Application of aspirin in enhancing curative effect of tumor infiltrating lymphocyte on treating liver cancer

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of aspirin in enhancing the curative effect of tumor infiltrating lymphocyte on treating liver cancer. The invention discloses application of aspirin in preparation of a medicine for enhancing the curative effect of tumor infiltrating lymphocyte on hepatocellular carcinoma. The invention discloses an application of aspirin in enhancing the curative effect of tumor infiltrating lymphocyte on treating liver cancer, the aspirin overcomes the problem of drug resistance of TIL therapy in HCC by adjusting the immune cell function in a tumor microenvironment, and the treatment effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of aspirin in enhancing the therapeutic effect of tumor-infiltrating lymphocytes in the treatment of liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, with persistently high incidence and mortality rates, posing a serious threat to human health. Current treatments for HCC include surgical resection, liver transplantation, local ablation, transarterial chemoembolization, and systemic drug therapy. However, due to the insidious onset and rapid progression of HCC, most patients are diagnosed at an advanced stage, and it easily develops resistance to traditional radiotherapy and chemotherapy, resulting in a poor overall prognosis and a persistently low five-year survival rate.

[0003] In recent years, tumor immunotherapy has brought new hope to patients with hepatocellular carcinoma (HCC). Among them, tumor-infiltrating lymphocyte (TIL) therapy is a personalized immunotherapy that involves isolating and expanding lymphocytes from the patient's tumor tissue and then reinfusing them into the body to attack tumor cells. Although this therapy has shown significant efficacy in some solid tumors, its effectiveness in HCC is often limited by the tumor immunosuppressive microenvironment. This microenvironment contains a large number of immunosuppressive cells (such as M2 tumor-associated macrophages) and inhibitory molecules, which severely weaken the proliferation, invasion, and killing functions of TILs, leading to low treatment response rates or drug resistance and relapse.

[0004] Aspirin (Acetylsalicylic Acid, ASA), a classic nonsteroidal anti-inflammatory drug, has garnered increasing attention for its antitumor potential in addition to its antipyretic, analgesic, and antiplatelet aggregation effects. Existing studies have shown that aspirin can influence tumor cell proliferation, apoptosis, and metastasis by inhibiting the cyclooxygenase (COX) pathway and regulating various signaling molecules. Furthermore, some studies suggest that aspirin may have certain immunomodulatory effects, such as affecting the function of immune cells like macrophages and T cells. However, whether aspirin can specifically regulate the immune microenvironment of hepatocellular carcinoma (HCC), particularly whether it can reverse its immunosuppressive state and thus enhance the efficacy of adoptive cellular immunotherapy, remains unclear, lacking definitive research reports and solutions. Summary of the Invention

[0005] This invention aims to provide the application of aspirin in enhancing the therapeutic efficacy of tumor-infiltrating lymphocytes in the treatment of liver cancer. Aspirin overcomes the drug resistance problem of TIL therapy in HCC by regulating the function of immune cells in the tumor microenvironment, thereby improving the therapeutic effect.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: The use of aspirin in the preparation of drugs to enhance the efficacy of tumor-infiltrating lymphocyte therapy for hepatocellular carcinoma.

[0007] Preferably, the drug enhances the therapeutic efficacy of tumor-infiltrating lymphocytes in treating hepatocellular carcinoma by promoting the transformation of immunosuppressive M2 macrophages into immunostimulatory M1 macrophages.

[0008] Preferably, the tumor-infiltrating lymphocytes are TIL-1 cells.

[0009] Preferably, the hepatocellular carcinoma is a hepatocellular carcinoma cell line that is resistant to tumor-infiltrating lymphocytes.

[0010] The present invention also provides a pharmaceutical composition for treating hepatocellular carcinoma, comprising the aspirin and tumor-infiltrating lymphocytes.

[0011] Preferably, the tumor-infiltrating lymphocytes are TIL-1 cells.

[0012] Preferably, the hepatocellular carcinoma is a hepatocellular carcinoma cell line that is resistant to tumor-infiltrating lymphocytes.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses the application of aspirin in enhancing the therapeutic efficacy of tumor-infiltrating lymphocytes (ASA) in treating hepatocellular carcinoma. For the first time, this invention reveals and verifies that aspirin can significantly enhance the therapeutic effect of ASA on hepatocellular carcinoma by reshaping the tumor immune microenvironment. ASA can polarize immunosuppressive M2 macrophages in the tumor microenvironment into immunostimulatory M1 macrophages, thereby effectively reversing the immunosuppressive barrier encountered by TIL therapy. This key mechanism is directly manifested in in vitro experiments as the combined treatment of ASA and TIL-1 synergistically inhibits the proliferation, invasion, and stem cell characteristics of drug-resistant HCC-1R cells, and promotes their apoptosis. In in vivo animal models, this combined treatment regimen exhibits a significant anti-tumor synergistic effect, strongly inhibiting tumor growth, reducing tumor angiogenesis, and ultimately significantly prolonging the survival of tumor-bearing mice. Therefore, this invention not only provides an effective strategy to overcome TIL therapy resistance but also offers a novel combination therapy for the clinical immunotherapy of hepatocellular carcinoma, possessing significant translational application prospects.

[0014] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0015] Figure 1 Cell morphology of HCC-1 cells treated with TIL-1 for 0-48 hours; Figure 2 Cell morphology of HCC-1R cells treated with TIL-1 for 0-48 hours; Figure 3 A statistical graph showing the effects of ASA and TIL-1 on the proliferation of HCC-1R cells in the CCK-8 assay. Figure 3 In the figure, A represents the statistical effect of ASA on the proliferation ability of HCC-1R cells. Figure 3 B in the figure represents the statistical graph of the effect of TIL-1 on the proliferation ability of HCC-1R cells; Figure 4 Statistical graph for evaluating the regulatory effects of ASA and TIL-1 on proliferation-related genes in HCC-1R cells using qPCR experiments; Figure 5 Statistical graph for evaluating the regulatory effects of ASA and TIL-1 on the expression of proliferation-related proteins in HCC-1R cells using ELISA experiments; Figure 6 Figure showing the effects of ASA and TIL-1 on the morphology of HCC-1R cells; Figure 7 HCC-1R and HM-1 cells were inoculated on day 0, and ASA and / or TIL-1 were injected intratumorally daily from day 7 to 9. Tumor volume was measured with calipers on days 6, 10, 13, 16 and 20. Figure 8 This is a tumor growth curve; Figure 9 The survival curve for mice; Figure 10 A statistical graph showing the regulatory effects of ASA and TIL-1 on M1 macrophage-related genes in mouse hepatocellular carcinoma xenografts as assessed by qPCR experiments. Figure 11 A statistical graph showing the regulatory effects of ASA and TIL-1 on genes related to M2 macrophages in mouse hepatocellular carcinoma xenografts, as assessed by qPCR experiments. Figure 12 Statistical graph for evaluating the regulatory effects of ASA and TIL-1 on M1 macrophage-related proteins in mouse hepatocellular carcinoma xenografts using ELISA assays; Figure 13 Statistical graph for evaluating the regulatory effects of ASA and TIL-1 on M2 macrophage-related proteins in mouse hepatocellular carcinoma xenografts using ELISA experiments. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0017] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0018] Source of experimental materials: Eagle medium (DMEM): Batch number: 11965092, Gibco.

[0019] Add the following reagents to RPMI 1640 medium (batch number: 12633012, Gibco) to prepare complete cell culture medium (CCM): 1% (v / v) 100 U / mL penicillin-streptomycin (Gibco), 1% (v / v) 1 mM sodium pyruvate (Invitrogen), 1% (v / v) non-essential amino acids (NEAA) (Gibco), 0.1% (v / v) 14.3 mM β-mercaptoethanol (Sigma-Aldrich), 1% (v / v) 2 mM L-glutamine (Gibco), 10% (v / v) Gibco fetal bovine serum, 1% (v / v) 25 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 10 μg / mL gentamicin, and 0.1% 50 μg / mL amphotericin B.

[0020] The following reagents were added to the CCM for amplification activation, which is REP Media I (Rapid Amplification Protocol Medium I): Interleukin-2 (IL-2) (PeproTech, Rocky Hill, NJ) (10 ng / mL), Interleukin-7 (IL-7) (PeproTech, Rocky Hill, NJ) (20 ng / mL), Interleukin-15 (IL-15) (PeproTech, Rocky Hill, NJ) (20 ng / mL), and 50 ng / mL Monoclonal Anti-Human CD3 Antibody, Mouse IgG2a (Clone: ​​OKT3) (ACRO, USA).

[0021] AIM V™ Medium lot number: 12055091, ThermoFisher.

[0022] REP Media II (Rapid Amplification Protocol Medium II): A 1:1 mixture of REP Media I and AIM V medium (lot number: 12055083, Invitrogen, California, USA).

[0023] CD45 (Common Leukocyte Antigen) Magnetic Beads: Batch No.: 130-118-780, MACS, Cologne, Germany.

[0024] Fcγ receptor III (CD16) +Mononuclear cell isolation kit: Batch number: 130-091-765, Miltenyi Biotec.

[0025] TRIzol reagent: Invitrogen, Carlsbad, California, USA.

[0026] BCA (Diquinoline carboxylic acid) protein concentration assay kit: Beyotime Biotechnology, Shanghai, China.

[0027] Malondialdehyde (MDA), oxygen free radical (OFR), 4-hydroxynonenal (4-HNE), glutathione (GSH), prolyl hydroxylase (PHD), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) kit: Nanjing Jiancheng Biotechnology Institute, Nanjing, China.

[0028] Enzyme-Linked Immunosorbent Assay (ELISA) Kit: Human IGF-1 (Insulin-like Growth Factor) ELISA Kit, Elabscience, Catalog No.: E-EL-H0086.

[0029] Human HGF (Hepatocyte Growth Factor) ELISA Kit, Elabscience, Catalog No.: E-EL-H0084.

[0030] Human EGF (Epidermal Growth Factor) ELISA Kit, Elabscience, Catalog No.: E-EL-H0059.

[0031] Human IL-6 (Interleukin-6) ELISA Kit, Elabscience, Catalog No.: E-EL-H6156.

[0032] Human IL-33 ELISA Kit, Elabscience, Catalog No.: E-EL-H2402.

[0033] Human G-CSF (granulocyte colony-stimulating factor) ELISA Kit, Elabscience, catalog number: E-EL-H0079.

[0034] Human IL-12 ELISA Kit, Elabscience, catalog number: E-EL-H0150.

[0035] Human IL-1β (Interleukin-1β) ELISA Kit, Elabscience, Catalog No.: E-EL-H0149.

[0036] Human TNF-α (Tumor Necrosis Factor-α) ELISA Kit, Elabscience, Catalog No.: E-EL-H0109.

[0037] Human NOS2 / iNOS (Inducible Nitric Oxide Synthase) ELISA Kit, Elabscience, Catalog No.: E-EL-H0753.

[0038] Human IL-23 ELISA Kit, Elabscience, Catalog No.: E-EL-H0107.

[0039] Human IL-10 ELISA Kit, Elabscience, catalog number: E-EL-H6154.

[0040] Human VEGF-A (Vascular Endothelial Growth Factor A) ELISA Kit, Elabscience, Catalog No.: E-EL-H0111.

[0041] Human PDGF-AA (Human Platelet-Derived Growth Factor AA) ELISA Kit, Elabscience, Catalog No.: E-EL-H6183.

[0042] Human ARG1 (arginase 1) ELISA Kit, Elabscience, catalog number: E-EL-H0497.

[0043] Human YM1 / Chitinase 3-like 3 ELISA Kit, MyBioSource, Catalog No.: MBS7269402.

[0044] Human TGF-beta 1 (Transforming Growth Factor β1) ELISA Kit, RayBiotech, Catalog No.: ELH-TGFb1.

[0045] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0046] Example 1 Animals and primary cells All animal experiments were conducted in strict accordance with the guidelines and regulations established by the Laboratory Animal Management and Use Committee (IACUC) of Tianjin Medical University, and all experimental protocols were approved by the committee.

[0047] Six-week-old NOD / SCID / IL2R- / - (NSG) mice were purchased from the Shanghai Model Organism Research Center.

[0048] Primary hepatocellular carcinoma (HCC-1) cells were isolated from a patient diagnosed with hepatocellular carcinoma (HCC) and cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Similarly, primary human liver macrophages (HM-1) were obtained from the same HCC patient and cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin. Primary tumor-infiltrating lymphocytes (TIL-1) isolated from the same HCC patient were cultured in REP Media I medium, followed by expansion in REP Media II medium. All cells were cultured in a humidified environment at 37°C with 5% carbon dioxide.

[0049] Isolation of HCC-1, TIL-1 and HM-1 cells: To isolate HCC-1 cells, tumor tissue from HCC patients was minced and enzymatically digested at 37°C using a digestion buffer containing 1 mg / mL collagenase A (Sigma-Aldrich) and 300 μg / mL DNase I (Sigma-Aldrich). The resulting cell suspension was filtered and then subjected to density gradient centrifugation.

[0050] To isolate TIL-1 cells, digested HCC tissue was processed into a single-cell suspension. TIL-1 cells were enriched by Percoll gradient density centrifugation and then sorted using CD45 magnetic beads.

[0051] Use CD16 + The mononuclear cell isolation kit sorted HM-1 cells from a single-cell suspension of HCC tissue. The sorted cells were cultured in DMEM supplemented with 20 ng / mL macrophage colony-stimulating factor (M-CSF) for 7–10 days to generate mononuclear cell-derived hepatic macrophages (HM-1).

[0052] Constructing TIL-resistant HCC cells (HCC-1R): To construct TIL-resistant HCC cells (HCC-1R), HCC-1 cells were first seeded in six-well plates and co-cultured with TIL-1 at an effector-to-target ratio of 1 for 48 hours. The surviving cells were re-digested, counted, and seeded again. Then, TIL-1 at an effector-to-target ratio of 2 was added, and the cells were co-cultured for another 48 hours. The surviving cells were then re-digested, counted, and seeded again. Finally, TIL-1 at an effector-to-target ratio of 20 was added, and the cells were co-cultured for another 48 hours. Finally, TIL-1 at an effector-to-target ratio of 50 was added, and the cells were co-cultured for another 48 hours. Single-cell clones were isolated from the TIL-1-exposed HCC cells and expanded for subsequent experiments.

[0053] Cytotoxicity assessment: For the cytotoxicity assay, HCC-1R cells were stored at 2 × 10⁶ cells per well. 3 Cells were seeded at a density of [missing information - likely a specific density] in 96-well plates. Cells were treated with different concentrations of TIL-1 and / or ASA for 24-72 hours. The specific experimental protocol is as follows: HCC-1R cells were cultured in DMEM (high glucose) medium containing 10% FBS and incubated at 37°C in a 5% CO2 incubator. 24 hours before the experiment, cells were passaged to the logarithmic growth phase to ensure good condition (no contamination, uniform morphology). HCC-1R cells were treated with TIL-1 (effect-to-target ratios of 1, 2, 5, 10, 20, 50) or ASA (0.01, 0.02, 0.05, 0.1, 0.2, 0.5) for 24-72 hours. 90 μL of supernatant was discarded from each well, and 100 μL of CCK-8 working solution was added (avoiding air bubbles). Cells were incubated at 37°C in the dark for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the data were recorded.

[0054] The cell growth rate was assessed according to the instructions for the CCK-8 kit (C0038, Beyotime).

[0055] Enzyme-linked immunosorbent assay (ELISA): Measured indicators: IGF-1, HGF, EGF, IL-6, IL-33, G-CSF, IL-12, IL-1β, TNF-α, iNOS, IL-23, IL-10, VEGFA, PDGFAA, ARG1, YM1 and TGFβ1.

[0056] The concentration of cytokines in tumor tissue homogenates or macrophages isolated from tumor tissues was determined by ELISA, and the procedure was strictly followed according to the kit instructions.

[0057] RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), and real-time RT-PCR: Total RNA was extracted from cells or tissues using TRIzol reagents according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using TransScript® First-Strand cDNA Synthesis Ultramix Reagent (TransGen). Reverse transcription polymerase chain reaction (RT-PCR) was performed using TransScript® Two-Step RT-PCR Ultramix Reagent (TransGen), and the PCR products were analyzed on a 1% agarose gel. Real-time RT-PCR was performed using TransStart® Top Green qPCR Ultramix Reagent (TransGen). Primer sequences are shown in Table 1 below.

[0058] Table 1 Primer Sequences

[0059] In vivo anti-tumor model: All animal protocols were approved by the Institutional Animal Care and Use Committee. On day 0, NSG mice were gently restrained using a restraint device specifically designed for tail vein injection. The tail was heated with warm water (40°C) for 1–2 minutes to dilate the vein. 100 μL of HM-1 cell suspension (5 × 10⁻⁶) was injected using a 29-gauge insulin syringe. 6 (One cell) is slowly injected into the tail vein. Successful injection is confirmed by the absence of resistance during injection and the visible return of blood to the syringe when the needle is withdrawn.

[0060] Immediately after HM-1 cell infusion, F-HCC-1R cell suspension (1×10⁻⁶ cells) was administered subcutaneously to the same mouse. 6 A mouse model of subcutaneous tumor xenograft was established using 100 cells. Approximately one week later, when the tumor reached about 50 mm... 3 Mice were randomly assigned to four treatment groups: PBS, TIL-1, ASA, and ASA / TIL-1. Treatment was administered via injection at the tumor site. Treatment was based on tumor weight and volume (L×W). 2 ) / 2) Quantify the anti-tumor effect.

[0061] When the tumor grows to 1000mm 3 Mice were anesthetized by inhalation of isoflurane. Euthanasia was performed using cervical dislocation. Results were as follows: Figures 1-9 As shown.

[0062] Depend on Figure 1 It can be seen that when sensitive HCC-1 cells are treated with TIL-1 at an effector-to-target ratio (TIL-1 / HCC-1 ratio) of 50 for 48 hours, the cells gradually become round and shrunken, showing obvious signs of death.

[0063] Depend on Figure 2 It can be seen that the drug-resistant HCC-1R cells treated with the same method showed less morphological change and better survival within 48 hours, proving that they have developed resistance to TIL-1 with a high target ratio.

[0064] Depend on Figure 3 It can be seen that, compared with single treatment (ASA or TIL-1), the combined treatment of ASA and TIL-1 can most significantly inhibit the proliferation of HCC-1R cells.

[0065] Depend on Figure 4 and Figure 5 It can be seen that combined treatment with ASA and TIL-1 can more effectively downregulate the expression of key proliferation-related genes and proteins in HCC-1R cells.

[0066] Depend on Figure 6 It can be seen that HCC-1R cells treated with ASA and TIL-1 showed more extensive morphological damage, membrane blistering, and increased cell debris, indicating that ASA and TIL-1 synergistically induced cell death.

[0067] Depend on Figures 7-9 It can be seen that the tumor volume growth in the mice treated with the combination of ASA and TIL-1 was the slowest, and the inhibitory effect was significantly better than that of each single treatment group. Figure 9 The mouse survival curves further demonstrate that combination therapy is the most effective way to prolong the survival time of tumor-bearing mice.

[0068] Depend on Figure 10 and Figure 12 It was found that ASA combined with TIL-1 significantly upregulated the expression of M1 macrophage-related genes and proteins in mouse hepatocellular carcinoma xenografts. Figure 11 and Figure 13 It can be seen that ASA combined with TIL-1 can significantly downregulate the expression of M2 macrophage-related genes and proteins in mouse liver cancer xenografts.

[0069] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The use of aspirin in the preparation of drugs for enhancing the efficacy of tumor-infiltrating lymphocyte therapy in hepatocellular carcinoma.

2. The application according to claim 1, characterized in that, The drug enhances the therapeutic efficacy of tumor-infiltrating lymphocytes in treating hepatocellular carcinoma by promoting the transformation of immunosuppressive M2 macrophages into immunostimulatory M1 macrophages.

3. The application according to claim 1, characterized in that, The tumor-infiltrating lymphocytes were TIL-1 cells.

4. The application according to claim 1, characterized in that, The hepatocellular carcinoma mentioned is a hepatocellular carcinoma cell line that is resistant to drugs from tumor-infiltrating lymphocytes.

5. A pharmaceutical composition for treating hepatocellular carcinoma, characterized in that, It comprises the aspirin and tumor-infiltrating lymphocytes as described in claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that, The tumor-infiltrating lymphocytes were TIL-1 cells.

7. The pharmaceutical composition according to claim 5, characterized in that, The hepatocellular carcinoma mentioned is a hepatocellular carcinoma cell line that is resistant to drugs from tumor-infiltrating lymphocytes.

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