Use of a substance for blocking the HIF-1α / EPO axis in the preparation of a medicament for treating tumor recurrence after radiofrequency ablation of liver cancer

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a substance for blocking HIF-1alpha / EPO axis in preparation of a medicine for treating tumor recurrence after radiofrequency ablation of liver cancer. The application firstly determines that the immune suppression microenvironment of residual tumor after incomplete radiofrequency ablation (IRFA) is a key driving factor for residual cancer progression, reveals that the increase in recruitment of immune suppression cells EDMCs is closely related to up-regulation of HIF-1alpha / EPO axis in the anoxic microenvironment after IRFA, and provides a new theoretical target and research direction for mechanism research on recurrence after RFA of liver cancer. The application further provides a medicine for treating tumor recurrence after radiofrequency ablation of liver cancer. The medicine is highly targeted, targets to block HIF-1alpha / EPO axis, can accurately reduce the recruitment of EDMCs immune suppression cells, effectively breaks the immune suppression microenvironment, restores the anti-tumor immune function of the body, thereby significantly inhibits the growth and progression of residual tumor after IRFA, and provides a brand-new precise treatment scheme for recurrence after RFA of liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of substances that block the HIF-1α / EPO axis in the preparation of drugs for treating tumor recurrence after radiofrequency ablation of liver cancer. Background Technology

[0002] Ultrasound-guided radiofrequency ablation (RFA) is particularly suitable for patients in the early stages and those who cannot tolerate surgical resection due to its advantages of being minimally invasive, having a definite curative effect, and causing minimal damage to the body. This technique uses high-frequency alternating current to induce ion oscillation and heat generation in tumor tissue, causing coagulative necrosis of cells and thus inactivating the tumor. However, its clinical application still faces the challenge of high recurrence rates. The main reason is that when the tumor is large, has indistinct borders, an irregular shape, or is near vital organs or large blood vessels, resulting in thermal sedimentation, residual tumor cells at the ablation margin are easily left, leading to incomplete radiofrequency ablation (IRFA), which in turn induces rapid tumor progression. Literature reports a 5-year recurrence rate as high as 50%–70% after RFA, with recurrence primarily occurring in localized progression, severely impacting long-term survival rates. Studies have shown that IRFA can activate the METTL1-TGF-β2-PMN-MDSC axis, induce the formation of an immunosuppressive microenvironment, and promote the proliferation of residual tumors. This is also an important molecular mechanism for the recurrence of liver cancer after RFA. However, the current technology still does not fully understand the core driving mechanism of the immunosuppressive microenvironment of residual tumors after IRFA, which makes it impossible to accurately grasp the core causes of residual cancer progression.

[0003] Currently, treatment methods for recurrence after radiofrequency ablation (RFA) are diverse, including repeat RFA, surgical resection, interventional embolization, targeted therapy, and immunotherapy. However, each method has significant drawbacks. Repeat RFA, while maintaining the advantages of minimally invasive procedures and suitable for patients with small, superficial recurrences, suffers from uneven electrode heat distribution. For recurrences larger than 3 cm, located deeper, or near large blood vessels, incomplete ablation due to thermal sedimentation can easily occur, inducing secondary recurrence. It also has poor suitability for multiple or complex lesions and cannot improve the immunosuppressive microenvironment surrounding residual tumors. Surgical resection is effective for patients with localized recurrences and good liver function reserve, but it is highly invasive. Many liver cancer patients have a history of cirrhosis, and most recurrence patients cannot tolerate a second surgery. Furthermore, it is difficult to completely remove micrometastases or poorly defined lesions, leaving a risk of recurrence post-surgery and potentially causing complications such as bleeding and bile duct injury. Interventional embolization inhibits tumor growth by blocking blood supply and is suitable for multiple recurrent lesions. However, it cannot directly inactivate residual tumor cells, has limited efficacy in highly vascularized or diffuse recurrent lesions, and is prone to adverse reactions such as liver damage and bone marrow suppression. Long-term use alone is difficult to control tumor progression, and it cannot break the closed loop of immunosuppression mediated by the hypoxic microenvironment. Targeted therapy and immunotherapy, based on the recurrence mechanism, intervene in related molecular pathways or the immune microenvironment, providing new options for patients with advanced recurrence. However, monotherapy has significant limitations. Existing targeted drugs mostly focus on angiogenesis and cell proliferation-related pathways, while immunotherapy has a slow onset of action and insufficient ability to rapidly control multiple recurrent lesions in advanced stages. Furthermore, both are unable to reverse the specific immunosuppressive microenvironment induced by IRFA, lack precise intervention guidance targeting the core driving mechanism, and combination therapy regimens also lack clear target support.

[0004] In summary, the high recurrence rate of RFA in treating liver cancer remains a clinical bottleneck. Current technologies suffer from insufficient understanding of the core mechanisms of residual cancer progression after IRFA and lack targeted interventions in this pathway. Existing treatment methods each have their limitations, and the unique characteristics of liver cancer recurrence necessitate targeted prevention and control strategies. Therefore, there is an urgent need to develop more efficient and precise recurrence intervention and treatment technologies to overcome the shortcomings of existing technologies in mechanism understanding and target intervention, and improve the long-term survival rate of liver cancer patients after RFA. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide the application of substances that block the HIF-1α / EPO axis in the preparation of drugs for treating tumor recurrence after radiofrequency ablation of liver cancer, so as to solve the problem of insufficient understanding of the core mechanism of residual cancer progression after IRFA in the prior art, which leads to the high recurrence rate of liver cancer treated by RFA.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] This invention provides the application of substances that block the HIF-1α / EPO axis in the preparation of drugs for treating tumor recurrence after radiofrequency ablation of liver cancer.

[0008] In some embodiments of the present invention, the drug reduces EDMC recruitment and reverses the immunosuppressive microenvironment by blocking the HIF-1α / EPO axis, thereby treating tumor recurrence after radiofrequency ablation of liver cancer.

[0009] In some embodiments of the present invention, the substance that blocks the HIF-1α / EPO axis is selected from one or more of small molecule inhibitors, neutralizing antibodies, soluble receptors, siRNA, shRNA, dsRNA, and antisense nucleic acids.

[0010] In some embodiments of the present invention, PX-478 2HCl, Kamebakaurin, LW6, Acriflavine, Topotecan (a topoisomerase I inhibitor), 17-AAG (an HSP90 inhibitor), soluble EPOR, anti-EPO antibody, neutralizing antibody against EPOR, Aminoquinolines, EZN-2698, EZN-2208, SN38, Irinotecan, Temsirolimus, Everolimus, Sirolimus, LY294002, Wortmannin, 2ME2, ENMD-1198, ENMD-1200, ENMD-1237, Radicicol, KF85833, SCH66336, Apigenin, and Hsp90 are included. One or more of the following: GA, 17-DMAG, TAK-17, Echinomycin, Doxorubicin, Daunorubicin, Chetomin, Bortezomib, and siRNAs targeting HIF-1α; The siRNA targeting HIF-1α consists of the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO: 2.

[0011] In some preferred embodiments of the present invention, the anti-EPO antibody includes an EPO neutralizing antibody.

[0012] In some preferred embodiments of the present invention, the substance blocking the HIF-1α / EPO axis is at least one of PX-478 2HCl, a HIF-1α-targeting siRNA or an EPO neutralizing antibody composed of the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO: 2.

[0013] The tumor microenvironment (TME) plays a central role in the growth, invasion, metastasis, and immune escape of hepatocellular carcinoma (HCC) through mechanisms such as regulating immunosuppressive cell infiltration and metabolic reprogramming. Myeloid-derived suppressor cells (MDSCs) play a crucial role in the formation of the immunosuppressive microenvironment after intraepithelial neoplasia (IRFA). However, the precise upstream signaling pathways driving the recruitment of immunosuppressive cells and their key subsets in the specific pathological process of IRFA remain unclear, limiting the development of targeted immune intervention strategies. Erythroid-differentiated myeloid cells (EDMCs), a subset of MDSCs, are immunosuppressive cells possessing both myeloid and erythroid characteristics. They exert a strong immunosuppressive function in the lung cancer microenvironment, influencing anti-tumor immune responses. Whether EDMCs participate in the recurrence process of HCC after IRFA and the specific regulatory mechanisms of their recruitment have not been reported domestically or internationally.

[0014] Hypoxia-inducing factor 1 alpha (HIF-1α) is a key transcription factor in cellular responses to hypoxic environments. Hypoxia is characteristic of most solid tumors, significantly impacting the biological behavior and phenotype of tumor cells and closely associated with poor patient prognosis. HIF-1α is known to promote tumor recurrence by regulating vascular endothelial growth factor (VEGF). IRF-1α causes intratumoral angiogenesis and forms inflammatory edema between normal and tumor tissues, leading to slowed blood flow and thrombosis, thereby exacerbating tumor hypoxia and upregulating HIF-1α. PX-478 2HCl is an HIF-1α inhibitor that has undergone phase I clinical trials and shown good tolerability in patients.

[0015] Erythropoietin (EPO) is an endogenous glycoprotein hormone that regulates the survival, proliferation, and differentiation of erythroid cells by activating its transmembrane receptor. Its secretion levels increase significantly in hypoxic environments. Studies have shown that tumor-secreted EPO may be a key reason why immune checkpoint blockade therapy is ineffective in more than half of patients. Currently, whether HIF-1α regulates the immune microenvironment after IRFA (immunoassay-induced immunodeficiency) by activating EPO, particularly driving the recruitment of specific immunosuppressive cells such as EDMCs, remains unknown.

[0016] The present invention also discloses a drug for treating tumor recurrence after radiofrequency ablation of liver cancer, wherein the active ingredients of the drug include the substance that blocks the HIF-1α / EPO axis and other pharmaceutically acceptable excipients.

[0017] In some embodiments of the present invention, the substance that blocks the HIF-1α / EPO axis is selected from one or more of small molecule inhibitors, neutralizing antibodies, soluble receptors, siRNA, shRNA, dsRNA, and antisense nucleic acids.

[0018] In some embodiments of the present invention, the substance blocking the HIF-1α / EPO axis is selected from PX-478 2HCl, Kamebakaurin, LW6, Acriflavine, topoisomerase I inhibitor Topotecan, HSP90 inhibitor 17-AAG, soluble EPOR, anti-EPO, neutralizing antibody against EPOR, Aminoquinolines, EZN-2698, EZN-2208, SN38, Irinotecan, Temsirolimus, Everolimus, Sirolimus, LY294002, Wortmannin, 2ME2, ENMD-1198, ENMD-1200, ENMD-1237, Radicicol, KF85833, SCH66336, Apigenin, Hsp90 One or more of the following: GA, 17-DMAG, TAK-17, Echinomycin, Doxorubicin, Daunorubicin, Chetomin, Bortezomib, and siRNAs targeting HIF-1α; The siRNA targeting HIF-1α consists of the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO: 2.

[0019] In some preferred embodiments of the present invention, the substance blocking the HIF-1α / EPO axis is selected from one or more of PX-478 2HCl, HIF-1α-targeting siRNA, or EPO neutralizing antibody.

[0020] In some embodiments of the present invention, the content of the substance that blocks the HIF-1α / EPO axis in the drug is 1wt% to 99wt%.

[0021] In some embodiments of the present invention, the drug is a tablet, granule, capsule, injection, or oral liquid preparation.

[0022] In some embodiments of the present invention, the drug further includes other pharmaceutically acceptable excipients.

[0023] In some embodiments of the present invention, the other pharmaceutically acceptable excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, anti-adhesion agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0024] In some embodiments of the present invention, the filler is selected from starch, sucrose, lactose, or microcrystalline cellulose; the binder is selected from cellulose derivatives, alginate, gelatin, or polyvinylpyrrolidone; the wetting agent is selected from water, glycerin, ethanol, methylcellulose, sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, or hydroxypropyl methylcellulose; the disintegrant is selected from sodium carboxymethyl starch, hydroxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, or sodium bicarbonate; the absorption promoter is a quaternary ammonium compound; the surfactant is selected from hexadecyl alcohol or sodium dodecyl sulfate; the adsorbent carrier is selected from kaolin or soap clay; the lubricant is selected from talc, calcium and magnesium stearate, micronized silica gel, or polyethylene glycol; and the flavoring agent is selected from sucrose, simple syrup, aromatic syrup, glycerin, sorbitol, or mannitol.

[0025] In some embodiments of the present invention, the drug further includes a drug carrier.

[0026] In some embodiments of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

[0027] Based on the technical solution of the present invention, the present invention has the following beneficial effects compared with the prior art: This invention reveals for the first time the reprogramming mechanism of the immunosuppressive microenvironment in residual tumors after incomplete radiofrequency ablation (IRFA) and demonstrates that it is a key factor driving residual cancer progression. Specifically, this invention discovers that the hypoxic microenvironment after IRFA specifically upregulates the expression of the HIF-1α / EPO axis, and the activation of this signaling axis is closely related to the increased recruitment of immunosuppressive endothelial cells (EDMCs). This discovery elucidates for the first time the specific cellular and molecular pathway connecting hypoxia and immunosuppression after IRFA—the "HIF-1α / EPO axis → EDMCs"—filling the gap in the existing understanding of the core mechanism of immune escape after IRFA and providing a novel theoretical target and direction for the study of the mechanism and treatment of recurrence after RFA in liver cancer.

[0028] Based on the above-mentioned mechanism, this invention provides a novel targeted treatment strategy. By using the HIF-1α inhibitor PX-478 2HCl to target and block the HIF-1α / EPO axis, the recruitment of EDMCs can be precisely reduced in animal models, effectively reversing the immunosuppressive microenvironment and restoring anti-tumor immune function, thereby significantly inhibiting the growth and progression of residual tumors after IRFA (radioactive immunoassay). This drug circumvents the limitations of existing treatment methods, possessing the advantages of strong targeting, definite efficacy, and low side effects, providing a novel precision treatment option for recurrent liver cancer after RFA, and is expected to significantly improve the long-term survival rate of patients. Attached Figure Description

[0029] Figure 1 The graph shows the results of EPO level measurements in different cell models; among them, Figure 1 In the figure, A represents the statistical results of EPO protein levels in the culture supernatant of Hepa1-6 cells in the control group and the IRFA simulation group; Figure 1 The figure B in the figure represents the statistical results of EPO mRNA expression in Hepa1-6 cells under different conditions.

[0030] Figure 2 The figure shows the results of HIF-1α expression level measurement in Hepa1-6 cells from different model groups; among them, Figure 2 In the figure, A represents the statistical results of HIF-1α mRNA expression levels in Hepa1-6 cells under different conditions; Figure 2 In the diagram, B represents the Western blot results of Hepa1-6 cells under different conditions; Figure 2 The figure shows the statistical results of the relative intensity of HIF-1α / β-ACTIN in Hepa1-6 cells under different conditions, represented by C.

[0031] Figure 3 The figure shows the results of EPO level measurement in different Hepa1-6 cell HIF-1α knockdown groups; among them, Figure 3 In the figure, A represents the statistical results of EPO mRNA levels in different treatment groups; Figure 3 In the figure, B represents the statistical results of EPO protein secretion in cells of different treatment groups.

[0032] Figure 4 Representative flow cytometry images of MDSCs and EDMCs in tumor tissues of the control group and IRFA group.

[0033] Figure 5 Quantitative statistical graphs of MDSCs and EDMCs in tumor tissues of the control group and IRFA group are shown; among them, Figure 5 The A in the text represents CD45. + Gr1 in cells + CD11b +The statistical results of the percentage (%) are shown in the graph; Figure 5 B in the text represents Gr1 + CD11b + Ter119 in cells + Statistical results of cell percentage (%).

[0034] Figure 6 Gr-1 in tumor tissues of the control group and IRFA group + Ter119 + Representative multiplex immunofluorescence images of cells.

[0035] Figure 7 Gr-1 in tumor tissues of the control group and IRFA group + Ter119 + Statistical graph of multiplex immunofluorescence in cells.

[0036] Figure 8 This indicates the levels of PD-L1 and CD8 in tumor tissues of the control group and the IRFA group. + Representative immunohistochemical (IHC) images of T cells.

[0037] Figure 9 PD-L1 and CD8 in tumor tissues of different treatment groups + A quantitative statistical graph of T cells; among which... Figure 9 In the figure, A represents the quantitative statistical graph of PD-L1 in tumor tissues of the control group and the IRFA group; Figure 9 In this context, B represents the CD8 concentration in tumor tissues of the control group and the IRFA group. + Quantitative statistical graph of T cells.

[0038] Figure 10 The figure shows the results of EPO expression level measurement in tumor tissues of the control group and IRFA group in a mouse liver cancer model; among them, Figure 10 In the figure, A represents the statistical results of EPO protein secretion in the serum of the control group and the IRFA group in a mouse liver cancer model. Figure 10 In the figure, B represents the statistical results of EPO mRNA levels in the control group and IRFA group in the tumor tissue of the mouse liver cancer model. Figure 10 In the image, C represents a representative immunohistochemical (IHC) image of EPO expression in tumor tissues of the control group and IRFA group in a mouse liver cancer model.

[0039] Figure 11 This is a statistical graph showing the quantitative analysis of EPO expression in tumor tissues of the control group and IRFA group in a mouse liver cancer model.

[0040] Figure 12 The figure shows the results of HIF-1α expression level measurement in tumor tissues of the control group and IRFA group in a mouse liver cancer model; among them, Figure 12 In the figure, A represents the statistical results of HIF-1α mRNA expression in tumor tissues of the control group and the IRFA group; Figure 12 In the image, B represents a representative immunohistochemical (IHC) image of HIF-1α expression in tumor tissues of the control group and the IRFA group; Figure 12 In the figure, C represents the quantitative statistical graph of HIF-1α expression in tumor tissues of the control group and the IRFA group.

[0041] Figure 13 This image shows the residual tumor growth in the control group and the EPO-NA group after treatment intervention with EPO neutralizing antibody (EPO-NA); where, Figure 13 In this context, A represents a representative gross image of residual tumor in the control group and the EPO-NA group after IRFA; Figure 13 In this context, B represents the tumor growth curves of different treatment groups; Figure 13 In the figure, C represents the final tumor weight statistics for different treatment groups.

[0042] Figure 14 This is a graph showing the detection results of immune infiltrating cell subsets in tumor tissue after EPO neutralizing antibody therapy; among them, Figure 14 In the diagram, A represents a representative flow cytometry image of MDSCs and EDMCs in the control group and the EPO-NA group; Figure 14 In this context, B represents CD45 in the control group and the EPO-NA group. + Gr1 in cells + CD11b + A quantitative statistical graph showing the percentage of cells (%); C in 14 represents Gr1 in the control group and the EPO-NA group. + CD11b + Ter119 in cells + CD71 + Quantitative statistical chart of the percentage of cells (%).

[0043] Figure 15 To measure PD-L1 expression and CD8 expression in tumor tissue after EPO neutralizing antibody therapy. + The results of the T cell infiltration detection are shown in the figure; among them, Figure 15 In this context, A represents the levels of PD-L1 and CD8 in tumor tissues from the control group and the EPO-NA group. + Representative immunohistochemical (IHC) images of T cells; Figure 15 In the figure, B represents the quantitative analysis statistics of PD-L1 in tumor tissues of the control group and the EPO-NA group; Figure 15 In this context, C represents CD8+ in tumor tissues from the control group and the EPO-NA group. + Statistical graph of quantitative analysis of T cell expression.

[0044] Figure 16 This is a graph showing the effect of PX-478 2HCl treatment on residual tumor growth; among them, Figure 16 In this context, A represents a representative gross image of residual tumor in the control group and the PX-478 2HCl group after IRFA; Figure 16 In the figure, B represents the tumor growth curves of the control group and the PX-478 2HCl group; Figure 16 In the figure, C represents the final tumor weight statistics of the control group and the PX-478 2HCl group.

[0045] Figure 17 This image shows the detection results of immune infiltrating cell subsets in tumor tissue after PX-478 2HCl treatment intervention; among them, Figure 17 In the image, A represents a representative flow cytometry image of the infiltration ratio of MDSCs and EDMCs in residual cancer tissues of mice in the control group and PX-478 2HCl group. Figure 17 In this context, B represents CD45 in the residual cancer tissue of mice in the control group and the PX-478 2HCl group. + Gr1 in cells + CD11b + A graph showing the quantitative statistical results of the percentage of cells (%); Figure 17 In this context, C represents Gr1 in the residual cancer tissue of mice in the control group and the PX-4782HCl group. + CD11b + Ter119 in cells + CD71 + A graph showing the quantitative statistical results of the percentage of cells (%).

[0046] Figure 18 Following PX-478 2HCl treatment intervention, the expression of PD-L1 and CD8 in tumor tissue was measured. + The results of the T cell infiltration detection are shown in the figure; among them, Figure 18 In this context, A represents the levels of PD-L1 and CD8 in residual cancer tissues of mice in the control group and the PX-478 2HCl group. + Representative immunohistochemical (IHC) images of T cells; Figure 18 In the figure, B represents the statistical graph of quantitative analysis of PD-L1 in residual cancer tissue of mice in the PX-478 2HCl group; Figure 18 The C in the figure represents CD8 in the residual cancer tissue of mice in the PX-478 2HCl group. + Statistical graph of quantitative analysis of T cell expression.

[0047] Figure 19 EDMC score and PD-L1 or depleted CD8 in residual tumors after IRFA in liver cancer patients + The results of Pearson correlation analysis of T-cell scores are shown in the figure; Figure 19In this context, A represents the correlation analysis between the EDMC score and the PD-L1 score; Figure 19 In this context, B represents the EDMC score and exhaustion CD8. + Correlation analysis of T cell scores.

[0048] Figure 20 This is a graph showing the results of Pearson correlation analysis between EDMC scores and EPO or HIF-1α scores in residual tumor cells after IRFA in liver cancer patients; among them, Figure 20 In this context, A represents the correlation analysis between the EDMC score and the EPO score; Figure 20 In the figure, B represents the correlation analysis between the EDMC score and the HIF-1α score.

[0049] Figure 21 Kaplan-Meier survival analysis of HCC patients with high and low EPO expression. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0051] The following description is based on specific embodiments.

[0052] Example 1: Effects of substances that block the HIF-1α / EPO axis on liver cancer cells I. Experimental Materials The Hepa1-6 mouse liver cancer cell line was obtained from the cell bank of the Shanghai Institute of Life Sciences and was stored in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2.

[0053] II. Cell Model Construction and Grouping Hepa1-6 liver cancer cells were divided into 5×10 4 Cells were seeded per well in 6-well plates and cultured in a standard incubator (37°C, 5% CO2). Once the cell confluence reached approximately 70%, subsequent grouping was performed.

[0054] (a) Model building grouping (used to observe the effect of IRFA-related conditions on the HIF-1α / EPO axis) (1) Control group (Ctrl): Cells were not subjected to heat damage and hypoxia treatment, and were placed in a conventional incubator (37℃, 5% CO2) and cultured synchronously with other groups until the corresponding time point when the cells were collected for use.

[0055] (2) Heat treatment group: Wash cells twice with 3 times the volume of sterile PBS, seal 6-well plates with plastic wrap, and immerse them in a 45°C water bath for 10 min to complete the sublethal heat injury treatment, simulating the sublethal heat stress of incomplete radiofrequency ablation (IRFA); remove the plastic wrap after removal, and quickly transfer them to a conventional incubator (37°C, 5% CO2) for 24 h of culture without hypoxia treatment, and keep them ready for use.

[0056] (3) Hypoxia group: No heat damage treatment was performed. The 6-well plate was directly transferred to a hypoxic incubator (1% O2, 5% CO2, 37℃) to simulate the hypoxic microenvironment and cultured for 24 h for later use.

[0057] (4) Simulate IRFA conditions: When the cells reach about 70%, wash twice with 3 times PBS, seal with plastic wrap, and soak in a 45℃ water bath for 10 min for heat treatment; after heat treatment, remove the plastic wrap and immediately transfer the plate to a hypoxic incubator (1% O2, 5% CO2, 37℃) for heat damage and hypoxia combined treatment for 24 h to obtain cells under simulated IRFA conditions for later use.

[0058] (ii) HIF-1α knockdown validation group (used to verify the regulatory effect of HIF-1α on EPO) Based on the simulated IRFA condition processing, the following groupings are set: (1) Knockout blank control group (Ctrl): Hepa1-6 liver cancer cells were cultured routinely for 24 hours, and then treated according to the above simulated IRFA conditions. Cells were collected after 24 hours for later use.

[0059] (2) Knockout negative control group (IRFA + si-NC, NC): Hepa1-6 liver cancer cells were transfected with NC siRNA (knockout sequence as shown in Table 1) in serum-free Opti-MEM medium using Lipofectamine 3000 transfection reagent to induce negative siRNA knockdown in liver cancer cells. After transfection, the cells were incubated at 37°C for 8 h, and then the medium was replaced with high-glucose DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin DMEM) for 24 h. The cells were then treated under the simulated IRFA conditions described above, and the cells were collected after 24 h for later use.

[0060] (3) HIF-1α knockdown group (si-HIF-1α): The Hepa1-6 liver cancer cells cultured in the above conventional culture were transfected with HIF-1α siRNA (the knockout sequence is shown in Table 1 si-HIF-1α) in Opti-MEM serum-free medium using Lipofectamine 3000 transfection reagent to knock down HIF-1α siRNA in liver cancer cells. After transfection, the cells were incubated at 37℃ for 8 h, and then cultured in high glucose DMEM complete medium for another 24 h. The cells were then treated under the above simulated IRFA conditions. Cells were collected after 24 h for subsequent molecular level detection and analysis.

[0061] Table 1 si-RNA transfection sequences III. Detection Methods 1. Molecular level detection (1) RNA extraction and qRT-PCR 1) Total RNA isolation: Total RNA was extracted from cells / tissues using an RNA extraction kit; 2) cDNA synthesis: Reverse transcription was performed using the Color Reverse Transcription Kit; 3) qRT-PCR reaction: The reaction system was prepared using 2×Color SYBR Green qPCR Master Mix, and the real-time quantitative PCR detection was completed. The PCR mouse primer sequences are shown in Table 2.

[0062] Table 2. Mouse primer sequences for real-time quantitative PCR 2. Western Blot (1) Protein extraction and separation: Total cell protein was extracted and separated by SDS-PAGE gel electrophoresis; (2) Transfer and antibody incubation: The proteins separated by electrophoresis were transferred to a PVDF membrane, incubated with primary antibody at 4°C overnight, and then incubated with HRP-conjugated secondary antibody at room temperature for 90 min; (3) Color development and imaging: ECL chemiluminescent substrate was used for color development, and protein band images were acquired using the Syngen G:BOX Chemi XT4 imaging system.

[0063] 3. EPO level determination (ELISA method) (1) Cell sample preparation: Collect cell culture supernatant, centrifuge at 1000×g and 4℃ for 10 min to remove cell debris, take the supernatant and store at -80℃ for later use; (2) Detection: Strictly follow the instructions of the EPO ELISA kit to measure the EPO level in serum.

[0064] 4. Data Processing All experiments were independently repeated three times. Experimental data are expressed as mean ± standard error (mean ± SEM), with a sample size of n=3 for each group. GraphPad Prism software was used for statistical analysis. For comparisons between groups, independent samples t-tests (for two groups) or one-way ANOVA (for multiple groups) were used depending on the experimental design. Pearson correlation analysis was used as needed for correlation analysis. The statistical significance criteria were as follows: p<0.05 (the difference was statistically significant); p<0.01 (the difference is statistically significant); p < 0.001 (the difference was statistically significant). Experimental data were used to generate relevant charts (such as bar charts, line charts, and protein band quantification graphs) using GraphPad Prism software to ensure clear charts and intuitive data.

[0065] IV. Experimental Results (I) Changes in the expression of EPO and HIF-1α under simulated IRFA conditions like Figure 1 As shown in Figure A, Hepa1-6 cells treated with simulated incomplete ablation showed a significantly increased EPO secretion level compared to the control group, approximately 2.2 times higher (107.6 vs 235.9, P < 0.0001). Figure 1 As shown in A), IRFA can significantly promote the secretion of EPO by liver cancer cells and tumor tissues.

[0066] like Figure 1 As shown in Figure B, compared with the control group, there was no statistically significant difference in the expression level of EPO mRNA in Hepa1-6 cells in the heat treatment group alone; however, the expression levels of EPO in the hypoxia treatment group alone and the heat treatment + hypoxia treatment group were significantly upregulated (expression increased by 14.71±3.490 in the hypoxia treatment group, P<0.05; expression increased by 16.05±6.118 in the heat treatment + hypoxia treatment group, P<0.05). This indicates that in IRFA, the hypoxic microenvironment plays a major inducing role in EPO secretion by Hepa1-6 cells.

[0067] like Figure 2 As shown in Figures A through C, there were no significant differences in HIF-1α mRNA (A) and protein expression (BC) in Hepa1-6 cells in the heat-treated group alone, indicating that heat treatment itself has no direct effect on HIF-1α expression; while the HIF-1α expression level was significantly upregulated in both the hypoxia-treated group alone and the heat-treated + hypoxia-treated group.

[0068] (II) Effect of HIF-1α knockdown on EPO expression like Figure 3 As shown in Figures A and B, knocking out HIF-1α significantly reduced the level of EPO secretion in Hepa1-6 cells. This indicates that HIF-1α is a key transcription factor that induces EPO upregulation in the hypoxic microenvironment following IRFA, and blocking the HIF-1α / EPO axis can significantly inhibit the abnormal secretion of EPO by liver cancer cells.

[0069] In summary, in simulated IRFA-residual hepatocellular carcinoma cells, the hypoxic microenvironment induces high EPO expression by upregulating HIF-1α; knocking down HIF-1α can effectively block this signaling axis and reduce EPO levels. The HIF-1α / EPO axis is an important adaptive regulatory pathway in IRFA-residual hepatocellular carcinoma cells and has potential interventional value.

[0070] Example 2: Effects of substances blocking the HIF-1α / EPO axis on an animal model of incomplete radiofrequency ablation I. Experimental Materials 1. Laboratory animals Healthy male C57BL / 6 mice (3–5 weeks old, 15–18 g) were purchased from the Laboratory Animal Center of Sun Yat-sen University. All in vivo experiments were conducted in accordance with guidelines approved by the International Association for the Protection and Use of Animals (IACUC) of Sun Yat-sen University.

[0071] 2. Medications EPO neutralizing antibody was purchased from R&D Systems (manufacturer); catalog number: AF959.

[0072] PX-478 2HCl was purchased from Selleck Chemicals (manufacturer), product number S7612, CAS number 685898-44-6.

[0073] II. Model Construction and Grouping 1. Animal model level experiments Animal experiments were conducted in accordance with the protocol approved by the Committee on the Management and Use of Laboratory Animals of Sun Yat-sen University (ethics number: SYSU-IACUC-2026-000167).

[0074] (1) Establishment of a subcutaneous hepatocellular carcinoma tumor model: Hepa1-6 cells (2 million) were mixed with Matrigel at a volume ratio of 1:1 and injected into the right subcutaneous tissue of C57BL / 6 mice. The tumor volume was measured with calipers every 2 days to monitor tumor growth.

[0075] (2) Construction of incomplete radiofrequency ablation (IRFA) model: When the subcutaneous hepatocellular carcinoma tumor in the subcutaneous hepatocellular carcinoma model reaches 300 mm 3 At approximately 10:00 pm, IRFA was performed using a bipolar RFA device (3W, 30 s), while the electrode tip temperature was kept ≤50℃ by a cooling system to complete the in vivo IRFA model construction.

[0076] 2. Grouping and administration 1. EPO neutralizing antibody therapy intervention IRFA model tumor-bearing mice were randomly divided into a control group and an EPO-NA group (n=5 in each group); the treatment regimen was to inject 2 μg of EPO neutralizing antibody into each tumor once every 5 days for a total of 3 doses; the control group was given an equal amount of solvent (PBS) as a control.

[0077] 2. PX-478 2HCl treatment intervention IRFA model tumor-bearing mice were randomly divided into a control group and a PX-478 2HCl drug group (n=5 in each group). The drug was administered via intraperitoneal injection. The control group was injected with saline once every 2 days, and the drug group was injected with PX-478 2HCl (120 mg / kg) once every 2 days for a total of 5 times. The control group was injected with an equal volume of saline intraperitoneally.

[0078] After drug administration, the tumor weight of mice in each group was measured, and the immune infiltration and histopathological characteristics of the tumor tissue in each group were analyzed.

[0079] III. Detection Methods 1. Tumor volume and weight detection Measure the long and short diameters of the tumor using vernier calipers, and calculate the tumor volume using the formula: Tumor volume (mm) 3 = Major axis × Minor axis 2 / 2. At the end of the experiment, the mice were sacrificed, the tumors were removed and weighed, and the tumor weight was recorded.

[0080] 2. Flow cytometry detection Mouse tumor tissue was collected and digested in a collagenase / protease mixture at 37°C for 1 h. After digestion, the tissue was filtered through a filter to prepare a single-cell suspension. The single-cell suspension was fluorescently labeled with CD11b, Gr-1, Ter119, and CD71 antibodies. The cells were then detected using a Beckman CytoFLEX flow cytometer, and the proportions of immune cell subsets such as MDSCs and EDMCs were analyzed using CytExpert software.

[0081] 3. Immunofluorescence (IF) staining detection (1) Pretreatment of paraffin sections: Bake at 60℃ for 2 h, and then perform dewaxing and hydration operations in sequence; (2) Antigen retrieval: High-pressure retrieval was performed using sodium citrate buffer for 15 min; (3) Antibody incubation and staining: Add Gr-1 primary antibody and incubate overnight at 4℃ → incubate with goat anti-rabbit secondary antibody for 30 min → TSA staining; then add Ter119 primary antibody and incubate overnight at 4℃ → secondary antibody incubation + TSA staining; (4) Counterstaining and imaging: Nuclear staining was performed using DAPI, and images were acquired using an Olympus FV3000 confocal microscope.

[0082] 4. Immunohistochemical (IHC) staining detection (1) Section pretreatment: After dewaxing, antigen retrieval was performed using sodium citrate buffer; (2) Antibody incubation: Primary antibody was incubated overnight at 4°C, and goat anti-rabbit secondary antibody was incubated at room temperature (RT) for 30 min; (3) Staining and imaging: DAB staining kit was used for staining, hematoxylin counterstaining, and images were acquired using a Nikon vertical microscope.

[0083] 5. qRT-PCR detection Total RNA was extracted from tumor tissue using an RNA extraction kit, and cDNA was synthesized using a reverse transcription kit. A reaction system was prepared using 2×Color SYBR Green qPCR Master Mix, and qRT-PCR was performed to analyze the mRNA expression levels of HIF-1α and EPO. Primer sequences are shown in Table 2 of Example 1.

[0084] 6. Western Blot Detection Total protein was extracted from tumor tissue, separated by SDS-PAGE gel electrophoresis, and transferred to a PVDF membrane. The protein was incubated with primary antibody overnight at 4°C, and then incubated with HRP-labeled secondary antibody at room temperature for 90 min. The protein bands were acquired by ECL chemiluminescence and the expression level of HIF-1α protein was detected by Syngen G:BOX Chemi XT4 imaging system.

[0085] 7. ELISA testing Mouse blood was collected, and serum was separated by centrifugation at 1000×g and 4℃ for 30 min and stored at -80℃. The EPO protein level in the serum was detected strictly in accordance with the EPO ELISA kit instructions.

[0086] IV. Measurement Results 1. The impact of incomplete radiofrequency ablation (IRFA) on residual tumor and related indicators (1) Effects on the tumor immune microenvironment: Compared with the control group, the infiltration ratio of MDSCs and EDMCs in the residual tumor tissue after IRFA was significantly increased ( Figure 4 and Figure 5 (A and B in the middle); Gr-1 + Ter119 + The number of infiltrating double-positive cells is significantly increased, and this difference can be clearly identified by multiplex immunofluorescence staining and quantitative statistics (e.g. Figure 6 and Figure 7 (As shown); simultaneously, PD-L1 expression levels were significantly increased in residual cancer tissue, CD8... + The number of infiltrating T cells was significantly reduced, which can be corroborated by immunohistochemical (IHC) staining and quantitative analysis (e.g. Figure 8 and Figure 9 As shown in A and B in the figure, this indicates that IRFA can induce residual cancer to form an immunosuppressive microenvironment.

[0087] (2) Effects on EPO expression and secretion: Compared with the control group, the level of circulating EPO protein in the serum of tumor-bearing mice after IRFA surgery was significantly increased (478.1 vs 935.5, P<0.01). Figure 10 (As shown in A in the figure); the mRNA expression level of EPO in the residual tumor tissue was significantly increased, increasing by 7.688±2.251 compared with the control group (P<0.05) (as shown in A in the figure). Figure 10 (As shown in B in the figure); EPO protein expression was also significantly increased in residual tumor tissue, with postoperative EPO protein levels increasing 6.4 times compared to the control group (1.803 vs. 11.60, P<0.001) (as shown in B in the figure). Figure 10 C in Figure 11 (As shown).

[0088] (3) Effect on HIF-1α expression: Compared with the control group, the mRNA expression level and protein level of HIF-1α in the residual tumor tissue after IRFA surgery were significantly increased, as shown by qRT-PCR detection results (e.g. Figure 12 (as shown in A), IHC staining images and quantitative statistics (such as...) Figure 12 B in Figure 12 The C) in the study clearly demonstrates this regulatory effect, indicating that IRFA can induce the upregulation of HIF-1α in residual tumor tissue.

[0089] In summary, IRFA can promote the recruitment of immunosuppressive cells such as EDMCs and MDSCs by upregulating the HIF-1α / EPO axis, increasing PD-L1 expression, and reducing CD8+ expression. + T cell infiltration was used to construct a residual cancer immunosuppressive microenvironment, which laid the foundation for two subsequent intervention experiments that blocked the HIF-1α / EPO axis.

[0090] 2. Results of EPO neutralizing antibody therapy intervention (1) Effect on residual tumor growth: Compared with the control group, residual tumor growth was significantly inhibited after EPO neutralizing antibody treatment (e.g. Figure 13 As shown in A), the tumor volume showed a significant decreasing trend (e.g., Figure 13 As shown in B), the final tumor weight was significantly reduced (e.g., Figure 13 As shown in Figure C), it intuitively demonstrates that EPO neutralizing antibodies can effectively inhibit the progression of residual tumors after IRFA.

[0091] (2) Effects on tumor tissue immune infiltrating cell subsets: Compared with the control group, the infiltration ratios of MDSCs and EDMCs in residual tumor tissue of the EPO-NA group were significantly reduced. Flow cytometry results and quantitative statistics clearly showed this difference (e.g. Figure 14 As shown in AC), this indicates that the EPO neutralizing antibody can inhibit IRFA-induced immunosuppression-related cell recruitment.

[0092] (3) Effects on PD-L1 expression and CD8 expression in tumor tissue + Effects on T cell infiltration: Compared with the control group, the expression level of PD-L1 in residual cancer tissue of the EPO-NA group was significantly decreased, while CD8 expression was significantly decreased. + The number of infiltrating T cells increased significantly, and immunohistochemical (IHC) staining images and quantitative statistical results corroborated this regulatory role (e.g. Figure 15 As shown in AC in the figure, this indicates that EPO neutralizing antibodies can reverse the IRFA-induced tumor immunosuppressive microenvironment.

[0093] 3. Results of PX-478 2HCl treatment intervention (1) Effect on residual tumor growth: Compared with the control group, the growth of residual tumor in the PX-478 2HCl treatment group was significantly inhibited (e.g., Figure 16 As shown in A), the dynamic growth of tumor volume was significantly inhibited (e.g., Figure 16 As shown in B), the final tumor volume and weight were significantly lower than those of the control group, and the gross morphology and quantitative statistical results of the residual tumor clearly demonstrated the inhibitory effect (e.g., Figure 16 (as shown in C).

[0094] (2) Effects on tumor tissue immune infiltrating cell subsets: Compared with the control group, the infiltration ratios of MDSCs and EDMCs in the residual cancer tissue of mice in the PX-478 2HCl group were significantly reduced. Representative images and quantitative statistical results detected by flow cytometry can verify this conclusion (e.g. Figure 17 As shown in AC), this suggests that targeting HIF-1α can inhibit IRFA-induced recruitment of EDMCs and MDSCs.

[0095] (3) Effects on PD-L1 expression and CD8 expression in tumor tissue +Effects on T cell infiltration: Compared with the control group, the expression level of PD-L1 in the residual tumor tissue of the PX-478 2HCl group was significantly decreased, while the expression level of CD8 was significantly decreased. + The level of T cell infiltration was significantly increased, and this difference was clearly shown in immunohistochemical (IHC) staining images and quantitative analysis results (e.g. Figure 18 As shown in AC), this indicates that PX-4782HCl can improve the tumor immunosuppressive microenvironment after IRFA and enhance the anti-tumor immune response.

[0096] Example 3: Correlation detection of EDMC recruitment, EPO expression, and immunosuppression and prognosis in hepatocellular carcinoma patients. I. Clinically relevant materials and research approval 1. Clinical genomic mapping data (1) Data download: Download liver cancer-related RNA-seq expression data from the TCGA-LIHC (liver cancer) cohort of The Cancer Genome Atlas (TCGA); at the same time, use the GEPIA (Gene Expression Profiling Interactive Analysis) database to obtain survival data related to EPO gene expression in liver cancer patients.

[0097] (2) Data processing and analysis: Based on the published gene sets, the gene abundances of EPO, HIF-1α, and PD-L1 were extracted from each case in the TCGA liver cancer dataset; Single sample gene set enrichment analysis (ssGSEA) was used to calculate the abundances of EDMC (erythroid-derived myeloid cells) and depleted CD8+ in each case in the TCGA liver cancer dataset. + T cell score; Pearson correlation coefficient was used to estimate the correlation between EDMC score and PD-L1, exhausted CD8. + Correlation between T cell scores, EPO, and HIF-1α.

[0098] 2. Research Approval All studies involving human clinical samples and clinical data in this embodiment have been approved by the Ethics Committee of Sun Yat-sen Memorial Hospital, Sun Yat-sen University (Ethics No.: SYSKY-2024-891-01), and all experimental procedures and data usage comply with ethical guidelines and relevant laws and regulations.

[0099] II. Experimental Detection and Analysis Methods (I) Bioinformatics Analysis 1. TCGA Data Extraction: RNA-seq expression data of EPO, HIF-1α, and PD-L1 were extracted from the TCGA-LIHC (liver cancer) cohort. Samples with missing expression data or incomplete clinical information were removed, and 424 qualified samples were finally obtained for subsequent analysis. 2. ssGSEA scoring analysis: The ssGSEA algorithm was used to calculate EDMC (erythroid-derived myeloid cells) and exhausted CD8. + T cell enrichment score; 3. Pearson Correlation Analysis: Pearson correlation analysis was used to explore the relationship between EDMC and PD-L1 and depleted CD8+. + The expression correlations among T cells, EPO, and HIF-1α were investigated, and the Pearson correlation coefficient and P-value were calculated. P < 0.05 was defined as statistically significant.

[0100] 4. Survival Analysis: Using EPO gene expression data and clinical follow-up survival data of liver cancer patients obtained from the GEPIA database, survival curves for patients in the high EPO expression group and low EPO expression group were plotted using the Kaplan-Meier method. The Log-rank test was used to compare the difference in overall survival between the two groups, with P<0.05 set as statistical significance, to analyze the association between EPO gene expression and prognosis of liver cancer patients.

[0101] V. Research and Approval VI. Experimental Results 1. EDMC and PD-L1, depleted CD8 in liver cancer patients + Correlation of T cells: In hepatocellular carcinoma patients, the Pearson correlation coefficient between EDMC and PD-L1 was 0.607 (P<0.001), and EDMC was associated with depleted CD8+. + The Pearson correlation coefficient for T cells was 0.819 (P<0.001). These results indicate that the recruitment of EDMCs in HCC is related to PD-L1 expression and CD8+ expression. + There is a significant positive correlation between T cell exhaustion and (e.g.) Figure 19 (As shown in A and B in the diagram).

[0102] 2. Correlation between EDMC and EPO / HIF-1α in HCC patients: The Pearson correlation coefficient between EDMC and EPO in HCC patients was 0.266 (P<0.001), and the Pearson correlation coefficient between EDMC and depleted HIF-1α cells was 0.357 (P<0.001). These results indicate a significant positive correlation between EDMC recruitment and EPO / HIF-1α expression in HCC (e.g., ...). Figure 20 (As shown in A and B in the diagram).

[0103] 3. Correlation between EPO gene expression and survival in liver cancer patients: Liver cancer patients with high EPO gene expression in the GEPIA (Gene Expression Profiling Interactive Analysis) database had significantly lower overall survival (e.g., Figure 21 (As shown).

[0104] The above results demonstrate that the recruitment of EDMC in hepatocellular carcinoma tissues is related to PD-L1 expression and CD8+. + T cell exhaustion-related immunosuppressive signaling is closely associated with the formation of an immunosuppressive microenvironment, which can promote the progression of liver cancer. The hypoxic microenvironment in the tumor after ablation activates the HIF-1α / EPO axis, resulting in abnormally high EPO expression in the ablated liver cancer tissue, which is associated with poor prognosis in liver cancer patients. Simultaneously, the HIF-1α / EPO axis can participate in the formation of an immunosuppressive microenvironment after ablation by regulating the recruitment of endocrine disrupted tumor cells (EDMCs), thus promoting post-ablation tumor progression.

[0105] This invention, through three levels of validation—cellular (Example 1), animal (Example 2), and clinical (Example 3)—clearly demonstrates that IRFA can activate the HIF-1α / EPO axis in liver cancer cells and tumor tissues by inducing a hypoxic microenvironment, promoting EPO secretion, recruiting immunosuppressive cells such as EDMCs and MDSCs, increasing PD-L1 expression, and reducing CD8+ expression. + T cell infiltration participates in the formation of a tumor immunosuppressive microenvironment, thereby promoting liver cancer progression and recurrence of residual cancer after IRFA (Intra-Invasive Therapy for Cancer). Blocking the HIF-1α / EPO axis with substances such as EPO neutralizing antibodies and PX-478 2HCl can significantly inhibit EPO secretion from liver cancer cells, reduce the recruitment of immunosuppressive cells, reverse the tumor immunosuppressive microenvironment, inhibit liver cancer growth, and improve the prognosis of liver cancer patients. Therefore, substances that block the HIF-1α / EPO axis can be used to prepare liver cancer therapeutic drugs, especially suitable for the treatment of residual cancer after IRFA, showing promising application prospects and clinical value.

[0106] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0107] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of substances that block the HIF-1α / EPO axis in the preparation of drugs for treating tumor recurrence after radiofrequency ablation of liver cancer.

2. The application as described in claim 1, characterized in that, The drug treats tumor recurrence after radiofrequency ablation of liver cancer by blocking the HIF-1α / EPO axis to reduce EDMC recruitment and reverse the immunosuppressive microenvironment.

3. The application as described in claim 1, characterized in that, The substance that blocks the HIF-1α / EPO axis is selected from one or more of the following: small molecule inhibitors, neutralizing antibodies, soluble receptors, siRNA, shRNA, dsRNA, and antisense nucleic acids.

4. The application as described in claim 3, characterized in that, The substances that block the HIF-1α / EPO axis are selected from PX-4782HCl, Kamebakaurin, LW6, Acriflavine, topoisomerase I inhibitor Topotecan, HSP90 inhibitor 17-AAG, soluble EPOR, anti-EPO antibody, neutralizing antibody against EPOR, Aminoquinolines, EZN-2698, EZN-2208, SN38, Irinotecan, Temsirolimus, Everolimus, and Siro. One or more of the following: limus, LY294002, Wortmannin, 2ME2, ENMD-1198, ENMD-1200, ENMD-1237, Radicicol, KF85833, SCH66336, Apigenin, Hsp90GA, 17-DMAG, TAK-17, Echinomycin, Doxorubicin, Daunorubicin, Chetomin, Bortezomib, and siRNAs targeting HIF-1α; The siRNA targeting HIF-1α consists of the sense strand shown in SEQ ID NO: 1 and the antisense strand shown in SEQ ID NO:

2.

5. A drug for treating tumor recurrence after radiofrequency ablation of liver cancer, characterized in that, The active ingredient of the drug includes the substance that blocks the HIF-1α / EPO axis as described in claim 1 and other pharmaceutically acceptable excipients.

6. The drug as described in claim 5, characterized in that, The substance that blocks the HIF-1α / EPO axis is selected from one or more of the following: small molecule inhibitors, neutralizing antibodies, soluble receptors, siRNA, shRNA, dsRNA, and antisense nucleic acids.

7. The drug as described in claim 5, characterized in that, The content of the substance that blocks the HIF-1α / EPO axis in the drug is 1wt%~99wt%.

8. The drug as described in claim 5, characterized in that, The drug is in the form of tablets, granules, capsules, injections, or oral liquid preparations.

9. The drug as described in claim 5, characterized in that, The drug also includes a drug carrier.

10. The medicament as claimed in claim 9, characterized in that, The drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

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