Plasma markers for evaluating efficacy of liver cancer immunotherapy and application thereof

Abstract

This invention discloses a plasma biomarker for evaluating the efficacy of immunotherapy for liver cancer, wherein the plasma biomarker is N1-acetylated spermidine. This invention also discloses the applications of the plasma biomarker. This invention discloses that N1-acetylated spermidine can be used for liver cancer detection, treatment, prognostic molecular targets, and immunotherapy efficacy evaluation, which can more accurately predict the recurrence time and immunotherapy efficacy of liver cancer patients, providing a targeted efficacy evaluation scheme for immunotherapy. Furthermore, the use of liquid chromatography combined with mass spectrometry to detect plasma N1-acetylated spermidine has the advantages of low detection volume, high efficiency, and high precision.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a plasma biomarker for evaluating the efficacy of immunotherapy for liver cancer and its application. Background Technology

[0002] Metabolic reprogramming is a hallmark feature of malignant tumors, with the selective utilization of aerobic glycolysis for cellular energy, supporting rapid tumor growth and proliferation, being particularly noteworthy. However, in addition to uncontrolled proliferation, most malignant tumors also exhibit other hallmark features such as invasion, metastasis, angiogenesis, immune evasion, and treatment resistance. Notably, the metabolic characteristics and preferences of malignant tumors change during disease progression. To date, how metabolic reprogramming supports the emergence of different cancer hallmarks remains unclear. Therefore, exploring the relationship between metabolic reprogramming and cancer hallmarks is crucial for developing anti-tumor strategies targeting metabolic reprogramming.

[0003] Polyamines are low-molecular-weight aliphatic polyvalent cations with high electrical charges. Natural polyamines such as putrescine, spermidine, and spermine participate in maintaining normal cellular function and replication. Therefore, in human cancers, excessive increases in polyamine levels, usually driven by MYC amplification or KRAS mutations, lead to uncontrolled cell proliferation. However, it is worth emphasizing that after N1-acetylation, intracellular polyamines can be effluxed into the extracellular environment via the SLC3A2 transporter. Although this is not directly related to the cancer immune microenvironment, recent studies have shown that polyamine metabolism inhibits the effector function of myeloid cells. This result suggests that tumor cells have a pathogenic effect through polyamine efflux. Currently, direct evidence supporting the role of polyamine efflux in the immunopathology of human cancer remains lacking. Furthermore, whether polyamine efflux modulates the cancer immune spectrum and thus affects the efficacy of cancer immunotherapy is an important question that urgently needs to be addressed.

[0004] Currently, liver cancer is the sixth leading cause of cancer death and the third leading cause of death worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of all liver cancer types, typically occurring in livers with inflammatory fibrosis and / or cirrhosis, accompanied by extensive leukocyte infiltration. Therefore, the immune microenvironment is a crucial factor determining tumor progression and treatment efficacy. Surgical treatment, represented by liver resection, remains the preferred treatment for liver cancer. However, the recurrence rate within 5 years after liver resection is as high as 60%-70%, and the mortality rate within 5 years is as high as 30%-40%. Furthermore, many patients with advanced-stage liver cancer are not suitable for liver resection due to factors such as extensive tumor metastasis, severe liver function impairment, serious comorbidities, and diffuse hepatocellular carcinoma. Therefore, immunotherapy or immunotherapy combined with radiotherapy and chemotherapy has become an important treatment for advanced liver cancer. However, the response rate of liver cancer patients to these treatments is less than 20%, and there is a lack of effective assessment methods to predict the efficacy of these immunotherapies and combined treatments.

[0005] Immunotherapy improves treatment efficacy by activating or enhancing the body's immune response to tumors, thereby altering the tumor microenvironment. However, the immune microenvironment of liver cancer, including regulatory T cells, tumor-associated macrophages, and immunosuppressive factors such as transforming growth factor β and interleukin-10, can not only reduce the overall efficacy of treatment by inducing immune escape but also participate in the malignant transformation of tumors, promoting hyperprogression. Therefore, it is urgent to discover effective biomarkers to predict and evaluate the efficacy of immunotherapy in liver cancer patients, develop more effective combined immunotherapy regimens, and alleviate the economic burden on patients.

[0006] Currently, the prediction of liver cancer progression and the efficacy of immunotherapy mainly relies on non-invasive detection using enzyme-linked immunosorbent assay (ELISA) by collecting peripheral blood serum. However, existing kits for this detection method cannot accurately distinguish between spermidine and N1-acetylated spermidine, and quantitative deviations are possible. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned technical problems by providing a technical solution for effectively predicting the progression of liver cancer and the efficacy of immunotherapy.

[0008] To achieve the above-mentioned objectives, the present invention provides a plasma biomarker for evaluating the efficacy of immunotherapy for liver cancer, wherein the plasma biomarker is N1-acetylated spermidine (N1-Ac-Spd).

[0009] On the other hand, the present invention also provides the application of the plasma biomarkers in predicting and evaluating the efficacy of immunotherapy for liver cancer.

[0010] On the other hand, the present invention also provides the application of the plasma biomarker in the preparation of products for predicting and evaluating the efficacy of immunotherapy for liver cancer.

[0011] On the other hand, the present invention also provides the application of the plasma biomarker in the preparation of products for predicting and assessing the progression of liver cancer.

[0012] On the other hand, the present invention also provides the use of the plasma biomarker in the preparation of a medicament for promoting the efficacy of immunotherapy for liver cancer.

[0013] On the other hand, the present invention also provides the application of reagents for detecting said plasma biomarkers in the preparation of products for predicting and evaluating the efficacy of immunotherapy for liver cancer.

[0014] On the other hand, the present invention also provides the application of reagents for detecting the plasma biomarkers in the preparation of products for predicting and assessing the progression of liver cancer.

[0015] On the other hand, the present invention also provides the use of reagents that block the synthesis and efflux of N1-acetylated spermidine in liver cancer in the preparation of drugs for promoting the efficacy of immunotherapy for liver cancer.

[0016] Preferably, the liver cancer is hepatocellular carcinoma.

[0017] Preferably, the detection is a combination of liquid chromatography and mass spectrometry.

[0018] This invention discloses the application of macrophage-mediated efflux of N1-acetylated spermidine (N1-Ac-Spd) in liver cancer cells as a molecular target for liver cancer detection, treatment, prognosis, and evaluation of immunotherapy efficacy. This invention finds that macrophage-mediated efflux of N1-acetylated spermidine (N1-Ac-Spd) in liver cancer cells is closely negatively correlated with the recurrence time and immunotherapy efficacy in liver cancer patients.

[0019] The plasma biomarkers of this invention can serve as important molecular markers for predicting liver cancer progression and the efficacy of immunotherapy. These biomarkers can be detected at both the tissue and plasma levels in patients, reflecting the tissue immunological characteristics of liver cancer patients. They can more accurately predict the recurrence time and the efficacy of immunotherapy, better guiding the establishment of clinical treatment protocols. This invention provides a targeted efficacy evaluation scheme for immunotherapy in liver cancer patients. By detecting the N1-acetylated spermidine content in the plasma of liver cancer patients, the immune microenvironment status and treatment regimen of liver cancer patients can be assessed: immunotherapy is given to patients with low N1-acetylated spermidine levels in plasma; for liver cancer patients with high N1-Ac-Spd levels in plasma, CCR8-targeting drugs are used to reduce the number of regulatory T cells infiltrating the tumor, thereby improving the efficacy of immunotherapy.

[0020] This study demonstrates that polyamine metabolism is a major metabolic pathway enriched in HCC tumors. N1-acetylated spermidine (N1-Ac-Spd) efflux plays a pathogenic role in shaping the immune microenvironment and weakening the efficacy of immunotherapy. Mechanistically, under the stimulation of inflammatory macrophages, endogenously expressed SAT1 in liver cancer cells catalyzes N1-acetylation of spermidine, thereby triggering SRC signaling-mediated polarization in CCL1+ M2b macrophages. This process depends on charge changes and ultimately recruits CCR8+ regulatory T cells, thus reducing the efficacy of immunotherapy. By blocking this pathogenic-prone interaction in vivo, the anti-tumor effect of immunotherapy for liver cancer can be enhanced.

[0021] Furthermore, the N1-acetylated spermidine (N1-Ac-Spd) detection method developed in this invention mainly utilizes liquid chromatography combined with mass spectrometry. It can be performed by extracting a small amount of sample from routine sampling (such as blood routine tests), and can distinguish different types of polyamines, eliminating interference from putrescine, spermidine, spermine, N1-acetylated spermidine, etc., on the content of N1-acetylated spermidine, thus achieving the goal of high efficiency and high precision with small sample volume. Attached Figure Description

[0022] Figure 1 This paper presents a technical approach for predicting and assessing liver cancer progression and the efficacy of immunotherapy by detecting the plasma biomarker N1-acetylated spermidine.

[0023] Figure 2 The results of collection and pretreatment of polyamines in tumor tissues and plasma for analysis based on high performance liquid chromatography-mass spectrometry (HPLC-MS) are shown.

[0024] Figure 3 The study demonstrated that increased N1-Ac-Spd levels and efflux weakened the efficacy of immunotherapy in liver cancer.

[0025] Figure 4 This demonstrates the key upstream and downstream components of the SATI1 / N1-Ac-Spd axis formed by macrophages.

[0026] Figure 5 This demonstrates that N1-Ac-Spd polarizes CCL1+M2b macrophages by activating the SRC signaling pathway.

[0027] Figure 6 This study demonstrated treatment resistance induced by macrophage CCL1-dominant N1-Ac-Spd.

[0028] Figure 7 This study demonstrates how macrophage CCL1 shapes an immune landscape dominated by regulatory T cells. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Unless otherwise specified, the reagents and instruments used in the embodiments of this invention are well known to those skilled in the art, and are commercially available. For brevity, the specific steps of certain experimental operations are not described in detail herein, but these experimental operations are well known to those skilled in the art, and even if the present invention does not describe all its details, those skilled in the art can implement them based on their conventional technical knowledge.

[0031] Example 1: Extraction and analysis of polyamines in samples

[0032] Figure 1 This paper presents a technical approach for predicting and assessing liver cancer progression and the efficacy of immunotherapy by detecting the plasma biomarker N1-acetylated spermidine.

[0033] 1. General experimental procedures and steps

[0034] 1.1 Extraction of polyamines from samples

[0035] Biological tissue: 2 mg of biological tissue was ground and resuspended in radioimmunoprecipitation assay (RIPA) lysis buffer (1:20 volume dilution, containing 1% (w / v) protease inhibitor), and sonicated for 5 minutes to fully dissolve. Then, it was centrifuged at 12000×g for 10 minutes and the supernatant was collected.

[0036] Plasma: Blood samples were collected using EDTA or heparin as anticoagulants and centrifuged at 4°C, 1000×g for 15 minutes within 30 minutes of collection. The supernatant was then collected.

[0037] 1.2 Sample Preparation and Pre-column Derivatization

[0038] (1) Mix the supernatant (30 μL) with acetonitrile (120 μL) containing 3 pmol internal standard (1,6-diaminohexane) and vortex for 30 seconds;

[0039] (2) Centrifuge at 4℃ and 3000×g for 5 min, then discard the precipitate;

[0040] (3) Evaporate the supernatant using a rotary evaporator / freeze-dry it to obtain a precipitate;

[0041] (4) Dissolve the precipitate with 150 μL of 0.1 M sodium tetraborate (pH 9.3), sonicate it, and react it with an equal volume of acetonitrile solution containing 40 mM MBD-F (4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole) at 60 °C for 30 min.

[0042] (5) The reaction mixture was passed through a Millex-LG membrane (replaced with a Sartoris vivacon). R 500, 10000MWCO HY (hydrophilic filter membrane) filtration, membrane pore size 0.2μm, inner diameter 4mm;

[0043] (6) Analyze an equal volume (5 μL) of filtrate using HPLC-MS.

[0044] 1.3 Drawing the Standard Curve

[0045] Polyamines of fixed concentrations (2–2000 nM) were prepared from the stock solution through serial dilution. After derivatization pretreatment, the working solution (30 μL) and the derivatized solution (5 μL) were analyzed by HPLC-MS, and linear regression curves (n = 5) of polyamine peak area ratio (y) versus polyamine concentration (x, ng / mL) were plotted.

[0046] 1.4 Calculation of plasma polyamine concentration

[0047] The concentration of polyamines in the patient's plasma was calculated based on the standard curve.

[0048] 2. Collection and pretreatment of polyamines in tumor tissues and plasma for analysis based on high performance liquid chromatography-mass spectrometry (HPLC-MS)

[0049] Tumor samples obtained from HCC patients or non-tumor tissue (2 mg) obtained from healthy donors were ground and resuspended in radioimmunoprecipitation (RIPA) lysis buffer (1:20 volume dilution, containing 1% (w / v) protease inhibitor) and sonicated for 5 minutes to fully dissolve. The samples were then centrifuged at 12000×g for 10 minutes, and the supernatant was transferred to a new collection tube. Blood samples were collected using EDTA or heparin as anticoagulants and centrifuged at 1000×g for 15 minutes at 4°C within 30 minutes of collection. The supernatant, i.e., plasma, was collected and transferred to a new collection tube. 30 μL of the supernatant or human plasma was mixed with 120 μL of acetonitrile containing the internal standard 1,6-diaminohexane (DAH, 3 pmol), vortexed for 30 seconds, and then the solvent was removed by centrifugation. The dried metabolite extract was redissolved in 150 μL of 0.1 M sodium tetraborate (pH 9.3), followed by the addition of an equal volume of acetonitrile solution containing 40 mM DBD-F, and reacted at 60 °C for 30 min. The reaction mixture was then passed through a Millex-LG membrane (Sartoris vivacon). R The samples were filtered using 500 or 10000MWCO HY (hydrophilic filter membrane, 0.2μm pore size, 4mm inner diameter) and finally analyzed using high performance liquid chromatography-tandem mass spectrometry (HPLC–MS).

[0050] Polyamines in HCC samples and healthy donors were analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS), including 10 healthy donors, 88 treatment-naïve patients, and 13 patients treated with TAI plus αPD-1. The experimental procedure was as follows: Figure 2 As shown in Figure A. Mass spectrometric peak analysis of polyamines and internal standard (DAH) is shown in [Figure A]. Figure 2 B in the middle. Figure 2In this context, Put represents putrescine, DAH represents standard 1,6-diaminohexane, N1-Ac-Spd represents N1-acetylspermidine, Spd represents spermidine, N1-Ac-Spm represents N1-acetylspermine, and Spm represents spermine.

[0051] Table 1 below also shows the high-performance liquid chromatography-mass spectrometry (HPLC-MS) parameters of the polyamines in the samples.

[0052] Table 1. High-performance liquid chromatography-mass spectrometry (HPLC-MS) parameters of polyamines

[0053]

[0054] Example 2: The impact of increased N1-Ac-Spd and efflux in liver cancer on the efficacy of immunotherapy

[0055] Figure 3 The study demonstrated that increased N1-Ac-Spd levels and efflux weakened the efficacy of immunotherapy in liver cancer.

[0056] Single-sample gene set enrichment analysis (ssGSEA) was used to compare the major metabolic changes in 20 human hepatocellular carcinoma (HCC) tumors with paired non-tumor liver tissues. Figure 3 Figure A shows a heatmap displaying the single-sample gene set enrichment analysis scores (GSE77509) of 33 metabolic pathways from paired non-tumor liver and tumor tissues of 20 HCC patients. Among all active metabolic pathways, polyamine metabolism was the most significantly altered pathway, in addition to the classic pentose phosphate pathway.

[0057] Figure 3 In Part B, 321 HCC patients from the TCGA dataset were divided into two groups based on their median polyamine metabolism (ssGSEA) score. The correlation between polyamine metabolism score and 5-year recurrence was statistically analyzed. A high polyamine metabolism score predicted early recurrence in HCC patients.

[0058] Figure 3 In study C, polyamines, including N1-acetylated spermidine (N1-Ac-Spd), putrescine (Put), spermidine (Spd), spermine (Spm), and N1-acetylspermine (N1-Ac-Spm), were quantitatively analyzed in paired non-tumor liver and tumor tissues from 10 HCC patients using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Among all the physiological polyamines analyzed, N1-acetylated spermidine (N1-Ac-Spd), a polyamine capable of extracellular emission, was found to accumulate only in tumor tissues, with no significant accumulation observed in non-tumor liver tissues.

[0059] Figure 3 In this study, the levels of polyamines in the plasma of 10 healthy donors and 10 HCC patients were quantitatively analyzed by HPLC-MS. N1-Ac-Spd was found to be the major physiological polyamine in plasma, and its level was significantly increased in HCC patients.

[0060] Figure 3 In the study, the correlation between plasma N1-Ac-Spd levels and N1-Ac-Spd content in paired tumor tissues from 10 HCC patients was analyzed. A positive correlation was found between plasma N1-Ac-Spd levels and N1-Ac-Spd content in paired tumor tissues from hepatocellular carcinoma patients.

[0061] Figure 3 In F and G, the correlation between plasma N1-Ac-Spd levels and TNM stage (F) and recurrence (G) was analyzed in 88 HCC patients. In G, patients were divided into two groups based on the median plasma N1-Ac-Spd content. Elevated plasma N1-Ac-Spd levels were found in patients with advanced HCC and served as an independent predictor of HCC recurrence with a significant hazard ratio. In contrast, the levels of other physiological polyamines (such as putrescine (Put), spermine (Spd), spermine (Spm), and N1-acetylspermine (N1-Ac-Spm)) in tumor tissue showed less variation. Figure 3 (C), and is almost undetectable in plasma ( Figure 3 Therefore, the efflux of N1-Ac-Spd may represent a process that promotes HCC progression.

[0062] Figure 3 In this study, NC (wild-type, WT) or Slc3a2 knockdown (Slc3a2KD) Hepa1-6 cells were inoculated into the liver tissue of C57BL / 6 and BALB / c mice for 15 days. Mice were then treated with PBS or N1-Ac-Spd (30 mg / kg) every 3 days for a total of 3 times. Tumor volume was analyzed (n=5 per group). It was found that silencing the polyamine transporter Slc3a2 inhibited liver cancer growth only in immunocompetent C57BL / 6 mice, but not in BALB / c nude mice.

[0063] Figure 3 In studies I and J, the correlation between plasma N1-Ac-Spd levels and patient response to TAI plus αPD-1 treatment was demonstrated. In study J, 13 HCC patients were divided into two groups based on the median plasma N1-Ac-Spd level. Notably, patients who did not respond to TAI plus αPD-1 treatment had significantly higher plasma N1-Ac-Spd levels than those who did respond to treatment. Figure 3Elevated plasma N1-Ac-Spd levels in HCC patients were negatively correlated with prolonged progression-free survival (PFS).

[0064] Figure 3 In the K and L groups, NC(WT) or Slc3a2 knockdown (Slc3a2KD) Hepa1-6 hepatocellular carcinoma C57BL / 6 mice or NC(WT) or Slc3a2 knockdown (Slc3a2KD) H22 hepatocellular carcinoma BALB / c mice were injected with αPD-L1 antibody (8 mg / kg) every 3 days for a total of 3 times (n=5 per group) in the presence or absence of N1-Ac-Spd. Tumor volume was analyzed. Consistent with the results in humans, αPD-L1 treatment inhibited tumor growth in Hepa1-6 or H22 hepatocellular carcinoma mice. This effect was further enhanced after silencing the polyamine transporter Slc3a2, but this effect was also attenuated by intraperitoneal injection of N1-Ac-Spd.

[0065] Figure 3 In M, the plasmids shown were injected into C57BL / 6 mice using hydrodynamic injection to induce spontaneous hepatocellular carcinoma. A mixture of plasmids containing 20 μg myr-AKT1 (pT3-myr-AKT-HA, Addgene), 20 μg N-RasV12 (pT / Caggs-NRASV12, Addgene), and 1.6 μg sleeping beauty transposase (pCMV / SB10, Addgene) was diluted in 0.9% saline to a final volume equal to 10% of the mouse body weight. The mice were subsequently treated with αPD-L1 antibody or equivalent isotype IgG (8 mg / kg) with or without N1-Ac-Spd. The maximum diameter of the gross tumor nodules was analyzed (n=5). ssGSEA, single-sample gene set enrichment analysis; TAI, transarterial infusion of the chemotherapy drugs oxaliplatin and 5-fluorouracil (oxaliplatin 130 mg / m²). 2 5-Fluorouracil 2400mg / m 2 Although αPD-L1 therapy did not show therapeutic effect in myr AKT / N-RasV12-induced spontaneous hepatocellular carcinoma models, intraperitoneal injection of N1-Ac-Spd further promoted hepatocellular carcinoma progression even in the presence of αPD-L1 antibodies.

[0066] Example 3: Macrophages form key upstream and downstream components of the SAT1 / N1-Ac-Spd axis

[0067] Figure 4 This demonstrates the key upstream and downstream components of the SATI1 / N1-Ac-Spd axis formed by macrophages.

[0068] Further investigation revealed how the immune environment facilitates SAT1-mediated N1-Ac-Spd efflux, and found significant accumulation of CD68+ macrophages, CD15+ neutrophils, CD3+ T cells, CD79a+ B cells, and CD56+ natural killer cells at the invasive border of liver cancer cells, which are mainly distributed with SAT1+ liver cancer cells. Figure 4 In Figure A, the density of CD68+ macrophages, CD15+ neutrophils, CD3+ T cells, CD79a+ B cells, and CD56+ NK cells (n=16) is shown in the non-tumor liver, invasive margin, and cancer nest of HCC.

[0069] In 321 HCC patients in the TCGA dataset, the correlation between SAT1 expression and the indicated genes was calculated. Figure 4 In Figure B, the analysis of SAT1 and lineage marker expression in HCC tissues showed a unique correlation between SAT1 and macrophage markers CD14 and CD68.

[0070] SAT1 expression in human PLC / PRF / 5 and Huh7 hepatocellular carcinoma cells was assessed by confocal microscopy after 20 hours of untreated culture or co-culture with immune cells isolated from HCC tumors. Figure 4 C). Scale bar, 20 μm. Analysis of N1-Ac-Spd formation in the supernatant by HPLC-MS ( Figure 4 The results showed that SAT1 was upregulated in PLC / PRF / 5 and Huh7 hepatocellular carcinoma cells only when co-cultured with CD14+-derived HCC macrophages, accompanied by significant N1-acetylated spermidine (N1-Ac-Spd) efflux.

[0071] Figure 4 In Figure E, the effect of SAT1 knockdown (SAT1KD) on N1-Ac-Spd production in human PLC / PRF / 5 (solid) and Huh7 (hollow) hepatocellular carcinoma cells seeded with tumor-associated macrophages (TAMs) (n=3 per group) was shown. The study found that SAT1 knockdown indeed inhibited tumor macrophage-mediated N1-Ac-Spd efflux from hepatocellular carcinoma cells, confirming that N1-acetylated spermidine (N1-Ac-Spd) efflux in hepatocellular carcinoma is SAT1-dependent.

[0072] Macrophages in the Hepa1-6 hepatocellular carcinoma model of C57BL / 6 mice were eliminated by injection of a specific anti-CSF1R antibody (αCSF1R) (8 mg / kg) after 15 days. The effect of macrophages on Sat1 expression in mouse hepatocellular carcinoma tissue was investigated. Figure 4 F) and N1-Ac-Spd generation ( Figure 4The effects of (G) were observed (n=5 per group). It was found that removing macrophages with a specific antibody targeting the CSF1 receptor (CSF1R) successfully inhibited the expression of Sat1 and the content of N1-acetylated spermidine (N1-Ac-Spd) in tumors, demonstrating the necessity of macrophages in SAT1-mediated N1-Ac-Spd efflux.

[0073] NC (wild-type, WT) or Sat1-overexpressing (Sat1) Hepa1-6 cells were inoculated into the liver tissue of C57BL / 6 mice for 15 days. Subsequently, the mice were injected with PBS or αCSF1R antibody (8 mg / kg) as described above. Tumor volume was analyzed (n=5 per group). Results are as follows. Figure 4 The figure is shown in H. Tumor volume was analyzed in mice inoculated with Hepa1-6 cells, either untreated or treated with N1-acetylated spermidine (N1-Ac-Spd) (30 mg / kg) with or without αCSF1R antibody (n=5 per group). Results are shown in H. Figure 4 As shown in Figure I, the results indicate that macrophage removal effectively inhibited the growth of hepatocellular carcinoma (HCC) in mice; however, overexpression of Sat1 or intraperitoneal injection of exogenous N1-Ac-Spd did not reverse the inhibition of HCC growth, although both treatments effectively accelerated HCC growth in mice with intact macrophages. This demonstrates that macrophages are not only an important upstream factor activating the Sat1 / N1-Ac-Spd axis but also a key downstream responder to the pro-tumor effect of this axis. Analysis of single-cell sequencing data from HCC (GSE140228), shown in Figure J as a heatmap, illustrates the expression of polyamine uptake genes in immune cells from the GSE dataset (GSE140228). Among them, CTL stands for cytotoxic T lymphocytes; DC stands for dendritic cells; ILC stands for intrinsic lymphoid cells; Mast stands for mast cells; Mφ stands for macrophages; Mo stands for monocytes; NK stands for natural killer cells; Th stands for helper T cells; Treg stands for regulatory T cells; TAM stands for tumor-associated macrophages; and TAN stands for tumor-associated neutrophils. Macrophages were found to be the main cell type expressing polyamine uptake genes in tumors.

[0074] Example 4: N1-Ac-Spd polarizes CCL1+M2b macrophages by activating the SRC signaling pathway

[0075] Figure 5 This demonstrates that N1-Ac-Spd polarizes CCL1+M2b macrophages by activating the SRC signaling pathway.

[0076] To further explore how N1-acetylated spermidine (N1-Ac-Spd) inversely regulates macrophages, thereby exerting a pro-tumorigenic effect. Tumor macrophages purified from HCC tissue were subjected to RNA sequencing (n=3) after incubation for 20 hours in untreated or co-incubated with 100 μM N1-acetylated spermidine (N1-Ac-Spd). Volcano plots showed changes in gene expression between N1-Ac-Spd-treated tumor macrophages and the untreated control group. Figure 5 (A). By analyzing RNA sequencing data, 132 genes that were upregulated or downregulated by at least two-fold in tumor macrophages treated with N1-acetylated spermidine (N1-Ac-Spd) were identified.

[0077] Further ssGSEA analysis revealed that N1-Ac-Spd primarily induced M2b polarization in tumor macrophages. Single-sample gene set enrichment (ssGSEA) analysis showed differences in macrophage differentiation between N1-Ac-Spd-treated and untreated tumor macrophages. Figure 5 (B). The heatmap shows the fold change in classic M2b macrophage markers in N1-Ac-Spd-treated macrophages compared to untreated macrophages in HCC patients ( Figure 5 C).

[0078] The production of CCL1 in untreated tumor macrophages or those incubated with 100 μM N1-acetylated spermidine (N1-Ac-Spd) for 20 hours was measured by ELISA (n=6). A significant increase in the chemokine CCL1 was detected in the culture supernatant of tumor macrophages exposed to N1-Ac-Spd. CCL1 is a characteristic functional marker of M2b macrophages. Figure 5 D).

[0079] CCL1 production in tumor macrophages co-cultured with supernatant of untreated or TNF-α-treated tumor cells (TNF-α-TSN) was analyzed in the presence or absence of the polyamine transport competitive inhibitor AMXT-1501 (n=6). A corresponding increase in CCL1 secretion was also detected when tumor macrophages were exposed to the culture supernatant of TNF-α-induced SAT1+ hepatocellular carcinoma cells, an effect that could be eliminated by inhibiting N1-Ac-Spd efflux from SAT1+ hepatocellular carcinoma cells using AMXT-1501. Figure 5 E).

[0080] This study investigated the effect of exogenous N1-acetylated spermidine (N1-Ac-Spd) on CCL1+ macrophage infiltration in mice with three different types of hepatocellular carcinoma (n=5). Injection of N1-Ac-Spd (30 mg / kg) significantly enhanced the polarization of CCL1+ macrophages from tumors in mice carrying the three different types of hepatocellular carcinoma. Figure 5 Therefore, the SAT1 / N1-Ac-Spd axis may promote the polarization of CCL1+M2b macrophages.

[0081] Confocal microscopy was used to analyze SATS1+ cells (green), CD68+ macrophages (white), and CCL1+ macrophages (red) in HCC tissue. Figure 5 (G, left). Scale bar, 50 μm. The correlation between CCL1 expression and SAT1 expression in 10 HCC tumors was analyzed using real-time PCR. Figure 5 (G, right). In human HCC tissue, CCL1 is expressed only by macrophages near SAT1+ hepatocellular carcinoma cells. In contrast, macrophages hardly express CCL1 in the HCC matrix lacking SAT1+ cells. In fact, SAT1 expression is positively correlated with CCL1 expression in HCC tissue.

[0082] This study investigated the effect of tumor-specific Sat1 deficiency on CCL1+ macrophage infiltration in mice with hepatocellular carcinoma (n=5). Scale bar, 50 μm. In mice carrying Hepa1-6 hepatocellular carcinoma, Sat1 knockdown successfully inhibited the polarization of CCL1+ M2b macrophages. Figure 5 H).

[0083] Further analysis was conducted on the enrichment of signaling pathways in N1-Ac-Spd-treated tumor macrophages. The gene set enrichment analysis of STAT, MAPK, NF-κB, and AKT signaling pathways in N1-Ac-Spd-treated tumor macrophages was compared with that in the untreated control group (n=3). Figure 5 Macrophages purified from non-tumor tissues or HCC tumor tissues were analyzed for activation of the STAT pathway (n=5) after 1 hour of incubation with N1-Ac-Spd, either untreated or completely. Figure 5 (J). It was found that although the STAT signaling pathways (especially the STAT3 and STAT6 signaling pathways) were selectively activated, the MAPK, NF-κB, or AKT signaling pathways were not activated.

[0084] Tumor macrophages were treated with different concentrations of STAT3 signaling inhibitor (μg / mL) or STAT6 signaling inhibitor (μM) for 20 hours in the presence or absence of STAT3 signaling inhibitor (μg / mL) or co-incubated with N1-Ac-Spd. CCL1 production was measured by ELISA (n=5). Figure 5 (K). Inhibitors that suppress STAT3 or STAT6 activation were found to have little effect on CCL1 secretion. Notably, the SRC-induced SYK / BTK signaling pathway is crucial for Fc receptor-mediated CCL1+M2b macrophage polarization.

[0085] The activation of the SRC-SYK-BTK signaling pathway was analyzed in tumor macrophages after 1 hour of incubation in untreated or co-incubated with N1-Ac-Spd (n=5). Figure 5 (L). It was found that SRC and its downstream SYK / BTK signaling pathway were significantly activated in N1-Ac-Spd treated tumor macrophages.

[0086] Tumor macrophages were incubated for 20 hours without treatment or co-incubated with N1-Ac-Spd, in the presence or absence of a specified inhibitor. CCL1 production was measured by ELISA (n=6). Figure 5 (M). Inhibitors targeting SRC or SYK / BTK signaling were found to significantly suppress CCL1 secretion in these cells.

[0087] Hepa1-6 hepatocellular carcinoma mice were treated with a specified inhibitor, and the infiltration of CCL1+ macrophages in the tumor was analyzed (n=5). Figure 5 In mice carrying liver cancer, inhibitors of the SRC / SYK / BTK signaling pathway also suppressed N1-acetylated spermidine (N1-Ac-Spd)-induced CCL1+M2b macrophage polarization.

[0088] The study investigated the effect of N1-Ac-Spd on SRC phosphorylation under conventional, high-salt culture, acetic acid (CH3COOH), control peptide (PepC), or carboxylated peptide (Pep1) conditions in a cell-free system (n=5). Figure 5 In a non-cellular system, P showed that SRC proteins were phosphorylated upon incubation with N1-acetylated spermidine (N1-Ac-Spd). Given that polyamines are positively charged at physiological pH and can bind to negatively charged proteins and phospholipids, the study further investigated whether this mechanism is involved in N1-Ac-Spd-mediated SRC activation. As expected, P demonstrated that removing the positive charge from N1-Ac-Spd by adjusting ionic strength, adding an equal amount of acetic acid, or using a negatively charged carboxylic peptide effectively inhibited SRC activation. Therefore, the SRC-mediated SYK / BTK signaling pathway dominates N1-Ac-Spd-mediated CCL1+M2b macrophage polarization, thereby promoting tumor-promoting effects in HCC.

[0089] Tumor macrophages were incubated for 1 hour with and without N1-acetylated spermidine (N1-Ac-Spd), and the level of N1-Ac-Spd was quantitatively analyzed by HPLC-MS (n=5). An increase in intracellular N1-Ac-Spd was observed in tumor macrophages treated with N1-Ac-Spd. Figure 5 O).

[0090] Example 5: Treatment resistance induced by macrophage CCL1-dominant N1-Ac-Spd

[0091] Figure 6 This study demonstrated treatment resistance induced by macrophage CCL1-dominant N1-Ac-Spd.

[0092] Since N1-Ac-Spd can induce CCL1+ macrophage polarization in hepatocellular carcinoma (HCC), is macrophage-derived CCL1 crucial for the N1-Ac-Spd-induced pro-tumor effect? ​​To explore this possibility, Hepa1-6 hepatocellular carcinoma mice were either untreated or injected intraperitoneally every 3 days with liposomes containing siCcl1 or siNC RNA to specifically deliver Ccl1 siRNA to mouse macrophages carrying hepatocellular carcinoma, and treated in the presence of N1-acetylated spermidine (N1-Ac-Spd) or an equivalent PBS. Figure 6 A). Analyze tumor volume ( Figure 6 B) and the infiltration and function of CTLs in tumors ( Figure 6 (C)(n=5). CTL stands for cytotoxic T lymphocytes; GB stands for granzyme B. This treatment effectively inhibited CCL1 expression and slowed liver cancer growth in both the presence and absence of N1-Ac-Spd. This treatment eliminated N1-Ac-Spd-mediated CD8+ T cell reduction and immunosuppressive effects in liver cancer.

[0093] NC (wild-type, WT) or Sat1-overexpressing (Sat1) Hepa1-6 hepatocellular carcinoma mice were either untreated or injected with N1-acetylated spermidine (N1-Ac-Spd) (30 mg / kg) or equivalent PBS. Figure 6 D). Analysis of CTL invasion and function in tumors ( Figure 6 (n=5). In a Sat1-overexpressing hepatocellular carcinoma model, deletion of Ccl1 expression in tumor macrophages successfully restored CD8+ T cell infiltration and function. Therefore, macrophage-derived CCL1 constitutes the main pro-tumorigenic factor induced by N1-Ac-Spd in HCC.

[0094] The effect of macrophage-derived CCL1 on N1-Ac-Spd-mediated resistance to anti-PD-L1 therapy was further investigated. Hepa1-6 hepatocellular carcinoma mice were either untreated or intraperitoneally injected with liposomes containing siCcl1 or siNC RNA, and treated in the presence of αPD-L1 or an equivalent isotype antibody. Figure 6 (F). Analyze tumor volume (G) and the invasion and function of CTLs in the tumor (H) (n=5). Scale bar is 0.5cm ( Figure 6 As expected, deletion of Ccl1 expression in tumor macrophages also completely eliminated immunotherapy resistance induced by intraperitoneal N1-Ac-Spd, accompanied by an increase in functional CD8+ T cell infiltration. Figure 6 H).

[0095] This study investigated the association between CCL1 expression in tumors and patient response to TAI plus αPD-1 therapy. Thirteen HCC patients were randomly assigned to two groups based on median plasma N1-Ac-Spd levels. TAI consisted of transarterial chemotherapy drugs oxaliplatin and 5-fluorouracil. CCL1 expression was found in HCC patients receiving anti-PD-1 therapy, and higher CCL1 expression was observed in tumor tissues of non-responders than in responders. Figure 6 ,I).

[0096] In summary, these findings suggest that targeting macrophage-derived CCL1 could be a strategy to overcome N1-Ac-Spd-induced immunotherapy resistance in HCC.

[0097] Example 6: Macrophage CCL1 shapes an immune landscape dominated by regulatory T cells

[0098] Figure 7 This study demonstrates how macrophage CCL1 shapes an immune landscape dominated by regulatory T cells.

[0099] After establishing the importance of macrophage-derived CCL1 in N1-Ac-Spd-mediated liver cancer progression and immunotherapy resistance, the study finally evaluated how macrophage-derived CCL1 exerts an immunosuppressive effect on tumors.

[0100] The heatmap shows the expression of chemokine receptor genes in immune cells from the GSE dataset (GSE140228). Figure 7 A), and a violin diagram of CCR8 expression in these immune cells (A). Figure 7(B). Among them, CTL represents cytotoxic T lymphocytes, DC represents dendritic cells, ILC represents innate lymphoid cells, Mast represents mast cells, Mφ represents macrophages, Mo represents monocytes, NK represents natural killer cells, Th represents helper T cells, and Treg represents regulatory T cells. Analysis of the chemokine receptor profile of HCC-infiltrating leukocytes, using single-cell sequencing data (GSE140228), revealed that the CCL1 receptor CCR8 is expressed only in regulatory T cells and not in any other immune cells. A violin diagram illustrates the expression of T cell-related genes in CCR8– or CCR8+ T cells (GSE140228). Figure 7 (B) CCR8+Th cells exhibit classic FOXP3hiIL2RAhiIL7RAlo transcriptional signatures, accompanied by high expression of co-repressor molecules CTLA4, GITR, and TIGIT.

[0101] The t-distributed random neighborhood embedding (t-SNE) plot illustrates the expression of CCR8 and FOXP3 genes in T cell clusters (GSE140228). Figure 7 (C). As a classic transcription factor for regulatory T cells, FOXP3 is also expressed only in CCR8+Th cells. Therefore, N1-Ac-Spd polarized CCL1+ macrophages may establish an immune microenvironment through regulatory T cells.

[0102] 321 HCC samples from the TCGA dataset were divided into two groups based on the median polyamine metabolism ssGSEA score. The Treg invasion index in PAhigh and PAlow tumors was analyzed using the CIBERSORT algorithm. Figure 7 D). D represents the positive correlation between regulatory T cell infiltration and polyamine metabolism score, confirmed by using the CIBERSORT algorithm.

[0103] Laser confocal microscopy was used to analyze SAT1+ cells (green), FOXP3+ cells (red), and CCL1+ macrophages (white) in HCC tissue. The density of FOXP3+ cells in SAT1high and SAT1low tumors (n=17) was analyzed. Scale bar, 50 μm ( Figure 7 (E). It was found that FOXP3+ regulatory T cells mainly accumulate in HCC regions containing SATI1+ and CCL1+ cells.

[0104] The effect of tumor-specific Sat1 deficiency on Treg invasion in Hepa1-6 hepatocellular carcinoma mice (n=5) was investigated. Figure 7 (F). In mice carrying Hepa1-6 hepatocellular carcinoma, knockdown of Sat1 in the hepatocellular carcinoma effectively reduced the infiltration of FOXP3+ regulatory T cells.

[0105] The effect of macrophage Ccl1 deficiency on N1-Ac-Spd-induced Treg invasion in Hepa1-6 hepatocellular carcinoma mice (n=5) was investigated. Figure 7 Deleting Ccl1 expression in tumor macrophages completely eliminated N1-Ac-Spd-induced infiltration of FOXP3+ regulatory T cells in hepatocellular carcinoma tissue.

[0106] Hepa1-6 hepatocellular carcinoma mice were used in experiments with or without N1-Ac-Spd αCD25 antibody in untreated mice. Tumor volume was analyzed (n=5). Figure 7 (H). It was found that removing regulatory T cells by injecting anti-CD25 antibodies can effectively eliminate the pro-tumor effect of exogenous N1-Ac-Spd.

[0107] This study investigated the association between CCL1 and FOXP3 expression in tumors of 13 HCC patients who received TAI plus αPD-1 therapy. TAI is a transarterial chemotherapy drug (... Figure 7 In HCC patients receiving anti-PD-1 therapy, FOXP3 expression was positively correlated with CCL1 expression.

[0108] This study investigated the association between FOXP3-CCL1 gene signature and progression-free survival (PFS) in 13 HCC patients treated with TAI plus αPD-1. Figure 7 Patients were divided into two groups based on the median FOXP3 and CCL1 gene signatures. "Double low" referred to patients with FOXP3lowCCL1low; "Others" included patients with FOXP3highCCL1low, FOXP3lowCCL1high, and FOXP3highCCL1high. Decreased expression of FOXP3 and CCL1 in tumor tissues of HCC patients was found to be positively correlated with prolonged progression-free survival after immunotherapy.

[0109] In summary, macrophage-derived CCL1 creates conditions that suppress the efficacy of immunotherapy by recruiting regulatory T cells into HCC.

Claims

1. A plasma biomarker for evaluating the efficacy of immunotherapy for liver cancer, characterized in that, The plasma biomarker is N1-acetylated spermidine.

2. The use of the plasma biomarker of claim 1 in the preparation of products for predicting and evaluating the efficacy of immunotherapy for liver cancer.

3. The use of the plasma biomarker of claim 1 in the preparation of products for predicting and assessing the progression of liver cancer.

4. The use of the plasma biomarker of claim 1 in the preparation of a medicament for promoting the efficacy of immunotherapy for liver cancer.

5. The use of the reagent for detecting the plasma biomarker of claim 1 in the preparation of products for predicting and evaluating the efficacy of immunotherapy for liver cancer.

6. The use of the reagent for detecting the plasma biomarker of claim 1 in the preparation of products for predicting and assessing the progression of liver cancer.

7. Application of reagents that block the synthesis and efflux of N1-acetylated spermidine in liver cancer in the preparation of drugs to enhance the efficacy of immunotherapy for liver cancer.

8. The application according to any one of claims 2 to 7, characterized in that, The liver cancer mentioned is hepatocellular carcinoma.

9. The application according to claim 5 or 6, characterized in that, The detection method is liquid chromatography combined with mass spectrometry.

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