Use of pak4 inhibitor comp55 in the manufacture of a medicament for inhibiting tumor associated fibroblast formation

Abstract

The application discloses application of PAK4 inhibitor comp55 in preparation of a medicine for inhibiting formation of tumor-related fibroblasts, and belongs to the technical field of medicine application. The application first proposes the application of PAK4 inhibitor comp55 in preparation of the medicine for inhibiting formation of tumor-related fibroblasts, and provides a new scheme for the medicine for inhibiting formation of tumor-related fibroblasts. The application first proposes that PAK4 inhibitor comp55 inhibits formation of CAFs by targeting PAK4. The application first proposes that PAK4 inhibitor comp55 and vitamin D analogues are used in combination to synergistically inhibit formation of CAFs. The application first proposes that PAK4 inhibitor comp55 and anti-PD-1 antibodies are used in combination to synergistically inhibit formation of CAFs. The application first proposes that PAK4 inhibitor comp55, vitamin D analogues and anti-PD-1 antibodies are used in combination to significantly inhibit tumor growth, inhibit formation of CAFs, promote CD8 + T cell infiltration and IFN-gamma secretion.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug application, and particularly relates to application of PAK4 inhibitor comp55 in preparation of a drug for inhibiting formation of tumor-related fibroblasts. BACKGROUND

[0002] Tumor is a common and frequently-occurring disease, is a new organism or neoplasm formed by abnormal clonal proliferation and differentiation caused by gene mutation under the long-term action of various tumor factors inside and outside the body. Tumor is divided into benign tumor and malignant tumor, and malignant tumor is further divided into three types of cancer derived from epithelial tissue, sarcoma derived from mesenchymal tissue and carcinosarcoma. The so-called "cancer" generally refers to all malignant tumors.

[0003] Malignant tumor is one of the most serious malignant diseases threatening human health, is the first cause of death in the world at present, and the latest data statistics show that there are nearly 20 million new cancer cases in the world in 2022, and 9.7 million deaths; as of 2022, cancer causes nearly one sixth (16.8%) of deaths and one fourth (22.8%) of non-communicable disease deaths in the world. In 2022, nearly half (49.2%) of the new cases and most (56.1%) of the deaths in the world occurred in Asia. It is predicted that the number of new cancer cases will reach 35 million per year by 2050, increasing by 77% compared with the level in 2022. The incidence rate and death toll of tumor are increasing year by year, which has become a common and frequently-occurring disease in modern society, seriously affects the life quality of patients, and brings heavy economic and spiritual burden to families and society, and is also a major social problem plaguing the world. The treatment and prevention of cancer has always been one of the most urgent problems in the world.

[0004] Tumor cells, immune cells, stromal cells and non-cellular components around the tumor cells together form a complex system of tumor microenvironment (TME). TME plays a key role in tumor proliferation, angiogenesis, immune escape and the like. Tumor cells can reprogram fibroblasts in TME to transform into tumor-related fibroblasts (CAF). CAF, as a core stromal component of TME, is transformed from various precursors such as resident fibroblasts, endothelial cells and mesenchymal stem cells, and the TGF-β1 signaling pathway is the main activation pathway. CAF plays an important role in proliferation, invasion and metastasis, drug resistance and immune escape of tumor cells by secreting growth factors, promoting epithelial-mesenchymal transition of tumor cells and the like. CAF can also shape the extratumoral stroma, and secrete a large amount of collagen, fibronectin and the like to promote solidification of the extratumoral stroma, form a penetration barrier for drugs or therapeutic immune cells, and then prevent drugs or cytotoxic CD8 +The deep penetration of immune cells such as T cells into tumor tissues reduces the effect of tumor treatment, and the regulation of TME on tumor immunity has attracted much attention. More and more studies have shown that the regulation of CAF or overcoming its barrier effect is a new means of tumor treatment. At present, the tumor treatment strategies targeting CAF mainly include: 1) interfering with CAF activation, 2) interfering with CAF function, 3) eliminating CAF, etc.

[0005] As a serine / threonine kinase downstream of Rho GTPase, p21-activated kinase 4 (PAK4) plays a key role in cytoskeleton reorganization, proliferation, metastasis and embryonic development. PAK family is divided into group I (PAK1-3) and group II (PAK4-6), among which PAK4 as a representative of group II mediates TGF-β1 signal regulation of Smad2 / 3 phosphorylation, and is abnormally highly expressed in many high-metastatic tumors, which is closely related to tumor proliferation, apoptosis and invasion. TGF-β1 is a core factor driving the transformation of fibroblasts into CAFs, and PAK4 is a downstream kinase of TGF-β1, but the effect of PAK4 on CAF formation has not been reported.

[0006] Although there are many PAK4 inhibitors such as PF-3758309 and KPT-9274, etc., but limited by toxic side effects and low target selectivity, there is no marketed drug at present. GNE-2861 is a selective inhibitor of group II PAKs, which has 870 times stronger selectivity for PAK4 than PAK1. Based on this, by optimizing the structure of GNE-2861 and retaining the propargyl alcohol group, a new PAK4 inhibitor comp55 with stronger selectivity is modified.

[0007] At present, there is no report on the inhibitory effect of PAK4 inhibitor comp55 on CAF formation. SUMMARY

[0008] In view of this, the purpose of the present application is to provide the application of PAK4 inhibitor comp55 in the preparation of a drug for inhibiting the formation of tumor-associated fibroblasts, and to provide a new scheme for the drug for inhibiting the formation of tumor-associated fibroblasts.

[0009] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: In a first aspect, the present application provides the application of PAK4 inhibitor comp55 in the preparation of a drug for inhibiting the formation of tumor-associated fibroblasts. The chemical name of comp55 is (S)-(1-(2-aminopyrimidin-4-yl)-6-((1-hydroxycyclohexyl)ethynyl)-1H-indol-3-yl)(3-methylpiperazin-1-yl)methanone.

[0010] Based on the above technical scheme, further, the tumor-related fibroblast is myCAF.

[0011] Based on the above technical scheme, further, the PAK4 inhibitor comp55 inhibits melanoma CAF.

[0012] In a second aspect, the present application provides a pharmaceutical composition for inhibiting the formation of tumor-related fibroblasts, the pharmaceutical composition comprising a PAK4 inhibitor comp55, a pharmaceutically acceptable salt of the PAK4 inhibitor comp55 or a solvate of the PAK4 inhibitor comp55.

[0013] Based on the above technical scheme, further, the pharmaceutical composition further comprises a vitamin D analogue.

[0014] Based on the above technical scheme, further, the pharmaceutical composition further comprises an anti-PD-1 antibody.

[0015] Based on the above technical scheme, further, the pharmaceutical composition further comprises a vitamin D analogue and an anti-PD-1 antibody.

[0016] Based on the above technical scheme, further, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, and the pharmaceutical composition is in a pharmaceutically acceptable dosage form.

[0017] Based on the above technical scheme, further, the human effective dose of the PAK4 inhibitor comp55 is in the range of 100-400 mg / day.

[0018] Based on the above technical scheme, further, the human effective dose of the PAK4 inhibitor comp55 is 200 mg / day.

[0019] Wherein, the human effective dose of 200 mg / day corresponds to 25 mg kg -1 day -1 Compared with the prior art, the present application has the following beneficial effects: 1. The present application first proposes the use of PAK4 inhibitor comp55 in the preparation of a drug for inhibiting the formation of tumor-related fibroblasts, providing a new scheme for the drug for inhibiting the formation of tumor-related fibroblasts.

[0020] 2. The present application first proposes that the PAK4 inhibitor comp55 inhibits the formation of CAFs by targeting PAK4.

[0021] 3. The present application first proposes that PAK4 inhibitor comp55 is combined with vitamin D analogues to synergistically inhibit the formation of CAFs.

[0022] 4. The present application first proposes that PAK4 inhibitor comp55 is combined with anti-PD-1 antibody to synergistically inhibit the formation of CAFs.

[0023] 5. The present application first proposes that PAK4 inhibitor comp55, vitamin D analogues and anti-PD-1 antibody are combined to significantly inhibit tumor growth, inhibit the formation of CAFs, promote CD8 + T cell infiltration and IFN-γ secretion. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application, the drawings involved in the embodiments will be briefly introduced below.

[0025] Figure 1 The results of PAK4 inhibitor comp55 of Example 1 inhibiting TGF-β1-induced CAF formation are shown in the following figures: A is the fluorescence localization result of α-SMA, B is the fluorescence localization result column chart of α-SMA, C is the fluorescence localization result of FN, and D is the fluorescence localization result column chart of FN; Figure 2 The results of PAK4 inhibitor comp55 and vitamin D analogue Calcipotriol synergistically inhibiting TGF-β1-induced CAF formation are shown in the following figures: Figure 3 The results of PAK4 inhibitor comp55 and Calcipotriol combined to inhibit CAF-induced proliferation of mouse melanoma B16-BL6 cells are shown in the following figures: Figure 4 The results of PAK4 inhibitor comp55 and Calcipotriol combined to inhibit CAF-induced migration of mouse melanoma B16-BL6 cells are shown in the following figures: Figure 5 The results of PAK4 inhibitor comp55 and Calcipotriol combined to inhibit CAF-induced proliferation of mouse melanoma B16-BL6 cells, and the synergistic effect with anti-PD1 are shown in the following figures: A is the administration flow chart, B is the tumor tissue picture of each group, C is the tumor weight of each group, and D is the tumor volume of each group. Figure 6 The results of PAK4 inhibitor comp55 and Calcipotriol combined to inhibit CAFs to increase CD8 +T cell infiltration, and synergistic effect with anti-PD1. A: Flow cytometry detection of tumor infiltrating T cells in each group, B: Flow cytometry quantification of tumor infiltrating CD8 + T cell infiltration, and synergistic effect with anti-PD1. A: Flow cytometry detection of tumor infiltrating T cells in each group, B: Flow cytometry quantification of tumor infiltrating CD8 + T cell infiltration, and synergistic effect with anti-PD1. A: Flow cytometry detection of tumor infiltrating T cells in each group, B: Flow cytometry quantification of tumor infiltrating CD8 + T cell infiltration, and synergistic effect with anti-PD1. A: Flow cytometry detection of tumor infiltrating T cells in each group, B: Flow cytometry quantification of tumor infiltrating CD8 + T cell infiltration, and synergistic effect with anti-PD1. A: Flow cytometry detection of tumor infiltrating T cells in each group, B: Flow cytometry quantification of tumor infiltrating CD8 Figure 7 T cell infiltration, and synergistic effect with anti-PD1. A: Flow cytometry detection of tumor infiltrating T cells in each group, B: Flow cytometry quantification of tumor infiltrating CD8 Note: Compared with the blank group, ### p<0.001; compared with the model group, ***p<0.001, **p<0.01, *p<0.05. DETAILED DESCRIPTION

[0026] The present application selects mouse fibroblast L929 and mouse melanoma cell B16-BL6 as research objects, activates L929 cells into CAFs by TGF-β1, and intervenes by PAK4 inhibitor comp55, and finds that it has an inhibitory effect on CAF formation. The present application designs a new combined drug regimen, which uses PAK4 inhibitor comp55 combined with vitamin D analogue Calcipotriol or PAK4 inhibitor comp55 combined with vitamin D analogue Calcipotriol and anti-PD-1, and finds that PAK4 inhibitor comp55 has an effect and research and development value in overcoming immunotherapy resistance by inhibiting CAF formation.

[0027] The application will be described in detail below in conjunction with the examples, but the embodiments of the application are not limited thereto. Obviously, the examples described below are only some of the embodiments of the application, and other similar embodiments obtained by those skilled in the art without creative labor are also within the protection scope of the application.

[0028] Example 1 In this embodiment, PAK4 inhibitor comp55 is used to inhibit the formation of CAFs induced by TGF-β1, and the inhibitory effect of PAK4 inhibitor comp55 on the in vitro CAF formation of the tested sample is evaluated.

[0029] Cell lines: mouse fibroblast L929, mouse melanoma B16-BL6.

[0030] The operation steps are as follows: 1. Cell culture: Cell line recovery: The cryopreservation tube stored in liquid nitrogen was quickly taken out and placed in 37°C warm water to dissolve. 153 g centrifugation for 3 min to remove the cryopreservation solution, carefully pour out the supernatant, add culture medium, and transfer it to a cell culture bottle, mark it and place it in a 37°C and 5% CO2 humidified incubator.

[0031] Cell line subculture: The B16-BL6 and L929 cell culture bottle was poured out, 1 mL trypsin was added to rinse the residual culture medium in the bottle, 3 mL trypsin was added and placed in a 37°C cell incubator for 2-3 min, then transferred to a centrifuge tube containing 3 mL medium, 153 g centrifugation for 3 min. Carefully pour out the supernatant and add 4 mL of culture medium to resuspend the cells, take one-fourth of the cells for subculture, and place them in the incubator for culture until they reach the logarithmic growth phase.

[0032] 2. CAFs induction method: TGF-β1 induction: After the mouse fibroblast L929 cells were recovered and grew to the logarithmic phase, the cell density was adjusted to 1×10 5 cells / mL, and inoculated into a 24-well plate. After the cells adhered, 10 ng / mL TGF-β1 was added and cultured for 24 h.

[0033] TGF-β1 dissolution method: Add sterile water to prepare a stock solution of 0.1 mg / mL, and then dilute to the desired concentration.

[0034] 3. Cell immunofluorescence experiment: L929 cells in the logarithmic growth phase were inoculated in a 24-well plate with round cell slides at a density of 1×10 5 cells / mL. After adhering in the incubator, they were divided into six groups for different treatments: Control group, TGF-β1 group, PAK4 inhibitor comp55 group (10, 40, 160, 640 nM). Continue to culture for 24 h.

[0035] The original culture medium in the suction plate was washed with PBS to remove the residual liquid. 4% paraformaldehyde was used for fixation for 20 min. 0.1% Triton X-100 was used for permeation for 10 min. 10% goat serum was used for blocking incubation at room temperature for 30 min. After incubation with the first antibody (VDBP antibody, 1:500; a-SMA antibody, 1:500; p53 antibody, 1:5000), it was transferred to 4°C overnight, keeping wet. After rewarming for 30 min, it was washed with TBST on a shaker for 5 times, 5 min each time. In a dark room, fluorescent secondary antibody (1:200) was added and incubated at room temperature for 2 h. After being wrapped with tin paper, it was placed on a shaker and washed with TBST for 5 times, 5 min each time. DAPI staining solution was added in a dark room for 15 min, and after avoiding light, it was washed with TBST for 5 times, 5 min each time. The cell climbing sheet was taken out, anti-fluorescence quencher was added to the cell attachment side, and the sheet was sealed. A laser confocal microscope was used for photography.

[0036] 4. Western blotting: Protein quantification: The standard and sample were added to the wells of a 96-well plate, and after the addition of the Coomassie brilliant blue reaction, the absorbance was measured at 590 nm by an enzyme-labeled instrument. The protein concentration of the sample was calculated according to the measurement results.

[0037] Preparation of protein sample: The total protein sample was taken, protein loading buffer was added, and the protein was denatured in a boiling water bath for 10 min. It was divided into 1.5 mL EP tubes and stored at -80°C.

[0038] Gel and loading: The glass plate, adhesive tape and comb were washed and dried. The glass plate was installed to make it tightly fit with the adhesive tape to avoid glue leakage. Separation glue was prepared and placed in the middle of the thin glass plate. Carefully seal the glue with isopropanol to make the glue surface flat. After it solidifies, prepare the concentrated glue, pour out the isopropanol, wash with distilled water until there is no alcohol smell, absorb the water with filter paper, pour in the concentrated glue, and insert the comb into the concentrated glue quickly and evenly. After the concentrated glue solidifies, pull out the comb and load.

[0039] SDS-PAGE polyacrylamide gel electrophoresis: Turn on the power of the gel electrophoresis instrument, set the initial voltage to 60 V, and after the Marker appears completely, set the voltage to 120 V and continue electrophoresis until the end.

[0040] Membrane transfer: The gel was removed from the glass plate and immersed in pre-chilled transfer buffer. The PVDF membrane was activated by soaking in methanol for 20 s. The gel was placed on filter paper with the PVDF membrane on top. Air bubbles between the membrane and the gel were gently rolled out with a glass rod. The transfer sandwich was assembled and the transfer cassette was placed in the electrophoresis tank in an ice water bath. The transfer was run at 100 mA constant current for 120 min.

[0041] Blocking: The PVDF membrane was immersed in 5% non-fat dry milk and blocked at room temperature for 2 h.

[0042] Primary antibody incubation: The membrane was washed with TBST for 3 times, 10 min each time. The antibody was diluted with the primary / secondary antibody dilution buffer and incubated on the PVDF membrane evenly. The membrane was transferred to 4°C overnight.

[0043] Secondary antibody incubation: The membrane was washed with TBST for 3 times, 10 min each time. The antibody was diluted with the primary / secondary antibody dilution buffer and incubated on the PVDF membrane evenly at room temperature for 2 h.

[0044] Imaging: The membrane was washed with TBST for 3 times, 10 min each time. The chemiluminescence reagent was mixed and coated on the membrane. The membrane was developed on the imager.

[0045] Quantification: The band gray value was quantified using Image J.

[0046] 5. Results: As a marker of CAFs, a-SMA plays an important role in the activation and function of CAFs. We detected the expression of a-SMA in L929 cells after the action of TGF-β1 by immunofluorescence staining. The results showed that after the addition of TGF-β1 induction, the content of CAFs specific marker protein in L929 cells increased, while the action of comp55 on L929 cells reduced the fluorescence intensity of CAFs specific marker protein, indicating that comp55 can inhibit the formation of CAFs in a concentration-dependent manner (Figure 1), that is, inhibiting PAK4 at the pharmacological level can inhibit the formation of CAFs induced by TGF-β1.

[0047] wherein nM is nanomoles per liter.

[0048] Example 2 This example evaluates the effect of PAK4 inhibitor comp55 in combination with vitamin D analogue Calcipotriol on the inhibition of TGF-β1 -induced CAFs formation in the tested samples. The tested samples and the experimental procedures involved are the same as in Example 1.

[0049] To investigate whether comp55 and Calcipotriol have a synergistic effect when used in combination, 10, 40 nM of comp55 and 25, 50 nM of Calcipotriol were selected for further experiments. The expression of a-SMA in TGF-β1 -induced L929 cells after the combination of the two drugs was detected by immunofluorescence. Figure 2 The combination index (CI) was calculated using CompuSyn software. When CI < 1, the two drugs showed a synergistic effect; when CI = 1, the two drugs showed an additive effect; and when CI > 1, the two drugs showed an antagonistic effect. By judging the CI value, it was found that the CI values of the four groups were all less than 1 when two different concentrations (10 and 40 μM) of comp55 were combined with two different concentrations (25 and 50 μM) of Calcipotriol for 24 h (Table 1). The results showed that the use of comp55 in combination with Calcipotriol can reduce the concentration of the drugs used and improve the drug efficacy.

[0050] Table 1 is the inhibition rate (%) and CI value of PAK4 inhibitor comp55 in combination with vitamin D analogue Calcipotriol on the inhibition of TGF-β1 -induced CAFs formation: Table 1 PAK4 inhibitor comp55 in combination with Calcipotriol on the inhibition of TGF-β1 -induced CAFs formation

[0051] Example 3 This example verifies that PAK4 inhibitor comp55 in combination with Calcipotriol can inhibit the proliferation and migration of mouse melanoma B16-BL6 cells induced by CAFs.

[0052] 1. Transwell cell migration experiment: L929 and B16-BL6 cells were starved for 12 h with serum-free medium to remove the promoting effect of serum on cell migration ability. Cells were collected in 2% FBS medium, and 200 μL of B16-BL6 cell suspension was added to the upper chamber with different concentrations of PAK4 inhibitor comp55 or Calcipotriol; 500 μL of medium containing 10% FBS or 2% FBS medium was added to the lower chamber with CAFs cell and drug solution mixture, and the ratio of B16-BL6 to CAFs cells was 1:1. Cultured for 24 h. Formaldehyde 20 min, crystal violet staining 10 min. Excess dye was washed off, and the cells on the inner side of the upper chamber were wiped off. Dry, use an inverted microscope to take random pictures, add 33% glacial acetic acid to dissolve crystal violet, and detect absorbance at 570 nm using a microplate reader.

[0053] 2. Transwell cell proliferation experiment: To determine the effect of PAK4 inhibitor comp55 and Calcipotriol combination therapy on CAFs-induced B16-BL6 cell proliferation, B16-BL6 cells were co-cultured with CAFs, and 10% complete medium was used to resuspend B16-BL6 cells and inoculated in the lower chamber, and 2% complete medium was used to resuspend CAFs cells and added to the upper chamber. Drug was given for 48 h, and MTT was used to detect B16-BL6 cells in the lower chamber.

[0054] 3. Results: To detect the effect of PAK4 inhibitor comp55 and Calcipotriol combination therapy on CAFs-induced B16-BL6 cell proliferation and migration, MTT detection and Transwell experiment results showed that the combination therapy could inhibit B16-BL6 cell proliferation ( Figure 3 ) and migration ( Figure 4 ) at a lower concentration. Therefore, PAK4 inhibitor comp55 and Calcipotriol combination therapy can inhibit the proliferation and migration ability of CAFs-promoted mouse melanoma B16-BL6 cells.

[0055] Example 4 This example verifies that PAK4 inhibitor comp55 and Calcipotriol combination therapy can inhibit the proliferation of mouse melanoma B16-BL6 cells by inhibiting CAFs, and has a synergistic effect with anti-PD1.

[0056] 1. Establishment of mouse melanoma model: A tumor-bearing mouse model was established by subcutaneous injection. The experiment used cells in the logarithmic growth phase, and the concentration of B16-BL6 cells was adjusted to 1.5×10 6The mice were inoculated with 5×106 SMMC-7721 cells / mL, and injected quickly into the right armpit of the mice, with 0.2 mL per mouse. The tumor volume of the mice was measured and recorded every day after the start of administration. The tumor volume calculation formula was: 0.5 × length × width 2 .

[0057] 2. Administration of PAK4 inhibitor comp55, Calcipotriol and anti-PD-1 treatment: After inoculation, when the average tumor volume of the mice reached 8-24 mm 3 , the mice were randomly divided into ① model group; ② PAK4 inhibitor comp55 (25 mg / kg); ③ anti-PD-1 (20 μg / mouse); ④ PAK4 inhibitor comp55 (25 mg / kg) + anti-PD-1 (20 μg / mouse); ⑤ Calcipotriol (6.5 μg / kg); ⑥ Calcipotriol (6.5 μg / kg) + PAK4 inhibitor comp55 (25 mg / kg); ⑦ PAK4 inhibitor comp55 (25 mg / kg) + Calcipotriol (6.5 μg / kg) + anti-PD-1 (20 μg / mouse).

[0058] The treatment groups were administered PAK4 inhibitor comp55 at a dose of 25 mg / kg via tail vein, Calcipotriol at a dose of 6.5 μg / kg via intraperitoneal injection, and anti-PD-1 at a dose of 20 μg / mouse via tail vein once every three days, for a total of 4 times; the control group and the model group were administered an equal volume of normal saline every day.

[0059] 3. Flow cytometry: Prepare the required solutions: Tumor lysis solution: 1 mg / mL collagenase IV + 100 U / mL hyaluronidase + 200 U / mL DNase I + 1% penicillin-streptomycin mixture + 10% fetal bovine serum + DMEM medium.

[0060] FACS solution: Add fetal bovine serum to PBS to a final concentration of 3%, and then add EDTA·Na2·2 H2O to a final concentration of 2 mM.

[0061] ① Transfer the tumor tissue or spleen tissue (0.02 g / mouse) to a 6-well plate, cut the tissue with scissors, and use a 1 mL pipette to suck it up. Add tumor lysis solution, 3 mL / well. Use the gun head to disperse the tissue powder, and place it in a 37°C incubator for 30 min. Every 5 min, take out the six-well plate and shake it once to mix.

[0062] ②Add complete medium to terminate the digestion, filter the single cell suspension with a filter screen into a 15 mL centrifuge tube, and remove the larger tissue blocks.

[0063] ③Centrifuge (56 g , 4°C, 5 min) and discard the supernatant, and collect the lower layer of cells; add the prepared FACS solution to resuspend, and divide into two equal volumes, one for staining surface antibodies and the other for staining intracellular antibodies, and place on ice for standby.

[0064] ④Incubate the antibodies: Stain the cell surface antibodies Centrifuge (56 g , 4°C, 5 min) to collect the cells, and discard the supernatant; after washing the cells, incubate in the dark for 30 min (4°C) in a CD3, CD4, and CD8 antibody staining buffer (1% FBS in PBS). Then wash the cells and use for machine operation.

[0065] Stain the intracellular antibodies 1) Centrifuge (56 g , 4°C, 5 min) to collect the cells, and discard the supernatant; incubate the tumor single cell suspension and the spleen lymphocytes in RPMI-1640 with 10% FBS and 0.2% cell stimulation mixture (plus protein transport inhibitors) at 37°C for 12 hours.

[0066] Collect the cells, centrifuge (56 g , 4°C, 5 min), and discard the supernatant; after washing the cells, incubate in the dark for 30 min (4°C) in a CD3 and CD8 antibody staining buffer (1% FBS in PBS). Then wash the cells 3 times with FACS.

[0067] Fix and permeate the cells with 0.1% Triton for 15 min. Centrifuge (56 g , 4°C, 5 min) to collect the cells, and discard the supernatant; incubate the cells in an IFN-γ antibody buffer at 4°C for 30 min, and then wash 3 times in a FACS buffer.

[0068] ⑤Machine operation: Add 1 mL of FACS solution, vortex, centrifuge (56 g , 4°C, 5 min) to collect the cells, and discard the supernatant. Add 400 μL of PBS, and detect on the machine. The number of tumor cells collected by the machine is 10 5 in each group.

[0069] 4. Immunohistochemistry: Paraffin section, baked section. Xylene I 15 min→xylene II 15 min; 100% alcohol I 5 min→100% alcohol II 5 min→95% alcohol 3 min→85% alcohol 5 min→75% alcohol 5 min; PBS washing 3 times, 5 min each time. Put into citric acid buffer and cook in microwave oven for 7 min at high fire and 13 min at low fire. Let it cool naturally, PBS washing 3 times, 5 min each time. Circle the tissue with immunohistochemical pen, drop 3% H2O2 for 15 min. PBS washing 3 times, 5 min each time. Block with fetal bovine serum for 45 min, drop the antibody after shaking off the blocking solution, incubate overnight at 4°C. Equilibrate at room temperature for 30 min, PBST washing 3 times, 5 min each time. Add secondary antibody, incubate at room temperature for 2 h, PBST washing 3 times, 5 min each time. DAB staining for 4 min, rinse with running water for 3 min. Hematoxylin staining for 2 min, rinse with running water, differentiate for 10 s. Blue with tap water for 2-3 min. 75% alcohol 5 min→85% alcohol 3 min→95% alcohol 5 min→100% alcohol I 5 min→100% alcohol II 5 min; xylene I 15 min→xylene II 15 min, neutral resin mounting.

[0070] 5. Results: The mouse melanoma B16-BL6 cell ectopic transplantation tumor model was constructed to explore the in vivo efficacy of the combination of the three drugs. The results showed that the inhibition effect of the combination of PAK4 inhibitor comp55 (25 mg / kg) and anti-PD-1 (20 μg / each), the combination of PAK4 inhibitor comp55 and Calcipotriol, and the combination of PAK4 inhibitor comp55 and anti-PD-1 and Calcipotriol (6.5 μg / kg) was more obvious compared with the model group, and the combination of PAK4 inhibitor comp55 and anti-PD-1 and Calcipotriol had the best effect. Figure 5 ).

[0071] The number of CD4 + and CD8 + T cells in the tumor was detected by flow cytometry. The results showed that PAK4 inhibitor comp55 + anti-PD-1 group, PAK4 inhibitor comp55 + Cal group, and PAK4 inhibitor comp55 + Cal + anti-PD-1 group could increase the infiltration of CD8+ T cells in the tumor, and the increase in PAK4 inhibitor comp55 + anti-PD-1 group and PAK4 inhibitor comp55 + Cal + anti-PD-1 group was the most significant.Figure 6 (A, B). Meanwhile, the PAK4 inhibitor comp55 + Cal + anti-PD-1 group significantly increased intratumoral CD4. + T cell infiltration ( Figure 6 C).

[0072] Changes in functional factors secreted by CD8+ T cells were detected, and it was found that, except for the Calcipotriol group, all groups showed increased levels of IFN-γ secreted by CD8+ T cells. Figure 6 (D, E). These results indicate that PAK4 inhibitors comp55 and anti-PD-1, alone or in combination, can activate CD8. + T cells increase the secretion of functional factors, thereby killing tumor cells.

[0073] Immunohistochemistry was used to detect α-SMA, a marker of fibroblasts (CAFs) in a mouse melanoma model. The results showed that the expression of CAF markers was significantly increased in the Model group, while drug treatment significantly inhibited their expression. Furthermore, the PAK4 inhibitor comp55 + anti-PD-1 + Calcipotriol group showed better inhibitory effects, indicating that B16-BL6 cells can promote the transformation of normal fibroblasts into CAFs in mice, and that the PAK4 inhibitor comp55, anti-PD-1, and Calcipotriol can synergistically inhibit CAF formation. Figure 7 ).

[0074] The above results indicate that the combination of PAK4 inhibitor comp55, anti-PD-1, and Calcipotriol can inhibit the proliferation of mouse melanoma B16-BL6 cells in C57BL / 6 mice by inhibiting CAF formation.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a PAK4 inhibitor comp55 in the manufacture of a medicament for inhibiting the formation of tumor-associated fibroblasts.

2. Use according to claim 1, characterized in that, The tumor-associated fibroblasts are myCAFs.

3. Use according to claim 2, characterized in that, The PAK4 inhibitor comp55 inhibits melanoma CAFs.

4. A pharmaceutical composition for inhibiting the formation of tumor-associated fibroblasts, characterized by, The pharmaceutical composition comprises the PAK4 inhibitor comp55, a pharmaceutically acceptable salt of the PAK4 inhibitor comp55, or a pharmaceutically acceptable solvate of the PAK4 inhibitor comp55.

5. The pharmaceutical composition for inhibiting formation of tumor-associated fibroblasts according to claim 4, wherein The pharmaceutical composition further comprises a vitamin D analog.

6. The pharmaceutical composition for inhibiting formation of tumor-associated fibroblasts according to claim 4, wherein The pharmaceutical composition further comprises an anti-PD-1 antibody.

7. The pharmaceutical composition for inhibiting formation of tumor-associated fibroblasts according to claim 4, wherein The pharmaceutical composition further comprises a vitamin D analog and an anti-PD-1 antibody.

8. The pharmaceutical composition for inhibiting formation of tumor-associated fibroblasts according to any one of claims 4 to 7, characterized by, The pharmaceutical composition comprises a pharmaceutically acceptable excipient, and the pharmaceutical composition is in a pharmaceutically acceptable dosage form.

9. The pharmaceutical composition for inhibiting the formation of tumor- associated fibroblasts according to any one of claims 4 to 7, characterized by, The human effective dose of the PAK4 inhibitor comp55 is in the range of 100-400 mg / day.

10. The pharmaceutical composition for inhibiting the formation of tumor- associated fibroblasts according to any one of claims 4 to 7, characterized by, The human effective dose of the PAK4 inhibitor comp55 is 200 mg / day.

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