Application of pentafluridol in preparation of medicine for inhibiting formation of CAF

Abstract

The invention provides an application of pentafluridol in preparation of a medicine for inhibiting formation of tumor-associated fibroblasts. Specifically, the invention finds that the pentafluridol can influence the formation process of the tumor microenvironment of the targeted tumor-related fibroblast cells, and the development of solid tumors is effectively inhibited from the source, so that the increase of the tumor volume is inhibited.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, and in particular relates to the application of pentafluridone in the preparation of drugs that inhibit CAF formation. Background Technology

[0002] Cancer is one of the most dangerous diseases to human life and health, and a large number of people die from it every year. Stomach cancer is a common tumor of the digestive system and is one of the most deadly malignant tumors worldwide. Its course is insidious, and early diagnosis is extremely difficult.

[0003] Stomach cancer exhibits significant geographical variations, with over 70% of new stomach cancer cases worldwide occurring in developing countries. Melanoma, also known as malignant melanoma, is a highly malignant tumor originating from melanocytes, primarily affecting the skin. Although melanoma accounts for only 10% of skin cancers, it accounts for 80% of skin cancer deaths. In Europe and the United States, melanoma ranks fifth in incidence among all malignant tumors. The number of newly diagnosed cases in the United States has nearly doubled annually over the past 30 years, while in the United Kingdom, the incidence rate has increased by 28% for men and 12% for women in the past five years. It metastasizes early, progresses rapidly, has a poor prognosis, and a high mortality rate.

[0004] Currently, the main treatments for stomach cancer and melanoma include surgery, chemotherapy, and radiation therapy. All these methods have limitations. Surgery is only suitable for early-stage patients and is a local treatment, ineffective against tumor cells that have metastasized to the bloodstream or other tissues. Furthermore, it often results in postoperative complications, further exacerbating patient suffering. Chemotherapy involves treating the disease with drugs that cannot specifically target cancer cells, killing only rapidly proliferating cells. This can lead to the destruction of rapidly proliferating normal cells such as bone marrow cells, digestive tract mucosa, and hair follicles, causing side effects such as bone marrow suppression, vomiting, diarrhea, and hair loss. Radiation therapy uses radiation to kill cancerous tissue. While radiation is more likely to damage cancer cells, it also damages normal cells, causing local or systemic radiation reactions.

[0005] As our understanding of cancer continues to expand, researchers have recognized the tumor microenvironment as a heterogeneous and complex system, primarily composed of tumor cells and non-malignant host components, including immune cells, the stroma, and the vascular system. In many tumors, such as pancreatic, lung, breast, and colorectal cancers, the stroma accounts for up to 90% of the tumor mass. The tumor stroma contains both cellular and non-cellular components, including collagen, fibroblasts, and mesenchymal matrix cells.

[0006] Fibroblasts are one of the most abundant and crucial cell types in tumor stroma, and are the main producers of extracellular matrix (ECM). In the tumor microenvironment, various inflammatory cytokines produced by tumor cells, host immune cells, and stromal cells can activate fibroblasts. These activated fibroblasts are called tumor-associated fibroblasts (CAFs). CAFs are an important structural component of solid tumors and play a crucial role in creating the tumor immunosuppressive environment. Compared to normal tissue fibroblasts, CAFs exhibit significant pro-tumorigenic properties, producing extracellular matrix, proteases, growth factors, and chemokines, which can promote tumor proliferation, angiogenesis, invasion, and metastasis, playing a vital role in tumor progression. Currently, CAFs are generally classified into three subgroups: myofibroblast-like CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs). Among these, myCAFs are located adjacent to tumor cells and express high levels of α-SMA; their formation influences tumor development and determines tumor size. In recent years, CAF-targeted therapy has made significant progress. The main objectives of these methods are: (1) to directly or indirectly consume CAF; (2) to reduce or eliminate the pro-tumor and immunosuppressive functions of CAF; and (3) to normalize or reprogram CAF to a resting state. However, existing methods are all targeted therapies for already formed CAF, and there are no reports on blocking the formation process of CAF (especially myCAF) to prevent or treat solid tumors.

[0007] Penfluridol (PF) is an organic compound with the chemical formula C60. 28 H 27 ClF5NO is an oral, long-acting antipsychotic approved by the U.S. Food and Drug Administration (FDA). It is primarily used to treat acute and chronic psychosis and seizures. Its main mechanism of action is the blocking of dopamine receptors, particularly postsynaptic D2 receptors. Currently, there are no reports linking cefoperazone (cF5NO) to tumors or the formation of coronary artery effusion (CAF).

[0008] Currently, although drugs with different mechanisms of action can target and eliminate CAF, there are no reported drugs that directly target CAF differentiation, nor are there publicly disclosed treatment strategies that directly target CAF (especially myCAF) differentiation for tumor treatment. It is foreseeable that effectively inhibiting CAF differentiation can fundamentally suppress the occurrence and development of solid tumors, which has significant practical implications for expanding tumor treatment approaches. Summary of the Invention

[0009] In view of this, the present invention aims to propose the application of pentafluridone in the preparation of drugs that inhibit CAF formation, thereby effectively inhibiting the occurrence and development of solid tumors by inhibiting the differentiation and formation of CAF from the root.

[0010] To achieve the above objectives, the technical solution of the present invention is implemented in the following manner:

[0011] In a first aspect, the present invention provides the use of penfluridone in the preparation of a drug that inhibits CAF formation. The structure of said penfluridone is shown in Formula I:

[0012]

[0013] Preferably, the CAF is myCAF.

[0014] Preferably, the inhibition of CAF formation includes the inhibition of melanoma CAF and / or gastric cancer CAF formation.

[0015] Our research shows that functional fibroblasts (CAFs) in tumors evolve from a type of stem cell precursor. Therefore, inhibiting the further differentiation of these precursor cells has the potential to suppress tumor formation. The inventors creatively discovered that CAFs differentiate from their precursor CD34+Pi16+ fibroblasts. Furthermore, through computational screening and experiments, we found that pendiflubenzuron can inhibit the differentiation of precursor CD34+Pi16+ fibroblasts, and verified its effectiveness as a drug that inhibits CAF formation and thus further suppresses tumors, achieving good results in cell experiments and mouse models.

[0016] Secondly, the present invention provides the use of pentafluridone in the preparation of a drug that inhibits the differentiation of CAF precursor CD34+Pi16+ fibroblasts into CAFs.

[0017] Thirdly, the present invention provides the use of pentafluridone in the preparation of medicaments for the prevention or treatment of solid tumors.

[0018] Preferably, the use of pentafluridone in the preparation of a medicament for the prevention or treatment of melanoma is provided.

[0019] Preferably, the use of pentafluridone in the preparation of medicaments for the prevention or treatment of gastric cancer is provided.

[0020] Preferably, the pentafluridone is an injectable or oral formulation.

[0021] Preferably, the dosage of pentafluridone is 1-20 mg / kg body weight.

[0022] More preferably, the dosage of pentafluridone is 5-10 mg / kg body weight.

[0023] More preferably, the dosage of pentafluridone is 10 mg / kg body weight.

[0024] Fourthly, the present invention provides the use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of solid tumors, said pharmaceutical composition comprising penfluridone, or a pharmaceutically acceptable salt thereof, or a solvation thereof.

[0025] Preferably, the solid tumor is melanoma or gastric cancer.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The inventors creatively discovered that CAFs differentiate from their precursor CD34+Pi16+ fibroblasts, which is of pioneering significance in the important research idea of ​​"blocking the CAF differentiation signaling pathway";

[0028] 2. Based on the discovery of the above differentiation signaling pathway, the inventors found that, unlike previous drugs that target and eliminate CAF, pendiflubenzuron can promote the growth and stemness maintenance of CD34+Pi16+ fibroblasts and inhibit the differentiation of CD34+Pi16+ fibroblasts into CAFs. Thus, by inhibiting the differentiation and formation of CAFs from the root, that is, blocking the formation process of CAFs before they are formed, it can more effectively inhibit the occurrence and development of solid tumors.

[0029] 3. The main contribution of this invention is that, based on the confirmation of different tumor CAF formation conditions and formation mechanisms, it was found that pendiflubenzuron can stably control the formation of tumor CAF at specific doses, thereby inhibiting tumor development. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0031] Figure 1 This is the condition 12 days after subcutaneous melanoma formation;

[0032] Figure 2 This is a curve showing the change in tumor volume of melanoma from 0 to 12 days. The tumor volume unit in the graph is mm. 3 ;

[0033] Figure 3 This is the condition 20 days after subcutaneous tumor formation of gastric cancer.

[0034] Figure 4 This is a curve showing the change in tumor volume of gastric cancer from 0 to 20 days. The tumor volume unit in the graph is mm. 3 ;

[0035] Figure 5 The changes in CD34, Pi16, α-SMA, etc., in melanoma tissue sections 12 days after subcutaneous tumor formation were observed after multiple fluorescence staining.

[0036] Figure 6 The statistical results are obtained from multiple fluorescence staining of melanoma tissue sections 12 days after subcutaneous tumor formation.

[0037] Figure 7 RNA was extracted from isolated tumor CD34+ fibroblasts after 7 days of in vitro differentiation culture, and the statistical results of transcriptional levels of CD34, Pi16, and α-SMA were obtained.

[0038] Figure 8 The expression of CD34, Pi16, and α-SMA in differentiated cells after multiple immunofluorescence staining;

[0039] Figure 9 This is a statistical result of the number of CD34, Pi16, and α-SMA positive cells in each field of view after multiplex immunofluorescence staining of differentiated cells. Detailed Implementation

[0040] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0041] In this invention, the term "CAF" refers to tumor-associated fibroblasts, which are an important component of the structure of solid tumors.

[0042] The term "myCAF" refers to a subpopulation of CAF that possesses myofibroblast characteristics.

[0043] The term "CD34" refers to a highly glycosylated type I transmembrane glycoprotein selectively expressed on the surface of hematopoietic stem / progenitor cells in humans and other mammals, and its expression gradually diminishes and disappears as cells mature. Current research indicates that CD34 molecules play an important role in mediating cell-cell adhesion, participating in the transport and colonization of hematopoietic stem cells, inflammatory responses, and lymphocyte homing.

[0044] The term "Pi16" stands for Peptidase Inhibitor 16 (PI16), a protein belonging to the peptidase inhibitor family. It is widely distributed in the human body, including tissues such as the lungs, kidneys, liver, and gastrointestinal tract. Pi16 regulates and controls proteolytic processes in the body by inhibiting the activity of specific peptidases. It can interact with and inhibit the activity of various peptidases, thereby regulating peptidase-mediated physiological and pathological processes. Multiple studies have shown that Pi16 is expressed in various stem and progenitor cells. The inhibitory effect of Pi16 can affect various biological processes, including inflammatory responses, immune regulation, cell proliferation, and apoptosis. Research indicates that peptidase inhibitor 16 may play an important role in the occurrence and development of tumors. Its expression level is closely related to the invasiveness and metastatic ability of tumors. Furthermore, Pi16 is also involved in the occurrence and development of other diseases, such as cardiovascular diseases, diabetes, and neurological disorders.

[0045] The term "α-SMA" stands for α-smooth muscle actin. α-SMA is a protein highly expressed in smooth muscle cells, involved in regulating smooth muscle cell contraction and relaxation. It is also an important marker of tumor-associated fibroblasts. Anti-α-smooth muscle actin antibodies can be used to detect the presence and distribution of myCAF using immunohistochemistry.

[0046] The term "merge" in immunofluorescence staining refers to the overlaying of images of different staining molecules to simultaneously display their positions and relationships. Typically, in immunofluorescence staining, different fluorescent dyes are used to label different target molecules or cellular structures; for example, green fluorescent dye is used to label one protein, and red fluorescent dye is used to label another. A merge image is a superimposed image of these different staining molecules, using different color channels to represent the different stainings. Merge images allow researchers to simultaneously display the positions and relationships of target molecules or cellular structures with different stainings, helping them observe and analyze interactions or co-localization. Merge images are usually presented in color, with different stainings represented by different colors. For example, green and red stainings are represented using green and red channels respectively, and when the two stainings are overlaid, they form a yellow color. By observing merge images, the relationships and interactions between target molecules or cellular structures with different stainings can be visually understood.

[0047] The term "DAPI" refers to a fluorescent dye, specifically 4',6-diamino-2-phenylpyridine. It is a DNA dye that binds to the double helix structure of DNA and emits blue fluorescence. DAPI can be used to stain cell nuclei, allowing for the observation and quantification of their number, morphology, and distribution. In immunofluorescence staining, DAPI is often used in conjunction with other fluorescent dyes to label different cellular structures or molecules using varying fluorescence colors.

[0048] The term "COL1A1" stands for the collagen Iα1 chain gene, which encodes the collagen Iα1 chain and is one of the important markers of fibroblasts. Collagen is a protein mainly found in connective tissue and is an important component of skin, bones, tendons, blood vessels, and other connective tissues. Mutations or abnormal expression of the COL1A1 gene are associated with various diseases and disease-related tissue fibrosis, such as osteoporosis and fracture healing.

[0049] The term "SPARC" refers to a protein, short for Secreted Protein Acidic and Rich in Cysteine. It is an extracellular matrix protein secreted by stromal cells and can also serve as a marker for stromal cells. SPARC plays a crucial regulatory role in the extracellular matrix. It participates in the synthesis, remodeling, and degradation of the extracellular matrix, regulating its structure and function. Furthermore, SPARC is involved in regulating cell-cell and cell-matrix interactions, influencing biological processes such as cell migration, proliferation, and differentiation.

[0050] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0051] It should be noted that all procedures in the experiments related to this invention have been approved by the relevant ethics committees and animal research institution committees, and comply with relevant regulatory requirements. All animal experimental procedures were conducted in accordance with the *Guideline for the Care and Use of Laboratory Animals*, issued by the National Institutes of Health (NIH), which has been approved by the Institutional Ethics Committee of Shanghai Xinhua Hospital. The animal experimental methods were approved by the Animal Research Institution Committee of Shanghai Jiao Tong University School of Medicine. Clinical samples from subjects were collected at Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. Written informed consent was obtained from all subjects prior to participation in this study. All clinical studies were conducted in accordance with the *Declaration of Helsinki* and have been approved by the Research Ethics Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine.

[0052] In this specific embodiment of the invention, data from experiments repeated ≥5 times were analyzed using the Kolmogorov-Smirnov normality test to determine data normality and the F-test to assess homogeneity of variance. Unpaired and two-tailed Student's t-tests were used to analyze the data from both groups. Data were expressed as mean ± standard deviation using GraphPad Prism 8 software. Comparisons among multiple groups were performed using one-way ANOVA and Bonferroni post-hoc analysis, with 5 replicates per group. The Kruskal-Wallis test was used to assess comparisons among multiple groups, with 3 replicates per group followed by Bonferroni post-hoc analysis. Appropriate significance was obtained at relatively small group sizes. P < 0.05 was considered statistically significant.

[0053] Example 1: Verification of the antitumor activity of pendiflubenzuron through animal experiments

[0054] 1. Establish a mouse tumor model

[0055] Eight-week-old C57BL / 6J,615 mice and Cd34-CreERT2;Rosa26-LSL-tdTomato mice (C57BL / 6J background, Shanghai Biomodel Biotechnology Development Co., Ltd.) were fed a normal laboratory diet. To avoid data variation caused by sex differences, the mice selected for the study were randomly assigned to different experimental groups in a male:female ratio of 1:1.

[0056] The mouse tumor cell line B16-F10-EGFP and the mouse foregut cancer cell line MFC-GFP were obtained from the Chinese Academy of Sciences Cell Bank Type Culture Collection Center and cultured according to its guidelines. Low-passaged cells were cultured at 1×10⁻⁶ cells / year. 5 Cells / ml were resuspended in a 1:1 mixture of PBS and matrix gel (Corning #356231) for B16F10 and MFC cells.

[0057] The fur on the right abdomen of C57BL / 6J mice (B16F10) or 615 mice (MFC) was shaved off beforehand. 100 μL of mouse tumor cell fluid B16-F10-EGFP was subcutaneously injected into the right back of C57BL / 6J mice to induce subcutaneous tumor formation and establish a melanoma mouse model. 100 μL of mouse forestomy carcinoma cell fluid MFC-GFP was subcutaneously injected into the right abdomen of 615 mice to induce subcutaneous tumor formation and establish a gastric cancer mouse model.

[0058] 2. Antitumor activity test of pentafluoride

[0059] 2.1 Test of penicillin's activity against melanoma

[0060] 2.1.1 Tumor volume measurement

[0061] The melanoma mouse model was divided into three groups of four mice each: a blank control group, a positive control group, and a penfluridone group. Starting from day 7 after subcutaneous tumor formation, the drug was injected directly into the melanoma in situ, with injections every other day.

[0062] The drug concentrations used in the three groups of melanoma mouse models are as follows:

[0063] a. Blank control group: injected with normal saline;

[0064] b. Positive control group: Erdafitinib was injected at a dose of 20 mg / kg body weight;

[0065] c. Penfluridone group: Penfluridone was injected at a dose of 10 mg / kg body weight.

[0066] On days 0, 2, 4, 6, 8, 10, and 12, the side length of the tumor on the mouse's back was measured using vernier calipers, and the tumor volume was calculated. On day 12, when the tumor had formed subcutaneously, the mouse was euthanized by cervical dislocation, and the tumor tissue was carefully dissected from the skin to measure the tumor volume.

[0067] like Figure 1 The image shows the tumor volume of three groups of mice at 12 days after the onset of subcutaneous melanoma, with four replicates in each group. It is evident that the tumor volume in the penfluridone group was significantly smaller than that in the blank control group.

[0068] like Figure 2 The figure shows the changes in tumor volume (in mm) of three groups of mice with subcutaneous melanoma from day 0 to day 12. 3 As can be seen, during the first 0-6 days of subcutaneous tumor formation, the tumor volumes in the three groups were basically the same due to the absence of drug injection; after drug injection began on day 7, the differences in tumor volume among the three groups gradually became apparent, with the tumor volumes in the pendiflubenzuron group and the positive control group being significantly smaller than those in the blank control group.

[0069] comprehensive Figure 1 and Figure 2 This indicates that pendiflubenzuron has an inhibitory effect on melanoma.

[0070] 2.1.2 Multiplex Fluorescence Staining Experiment of Melanoma Tissue Sections

[0071] Tumor tissue excised from the skin of euthanized mice was frozen in liquid nitrogen: samples were further fixed in 4% PFA for 2–3 hours, then dehydrated overnight in 30% sucrose. The samples were then immersed in an optimal cutting temperature compound (OCT, Tissue-Tek) at room temperature for 30 minutes, embedded in OCT at -80°C, and frozen. For paraffin sectioning, samples were fixed in 4% PFA for 24 hours, then dehydrated using a gradient of ethanol (50%–100%) and xylene to achieve clarity, and finally embedded in paraffin. 3 μm sections were collected. Frozen sections were immersed in PBS to dissolve OCT, and paraffin sections were immersed in xylene and a gradient of ethanol to dissolve paraffin. Subcutaneous melanoma sections from mice were treated with 0.2% Triton X-100 in PBS for 15 minutes, then blocked with 5% donkey serum at room temperature. The slides were then incubated overnight at 4°C with primary antibody solution. After washing three times with PBS, secondary antibody was applied at 37°C for 1 hour, followed by three more washes with PBS. Cell nuclei were then stained with DAPI. All slides were mounted on antifluorescent mounting media. Images were acquired using a Leica SP5 confocal microscope and analyzed using ImageJ software. The following antibodies were used: anti-α-smooth muscle actin (SMA) ab7817; anti-C-kit ab283653 (Abcam); anti-Cd34 ab81298 (Abcam); and anti-Pi16, AF4929 (R&D system). Cell nuclei were reverse-stained with DAPI (4′, 6-diamidinyl-2-phenylindole). The stained tissue was mounted on slides and images were captured using a Leica SP5 confocal microscope. The results are shown below. Figure 5 As shown.

[0072] like Figure 5 The image shows the changes in CD34, Pi16, and α-SMA in melanoma tissue sections 12 days after subcutaneous tumor formation, observed after multiple fluorescence staining. Figure 6 The results show the statistical results of the proportion of positive cells in the five figures. The expression levels of CD34 and Pi16 represent the levels of precursor cells CD34+Pi16+ fibroblasts.

[0073] Combination Figure 5 and Figure 6 It was found that the expression levels of CD34 and Pil6 in the pendiflubenzuron group were significantly higher than those in the positive control group, indicating that pendiflubenzuron promoted the generation of CAF precursor CD34+Pi16+ fibroblasts. At the same time, the expression levels of Col1a1 and α-SMA in the pendiflubenzuron group were significantly lower than those in the blank control, indicating that pendiflubenzuron inhibited the generation of myCAF.

[0074] 2.2 Test of pentafluoropropyl's activity against gastric cancer

[0075] 2.2.1 Tumor volume measurement

[0076] The gastric cancer mouse model was divided into three groups of five mice each: a blank control group, a positive control group, and a penfluridone group. For the gastric cancer mouse model, the drug was administered intraperitoneally every other day on day 7 after subcutaneous tumor formation.

[0077] The drugs and their concentrations used in the three groups of gastric cancer mouse models are as follows:

[0078] a. Blank control group: injected with normal saline;

[0079] b. Positive control group: injected with 20 mg / kg erdatinib;

[0080] c. Penfluridone group: 10 mg / kg penfluridone was injected.

[0081] like Figure 3 The figure shows the tumor volume of gastric cancer in three groups of mice 20 days after subcutaneous gastric carcinoma formation, with five replicates in each group. It is evident that the tumor volume in the pentaflulide group and the positive control group was smaller than that in the blank control group, and the tumor volume in the pentaflulide group was significantly smaller than that in the positive control group.

[0082] like Figure 4 The figure shows the changes in tumor volume in three groups of mice from day 0 to day 20 of subcutaneous gastric cancer tumor formation. It can be seen that from day 0 to day 7 of subcutaneous tumor formation, the tumor volumes in the three groups were basically the same due to the absence of drug injection. From day 7 onwards, after drug injection, differences in tumor volume gradually became apparent among the three groups. The gastric cancer tumor volume in the penfluridone group was significantly smaller than that in the blank control group and also significantly smaller than that in the positive control group.

[0083] comprehensive Figure 3 and Figure 4 This indicates that pentaflulide has an inhibitory effect on gastric cancer tumors, and its inhibitory effect is significantly better than that of erdatinib in the positive control group.

[0084] Example 2: Verification of the antitumor activity of pentaflulide through cell experiments

[0085] 1. Sample collection

[0086] This study included 10 patients pathologically diagnosed with gastric cancer. None of the patients had received chemotherapy, radiotherapy, or any other anti-tumor drugs prior to tumor resection. Immediately after surgical resection, pairs of freshly removed tumor tissue and adjacent normal tissue were obtained. The adjacent normal tissue was at least 5 cm away from the tumor tissue.

[0087] 2. Isolation, culture, and grouping of primary fibroblasts

[0088] The differentiation medium is prepared and consists of the following components: DMEM. + 2% chicken embryo extract, 1% FBS (10099141, Thermo Fisher Scientific, Waltham, MA, USA), 1% N2, 2% B27, 100 nM retinoic acid, 50 nM 2-mercaptoethanol (2-ME:M6250, Sigma Aldrich, St. Louis, MO, USA), 1% P / S and 20 ng / ml bFGF.

[0089] Prepare S / P stem cell culture medium, wherein the S / P cell culture medium consists of the following components: DMEM + 2% chicken embryo extract, 1% FBS, 1% N2, 2% B27, 100 nM retinoic acid, 50 nM 2-mercaptoethanol, 1% P / S, 20 ng / ml bFGF and 0.01% leukemia inhibitory factor (10 ng / ml) (LIF: ESG1107, EMD Millipore, Burlington, MA, USA).

[0090] Fresh gastric cancer tissue was cut into fragments and prepared with phosphate-buffered saline (PBS; Gibco). TM Cells were isolated from PBS (United States). The single-cell suspension was the same as previously described. The isolated cells were pelleted by centrifugation at 1200 rpm for 5 min and resuspended in PBS. These cells were purified using a Cd34 microbead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in S / P cell culture medium. For differentiation assays, Cd34 cells were cultured in a medium obtained by mixing differentiation medium and S / P medium at a 3:1 volume ratio. + Fibroblasts.

[0091] The tumor culture medium is the culture supernatant of the gastric cancer cell line BGC: high glucose medium (DMEM) + 10% fetal bovine serum (FBS) + 1% P / S, cultured for 2 days, collect the supernatant, centrifuge at 1000g for 5 minutes, and collect the supernatant, which is the tumor culture medium.

[0092] Cd34 will be cultivated + Fibroblasts were divided into 5 groups. The drug was added directly to the differentiation medium, and the medium was changed every other day. The grouping and the drug concentration added to each group are as follows:

[0093] a. Blank control group: supplemented with physiological saline;

[0094] b. Tumor culture medium group: No drugs added;

[0095] c. Positive control group: Tumor culture medium supplemented with 0.01 μM erdatinib;

[0096] d. 10 μM pentaflulide group: Tumor culture medium supplemented with 10 μM pentaflulide;

[0097] e. 1 μM penfluridone group; tumor culture medium supplemented with 0.1 μM penfluridone.

[0098] All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Primary fibroblasts were used up to the 5th generation.

[0099] 4. PCR experiment

[0100] Total RNA was extracted using the RNeasy Mini kit (QIAGEN). The concentration and quality of total RNA were assessed using a Nanodrop spectrophotometer (ThermoFisher Scientific). cDNA synthesis was performed using the Prime-Script RT master mix (TaKaRa). Primers were obtained from Sangon Biotech (Shanghai) Co., Ltd. qRT-PCR analysis was performed in triplicate using a SYBR PremixEx Taq (TaKaRa) system on a 7900HT real-time PCR system (Applied Biosystems). The primers are as follows:

[0101]

[0102] like Figure 7 The image shows the results of in vitro differentiation culture of isolated tumor CD34+ fibroblasts. After 7 days of culture, cells were collected and RNA was extracted. The transcriptional levels of CD34, Pil6, and α-SMA were statistically analyzed. It can be seen that after 7 days of differentiation, the relative RNA expression levels of CD34 and Pil6 in the positive control group were significantly higher than those in the blank tumor culture group. 10 μM fenfluridine significantly increased the relative RNA expression levels of CD34 and Pil6. Even when the concentration of fenfluridine was reduced to 1 μM, the relative RNA expression levels of CD34 and Pil6 were still significantly higher than those in the blank tumor culture group. This indicates at the gene level that fenfluridine promotes the maintenance of stemness in CAF precursor CD34+Pi16+ fibroblasts.

[0103] Meanwhile, the relative RNA expression levels of α-SMA in the 10 μM and 1 μM penfluridone groups were significantly lower than those in the blank tumor culture medium group, while the relative RNA expression level of Col1a1 was comparable to that in the blank tumor culture medium group, indicating that penfluridone inhibited the generation of myCAF.

[0104] 5. Immunofluorescence staining

[0105] Gastric cancer tissue from patients was treated with 0.2% Triton X-100 in PBS for 15 minutes, followed by blocking with 5% donkey serum at room temperature. Slides were then incubated overnight at 4°C with primary antibody solution. After washing three times with PBS, secondary antibody was applied at 37°C for 1 hour, followed by three washes with PBS. Cell nuclei were then stained with DAPI. All slides were mounted on antifluorescent mounting media. Images were acquired using a Leica SP5 confocal microscope and analyzed using ImageJ software. The following antibodies were used: anti-type I collagen, ab138492 (Abcam); anti-α-smooth muscle actin, ab7817; anti-C-kit, ab283653 (Abcam); anti-Cd34, ab81298 (Abcam); and anti-Pi6, AF4929 (R&D system). Cell nuclei were reverse stained with DAPI (4′,6-diamidin-2-phenylindole). The stained tissue was mounted on a glass slide, and images were taken using a Leica SP5 confocal microscope. The results are as follows: Figure 8 As shown.

[0106] like Figure 8 The image shows the expression of CD34, Pil6, and α-SMA in differentiated cells after multiple immunofluorescence staining. Figure 9 To calculate the statistical results of the number of CD34, Pil6, and α-SMA-positive cells in each field of view under multiplex immunofluorescence staining. It was observed that the expression levels of CD34 and Pil6 in the pendiflubenzuron group were higher than or comparable to those in the positive control group, indicating at the cellular level that pendiflubenzuron promoted the maintenance of stemness in CAF precursor CD34+Pi16+ fibroblasts. Simultaneously, the expression level of α-SMA in the pendiflubenzuron group was significantly lower than that in the blank control, indicating at the cellular level that pendiflubenzuron inhibited the generation of myCAF.

[0107] In summary, animal and cell experiments have demonstrated that pendiflubenzuron can promote the growth and stemness maintenance of CAF precursor CD34+Pi16+ fibroblasts, inhibit their transformation into CAF, and thus inhibit the increase in the volume of solid tumors dominated by CAF, playing a positive role in the prevention and treatment of solid tumors.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of piflutixol in the preparation of a drug for inhibiting the formation of CAF.

2. The use according to claim 1, wherein the CAF is myCAF.

3. Use according to claim 1, characterized in that, The inhibition of the formation of CAF includes inhibiting the formation of melanoma CAF and / or gastric cancer CAF.

4. Use of piflutixol in the preparation of a drug for inhibiting the differentiation of CD34+Pi16+ fibroblasts into CAF.

5. Use of piflutixol in the preparation of a drug for preventing or treating solid tumors.

6. Use according to claim 5, characterized in that, The solid tumor is melanoma or gastric cancer.

7. The use according to any one of claims 1 to 6, characterized in that, The piflutixol is an injection or an oral preparation.

8. Use according to any one of claims 1 to 6, characterized in that, The dose of piflutixol is 1-20 mg / kg body weight.

9. Use according to claim 8, characterized in that, The dose of piflutixol is 5-10 mg / kg body weight.

10. Use of a pharmaceutical composition for the manufacture of a medicament for the prevention or treatment of a solid tumor, characterized in that, The pharmaceutical composition comprises piflutixol, or a pharmaceutically acceptable salt thereof, or a solvate thereof.

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