A high-throughput drug screening method based on primary head and neck squamous cell carcinoma cells and anti-head and neck squamous cell carcinoma drugs
Through a high-throughput drug screening method based on primary head and neck squamous cell carcinoma cells, new drugs with anti-head and neck squamous cell carcinoma activity were screened out, solving the problem of lack of targeted drugs for head and neck squamous cell carcinoma, shortening the time from drug discovery to clinical transformation and broadening the therapeutic effect.
Patent Information
- Application Number
- CN202210909960.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-29
- Publication Date
- 2025-09-16
- Estimated Expiration
- 2042-07-29
AI Technical Summary
There is a lack of targeted drug treatment strategies for head and neck squamous cell carcinoma in existing technologies, and the cost of new drug research and development is high, the cycle is long, the failure rate is high, and there is a lack of rapid and effective drug screening methods.
A high-throughput drug screening method based on primary head and neck squamous cell carcinoma cells was used. By constructing patient-derived primary head and neck squamous cell carcinoma cells and commercially purchased cell lines as carriers, drugs with a killing effect of more than 50% and concentration gradient dependence were screened. Combined with pharmacogenomic analysis, anti-head and neck squamous cell carcinoma drugs were screened.
It shortens the time from drug discovery to clinical transformation, broadens the range of drug choices for head and neck squamous cell carcinoma, and provides important clinical treatment significance.
Smart Images
Figure CN115109824B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a high-throughput drug screening method based on primary head and neck squamous cell carcinoma cells and an anti-head and neck squamous cell carcinoma drug. Background Art
[0002] Head and neck cancer (HNSCC) is the sixth most common cancer type worldwide. It encompasses epithelial malignancies originating in the paranasal sinuses, nasal cavity, oral cavity, pharynx, and larynx. Globally, it causes over 550,000 cases and over 300,000 deaths annually. Over 95% of these cases are HNSCC (head and neck squamous cell carcinoma). Currently, surgery, radiotherapy, and chemotherapy remain the mainstays of treatment for HNSCC, but they can also damage head and neck organs and function, leading to a decrease in patients' quality of life. In recent years, advances in sequencing and multi-omics analysis technologies have enabled targeted therapies that specifically target key molecules and the signaling pathways they regulate, playing a key role in tumorigenesis and progression. These therapies have demonstrated high efficacy and low toxicity, becoming a mainstream treatment for various cancer types. However, the genetic profile of HNSCC reveals that mutations in tumor suppressor genes are predominant, leading to a significant lack of targeted drug treatment strategies.
[0003] Considering the high cost, long cycle and extremely high failure rate of new drug development, repurposing drugs with known indications to treat other diseases is becoming an increasingly promising approach to drug development, commonly known as "repurposing old drugs for new uses". Compared with the development of new drugs, "repurposing old drugs for new uses" has the following advantages: (1) a higher success rate, as marketed drugs have undergone systematic safety evaluation; (2) a shorter development cycle, as marketed drugs have comprehensive preclinical pharmacokinetic and toxicological parameters, and the development of new indications based on previous research can greatly shorten the development cycle; (3) a lower development cost, as compared with the development of new drugs for the same indication, "repurposing old drugs for new uses" can significantly save development costs in preclinical and clinical phases I and II.
[0004] Despite the increasing attention paid by research institutions and pharmaceutical companies to the field of "repurposing old drugs," few studies have used this strategy to discover drugs that can be rapidly promoted for clinical use in head and neck squamous cell carcinoma. Summary of the Invention
[0005] The purpose of the present invention is to provide a high-throughput drug screening method based on primary head and neck squamous cell carcinoma cells, as well as anti-head and neck squamous cell carcinoma drugs obtained according to the screening method, in view of the fact that there are few drugs for treating head and neck squamous cell carcinoma in the existing technology. The anti-head and neck squamous cell carcinoma drugs provided by the present invention are all old drugs with new uses, which can greatly shorten the time from drug discovery to clinical transformation and have important clinical therapeutic significance.
[0006] To solve the above problems, the present invention first provides a high-throughput drug screening method based on primary head and neck squamous cell carcinoma cells, comprising the following steps:
[0007] S1, construction of primary head and neck squamous cell carcinoma cells from different patients;
[0008] S2, randomly selecting at least a portion of the primary head and neck squamous cell carcinoma cells constructed in step S1 as carriers, adding a single concentration of the drug to be screened and culturing the cells, measuring cell proliferation activity, and screening for drugs that have a killing effect of more than 50% on the primary head and neck squamous cell carcinoma cells of at least one source, to obtain a first candidate drug;
[0009] S3, randomly taking at least a portion of the primary head and neck squamous cell carcinoma cells constructed in step S1 as carriers, adding different concentrations of the first candidate drug to co-culture, measuring cell proliferation activity, and screening for a drug that has a killing effect of more than 50% on the primary head and neck squamous cell carcinoma cells of at least one source and is concentration-dependent, to obtain a second candidate drug;
[0010] S4, using head and neck squamous cell carcinoma primary cells and cell lines as carriers, adding different concentrations of the second candidate drug and co-culturing for 72 hours, measuring the absorbance of the culture medium at 420-480nm, and calculating the IC50, Emax and AUC, as well as the corresponding GR 50 , GRmax and GR AOC value, evaluate the potential and effectiveness of the drug, and obtain anti-head and neck squamous cell carcinoma drugs.
[0011] Preferably, in step S4, all the primary head and neck squamous cell carcinoma cells constructed in step S1 and multiple commercially purchased head and neck squamous cell carcinoma cell lines are used as vectors.
[0012] Preferably, the specific operations of step S1 include:
[0013] S1.1. The obtained head and neck squamous cell carcinoma tumor tissue was isolated and minced, centrifuged and the supernatant removed, then resuspended in tissue digestion buffer and incubated on a shaker at 37°C for 30-60 min.
[0014] S1.2, neutralizing and digesting the cells by adding culture medium containing 10% fetal bovine serum, and sequentially passing the cells through filters of varying pore sizes, collecting the tissue fragments on the filters and the cell suspension below the filters; in some embodiments, separation is performed by sequentially passing through filters of 100 μm and 40 μm pore sizes;
[0015] S1.3, transfer the tissue block obtained in step S1.2 to a culture flask for adherent culture; after centrifugation of the cell suspension, add complete culture medium and resuspend it for adherent culture. Cells that have been successfully passaged for more than 5 times and still maintain high proliferation activity are screened, which means that the said head and neck squamous cell carcinoma primary cells have been successfully constructed.
[0016] Preferably, the tissue digestion fluid formula is as follows:
[0017] Collagenase type IV 1 mg / mL, hyaluronidase 200 U / mL, DNase type I 200 U / mL, phenol red-free trypsin 1×.
[0018] Preferably, the complete culture medium formula is as follows:
[0019] DMEM / F12 medium, insulin 5 μg / ml, amphotericin B 250 ng / ml, gentamicin 10 μg / ml, cholera toxin 0.1 nM, EGF 0.125 ng / ml, hydrocortisone 25 ng / ml, ROCK inhibitor Y-27632 10 μM.
[0020] Preferably, the high-throughput drug screening method further comprises:
[0021] Step S5: screening the efficacy biomarkers of the anti-head and neck squamous cell carcinoma drug based on pharmacogenomic analysis.
[0022] Another aspect of the present invention further provides an anti-head and neck squamous cell carcinoma drug obtained according to any of the above screening methods, and the anti-head and neck squamous cell carcinoma drug is shown in the following table:
[0023]
[0024]
[0025] Another aspect of the present invention provides a use of the aforementioned anti-head and neck squamous cell carcinoma drug.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] (1) The present invention provides a method for screening drugs for head and neck squamous cell carcinoma. The method uses patient-derived primary head and neck squamous cell carcinoma cells and multiple commercially purchased head and neck squamous cell carcinoma cell lines as carriers, and high-throughput screening of drugs known to target other indications is performed to ultimately obtain drugs with anti-head and neck squamous cell carcinoma activity, providing a new approach for broadening the range of drug selection for head and neck squamous cell carcinoma.
[0028] (2) Drug research takes an average of 8 to 10 years from the time a compound molecule is developed to the time it takes to actually enter the clinic, and this requires a lot of manpower and material resources, resulting in huge time and economic costs. This invention achieves "new uses for old drugs," significantly shortening the time from drug discovery to clinical translation, and has important clinical therapeutic significance. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 The results of high-throughput drug screening of 2248 compounds against 13 primary head and neck squamous cell carcinoma cells are presented.
[0030] Figure 2 The results of high-throughput drug screening of 300 compounds against 5 primary head and neck squamous cell carcinoma cells are presented.
[0031] Figure 3 These are the results of high-throughput drug screening of 129 compounds against 40 primary head and neck squamous cell carcinoma cells and 14 commercial head and neck squamous cell carcinoma cell lines. The drug response evaluation indicator is GRmax. DETAILED DESCRIPTION
[0032] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also considered to be within the scope of protection of the present invention.
[0033] In the description of the present invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. In addition, unless otherwise specified, "plurality" means two or more.
[0034] Unless otherwise specified in the examples, conventional conditions or manufacturer-recommended conditions were followed. All reagents and instruments used, without manufacturer's indication, are commercially available. The 14 commercially available head and neck squamous cell carcinoma cell lines used in this invention are: UPCISCC-172, SCC4, PECA-PJ41, PECA-PJ15, HN6, HN30, FADU, Detroit562, CAL33, CAL27, HSC2, SCC9, TU212, and UPCISCC-131.
[0035] Example 1 Construction of patient-derived head and neck squamous cell carcinoma primary cells (PDC)
[0036] The head and neck squamous cell carcinoma tumor tissues used in this example were all obtained from the Department of Oral and Maxillofacial Head and Neck Oncology of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University, and all patients were newly diagnosed with head and neck squamous cell carcinoma.
[0037] Build process:
[0038] (1) Prepare tissue digestion solution: collagenase type IV 1 mg / mL, hyaluronidase 200 U / mL, DNase type I 200 U / mL, phenol red-free trypsin 1×;
[0039] Prepare complete culture medium: DMEM / F12 medium (3:1), insulin 5 μg / ml, amphotericin B 250 ng / ml, gentamicin 10 μg / ml, cholera toxin 0.1 nM, EGF 0.125 ng / ml, hydrocortisone 25 ng / ml, and ROCK inhibitor Y-27632 10 μM. Sterilize the solution with a 0.22 μm sterile filter and store at 4°C until needed. Prepare fresh after a maximum of 2 months.
[0040] (2) After transporting head and neck squamous cell carcinoma tumor tissues from 40 patients back to the laboratory at 4°C, the outer packaging was disinfected and the tissues were transferred to the cell room through a sterile transfer chamber. Sterile microscissors and microtweezers were used to separate and mince the tissues.
[0041] (3) After the minced tissue is centrifuged at 1500 rpm for 3 minutes, it is resuspended in tissue digestion solution (the volume is about 5-10 times the tumor volume) and added to the tissue dissociation tube. It is placed in a Miltenyi tissue processor and separated by different separation modes according to the toughness of the tissue. After the tissue is crushed, it is placed on a shaker in a 37°C environment and digested for 30-60 minutes. During this period, the tissue dissociation tube is shaken up and down every 10 minutes to mix the tissue and avoid tissue clumping, which affects digestion. When the digestion solution becomes turbid and no obvious tissue remains, the digestion can be terminated.
[0042] (4) Neutralize and digest the cells using a culture medium containing 10% fetal bovine serum, and collect the tissue blocks on the filter and the cell suspension under the filter after passing through 100 μm and 40 μm filters, respectively. Continue to culture the tissue blocks and cell suspension until cells that have been successfully passaged for more than 5 times and still maintain high proliferation activity are screened. This is the successful construction of the head and neck squamous cell carcinoma primary cell (PDC), and each PDC cell is named PDC_N (N is an integer greater than zero). Specifically:
[0043] For the cell suspension, centrifuge at 1500 rpm for 3 minutes. If excessive red blood cell contamination is detected, add 1 ml of red blood cell lysis buffer. Let it stand for 3 minutes, then dilute with 10 ml of PBS and centrifuge at 1500 rpm for 3 minutes. Discard the supernatant, resuspend in 5 ml of complete culture medium, and continue incubating in a 37°C, 5% CO2 humidified incubator. PDCs that maintain high cell proliferation activity after 5 or more successful passages are considered successful.
[0044] For tissue blocks, transfer them to a T25 culture flask rinsed with complete culture medium in advance, spread them out and stick them to the bottom wall of the flask. Aspirate the culture medium as much as possible to prevent the tissue blocks from sliding and be careful not to aspirate the tissue blocks. Place the T25 culture flask in a 37°C, carbon dioxide incubator and culture it upside down. After 24 hours, observe whether the tissue blocks in the tissue block culture group have not slipped or fallen off and whether tumor cells have grown from the tissue blocks. If there is no sliding and tumor cells can be observed adhering to the wall at the boundary of the tissue block, carefully add complete culture medium and culture them upright. If the wall is not firmly attached, delay adding complete culture medium, aspirate the excess culture medium, and continue to culture inverted for 24 hours until the tissue blocks are firmly attached. PDC cells that have been successfully passaged for more than 5 times and still maintain high cell proliferation activity are considered to be successfully constructed.
[0045] Example 2 High-throughput drug screening for the purpose of "new uses of old drugs" using primary head and neck squamous cell carcinoma cells and cell lines
[0046] In order to explore the strategy of "new uses of old drugs" for head and neck squamous cell carcinoma, the present invention selected 2248 small molecule compounds, mainly drugs approved by the U.S. Food and Drug Administration (1800 / 2248), and also included 319 clinical trial evaluation drugs and 129 preclinical compounds. Non-antitumor drugs accounted for more than 50% (1419 / 2248), and the remaining tumor-related compounds targeted some popular mechanisms of action, with 718 targeted drugs and 111 chemotherapy drugs. It includes inhibitors targeting popular oncogene targets, including PI3K inhibitors, mTOR inhibitors, CDK inhibitors, and HDAC inhibitors, etc., covering a total of 286 different drug targets.
[0047] First, 13 patient-derived head and neck squamous cell carcinoma primary cells constructed in Example 1 were selected and 2248 compounds were screened three times with a single dose (1 μM). Cell proliferation activity was measured 72 hours after drug exposure. Compounds with a killing effect of more than 50% on at least one source of head and neck squamous cell carcinoma primary cells were screened to obtain the first candidate drug. The results are as follows: Figure 1 As shown, according to the initial screening results, 300 compounds can cause 50% cell killing effect in more than one head and neck squamous cell carcinoma primary cell model;
[0048] The above 300 compounds were screened. Five primary head and neck squamous cell carcinoma cells derived from patients constructed in Example 1 were selected and the first candidate drug was rescreened at three concentrations (5 μM, 1 μM, and 0.2 μM). Figure 2 As shown in the figure, a total of 171 compounds were excluded because they had no concentration gradient dependence or could not reduce the number of viable cells by at least 50% in more than one cell model. After rescreening, a total of 129 compounds were obtained as second candidate drugs.
[0049] Continue to perform terminal screening on 129 second candidate drugs. Take 54 head and neck squamous cell carcinoma cell models (40 patient-derived primary tumor cells and 14 commercially purchased cell lines constructed in Example 1) for response curve analysis (10 concentration gradient). The cells were seeded into 384-well plates at a predetermined density (volume of 70 μl). After overnight incubation, the compounds were diluted 3 times and added to the cell culture plates in sequence. Each compound and each concentration used 4 replicate wells, and the 384-well culture plates were incubated continuously for 72 hours. At the end of drug treatment, 8 μl Cell Counting Kit-8 was added to each well, incubated for another 2 hours, and the absorbance at 450 nm was measured with a microplate reader. The IC50, Emax and AUC, as well as the corresponding GR50, GRmax and GR were calculated using the program GRmetrics in the R language. AOC .
[0050] It should be noted that the 54 cell lines used for drug screening have a wide range of cell doubling times (13.47 to 50.99 hours), and previous studies have shown that changes in doubling time can seriously affect the evaluation of drug response. Therefore, this study used the R package GRmetrics to perform multi-parameter analysis of drug response curves to obtain the values of traditional indicators (IC50, Emax and AUC) and growth rate inhibition indicators (GR50, GRmax and GRAOC). In order to determine the maximum drug response variation of each drug among the 54 cell lines, we calculated the IC50, Emax, AUC, GR50, GRmax and GR AOC The coefficient of variation of the efficacy parameters including GR and GRmax was used (for the calculation method, see Clark NA, Hafner M, Kouril M, et al. GR calculator: an online tool for calculating and mining dose–response data [J]. Bmc Cancer, 2017, 17(1): 698). Among these six parameters, GRmax has the strongest coefficient of variation, indicating that this parameter can reflect the differentiated response patterns of various drugs in different cell models. In this example, GRmax was ultimately selected for drug response evaluation.
[0051] The results are as follows Figure 3 As shown in Figure 1, after final screening, 129 compounds showed good anti-HNSCC activity. These compounds form the "old drugs with new uses" drug collection for HNSCC of the present invention, including 18 chemotherapeutic drugs, 19 non-tumor drugs, and 92 targeted drugs. The specific drug names and the pathways and targets involved are shown in Table 1:
[0052] Table 1 Information on 129 compounds
[0053]
[0054]
[0055]
[0056]
[0057]
[0058]
[0059] Based on the above research, the inventors of this application are currently screening the efficacy biomarkers of the anti-head and neck squamous cell carcinoma drugs through pharmacogenomics analysis, in order to develop personalized treatment plans for different patients, improve treatment effects, and improve the patients' quality of life.
[0060] In summary, the present invention provides a method for screening anti-head and neck squamous cell carcinoma drugs. The method is based on patient-derived head and neck squamous cell carcinoma primary cells and multiple commercially purchased head and neck squamous cell carcinoma cell lines as carriers, and high-throughput screening of drugs known to have other indications is carried out, ultimately achieving "new uses for old drugs" and obtaining drugs with anti-head and neck squamous cell carcinoma activity. On the one hand, it provides new ideas for broadening the range of drug selection for head and neck squamous cell carcinoma; on the other hand, the research of the present invention can greatly shorten the time from drug discovery to clinical transformation, and has important clinical therapeutic significance.
[0061] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.
Claims
1. Application of Cetylpyridinium chloride monohydrate in the preparation of drugs for treating head and neck squamous cell carcinoma.