CN117398386A

CN117398386A - New use of synergistic pharmaceutical composition in treating liver cancer

Info

Publication number: CN117398386A
Application number: CN202311396716.3A
Authority: CN
Inventors: 齐峰; 夏景林; 孔秀燕; 张简
Original assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Current assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Priority date: 2023-10-25
Filing date: 2023-10-25
Publication date: 2024-01-16

Abstract

The invention discloses a new application of a synergistic pharmaceutical composition in treating liver cancer, wherein the synergistic pharmaceutical composition contains thalidomide, cantharidin and carmofur, and the synergistic pharmaceutical composition is applied in preparing medicines for treating liver cell liver cancer. The synergistic pharmaceutical composition can effectively inhibit the progress of liver cancer of liver cells, and provides a thought for exploring a specific mechanism of the synergistic pharmaceutical composition in treating liver cancer of liver cells. The invention cooperates with the drug combination to enable the SAMD4B to inhibit the tumor immune escape of the liver cancer of the liver cells by regulating the stability of the APOA2 mRNA.

Description

New use of synergistic pharmaceutical composition in treating liver cancer

Technical Field

The invention relates to the technical field of medicines, in particular to a novel application of a synergistic medicine composition in treating liver cancer.

Background

Primary liver cancer (hereinafter referred to as liver cancer) is the common cause of malignant tumor and mortality in China. Because of the hidden onset, no symptoms or insignificant symptoms at early stage, rapid progress, although surgery is the first treatment of liver cancer, most patients have reached middle and late stages at the time of diagnosis, often losing the opportunity for surgery, and only about 20% of patients are statistically suitable for surgery. Thus, there is a need for aggressive non-surgical treatment that may lead to reduced symptoms, improved quality of life and increased survival in a significant proportion of patients. Interventional (TACE) therapy is an important method for comprehensive treatment of liver cancer as a minimally invasive therapy, and has obvious effects in inhibiting tumor growth, improving survival rate of patients and the like.

Although the traditional treatment methods of liver cancer are more, such as surgery, intervention, chemotherapy, radiotherapy, liver transplantation and the like, less than 30% of patients can receive curative treatment such as surgery or liver transplantation and the like, the postoperative recurrence and transfer rate are very high, most of the remaining patients are late patients, the chance of surgery or liver transplantation is lost, and systemic chemotherapy drug treatment can be the only effective treatment method. Therefore, there is a global need to enhance the research in the medical field related to hepatocellular carcinoma.

TACE surgery is a common method in interventional therapy of liver cancer, but tumor blood vessels may still regenerate after TACE surgery, resulting in local recurrence, so how to block the regeneration of blood vessels is a key place for controlling tumors. The primary angiogenic factor is VEGF, which not only promotes division and migration of tumor vascular endothelium, but also supports survival of endothelial cells, and therefore, elevated concentrations tend to be predictive of poor patient prognosis. Studies have shown that elevated concentrations of VEGF after TACE surgery may be present as an indicator of the effectiveness of TACE.

From the above description, it is clear that primary liver cancer is a malignant digestive tract tumor, and global data shows that the morbidity and mortality of the primary liver cancer are the sixth and fourth malignant tumors, respectively, with hepatocellular carcinoma (hepatocellular carcinoma, HCC) accounting for 90%. Because of the hidden onset, about 70% of patients are at mid-late stage when diagnosed, losing the opportunity for radical surgery, and for these patients, arterial chemoembolization (TACE) is an effective treatment because of its good therapeutic efficacy compared to conservative or systemic therapies, which are widely used clinically. Although TACE is one of the preferred options for the radical treatment of HCC, the survival time of patients after TACE surgery is still limited. To further extend patient survival, many studies have combined TACE with systemic therapy. TACE in combination with sorafenib is one of the strategies for first-line systemic treatment, but its therapeutic effect remains controversial, such as patients showing longer Progression Free Survival (PFS) in the tacic trial, but only very limited benefit was observed in the STAH trial. In addition, studies on TACE and other targeted drugs (such as Brivanib and Orantinib) have also ended with failure. The results of these combination regimens are not satisfactory, indicating that a new oral drug is needed to be used in combination with TACE, to participate in the treatment, and to produce good results.

Disclosure of Invention

The invention provides a new application of a synergistic pharmaceutical composition in treating liver cancer.

The scheme of the invention is as follows:

the novel application of a synergistic pharmaceutical composition in treating liver cancer, wherein the synergistic pharmaceutical composition contains thalidomide, cantharidin and carmofur, and the synergistic pharmaceutical composition is applied in preparing medicines for treating liver cancer of liver cells.

As a preferable technical scheme, the synergistic medicine combination is applied to the preparation of medicines for inhibiting liver cell and liver cancer.

As a preferable technical scheme, the synergistic medicine combination is applied to the preparation of medicines for prognosis of the chemoembolization by hepatic artery.

As a preferable technical scheme, the synergistic medicine composition is applied to the preparation of medicines for inhibiting the immune escape of liver cell and liver cancer tumors.

As a preferred technical scheme, the synergistic pharmaceutical composition is applied to the preparation of a medicament containing a 4B sterile alpha motif domain for regulating the stability of apolipoprotein A II.

The invention also discloses a synergistic pharmaceutical composition, which contains thalidomide, cantharidin and carmofur, and is an oral pharmaceutical formulation.

As a preferable technical scheme, the mass ratio of thalidomide, cantharidin and carmofur in the synergistic pharmaceutical composition is 0.1:1.5:0.3.

As an optimal technical scheme, the cantharidin is a compound cantharidin capsule.

Due to the adoption of the technical scheme, the novel application of the synergistic medicine combination in treating liver cancer is provided, wherein the synergistic medicine combination contains thalidomide, cantharidin and carmofur, and the application of the synergistic medicine combination in preparing medicines for treating liver cell liver cancer is provided.

The invention has the advantages that: the synergistic pharmaceutical composition can effectively inhibit the progress of liver cancer of liver cells, and provides a thought for exploring a specific mechanism of the synergistic pharmaceutical composition in treating liver cancer of liver cells.

The invention cooperates with the drug combination to enable the SAMD4B to inhibit the tumor immune escape of the liver cancer of the liver cells by regulating the stability of the APOA2 mRNA.

Drawings

FIG. 1 is a graph showing the Kaplan-Meier survival analysis of the present invention; wherein FIG. 1a is a graph of survival analysis of study cohorts with and without TCC treatment, FIG. 1b is a statistical graph of survival at 6 months, 12 months, 24 months for cohorts with and without TCC treatment, respectively, FIG. 1c is a graph of changes in AFP and PIVKA-II expression for a patient in a cohort with TCC treatment, FIG. 1d is a graph of CT comparison of a patient in a cohort with TCC treatment after 5 courses of TCC treatment, wherein THA is thalidomide; CAN is cantharidin; CAR is carmofur; TC is thalidomide combined carmofur; TCC is a synergistic pharmaceutical combination of three combinations of thalidomide, cantharidin, carmofur; * P <0.05, < P <0.01, < P <0.001; FIG. 1e is a graph of CT contrast after 3 courses of TCC treatment for a patient in a cohort receiving TCC treatment, FIG. 1f is a graph of model of 36 PDX models established and co-drug combinations and single drug treatments following implantation of patient-derived tumor cells subcutaneously in the abdomen of BALB/C mice, FIGS. 1g, 1h and 1i are graphs of tumor volume and weight statistics of co-drug combinations and single drug groups, FIGS. 1j and 1k are graphs of results and quantitative statistics of experiments of co-drug combinations and single drug treatments on colony formation of various human liver cancer cell lines;

FIG. 2 is a single cell sequencing analysis chart of PDX by comparing single drugs with the synergistic drug combination of the invention, wherein FIG. 2a is a schematic diagram of a single cell sequencing program, and FIG. 2b, FIG. 2d, FIG. 2e and FIG. 2f are the variation charts of the number of 20 malignant tumor cells treated by the synergistic drug combination and single drugs; FIG. 2g and FIG. 2h are graphs of gene enrichment analysis and pathway enrichment analysis of differentially expressed genes;

FIG. 3 is a graph showing the gene expression of significant differences in the PDX model of the effect found by the single cell transcriptome analysis of the present invention; wherein, fig. 3a, 3c, 3e and 3f are tSNE plots of DMSO group and synergistic drug combinations and single drug treatment; FIGS. 3b, 3d, 3f and 3h are cell fraction graphs of DMSO groups in combination with synergistic drug combinations and single drug treatments; FIGS. 3i, 3j, 3m, 3n and 3o are volcanic charts of significant differential expressed genes for DMSO groups in combination with synergistic drug combinations and single drug treatments; FIGS. 3k and 3l are graphs of the expression analysis of SAMD4B in DMSO group, TC group and TCC group; FIG. 3p is a Venn diagram of a differentially expressed gene common to the single drug group;

FIG. 4 is a graph showing the tumor suppression effect of SAMD4B of the present invention by targeting APOA2 oncogenes; wherein FIG. 4a is a tSNE plot of the DMSO, TC, and TCC groups APOA2 and SAMD 4B; FIG. 4B is a graph of the expression level of APOA2 versus the expression level of SAMD 4B; FIGS. 4c and 4d are immunohistochemical and quantitative analysis charts of SAMD4B in the paracancerous and cancerous tissues of 158 HCC patients; FIG. 4e is a graph of survival analysis of patients with high and low levels of SAMD4B expression in the tissue; FIGS. 4f and 4g are immunohistochemical and quantitative analysis plots of paracancerous and cancerous tissue; FIG. 4h is a graph of survival analysis of patients with high and low levels of APOA2 expression in tissues; FIGS. 4i, 4k and 4l are graphs of APOA2 and SAMD4B expression versus occurrence of tumor metastasis, tumor size and AFP levels, prognosis for survival; FIG. 4j is a graph showing the relationship between SAMD4B and APOA2 expression;

FIG. 5 is a diagram illustrating the 2' -O-methylation of APOA2mRNA by SAMD4B of the present invention; wherein FIG. 5a is a validation graph of over-expression and knockout of SAMD 4B; FIG. 5B is a half-life plot of APOA2mRNA after overexpression and knockdown of SAMD 4B; FIG. 5c is a graph of liquid chromatography-mass spectrometry detection of all known RNA modification types; FIG. 5d is a partial map of the occurrence of 2' -O-methylation modification in the co-therapy group; FIG. 5e is an analytical plot of changes at two modification sites of APOA 2; FIG. 5f is a WB plot of a change in SAMD4B to detect the expression of downstream APOA2 and 4 immune checkpoints; FIG. 5g is a validation graph of the detection of the direct interaction of APOA2 with PD-L1 by Co-immunoprecipitation (Co-IP) method;

FIG. 6 is a graph illustrating that high expression of SAMD4B of the present invention reduces PD-L1, thereby impairing the immune escape of tumor cells to primary CD29+CD8+T cells; wherein; FIG. 6a is a tSNE diagram of unsupervised aggregation of T cells and NK cells; FIG. 6B is a graph showing the ratio of different immune cells in tumor tissue with high and low SAMD4B expression; FIG. 6c is a plot of the proportion of CD4+ T cells and CD8+ T cells in SAMD4B highly and lowly expressing tumor tissue for breast and colon cancer samples; FIG. 6d is a pseudo-time analysis of CD8+ T cells; FIG. 6e is a graph showing the trend of expression of depletion signals and cytotoxicity model; FIG. 6f is a transcriptional change of CD8+ T cells associated with a transitional state; FIG. 6g is a volcanic chart of differential gene analysis of SAMD4B high and low expression; FIG. 6h is a graph of ITGB1 (CD 29) expression versus PDCD1 expression in CD8+ and CD8+ naive T cells; FIG. 6i is a graph of the co-localization relationship of PD-L1, CK18, SAMD4B, PD1, CD29, CD3, CD 8; FIG. 6j is a plot of the ratio of CD29+CD8+T cells in a sample of high and low SAMD4B expression; FIG. 6k is a graph of CD29+CD8+ T cell expression levels versus prognosis for survival of HCC patients;

FIG. 7 is a graph depicting the efficacy and mechanism of TCC treatment in immunized normal mice according to the present invention; wherein, FIG. 7a is a graph of in situ tumor model and drug treatment pattern of C57BL6/J mice; FIGS. 7b, 7c and 7d are graphs of tumor volume and weight of synergistic drug combinations and single drug groups; FIG. 7e is a statistical plot of the CD29+CD8+ T cell ratio of the synergistic drug combination to the single drug group; FIGS. 7f and 7g are diagrams of immunohistochemistry and quantitative analysis of SAMD4B, APOA2, PD-L1, NOTCH1 and NOTCH2 in a synergistic drug combination with a single drug group of mouse tumor tissue;

FIG. 8 is a graph of liver images before and after treatment of a TCC treated patient of the present invention.

FIG. 9 is a graph showing the effect of a synergistic combination of drugs and single drugs of the present invention on murine immune cells in the PDX model; wherein, fig. 9a and 9b are a thermal diagram and tSNE diagram of murine cells; FIGS. 9c and 9d are graphs of analyses of the distribution of 6 marker genes to known cell lineages; FIG. 9e is a plot of the cell cycle tSNE of mouse-derived cells; FIG. 9f is a cell cycle heat map of murine cells; FIG. 9g is a plot of cell cycle tSNE of mouse-derived cells in single and synergistic drug combinations;

FIG. 10 is a diagram showing the mechanism of the synergistic pharmaceutical combination of the present invention for inhibiting tumor immune escape through the SAMD4B-APOA2 axis and exerting an effective anti-hepatocellular carcinoma effect.

Detailed Description

The invention is further described in connection with the following embodiments in order to make the technical means, the creation features, the achievement of the purpose and the effect of the invention easy to understand.

Example 1

The synergistic pharmaceutical composition comprises 100mg of thalidomide, 1.5g of cantharidin and 300mg of carmofur, wherein the cantharidin is a compound cantharidin capsule.

Participation in experiments

The combined use of interventional hepatic arterial chemoembolization (TACE) therapy with synergistic treatment with Thalidomide (THA), compound cantharides Capsules (CAN), carmofur (CAR) in advanced HCC patients, and the results of retrospective cohort analysis by earlier stage also suggest that patients with TACE in combination with oral TCC (tha+can+car) exhibit a better prognosis than patients with TACE alone (fig. 1a, fig. 1 b), this therapeutic strategy more effectively inhibits HCC progression (fig. 1c, fig. 1d, fig. 1 e). Thus, a specific mechanism of the synergistic pharmaceutical combination of the present application in the treatment of HCC is necessary.

The (one) triple synergistic pharmaceutical combination can exert the inhibition effect on HCC by inhibiting tumor immune escape

In the early research results, single-cell sequencing analysis is carried out on the tumor tissue of the constructed humanized liver cancer transplantation tumor nude mouse model (PDX), and the KEGG channels with the enrichment of differential genes are related to immune response and immune escape (FIG. 2g and FIG. 2 h); furthermore, in addition to the majority of human tumor cells, a portion of murine cells was detected, and after cell type identification of this portion of cells, it was found that the majority of murine immune cells were the largest number of immune cells in the TCC group (fig. 9a, 9b, 9c, 9 d), whereby the use of TCC could suppress immune escape from the tumor and further activate the immune response of the body to the tumor, and therefore, in the following studies, further studies will be conducted on how the immune escape specifically functions in the treatment of HCC.

(II) 4B-containing sterile alpha motif domain (SAMD 4B) may play a role in triple synergic drug therapy of HCC by affecting the stability of apolipoprotein A II (APOA 2) mRNA

SAMD4B protein, which is not expressed at high levels in normal liver tissue, may play an important role in regulating the development and progression of HCC. SAMD4B as a homolog of Smaug, although it has been demonstrated to affect the stability of post-transcriptional RNA;

the triple drug sensitive HCC cell subset C16 was subsequently identified using single cell sequencing technology, and the SAMD4B gene was screened for a significant increase in expression level in the triple drug (fig. 3 j-l), and in the single drug application of THA, CAN, CAR, three groups of genes with different and most significant expression compared to the control were APOA2 (fig. 3 m-o), and it was verified that SAMD4B and APOA2 expression exhibited a negative correlation under the effect of the triple drug (fig. 4B). Based on this, SAMD4B influences APOA2 expression by modulating post-transcriptional RNA methylation modification, and further mechanisms are further verified by subsequent experiments.

Taken together, SAMD4B inhibited tumor immune escape of HCC by modulating the stability of APOA2mRNA (fig. 5 a-B). Work is performed from the aspect: 1) Verifying the correlation of SAMD4B with APOA2 among HCC; 2) SAMD4B regulates the molecular mechanism of APOA2mRNA stability; 3) APOA2 affects the molecular mechanisms of HCC tumor immune escape.

Experiment 1

Clinical data of non-resectable liver cancer patients diagnosed in the auxiliary Zhongshan hospital of the double denier university from 5 months 2017 to 5 months 2020 are collected, and the curative effect of TACE combined triple drug (TCC) in clinical practice is evaluated. All patients initially underwent hepatic arterial chemoembolization (TACE); on this basis, the study group was given thalidomide (THA, 100mg, 1 time per night), carmofur (CAR, 300mg, 1 time per day) and compound cantharis capsule (CAN, 1.5g, 1 time per day) in combination with oral treatment until tumor progression or intolerable adverse effects occurred. Kaplan-Meier survival analysis showed (fig. 1), study group showed better overall survival.

Next, the underlying mechanisms of TCC treatment continue to be explored. One of the patients is transplanted with liver cell liver cancer tumor cells from a source into nude mice subcutaneously, and 36 PDX models are established. Mice were divided into 6 groups, injected DMSO, THA, CAR, CAN, TC (tha+car) and TCC, respectively.

The nude mice PDX model method comprises the following steps: fresh tumor tissue was isolated from HCC patients, and after anesthetizing NOG mice, liver cancer tissue was subcutaneously transplanted into the right upper abdomen of the mice. After 2 months, when the tumor diameter reached 1cm, the subcutaneous PDX tumor was resected, cut into 3 blocks with the size of 2X 2mm, transplanted again to the abdomen of a nude mouse and grown for about 30 days. Mice were euthanized no more than 5 weeks or when the tumor diameter reached 10 mm. The drug treatment group was injected with 18mg/kg of thalidomide, 225mg/kg of carmofur, 45mg/kg of cantharidin, 18mg/kg of thalidomide + 225mg/kg of carmofur (TC) and 225mg/kg of thalidomide + carmofur + 45mg/kg of cantharidin (TCC) every 2 days tail vein on day 8 post-implantation.

Regarding the Hep1-6 mouse hepatoma carcinoma in situ model, we suspended 5×107Hep 1-6 cells in 100 μl serum-free DMEM and Matrigel (1:1) and injected subcutaneously in the right flank of C57 mice. The tumor with diameter of 1cm was cut into small pieces (2X 2 mm) ³ ). Livers of the living mice (6 per group) were then transplanted. Animals were sacrificed 35 days after implantation. The drug treatment group was injected with 18mg/kg of thalidomide, 225mg/kg of carmofur, 45mg/kg of cantharidin, 18mg/kg of thalidomide + 225mg/kg of carmofur (TC) and 225mg/kg of thalidomide + carmofur + 45mg/kg of cantharidin (TCC) every 2d tail vein on day 8 post-implantation.

It was found that single drug treatment inhibited tumor growth to some extent, but the combination, especially TCC, further retarded tumor growth. However, colony formation experiments show that TCC has no effect on mouse and human hepatoma cells in vitro, and it is speculated that TCC does not directly kill cells, but rather modulates tumor immune microenvironment to produce an anti-tumor effect.

Experiment 2

To explore the relationship between TCC and tumor immune microenvironment, tumors were excised and single cell RNA sequencing was performed in the constructed nude mouse PDX model. 20 main cell clusters were identified in 6 groups of cells expressing specific marker genes, and these defined cell clusters were classified into known cell lines including malignant cells, immune cells (B cells, plasma, T cells, NK cells, and myeloid cells) and epithelial cells according to the marker genes. After observing the change of the cell number in each cluster after single drug or combined drug treatment, the different clusters are found to show multi-effect change, and the actual effect of the drug on tumor cells in vivo is more complex, so that the effectiveness of the drug cannot be verified only through in vitro experiments. In addition, enrichment analysis of the 6-group differential expression genes revealed that TCC-group tumor-associated signaling pathways were up-regulated and immune-associated signaling pathways were up-regulated, suggesting that TCC may activate immune responses to tumors. (see FIG. 2)

Experiment 3

To further determine the role of TCC in immune response to tumors in humans, comparative analysis was performed on group 6, where only the c16+c18 cell cluster was the primary target cell cluster for TCC, i.e., the group was sensitive to combination therapy, but not significantly responsive to three single drug therapies. Thus, HCC patients with the c16+c18 gene cluster are considered the target population for TCC treatment.

In the gene enrichment analysis of the combination group, enrichment of immune-related pathways and enrichment of tumor-related pathways were found, confirming the presumption that the inhibition of TCC on tumors may be related to tumor immune microenvironment. To investigate the mechanism of action of TCC on c16+c18 cells, the differentially expressed genes of the combination treatment group were analyzed and the sterile alpha motif domain containing 4B (sterile alpha motif domain containing B, samd 4B) was found to be the most significant differentially expressed gene whose expression levels were progressively increased in DMSO, TC and TCC groups, consistent with the therapeutic trend in the PDX model. In addition, the cause of resistance of c16+c18 cells to single drug therapy was studied, and by analyzing the differentially expressed genes of the three single drug groups, it was found that apolipoprotein AII (APOA 2) was the most significant gene. (see FIG. 3)

Experiment 4

To explore the association between SAMD4B and APOA2, their expression levels were assessed using single cell RNA sequencing. In DMSO, TC, and TCC groups, the expression level of APOA2 gradually decreased, while the expression level of SAMD4B gradually increased, and there was a significant negative correlation between the two. Then, the expression levels of SAMD4B and APOA2 in the liver cancer queue were studied retrospectively, and the cancer tissues and the paracancerous tissues of 158 liver cancer patients were collected for immunohistochemical staining detection, and it was found that SAMD4B was highly expressed in the paracancerous tissues, and that the prognosis of the patient with the high expression of SAMD4B in the cancer tissues was good, whereas APOA2 was significantly low-level expressed in the paracancerous tissues, and that the prognosis of the patient with the low expression of APOA2 only in the cancer tissues was good, suggesting that SAMD4B might be an oncogene and APOA2 might be an oncogene. Two genes were also examined for correlation with clinical features and it was found that patients with high APOA2 expression levels were more susceptible to metastasis, while patients with high SAMD4B expression levels were less likely to undergo metastasis. There is a significant correlation between the two genes in HCC patients; i.e. patients with high SAMD4B expression are more likely to develop low APOA2 expression. Furthermore, patients with high APOA2 and low SAMD4B expression levels were found to have larger tumors, higher AFP levels and the worst prognosis, while the low APOA2 and high SAMD4B expression level groups had the best prognosis. Thus, TCC has good therapeutic effects because it is capable of activating SAMD4B and inhibiting APOA2. (see FIG. 4).

Experiment 5

Next, the interaction mechanism of SAMD4B with APOA2 was further verified. Transfection of SAMD4B and APOA2 plasmids in HEK293T cells revealed that overexpression of SAMD4B down-regulated APOA2 expression and upregulated APOA2 expression after SAMD4B knockout, indicating that they do have a negative regulatory relationship at the transcriptome level. Furthermore, overexpression of SAMD4B increased the instability of APOA2mRNA, which was reduced by SAMD4B knockdown, consistent with previously reported SAMD4B being able to modulate mRNA instability.

Plasmid transfection of HEK293T cells, control over-expressing APOA2, treatment over-expressing SAMD4B and APOA2 simultaneously, and evaluation of all known RNA modification types, i.e. 2 '-O-methylation, m1I and m6A, by liquid chromatography-mass spectrometry revealed that only the 2' -O-methylation results were consistent with what was expected, i.e. that C-terminal modification increased significantly after over-expression of SAMD4B by the transfected plasmid. Thus, SAMD4B regulates APOA2 through C-terminal 2' -O-methylation.

The 2 '-O-methylation modification of the treated group was then sequenced and was found to occur predominantly in the coding region, and the two 2' -O-methylation modification sites of APOA2 were varied between the control and treated groups. The chr1_161192742_a site is located in the exon region of APOA2, and is significantly changed in the treatment group, so that the site is considered to be the site of 2' -O-methylation of APOA2 by SAMD4B, thereby affecting the instability of APOA2mRNA and reducing the abundance thereof.

TCC can inhibit the expression of the oncogene APOA2 by activating the tumor suppressor SAMD 4B. Ectopic expression of Myc-tagged SAMD4B, flag-tagged APOA2 and HA-tagged immune checkpoints, including PD-L1, PD-L2, FGL1 or HMGB1, and demonstrated that overexpression of SAMD4B could reduce protein levels of APOA2, only PD-L1 was affected by SAMD4B and APOA2 in immune checkpoints expressed in tumors and its expression was positively correlated with APOA2. To verify whether APOA2 directly regulates PD-L1, CO-immunoprecipitation (CO-IP) experiments were performed in HEK293T cells, which found that APOA2 physically bound to PD-L1, indicating that APOA2 regulates PD-L1 by direct interaction. (see FIG. 5).

Experiment 6

Next, unsupervised clustering of T cells and NK cells was performed, and refolding showed 11 cell populations including 2 NK subtypes (NK CD16 and CD 160), 3 cd4+ T cell subtypes (CD 4 SOX5, CD4 LEF1 and Treg), 3 cd8+ T cell clusters (CD 8 TRBC1, CD8 NR4A2 and CD8BRCA 1), plzf+ T cells and others.

Cd8+ T cells were found to be enriched in tumor tissue of the SAMD4B low expression group, and also cd8+ T cells were found to be significantly enriched in the SAMD4B low expression group in the breast and colon cancer dataset, without a difference in the proportion of cd4+ T cells. Next, the dynamic immune status and cellular transformation of liver cancer infiltrating cd8+ T cells was explored using monocles to infer the status trajectories. Pseudo-time analysis showed that cd8+rmp2 cells occurred at the beginning of the trajectory path, while cd8+nr4a2 cells were in the final state. This switch is initiated by cd8+rmp2 cells, eventually reaching a depleted state characterized by cd8+nr4a2 cells, by an intermediate cytotoxic state characterized by cd8+eef1a1, cd8+itgae and cd8+ubash3B cells. It was demonstrated that depletion tags were upregulated, whereas cytotoxic tags did not have a significant downregulation or upregulation trend.

To understand the specific mechanisms of their immunomodulation, 4 patients were selected from the SAMD4B low and high expression groups for single cell RNA sequencing. In addition to epithelial/cancer cells, immune cells, including myeloid-derived cells, T cells, B cells, stromal cells, and NK cells have been identified. The differences in immune cell clusters between the patient and the SAMD4B low/high expression group were found, revealing heterogeneity in immune cell composition in liver cancer patients. Then, we studied transcriptional changes associated with transitional states and observed that cd8+ T cell clusters can be divided into 3 phases: CD8 RRM2 cells are mainly first stage cells characterized by up-regulated CCR8, RRM1, RRM2 and CDC45 expression, while TIGIT, TOX and TOX2 expression are low, suggesting that these cells have minimal cytotoxic capacity. Pathway analysis showed that the p53 signal pathway was enriched in the first phase; the second stage is characterized by an increased expression level of classical cytotoxic Genes (GZMA). Pathway analysis showed that cholesterol metabolism was increased, playing an important role in activation, clonal proliferation and effector function of cd8+ T cells; the third stage is characterized by high levels of T cell depletion associated TF, including TOX, TOX2, ARNT and ETV1, and genes involved in PD-1 and cytotoxic pathways, further confirming the depleted status of these cells. It was found that although the SAMD4B low expressing group had more cd8+ T cells, most of them were naive and therefore unable to play a role in immunomodulation. To further investigate the unique transcriptional status of the SAMD4B low and high expression sets cd8+ T cells, by differential expression gene analysis, ITGB1 (CD 29) was found to be the highest up-regulated gene in cd8+ T cells in SAMD4B low, and ITGB1 (CD 29) expression was significantly positively correlated with PDCD1 in cd8+ T cells and cd8+ naive T cells, suggesting that cd29+cd8+ T cells might be involved in this immunomodulatory pathway. To verify that cancer cells (CK 18), T cells (CD 3, CD4, CD8 and CD 29), SAMD4B, PD1 and PD-L1 were subjected to polychromatic immunofluorescence, indicating that PD-L1, CK18 and SAMD4B co-localize and PD1 co-localize with CD29, CD3 and CD 8. By tissue chip analysis of the retrospective cohort (158 HCC patients), samples under expressing SAMD4B contained more cd29+cd8+ T cells, while the prognosis of high expressing cd29+cd8+ T cells was worse. (see FIG. 6)

The above patients were obtained by collecting clinical data of non-resectable liver cancer patients diagnosed in the auxiliary Zhongshan hospital of the double denier university from 5 months in 2017 to 5 months in 2020, and the clinical data comprises 27 study groups and 65 control groups. The initial treatment with hepatic arterial chemoembolization (TACE) was performed on patients, on which the study group was subjected to TCC treatment, and the clinical profile of the inclusion of patients is illustrated in experiment 1, with only Kaplan-Meier survival analysis data shown in fig. 1a to e, and with a graph of relevant clinical data for significant reduction in tumor volume during TCC treatment in 2 patients in the study group.

Experiment 7

5X 107Hepa1-6 cells were injected subcutaneously into immunized C57BL6/J mice, and after 3 weeks, the mice were euthanized, tumor excised and cut into small pieces, which were then transplanted in situ into the livers of recipient mice (C57 BL 6/J). After 7 days, mice were injected with DMSO, THA, CAR, CAN, TC and TCC therapy, respectively, and the tumor volume of the TCC treatment group was found to be smaller, indicating that the tumor suppression effect of TCC was strongest. And flow cytometry analysis was performed to find that TCC group cd29+cd8+ T cells were the lowest in proportion. Expression of genes involved in regulatory pathways was also examined by immunohistochemistry and it was found that TCC three drug combination therapy upregulated SAMD4B expression and downregulated APOA2, PD-L1, NOTCH1 and NOTCH2 expression.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. The novel application of the synergistic pharmaceutical composition in treating liver cancer is characterized in that the synergistic pharmaceutical composition contains thalidomide, cantharidin and carmofur, and the synergistic pharmaceutical composition is applied in preparing medicines for treating liver cancer of liver cells.

2. The novel use of a synergistic pharmaceutical combination as claimed in claim 1 for the treatment of liver cancer, characterized in that: the application of the synergistic medicine combination in preparing medicines for inhibiting liver cell and liver cancer.

3. The novel use of a synergistic pharmaceutical combination as claimed in claim 1 for the treatment of liver cancer, characterized in that: the synergistic medicine composition is applied to the preparation of medicines for prognosis of embolism by hepatic artery chemotherapy.

4. The novel use of a synergistic pharmaceutical combination as claimed in claim 1 for the treatment of liver cancer, characterized in that: the synergistic medicine composition is applied to the preparation of medicines for inhibiting the immune escape of liver cell and liver cancer tumors.

5. The novel use of a synergistic pharmaceutical combination as claimed in claim 1 for the treatment of liver cancer, characterized in that: the application of the synergistic pharmaceutical composition in preparing a medicine containing a 4B sterile alpha motif domain for regulating the stability of apolipoprotein A II.

6. A synergistic pharmaceutical combination, characterized in that: the synergistic medicine combination contains thalidomide, cantharidin and carmofur, and is an oral medicine formulation.

7. A synergistic pharmaceutical combination as claimed in claim 6, wherein: the mass ratio of thalidomide, cantharidin and carmofur in the synergistic pharmaceutical composition is 0.1:1.5:0.3.

8. A synergistic pharmaceutical combination as claimed in claim 6, wherein: the cantharidin is compound cantharidin capsule.