CN119606961A

CN119606961A - Application of sorafenib in the preparation of drugs for treating lung metastatic tumors

Info

Publication number: CN119606961A
Application number: CN202411866676.9A
Authority: CN
Inventors: 沈颖颖; 郭巨锋; 王涵靓
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2024-10-23
Filing date: 2024-10-23
Publication date: 2025-03-14
Also published as: CN118995604A; CN118995604B

Abstract

The invention discloses an application of sorafenib in preparing a medicine for resisting lung metastatic tumor, and relates to the technical field of biological medicine. When the CD4 ⁺ T cells are differentiated into the Th9 cells, the CD4 ⁺ T cells are treated by the sorafenib, and the sorafenib is found to be capable of remarkably improving the differentiation efficiency of the Th9 cells in vitro, so that the sorafenib can be used for expanding a large amount of Th9 cells with anti-tumor effect in vitro, and a new thought and a new way are provided for the ACT treatment method of clinical tumors.

Description

Application of sorafenib in preparing medicines for resisting lung metastatic tumors

The application relates to an in vitro treatment method for improving differentiation efficiency of Th9 cells, which is a divisional application of a parent application with the application number of 2024114799244 and the application date of 2024, 10, 23 days.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of sorafenib in preparing medicines for resisting lung metastatic tumors.

Background

Adoptive cell therapy (Adoptive CELL THERAPY, ACT) has made remarkable progress in tumor therapy in recent years as a leading branch of the field of cancer immunotherapy, providing new therapeutic hopes for many patients. The rationale for this therapy is to identify and destroy tumor cells by activating, expanding, in vitro, the patient's own immune cells, particularly tumor-specific T cells, and then reinfusion these engineered cells back into the patient. Traditionally, ACT has focused primarily on CD8 ⁺ cytotoxic T Cells (CTLs), which are capable of directly killing recognized cancer cells, key effector cells in tumor immune responses. However, with the intensive research, the role of CD4 ⁺ T cells in anti-tumor immunity has also become increasingly prominent, especially its subpopulations such as Th1 and Th17 cells, which are indirectly or directly involved in anti-tumor processes by modulating immune responses, promoting activation and maintenance of CTLs, and modulating tumor microenvironment.

Of the numerous CD4 ⁺ T cell subsets, th9 cells are a relatively new and potential-filled population. Since 2008 was first discovered and reported, th9 cells have been attracting attention because of their unique cytokine secretion pattern (mainly IL-9 is secreted, while IL-21 and IL-3 are also produced) and important roles in various diseases. In tumor immunology, th9 cells exhibit unique anti-tumor potential. Through cytokines such as IL-9 and the like secreted by the tumor cell, the tumor cell can be directly acted, the growth and proliferation of the tumor cell can be inhibited, other immune cells such as Dendritic Cells (DCs), CD8 ⁺ CTLs, natural killer cells (NK cells) and mast cells can be activated and regulated, a complex immune network is formed, the complex immune network acts on the tumor microenvironment together, and the enhancement of anti-tumor immune response is promoted.

Of particular note, recent studies have shown that melanoma can be eradicated completely in a mouse model by reinfusion of Th9 cells, a result that not only demonstrates the effectiveness of Th9 cells in anti-tumor therapy, but also reveals that it may have a significant therapeutic effect over other T cell subsets (e.g., th1 and Th 17) in certain types of tumors (e.g., melanoma, which are commonly known as "cold tumors"). Cold tumors refer to those tumors that have less immune cell infiltration and a weaker immune response, which often do not respond well to traditional immunotherapy, while the discovery of Th9 cells provides a new strategy for the treatment of such tumors.

However, although Th9 cells exhibit great therapeutic potential, how to effectively expand these cells in vitro to meet the needs of clinical ACT therapy remains a current challenge. Conventional cell expansion methods may not efficiently induce and maintain Th9 cell differentiation, and may be accompanied by problems such as decreased cell function or deviation of differentiation direction. Therefore, the development of a novel and efficient Th9 cell expansion technology has important significance for promoting the application of ACT in tumor treatment.

Sorafenib (Sora, abbreviated as sorafenib), on the other hand, has been shown in clinical practice to be effective against a variety of tumors including hepatocellular carcinoma, renal cell carcinoma, and specific types of thyroid cancer as a multi-target, multi-kinase inhibitor. It achieves dual efficacy in inhibiting tumor proliferation and angiogenesis by inhibiting key kinases on tumor cells and their blood vessels, such as serine/threonine kinase and receptor tyrosine kinase. Although sorafenib was mainly used as a monotherapy, there has been a search in recent years to explore the possibility of using it in combination with other therapeutic means (such as immunotherapy) in order to achieve better therapeutic effects.

However, whether sorafenib has a correlation with Th9 cells has not been reported.

Disclosure of Invention

Based on the defects in the prior art, the invention provides the application of sorafenib in preparing the medicines for resisting lung metastatic tumors.

The naive CD4 ⁺ T cells can be induced in vitro by TGF-beta in combination with IL-4, thereby differentiating into a class of Th9 cells that secrete IL-9, IL-10, and IL-21. On this means, the applicant can further improve the differentiation efficiency of Th9 cells, and the applicant screens whether the clinically common kinase inhibitor can improve the differentiation efficiency of Th9 cells, and finds that sorafenib can remarkably promote the differentiation of Th9 cells.

Previous research data of the applicant shows that sorafenib can significantly increase the differentiation efficiency of Th9 cells in vitro. Analysis of the relevant mechanisms, sorafenib promoted Th9 cell differentiation was dependent on ERK signaling pathways.

Thus, in vitro sorafenib-treated and untreated Th9 cell tail veins were infused back into other cold tumors such as breast cancer and osteosarcoma lung metastasis mice, and the results showed that the antitumor immunity of the breast cancer and osteosarcoma lung metastasis mice infused back with sorafenib-treated Th9 cells was enhanced and the tumor progression was also slow compared to those of the breast cancer and osteosarcoma lung metastasis mice infused back with sorafenib-untreated Th9 cells.

First, the present invention provides an in vitro treatment method for improving the differentiation efficiency of Th9 cells, wherein CD4 ⁺ T cells are treated with sorafenib when the CD4 ⁺ T cells are differentiated into Th9 cells.

Preferably, sorafenib is used at a concentration of 0.2 to 2 μm. Th9 cells were also induced by plating with 10. Mu.g/ml anti-IFNγ,20ng/ml IL4 and 10ng/ml TGF- β.

The invention also provides application of sorafenib in preparing a medicine for resisting lung metastatic tumor, wherein the sorafenib is sorafenib itself or a pharmaceutically acceptable salt.

Sorafenib is used clinically to treat unresectable hepatocellular carcinoma, advanced renal cell carcinoma, and thyroid carcinoma that is locally recurrent or metastatic, progressive, differentiated, and refractory to radioiodine therapy. Therefore, sorafenib is extremely safe as a ready-to-use drug for treating lung metastatic tumors. Meanwhile, in vivo test results show that Th9 cells after the treatment of reinfusion sorafenib obviously prolong the survival time of a mouse 4T1-OVA (mouse breast cancer cells) or K7M2-OVA (mouse osteosarcoma cells) lung metastasis tumor model. The mechanism is that sorafenib can significantly increase the expression level of the protein kinase p-ERK in Th9 cells.

Preferably, the lung metastatic tumor is caused by breast cancer or osteosarcoma.

Sorafenib is added when the CD4 ⁺ T cells differentiate into Th9 cells, and the concentration of sorafenib is 0.2-2 mu M.

In vivo experiments show that Th9 cells treated by reinfusion sorafenib obviously increase the proportion of CD8 ⁺CD45⁺ cells in spleen of a mouse 4T1-OVA or K7M2-OVA lung metastasis tumor model, reduce the tumor areas of the mouse lung 4T1-OVA and K7M2-OVA, and further prolong the survival time of the mouse 4T1-OVA or K7M2-OVA lung metastasis tumor model.

The invention also provides a medicine for resisting lung metastatic tumor, the active ingredient comprises Th9 cells, the Th9 cells are obtained by adding sorafenib stimulation in the differentiation process of CD4 ⁺ T cells to the Th9 cells, and the sorafenib is sorafenib itself or pharmaceutically acceptable salt. The concentration of sorafenib is 0.2-2 mu M.

Preferably, the dosage form of the medicament is a liquid injection. In the application, th9 cells are returned into mice of a 4T1-OVA or K7M2-OVA lung metastasis tumor model through tail veins. The dose of the liquid injection was 3×10 ⁶ Th9 cells infused back per mouse.

In conclusion, the research of the invention discovers that sorafenib can obviously improve the differentiation efficiency of Th9 cells in vitro, which indicates that sorafenib can expand a large amount of Th9 cells with anti-tumor effect in vitro, and provides a new thought and a new way for ACT treatment method of clinical tumor.

Drawings

FIG. 1 is a normal C57 mouseCD4 ⁺ T cells are subjected to Th9 cell polarization capacity, with or without sorafenib treatment, specifically, the ratio of Th9 cells is detected by flow analysis, a is a chart of Th9 cells detected by flow, and b is a statistical analysis of results of a chart. "" means P <0.005 "" means P <0.001 ".

FIG. 2 shows the detection of cytokine IL9 expression levels in Th9 cell supernatants by enzyme-linked immunosorbent assay (ELISA). "+" means P <0.05 "+" means P <0.005 "", and "" means P <0.001 ".

FIG. 3 shows the detection of the expression level of the Il9 gene in Th9 cells by real-time fluorescent quantitative analysis. "+" means P <0.05 "+" means P <0.005 "", and "" means P <0.001 ".

FIG. 4 is a normal C57 mouseERK signal channel protein expression level in the polarization process of CD4 ⁺ T cells to Th9 cells, and in addition or no sorafenib treatment, in particular, western Blotting (WB) detection of the p-ERK, ERK and beta-Actin protein expression level of Th9 cells.

FIG. 5 is a normal C57 mouseCD4 ⁺ T cells were treated with or without sorafenib or U0126 (MEK 1/MEK2 inhibitor) to determine the proportion of Th9 cells by flow assay, a is a plot of flow assay Th9 cells, and b is a statistical analysis of the results of plot a. ns indicates no significant difference, "x" indicates P <0.05, "x" indicates P <0.005.

Fig. 6 shows lung metastasis tumor model induced by tail vein injection of 4T1-OVA cells in normal C57 mice, tail vein back transfusion or no back transfusion of sorafenib treated or untreated Th9 cells, specifically, lung section tumor area ratio and survival curve of 4T1-OVA lung metastasis tumor model detected by hematoxylin-eosin staining method, a is hematoxylin-eosin staining picture of lung, b is lung section tumor area ratio statistics for a graph, C is survival curve of tumor mice. "+" means P <0.05 "+" means P <0.005 "", and "" means P <0.001 ".

Fig. 7 shows lung metastasis tumor model induced by tail vein injection of K7M2-OVA cells in normal C57 mice, tail vein back transfusion or no back transfusion of sorafenib treated or untreated Th9 cells, specifically, lung slice tumor area ratio and survival curve of K7M2-OVA lung metastasis tumor model detected by hematoxylin-eosin staining method, a is hematoxylin-eosin staining picture of lung, b is lung slice tumor area ratio statistics for a graph, C is survival curve of tumor mice. "+" means P <0.05 "+" means P <0.005 "", and "" means P <0.001 ".

FIG. 8 shows the CD8 ⁺ T cell fraction in the tumor microenvironment of mice tested by flow analysis under conditions in which normal C57 mice induced lung metastasis by tail vein injection of 4T1-OVA cells with or without tail vein reinfusion of sorafenib-treated or untreated Th9 cells. "x" means P <0.05; "x" means P <0.005.

FIG. 9 shows the CD8 ⁺ T cell fraction in the tumor microenvironment of mice tested by flow analysis under conditions in which normal C57 mice induced lung metastasis by tail vein injection of K7M2-OVA cells, tail vein reinfusion or no reinfusion of sorafenib-treated or untreated Th9 cells. "" means P <0.005 "" means P <0.001 ".

Detailed Description

The C57 mice used in the present invention were purchased from Jiangsu Jiugang Biotech.

Example 1

T cell sorting:

(1) Taking spleen and lymph node of C57 mouse, squeezing with syringe piston to obtain tissue suspension;

(2) Transferring into a 15ml conical test tube, and precipitating the large block at the bottom of the test tube or filtering by a nylon filter screen to obtain single cell suspension;

(3) Centrifuging at 1500rpm at 4℃to pellet the cell suspension for 5min, and discarding the supernatant;

(4) Resuspension the samples with 2ml PBS buffer, dilute with 10 μl of 3% glacial acetic acid by mass concentration, and perform cell count;

(5) Centrifuging the cells again, discarding the supernatant, and re-suspending the cells with a sorting buffer according to the counting result to adjust the density to 1X 10 ⁸/ml;

(6) CD4 ⁺ T cells were sorted out using the CD4 negative selection kit (EASYSEPTM MOUSE CD4 ⁺ T Cell Isolation Kit, # 19765) for use in subsequent experiments.

Example 2

Th9 cell in vitro differentiation anti-CD3 (145-2C 11, bio X cell) and anti-CD28 (PV-1, bio X cell) were diluted with autoclaved PBS to a final concentration of 2. Mu.g/ml in 96 well plates, plated at 200. Mu.l/well, and allowed to stand at 37℃for 2 hours or more.

CD4 ⁺ T cells, 4X 10 ⁵ cells/200. Mu.l/well were isolated and Th9 induced differentiation was performed as described in example 1. Th9 cell differentiation conditions are shown below 10. Mu.g/ml anti-IFNγ (BE 0054, bio X cell), 20ng/ml IL4 (130-094-061,Miltenyi Biotec) and 10ng/ml TGF- β (130-095-067,Miltenyi Biotec). With or without sorafenib (HY-10201, medChemExpress) stimulation, with sorafenib concentrations of 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, and 2. Mu.M, respectively, were plated, i.e., day 0 differentiation. And waiting until the 4 Th day is Th9 cells, and waiting for subsequent experiments.

Example 3

Flow cytometry to detect the intracellular cytokine IL-9 in Th9 cells, cells were treated prior to staining, 50ng/mL phorbol ester (PMA, IP1010, solarbio), 1 μg/mL Ionomycin (Ionomycin, I8800, solarbio) and 3 μg/mL Brefeldin A (00-4506-51, thermosusher) were stimulated for 4h and harvested. Cells to be detected were washed 1 time with PBS and resuspended in PBS after centrifugation. After adding CD4-PE (12-0041-82, thermofilter) flow antibody, incubating on ice or at 4℃for 30min in the absence of light, neutralizing with PBS for 2 times, discarding the supernatant, adding 200 μl of 1 Xflow intracellular fixation buffer (00-8222-49, thermofilter) per tube, and keeping out of light for 20min at room temperature. Adding into flow type membrane rupture buffer (00-8333-56, thermofilter) for neutralization and centrifuging. After centrifugation, 100. Mu.l of 1 Xflow rupture buffer was used to resuspend cells, IL9-APC (50-8091-82, invitrogen) flow antibody was added, incubated at 4℃for 30min in the dark, and then 1 Xflow rupture buffer was added for neutralization and washing 2 times. The supernatant was discarded, the cells were resuspended in an appropriate amount of PBS, flow cytometric analysis was performed using NovoCyte flow cytometer (ACEA), and the data was analyzed using FlowJo software.

If the T cell surface antigens CD45 and CD8 are to be detected, the cells to be detected are washed 1 time with PBS, centrifuged and resuspended with PBS. After adding CD45-PB (MCD 4528, thermofisher) and CD8-APC (MHCD 0805, thermofisher) flow antibodies, incubating for 30min on ice or at 4℃in the dark, neutralizing with PBS for centrifugation 2 times, discarding the supernatant, and re-suspending cells with an appropriate amount of PBS for flow cytometry. The results in fig. 1 show that sorafenib significantly increases the number of IL9 ⁺CD4⁺ T cells (Th 9 cells). The results in FIG. 5 show that the effect of sorafenib on the number of IL9 ⁺CD4⁺ T cells (Th 9 cells) was eliminated after addition of U0126 (0.5. Mu.M, HY-12031A, medChemExpress).

Example 4

ELISA, namely collecting the supernatant of Th9 cells obtained in the example 2, centrifuging and taking the supernatant as a sample. Using a kit (88-8092-88, thermofiser), the pre-coated plates and the required reagents were equilibrated to room temperature, 350 μl wash buffer was added to the well plates, left to stand for 40s, the supernatant discarded and the well plates were tapped on absorbent paper and repeated three times. The standard substance freeze-dried powder is subjected to gradient dilution by using sample diluent, standard substances with different concentrations and prepared samples are added into a pore plate according to 100 mu l/hole, 100 mu l of sample diluent is added into a blank hole, and 3 compound holes are repeated for each sample. The wells were blocked with a plate membrane, incubated at 37℃for 2h, then the wells were discarded, washing was repeated 3 times, 100. Mu.l of streptavidin-horseradish peroxidase working solution was added to the wells, and incubation was performed at 37℃for 30min after plate membrane sealing. The wells were discarded and the wash repeated 3 times. 100 μl of tetramethylbenzidine substrate was added to the wells, incubated at 37℃in the dark for 15-20min, 50 μl of stop solution was added after the liquid in the wells turned blue, and OD was measured at room temperature using an ELISA reader. The cytokine content of the sample was calculated from the OD values measured. The results in fig. 2 show that sorafenib significantly increases the content of IL9 in Th9 cell supernatants and increases with increasing sorafenib concentration.

Example 5

RNA reverse transcription Sorafenib-treated (0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M) and untreated total cellular RNA were extracted according to the instructions of TaKaRa company RNAiso Plus. cDNA was synthesized by reverse transcription using 1. Mu.g of the total amount of RNA as a template according to the TaKaRa reverse transcription kit instructions (#RR037A). Reverse transcription was performed for 15min at 37℃with the following ：5×PrimeScript Buffer(2μl)、Oligo dT Primer(0.5μl)、Random 6mers(0.5μl)、PrimeScript RT Enzyme Mix I(0.5μl)、Total RNA(500ng)、Rnase Free H₂O( -fold make-up to 10. Mu.l) and heat-inactivated enzyme activity at 85 ℃.

Real-time fluorescent quantitative PCR (quantitative PCR) the synthesized cDNA can be used as a template for reaction, and the corresponding fluorescent quantitative PCR primers are respectively used for quantitative PCR amplification by adopting SYBR Premix Ex TaqTM (#RR041A). The fluorescent quantitative PCR system was 10. Mu.l SYBR Mix (5. Mu.l), cDNA template (1. Mu.l), primer (0.6. Mu.l), ddH ₂ O (filled to 10. Mu.l). The following two-step PCR amplification standard procedure was used. The first step, pre-denaturation at 95℃for 30s, and the second step, PCR reaction (40 cycles), 95℃for 5s,60℃for 30s.

The PCR primer sequences were as follows:

Actb-F:

Actb-R:

Il9-F:

Il9-R:

the results in fig. 3 show that sorafenib significantly increases the expression level of Il9 in Th9 cells and increases with increasing sorafenib concentration.

Example 6

Protein extraction 2. Mu.M sorafenib-treated and untreated Th9 cells were collected in 1.5mL EP tubes, washed twice with PBS, 30. Mu.L of RIPA lysate (R0020, solarbio) containing the protease inhibitor PMSF (P0100, solarbio) was added, mixed well and lysed on ice for 30min. Centrifuge at 12000 Xg for 10min at 4℃and take the supernatant to a new EP tube, add 5X SDS Loading Buffer (P1040, solarbio), bath with boiling water for 10min, and obtain protein sample for use.

Protein immunoblotting, namely placing the prepared gel on an electrophoresis core, pouring electrophoresis liquid (Tris 3g/L, glycine 14.4g/L and SDS1 g/L), adding Protein Ladder (26616, solarbio) and the prepared Protein sample into the holes one by one, starting at 90V, and regulating the voltage to 180V after the Protein Ladder is separated. After the end, the gel was removed and cut into appropriate size bubbles and placed in transfer solution (Tris 3g/L, glycine 14.4g/L,20% methanol). PVDF membrane (IPVH 00010, millipore) of corresponding size was cut and activated in methanol for more than 30 s. Placing the membrane transferring clamps in the order of a black clamp plate, a black sponge, thick filter paper, gel, PVDF membrane, thick filter paper, black sponge and a transparent clamp plate, placing the membrane transferring clamps in a frame, pouring a membrane transferring liquid, and transferring the membrane for 90 minutes at 220 mA. PVDF membranes were removed and incubated in 5% skim milk in a shaker at room temperature for 1h, 3 times for 5min each with PBST, and then incubated overnight with the corresponding primary antibodies (p-ERK, #4370; ERK, #9102; β -Actin, #4967,Cell signaling technology) in a shaker at 4 ℃. The next day the primary antibody was discarded, the membrane was washed 3 times with PBST for 10min each and the secondary antibody (# 7074P2,Cell signaling technology) was incubated for 1h at room temperature in a shaker. The secondary antibody was discarded and the membrane was washed 3 times with 10min each time with PBST. The film was gently wiped with absorbent paper, and ECL (4 AW011-500A/B, sizhengbai) was added dropwise, followed by exposure using an exposure apparatus.

The results in FIG. 4 show that sorafenib significantly increases the expression level of the protein kinase p-ERK in Th9 cells.

Example 7

In the lung metastasis tumor model of the mice, day0 respectively collects 4T1-OVA and K7M2-OVA tumor cells, the cells are washed by PBS for 2 times and counted under a microscope, the cell density is adjusted to 1X 10 ⁷/mL, 100 mu l of tumor cell suspension is injected into each tail vein of the C57 mice, and the cells are blown and evenly mixed before injection.

T cell feedback treatment 2. Mu.M sorafenib treated and untreated Th9 cells were obtained as in example 2, resuspended in PBS, and 3X 10 ⁶ cells or an equal volume of PBS were returned to the tail vein of mice in lung metastasis model using insulin needles at Day7 and Day14, respectively, and mice were observed daily for survival. The results of c in FIG. 6 and c in FIG. 7 show that the reinfusion of Th9 cells significantly prolonged the survival time of the mouse 4T1-OVA or K7M2-OVA lung metastasis tumor model, and that the reinfusion of Th9 cells stimulated with sorafenib significantly enhanced this effect.

Example 8

Mice were treated by day 17 and their lungs were H & E stained as in example 7. The results of a and b in FIG. 6 and a and b in FIG. 7 show that the reinfusion of Th9 cells significantly reduces the tumor area of 4T1-OVA and K7M2-OVA in the lungs of mice, prolonging the survival time, and the reinfusion of Th9 cells after sorafenib stimulation further enhances the effect.

Example 9

Lung flow cytometry mice were modeled for lung metastasis as in example 7, treated on day 17, lung was taken, ground, filtered through a filter screen, the filtrate centrifuged and resuspended in 1mL of erythrocyte lysate (R1010, solarbio), allowed to stand at room temperature for 5min, the supernatant centrifuged and the flow cytometry was performed as in example 3. The results in figures 8 and 9 show that the reinfusion of Th9 cells significantly increases the proportion of CD8 ⁺CD45⁺ cells in the spleen of a mouse 4T1-OVA or K7M2-OVA lung metastasis tumor model, and that the reinfusion of Th9 cells after sorafenib stimulation significantly enhances this effect.

Claims

1. Use of sorafenib in the preparation of a drug for treating lung metastatic tumors, wherein the sorafenib is sorafenib itself or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that the lung metastatic tumor is caused by breast cancer or osteosarcoma.

3. The use according to claim 1, characterized in that sorafenib is added when CD4 ⁺ T cells differentiate into Th9 cells, and the concentration of sorafenib used is 0.2-2 μM.