

[0145] MT-COⅠ Fw AATAGGAGCTGTATTTGCCAT(SEQ ID NO.14)

[0146] MT-COⅠRvAGAAAGTTAGATTTACGCCGAT(SEQ ID NO.15)

[0147] This invention employs Pearson correlation analysis to compare the expression data of mitochondrial protein-coding genes MT-ND6, MT-ND5, MT-COⅠ, and MT-ND4 from seven lung squamous cell carcinoma cell lines with the sensitivity data (i.e., IC50 values) of platinum-based drugs (CIS and CBP). The invention found that the transcriptional levels of these four mitochondrial genes were negatively correlated with the IC50 value of CIS, with MT-ND6 showing the strongest negative correlation (see...). Figure 3 (B, D). However, in the correlation analysis with CBP IC50 values, only MT-ND6 showed a negative correlation, while other genes showed a positive correlation (see B, D). Figure 3 (C, D). Protein levels further confirmed that MT-ND6 protein expression (MT-ND6 antibody: supplier: ABClonal, catalog number: A17991; internal control beta-tubulin, supplier: ABClonal, catalog number: AC021) was negatively correlated with cisplatin and carboplatin, with correlation coefficients of -0.18 and -0.5, respectively (see C, D). Figure 4 (A, B)

[0148] Based on these analyses, this invention observed a general negative correlation between mitochondrial gene transcription levels and the IC50 value of CIS, consistent with the results found in the database. This finding indicates that high expression levels of the mitochondrial gene MT-ND6 are associated with lower IC50 values / higher sensitivity, suggesting that mitochondrial gene transcription levels may be a potential indicator of sensitivity to platinum-based chemotherapy. Furthermore, these results highlight the possibility that the mitochondrial gene MT-ND6 is a key molecule driving sensitivity to platinum-based chemotherapy.

[0149] Example 3 Verification of biological effects

[0150] The present invention conducted the following experiments to verify the biological effects:

[0151] (1) The effect of CIS and CBP on the proliferation of lung squamous cell carcinoma cells was investigated by inhibiting mitochondrial gene expression. The results showed that compared with the control group treated with CIS or CBP alone, the cytotoxicity of lung squamous cell carcinoma cell lines to these two platinum-based drugs was significantly reduced after inhibiting mitochondrial gene expression. This result indicates that the inhibition of mitochondrial gene expression, including MT-ND6, can significantly weaken the cytotoxic effects of CIS and CBP and reduce the sensitivity of cells to CIS and CBP.

[0152] To further investigate the role of mitochondrial genes in the sensitivity of platinum-based chemotherapy drugs, this invention used a 25 μM concentration of a mitochondrial RNA polymerase (POLRMT) inhibitor (referred to as IMT1) in combination with a pre-defined concentration gradient of platinum-based drugs (CIS and CBP) to treat lung squamous cell carcinoma cell lines for 96 hours, aiming to observe the effect of this combined treatment on the proliferation ability of lung squamous cell carcinoma cells. The 25 μM mitochondrial inhibitor concentration was chosen because this concentration can significantly inhibit the transcription of mitochondrial genes, including MT-ND6, and no significant cytotoxicity was observed in other NSCLC cell lines in this experiment.

[0153] Results regarding the survival rate of NSCLC cells treated with the combination of cisplatin (CIS) and IMT1 showed that in the CIS+IMT1 group, in lung squamous cell carcinoma cell lines H520, SK-MES-1, H226, and H2170, cell survival was significantly higher from the lowest dose of combination therapy than in the group treated with CIS alone. These differences were statistically significant (p<0.05), see [link to relevant documentation]. Figure 5a In the H226 lung squamous cell carcinoma cell line, the cell survival rate in the combination therapy group was also observed to be higher than that in the CIS group alone, but the difference was not statistically significant. Results from four lung squamous cell carcinoma cell lines suggest that extensive inhibition of mitochondrial expression induces cisplatin insensitivity to chemotherapy.

[0154] Results regarding the survival rate of NSCLC cells treated with the combination of carboplatin (CBP) and IMT1 showed that in the two cell lines tested (H520 and H226), the cell survival rate of the CBP+IMT1 combination group was significantly higher than that of the CBP-only group. Figure 5b B. This suggests that widespread inhibition of mitochondrial expression induces insensitivity to carboplatin chemotherapy.

[0155] The above experimental results indicate that, in the four lung squamous cell carcinoma cell lines tested, inhibiting the transcription of mitochondrial genes (including MT-ND6) can significantly reduce the sensitivity of lung squamous cell carcinoma cells to platinum-based drugs (CIS and CBP).

[0156] (2) Further investigation was conducted by overexpressing the MT-ND6 gene in lung squamous cell carcinoma cell lines to examine the effect of CIS on the proliferation ability of lung squamous cell carcinoma cells. The experimental results showed that, compared with the control group, the lung squamous cell carcinoma cell lines overexpressing the MT-ND6 gene showed significantly improved sensitivity to CIS chemotherapy.

[0157]

[0158] 1. Plasmid Construction

[0159] 1.1 Primers for target gene amplification

[0160] The target gene is ND6, and the vector is pCDH-3×Flag+TurboRFP+Puro. The design map is as follows: Figure 13 As shown.

[0161] COX8 is the MTS sequence, and ND6 is the target sequence. The COX8-ND6 fragment was synthesized using overlap extension PCR and used as the amplification template. The PCR primer sequences are as follows:

[0162] F: GATTCTAGAGCTAGCGAATTCGCCACCATGTCCGTCCTGACGC (SEQ ID NO. 2)

[0163] R: ATGGTCTTTGTAGTCGGATCCATTCCCCCGAGCAATCTC (SEQ ID NO.3)

[0164] Note: Figure 13 In the diagram, the underlined part represents the homologous arm, the red part represents the restriction enzyme site, the green part represents the Kozak sequence, the upstream part represents EcoRI, and the downstream part represents BamHI.

[0165] 1.2 Enzyme digestion and recovery of the vector

[0166] The pLV3-CMV-MCS-3×FLAG-turboRFP-Puro vector was digested with both EcoRI and BamHI. The digestion system is as follows:

[0167] 10×Buffer: 5μL

[0168] Vector: 3μL

[0169] EcoRI: 1.5μL

[0170] BamHI: 1.5 μL

[0171] ddH2O: 39μL

[0172] Total volume: 50 μL, digested at 37 °C for 30 minutes. The vector was then detected and recovered after digestion.

[0173] 1.3 Multi-segment homologous recombination reaction

[0174] The connection reaction is carried out according to the following reaction system:

[0175] Exnase II enzyme: 1 μL

[0176] 5×CE II Buffer: 2μL

[0177] Vector fragment: 3μL

[0178] Target fragment: 1 μL

[0179] ddH2O: 3μL

[0180] Total volume: 10 μL, react at 37 °C for 30 minutes.

[0181] 1.4 Transformation of Ligation Products into Competent Cells

[0182] BL21(DE3) competent cells were mixed with the recombinant product and incubated on ice for 30 minutes. Then, they were heat-shocked (42°C, 45 seconds) and cooled for 2 minutes. 900 μL of antibiotic-free LB medium was added, and the cells were incubated at 30°C and 180 rpm for 30 minutes. The cells were centrifuged at 10,000 rpm for 1 minute, the supernatant was discarded, and the mixture was gently spread onto LB agar plates containing 100 μg / mL ampicillin. The cells were allowed to absorb at room temperature and then incubated overnight at 37°C.

[0183] 1.5 Colony PCR Identification

[0184] Single colonies were selected for PCR identification using the following primers:

[0185] CMV-F: GTAGGCGTGTACGGTGGGAG (SEQ ID NO.4)

[0186] JD-R:CTACCTCCATCGCTAACCC(SEQ ID NO.5)

[0187] CMV-F is located at the CMV promoter of the pLV3-CMV-MCS-3×FLAG-turboRFP-Puro vector, and JD-R is located on the ND6 gene, with a product size of 502 bp. After PCR, the results were detected by 1% agarose gel electrophoresis. Clones matching the target fragment were selected for overnight culture, plasmids were extracted, and sequenced.

[0188] 2. Plasmid detoxification and large-scale extraction

[0189] Take portions from 150 mL of overnight culture medium, centrifuge at 12000 rpm for 2 minutes, and remove the supernatant. Add 12 mL of Buffer P1 and mix well, then add 12 mL of Buffer P2 and mix gently, allowing to stand for 5 minutes. Add 2 mL of Buffer PS to the adsorption column to equilibrate, centrifuge at 12000 rpm for 2 minutes, and discard the waste liquid.

[0190] Then add 12 mL of Buffer E3, centrifuge at 12000 rpm for 10 minutes, and transfer the supernatant. After mixing with isopropanol, collect the DNA using an adsorption column. Wash the adsorption column sequentially with Buffer PW, and finally elute the plasmid with Endo-Free Buffer EB, and store at -20℃.

[0191] 3. Lentiviral Packaging and Infection Verification

[0192] Cell plating: 293T cells were collected by trypsin digestion and seeded in 10cm culture dishes. They were cultured at 37℃ and 5% CO2 until 50-60% confluence.

[0193] Preparation of plasmid-transfection reagent complex: Mix ND6 overexpression vector, psPAX2, and Pmd2.G plasmid in a 1:1:1 ratio, add transfection reagent, and let stand at room temperature for 15 minutes.

[0194] Transfecting cells: Add the complex to the cells and incubate at 37°C and 5% CO2.

[0195] Collect viral supernatant: Collect the supernatant 48 hours after transfection and store at 4°C. Collect again after 72 hours and centrifuge and filter.

[0196] Lentiviral concentration: Viral fluid is treated with a concentration reagent and the virus is further purified and concentrated as needed.

[0197] 4. Cell transfection

[0198] Cells were seeded into 6-well plates and cultured at 37°C in a 5% CO2 incubator, ensuring a cell density of 30-50% at infection on the second day. After 24 hours, once cells had adhered, they were labeled and grouped, and transfection reagent was added to each well. After another 24 hours, 1 mL of complete culture medium was added to each well. After 48 hours, fluorescence intensity was observed to determine infection efficiency, and the infection period was extended to 72 hours as needed. Based on the infection efficiency, 2 mL of culture medium containing a sufficient concentration of puromycin was added to kill all control cells. After another 48 hours of culture, 2 mL of complete culture medium was added, and transfection efficiency was observed using an inverted fluorescence microscope.

[0199] This invention employed both Western blot (WB) and immunofluorescence staining (IF) to verify the expression of MT-ND6 protein and its localization in cells. Western blot analysis of MT-ND6 protein and its fused 3×Flag tag protein showed that in the H226 lung squamous cell carcinoma overexpression (OE) group, both MT-ND6 protein and its Flag tag were located around 25 kDa (see [link to article]). Figure 6 (A) This study confirmed successful protein expression and the correct molecular size. The present invention detected the co-localization of the TurboRFP-tagged protein (red fluorescence) inherent in the MT-ND6 plasmid vector and the immunofluorescence-stained mitochondrial marker protein MT-COⅠ (green fluorescence) in cells. IF (immunofluorescence) results showed that these two proteins co-localized in cytoplasmic regions, such as... Figure 6As shown in Figure B, this invention confirms the successful construction of an NSCLC cell line that overexpresses MT-ND6 in mitochondrial organelles.

[0200] Cellular immunosorbent assay (CIS) at preset drug concentration gradients (10, 5, 2.5, 1.25, 0.625, 0.3125 μM) was applied to MT-ND6 protein-overexpressing H226(OE) and control H226(NC) cell lines for 48 hours to evaluate the effect of CIS on the cell proliferation capacity of the two groups. Results showed that, starting from the lowest dose, the cell viability of the H226(OE) group was significantly lower than that of the H226(NC) group. Figure 7 As shown in Figures A and B. The above experimental results indicate that upregulating MT-ND6 protein expression in lung squamous cell carcinoma cell lines can increase the sensitivity of CIS to chemotherapy.

[0201] The main mechanism by which cisplatin-induced tumor cytotoxicity is through its action on DNA, inducing cellular DNA damage and promoting cell death. Furthermore, this invention uses Gamma-H2AX (clone JBW30, Millipore) immunofluorescence (IF), a marker indicating DNA damage, to investigate the effect of cisplatin on MT-ND6 overexpressing cells (H226-ND6-OE) DNA damage compared to the empty vector group (H226-ND6-NC). Figure 8 (Left figure). Quantification of the degree of DNA damage in the cell nucleus of each group was based on the number of foci (spots) stained with the DNA damage marker Gamma-H2AX. Figure 8 The right figure shows that, compared to normal NSCLC cells (NC group), cisplatin treatment of NSCLC cells overexpressing MT-ND6 significantly induced more DNA damage. This result suggests that, compared to control cells, cisplatin treatment of MT-ND6-overexpressing NSCLC cells can induce more DNA damage, exacerbate cisplatin's cytotoxicity to NSCLC tumor cells, and thus increase drug sensitivity. This study provides a molecular basis for the biological effect of mitochondrial-encoded protein MT-ND6 expression as an indicator of cisplatin chemosensitivity.

[0202] Example 4: Validation of Clinical Samples

[0203] The present invention has conducted the following experimental verification of clinical samples:

[0204] (1) To investigate the association between MT-ND6 protein expression and survival in patients with squamous cell carcinoma of the lung, this invention detected MT-ND6 expression in paraffin-embedded tumor tissue samples from 87 patients with complete clinical follow-up data from Sichuan Cancer Hospital, as well as in adjacent normal tissues of the tumor patients, using immunohistochemistry. Figure 9AThe expression of MT-ND6 protein in tumor tissue of patients with squamous cell carcinoma of the lung was significantly higher than that in adjacent normal tissue, and the difference was statistically significant (P = 7.6 × 10⁻⁶). -16 The surv-cutpoint function was used to determine the MT-ND6 H-Score in tumor and normal tissues; the optimal expression cutoff point for the MT-ND6 H-Score in tumor tissue was also determined, and patients were divided into high-expression and low-expression groups based on this cutoff value (78.6 points). MT-ND6 levels in NSCLC tumor tissues were significantly higher than in adjacent normal tissues; the overall survival (OS) in the high-expression group was significantly longer than that in the low-expression group (P = 0.044). Figure 9B MT-ND6 serves as a diagnostic marker that significantly distinguishes between NSCLC tumors and normal tissues; MT-ND6 in tumor tissues has value as a potential prognostic biomarker for NSCLC, with high expression of MT-ND6 in patients with squamous cell carcinoma of the lung suggesting a better prognosis.

[0205] (2) Among the 87 patients with squamous cell carcinoma of the lung mentioned above, this invention screened out patients who only received systemic adjuvant therapy with platinum-based chemotherapy after surgery and had a survival period. The analysis included 33 patients with squamous cell carcinoma of the lung who had not received neoadjuvant therapy and received adjuvant therapy with platinum-based chemotherapy after surgery (Table 1: lists the patients' treatment information). The survival analysis results showed that compared with patients with low MT-ND6 expression, patients with high MT-ND6 IHC expression in their tumor tissue had significantly longer survival periods. Figure 10 The results suggest that NSCLC squamous cell carcinoma patients with high MT-ND6 expression in tumor tissue have a better prognosis after receiving systemic treatment with platinum-based chemotherapy drugs. MT-ND6 expression can be a predictive biomarker for the sensitivity of squamous cell carcinoma patients who have not received neoadjuvant therapy and have received platinum-based adjuvant therapy after surgery to systemic treatment with platinum-based chemotherapy drugs.

[0206] Table 1 shows information on 133 non-small cell lung cancer patients who did not receive platinum-based chemotherapy as adjuvant therapy after surgery.

[0207]

[0208]

[0209] (3) To investigate whether the difference in efficacy of platinum-based therapy in patients with squamous cell lung cancer is related to the expression level of MT-ND6 protein, this study selected two representative cases (NSCLC patients with squamous cell lung cancer who received neoadjuvant platinum-based chemotherapy combined with immunotherapy) for analysis. Both cases had a tumor stage of cT4N2M0 before treatment, and the treatment regimen consisted of two cycles of cisplatin-based chemotherapy combined with tislelizumab. This invention retrospectively detected and analyzed the expression of MT-ND6 protein in paraffin-embedded tissue samples taken before treatment. Based on previous ( Figure 9A The evaluation criteria used in the detection and analysis of MT-ND6 protein expression in tumors of 87 patients with squamous cell carcinoma of the lung were as follows: the surv-cutpoint function determined the optimal expression cutoff value of 78.6 points for the MT-ND6 H-Score in tumor tissue. Patients with values ​​higher than this value were classified as high expression group, and those with values ​​lower than this value were classified as low expression group. This invention observed that Case 1 (good efficacy) had high MT-ND6 expression (H-Score = 108), while Case 2 (poor efficacy) had low expression (H-Score = 69.8). Combined with CT imaging results, Case 1, with higher MT-ND6 expression, achieved partial response (PR) after treatment, while Case 2, with lower expression, had stable disease (SD) (see...). Figure 11 (A and B in the original text). This result suggests that high expression of the biomarker MT-ND6 alone is a potentially effective biomarker for predicting the sensitivity of patients with squamous cell lung cancer to platinum-based chemotherapy combined with immunotherapy.

[0210] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0211] As used in this invention, the term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects.

[0212] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0213] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

[0214] References:

[0215] 1.Bray,F.,et al.,Global cancer statistics 2022:GLOBOCAN estimates ofincidence and mortality worldwide for 36cancers in 185countries.CA:A CancerJournal for Clinicians,2024.74(3):p.229-263.

[0216] 2.Zheng,R.S.,et al.,[Cancer incidence and mortality in China,2022].Zhonghua Zhong Liu Za Zhi [Chinese Journal of Oncology],2024.46(3):p.221-231.

[0217] 3.Barta,J.A.,C.A.Powell,and J.P.Wisnivesky,Global Epidemiology ofLung Cancer.Annals of Global Health,2019.85(1):p.8.

[0218] 4.Lau,S.C.M.,et al.,Squamous cell lung cancer:Current landscape andfuture therapeutic options.Cancer Cell,2022.40(11):p.1279-1293.

[0219] 5.Zhao,S.,et al.,Progress of PD-1 / PD-L1 inhibitor combination therapyin immune treatment for HER2-positive tumors.European Journal of ClinicalPharmacology,2024.80(5):p.625-638.

[0220] 6.Metallomics,13,2021,mfab029 https: / / doi.org / 10.1093 / mtomcs / mfab029.

[0221] 7.Kuang et al.Journal of Biomedical Science(2024)31:50https: / / doi.org / 10.1186 / s12929-024-01041-6RESEARCH.

Claims

1. A biomarker for predicting the sensitivity / prognosis of platinum-based chemotherapy systemic therapy for non-small cell lung cancer, characterized in that, The biomarker is MT-ND6, and its amino acid sequence is shown in SEQ ID NO.

1.

2. Application of the biomarker MT-ND6 as a drug target in the preparation of drugs for the prevention / delay / treatment / diagnosis of non-small cell lung cancer.

3. The application as described in claim 2, characterized in that, The amino acid sequence of MT-ND6 is shown in SEQ ID NO.

1.

4. The substance described in any one of the following, characterized in that, The substance includes: (1) A detection reagent / kit for detecting the expression of the biomarker MT-ND6 as described in claim 1; (2) A modulator / inhibitor / antagonist, said modulator / inhibitor / antagonist comprising IMT1; (3) A drug / drug composition comprising the modulator / inhibitor / antagonist as described in (2) above, and / or a pharmaceutically acceptable carrier.

5. The substance as described in claim 4, characterized in that, The diagnostic reagent / kit is used to prepare products for the diagnosis of non-small cell lung cancer; and / or, Products used for the preparation of prognostic biomarkers for non-small cell lung cancer; and / or, For the preparation of products used to predict the sensitivity / prognosis of platinum-based chemotherapy systemic therapy for non-small cell lung cancer; and / or, This product is used to prepare a product that targets and regulates MT-ND6 expression for the treatment of non-small cell lung cancer.

6. The substance as claimed in claim 4, characterized in that, The drug / drug composition may be used alone and / or in combination with other substances.

7. The method according to any one of the following, characterized in that, The method includes: (1) A method for screening biomarkers as described in claim 1, the method comprising the following steps: The first step involved obtaining data on the half-maximal inhibitory rate (IC50) of platinum-based drugs against non-small cell lung cancer (NSCLC) cells, as well as the expression levels of mitochondrial genes in NSCLC cells, from databases. Preliminary screening was conducted to identify mitochondrial gene-encoded biomarkers that could potentially predict the sensitivity of NSCLC to platinum-based chemotherapy systems. A hypothesis was proposed that genes encoding mitochondrial proteins could indicate the sensitivity of NSCLC to platinum-based chemotherapy systems. The second step involved detecting the sensitivity of non-small cell lung cancer (NSCLC) cell lines to systemic treatment with platinum-based chemotherapy drugs. RT-qPCR was used to quantitatively analyze the gene expression levels of relevant biomarkers. The experiment confirmed that the gene expression level of the mitochondrial protein-encoded MT-ND6 was negatively correlated with the half-maximal inhibitory concentration (50% inhibition) of NSCLC patients treated with platinum-based chemotherapy drugs. Western blotting was then used to quantitatively analyze the protein expression level of MT-ND6 in NSCLC cell lines, further confirming the negative correlation between the protein expression level of the mitochondrial protein-encoded MT-ND6 and the 50% inhibition rate of NSCLC patients treated with platinum-based chemotherapy drugs. The third step is to verify the biological effects. Through functional experiments of inhibiting and overexpressing the biomarker MT-ND6, it is confirmed that the level of the biomarker MT-ND6 is directly related to the degree of nuclear DNA damage induced by platinum-based chemotherapy drugs in non-small cell lung cancer, and the cellular and molecular mechanisms by which the biomarker MT-ND6 regulates the sensitivity of non-small cell lung cancer to systemic treatment with platinum-based chemotherapy drugs are revealed. The fourth step involved proposing clinical diagnostic biomarkers. Immunohistochemical detection of the biomarker MT-ND6 revealed that MT-ND6 levels in tumor samples from non-small cell lung cancer (NSCLC) patients were significantly higher than in adjacent normal tissues. High expression of MT-ND6 in tumor tissues of NSCLC patients indicated better prognosis, with patients exhibiting high MT-ND6 expression having significantly longer survival than those with low expression. The optimal expression cutoff point for the MT-ND6 H-Score in tumor tissues was determined using the surv-cutpoint function, and patients were divided into high-expression and low-expression groups based on this cutoff value. The overall survival (OS) of the high-expression group was significantly longer than that of the low-expression group (P = 0.044). Step 5: Validation of biomarkers for predicting clinical efficacy. In patients with non-small cell lung cancer who received systemic therapy with platinum-based drugs after surgery, the survival of patients with high expression of the biomarker MT-ND6 was significantly higher than that of patients with low expression. High expression of the biomarker MT-ND6 can be an effective indication for better sensitivity of non-small cell lung cancer to systemic therapy with platinum-based drugs after surgery. Step 6: Further validation of clinical efficacy prediction biomarkers. For non-small cell lung cancer patients who received platinum-based systemic therapy (platinum-based chemotherapy combined with immunotherapy) before surgery, high expression of the biomarker MT-ND6 after imaging evaluation can effectively indicate better sensitivity of non-small cell lung cancer patients who received platinum-based systemic therapy before surgery. (2) A method for predicting / evaluating the sensitivity / prognosis of platinum-based chemotherapy system therapy for non-small cell lung cancer, wherein the method predicts / evaluates the sensitivity / prognosis of platinum-based chemotherapy system therapy for non-small cell lung cancer by detecting the expression of the biomarker as described in claim 1. (3) A method for screening candidate drugs for treating non-small cell lung cancer, alleviating or preventing non-small cell lung cancer metastasis and / or improving the prognosis of non-small cell lung cancer, the method comprising detecting the effect of the candidate drug on the level of anti-MT-ND6 autoantibody in a subject or a sample obtained from the subject, wherein a decrease in the level of anti-MT-ND6 autoantibody after using the candidate drug indicates that the candidate drug has the effect of treating non-small cell lung cancer, alleviating or preventing non-small cell lung cancer metastasis and / or improving the prognosis of non-small cell lung cancer; (4) A method for preventing / delaying / treating non-small cell lung cancer, the method comprising administering to the individual an effective amount of the modulator as described in claim 3, or the drug / drug composition as described in claim 3.

8. An application characterized in that, The applications include the use of the biomarkers as described in claim 1, or the substances as described in any one of claims 4-6, or the methods as described in claim 7 in the preparation of drugs for the prevention / delay / treatment / diagnosis of non-small cell lung cancer, products for the diagnosis of non-small cell lung cancer, products for the sensitivity prediction / prognosis of platinum-based chemotherapy drug systems for the treatment of non-small cell lung cancer, products for the treatment of non-small cell lung cancer by targeting MT-ND6 regulated expression, and drugs for the prevention / delay / treatment / diagnosis of non-small cell lung cancer.

9. The biomarker, substance, method, or application according to any one of claims 1-8, characterized in that, The non-small cell lung cancer includes lung adenocarcinoma, squamous cell carcinoma, and large cell carcinoma; And / or, the platinum is selected from one or more of cisplatin, carboplatin, nedaplatin, and lobaplatin; And / or, The platinum-based chemotherapy treatment includes combination therapy with platinum-based drugs and other chemotherapy drugs, combination therapy with platinum-based drugs and immunotherapy, combination therapy with platinum-based drugs and radiotherapy, or a combination thereof; and / or, The amino acid sequence of MT-ND6 is shown in SEQ ID NO.

1.

10. The biomarker, substance, method, or application according to any one of claims 1-8, characterized in that, The biomarker MT-ND6 is used to predict the sensitivity of non-small cell lung cancer patients to systemic platinum-based chemotherapy, thus avoiding unnecessary treatment: For non-small cell lung cancer patients receiving neoadjuvant systemic platinum-based chemotherapy, the biomarker MT-ND6 is used to guide the screening of sensitive patients receiving systemic platinum-based chemotherapy, increasing the response rate of these patients to neoadjuvant systemic platinum-based chemotherapy and reducing the risk of missing the surgical window; And / or, For adjuvant therapy with platinum-based chemotherapy after radical resection of non-small cell lung cancer (NSCLC) patients, the biomarker MT-ND6 is used to guide the screening of NSCLC patients who are sensitive to systemic adjuvant therapy with platinum-based chemotherapy, thereby improving the treatment prognosis of these patients and avoiding unnecessary treatment and toxic side effects; and / or, For patients with advanced non-small cell lung cancer (NSCLC) who have poor physical condition and face significant toxicity from platinum-based chemotherapy drugs, the MT-ND6 is used to guide the screening of these patients who may be insensitive to systemic treatment with platinum-based chemotherapy drugs, thus avoiding exacerbation of disease progression; and / or, For patients who have never received platinum-based chemotherapy, the biomarker MT-ND6, as a single biomarker, is highly expressed in patients and can effectively indicate a good survival prognosis for NSCLC patients. It is a potentially good prognostic biomarker for the diagnosis and treatment of non-small cell lung cancer patients.

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