Application of TDO2 inhibitor in preparation of medicine for treating skin squamous cell carcinoma

Abstract

The invention relates to application of a TDO2 inhibitor in preparation of a medicine for treating skin squamous cell carcinoma. Compared with the prior art, the invention not only discloses the specific action mechanism of TDO2 in cSCC, but also provides a new targeted treatment strategy, and is expected to obviously improve the treatment effect of cSCC.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of a TDO2 inhibitor in the preparation of a drug for treating skin squamous cell carcinoma. Background Art

[0002] Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer after basal cell carcinoma, and it is mainly manifested as malignant proliferation of squamous epithelial cells. cSCC usually occurs in exposed parts of the elderly, is highly invasive, and may metastasize in the early stages. According to the Global Cancer Burden Report, the incidence of non-melanoma skin cancer (NMSC) ranks first in the world. Despite its relatively low incidence in the yellow race, cSCC still ranks sixth in the incidence of skin cancer, with as many as 1.2 to 1.8 million new cases worldwide each year. Early treatment of cSCC mainly relies on surgical resection, but surgery is often destructive and prone to recurrence, and treatment after recurrence becomes more complicated and difficult. For multiple or advanced cSCC, surgery is usually not feasible, and the disease does not respond well to radiotherapy and chemotherapy. According to the latest study in the New England Journal of Medicine, the complete remission rate of advanced cSCC treated with immune checkpoint inhibitor PD-1 antibody is only 51%, which means that many patients with advanced cSCC find it difficult to find effective treatments. Although some targeted therapies, such as epidermal growth factor receptor (EGFR) inhibitors, have entered the clinical trial stage, their efficacy in cSCC may be limited based on their performance in the treatment of other tumors. Therefore, in-depth research on the pathogenesis of cSCC and the discovery of new, efficient, safe, and targeted therapeutic targets are imperative to optimize the treatment strategy for cSCC. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide an application of a TDO2 inhibitor in the preparation of a drug for treating cutaneous squamous cell carcinoma.

[0004] The present invention provides an application of a TDO2 inhibitor in the preparation of a drug for preventing and / or treating cutaneous squamous cell carcinoma (cSCC).

[0005] Use of the TDO2 inhibitor in the preparation of targeted drugs for preventing and / or treating cutaneous squamous cell carcinoma (cSCC).

[0006] The present invention provides an application of a TDO2 inhibitor in preparing a drug for inhibiting the growth and metastasis of cutaneous squamous cell carcinoma cSCC.

[0007] The present invention provides an application of a TDO2 inhibitor in the preparation of an anti-tumor immune response drug.

[0008] The application of the TDO2 inhibitor in the preparation of a drug for resisting tumor immune response of cutaneous squamous cell carcinoma cSCC.

[0009] The TDO2 inhibitors include agents that inhibit the expression or activity of TDO2.

[0010] The medicine also includes single-ingredient preparations or compound preparations.

[0011] TDO2: Tryptophan 2,3-Dioxygenase (TDO2).

[0012] The present invention has carried out:

[0013] 1. Data analysis and verification:

[0014] The public dataset GSE193304 was used for scRNA-seq analysis to determine the expression of TDO2 in various cell subsets of cSCC.

[0015] In situ hybridization was used to verify the expression and cellular localization of TDO2 in cSCC tissues.

[0016] Immunofluorescence and WB experiments were used to detect the expression of TDO2 in CAFs.

[0017] 2. Clinical specimen analysis:

[0018] IHC and mIF staining were performed to analyze the relationship between TDO2 expression and immune cell infiltration in cSCC clinical specimens.

[0019] 3. Mouse model experiments:

[0020] A UV-induced cSCC mouse model was established and TDO2 inhibitor intervention was performed.

[0021] FCM and IHC were used to detect the infiltration of immune cells in mouse tumor tissues.

[0022] 4. Gene expression and pathway analysis:

[0023] RNA-seq was performed on cSCC tissues of mice treated with TDO2 inhibitors, combined with GO, KEGG, and GSEA analyses to identify the key genes and pathways of TDO2-induced immune escape.

[0024] Based on the key role of TDO2 in tumor immune escape, the present invention proposes to enhance CD8+T cell infiltration in cSCC by inhibiting TDO2, thereby improving the effectiveness of anti-tumor immune response. TDO2 is highly expressed in CAFs, and by regulating immune-related pathways, it inhibits the killing function of CD8+T cells and mediates immune escape in the tumor microenvironment. By inhibiting TDO2, this process can be reversed, the infiltration and killing of CD8+T cells in tumors can be increased, and the progression of tumors can be inhibited.

[0025] The present invention provides an application of a TDO2 inhibitor in the preparation of a drug for treating cutaneous squamous cell carcinoma (cSCC), which shows that TDO2 is highly expressed in cancer-associated fibroblasts (CAFs) of cSCC, and this high expression is closely related to the immune escape mechanism of the tumor. The present invention proposes that by inhibiting the expression or activity of TDO2, the infiltration of CD8+T cells in the tumor microenvironment can be significantly increased, and their killing function can be enhanced, thereby effectively inhibiting the growth and metastasis of cSCC, providing a new direction and molecular target for the targeted treatment of patients with cutaneous squamous cell carcinoma.

[0026] The TDO2 inhibitor of the present invention is a small molecule inhibitor, which can intervene in the activity of TDO2, block the immune escape induced by TDO2, and restore the immune system's ability to recognize and attack tumor cells.

[0027] In the present study, TDO2 inhibitors were applied to a skin squamous cell carcinoma model, and the results showed that TDO2 inhibitors could significantly reduce tumor volume and number and increase CD8+T cell infiltration. In addition, TDO2 inhibitors could also inhibit tumor progression by regulating related immune pathways.

[0028] The TDO2 inhibitor of the present invention can be used to prepare drugs for treating cSCC, which can significantly improve the therapeutic effect. The treatment method provides a new treatment option for patients with advanced or refractory cSCC and has broad clinical application prospects.

[0029] The present invention effectively solves the immune escape problem existing in existing treatment methods by inhibiting the expression and function of TDO2 in cutaneous squamous cell carcinoma, and provides a new targeting strategy and clinical application method for the treatment of cSCC.

[0030] Beneficial Effects

[0031] The present invention proposes a treatment method for enhancing anti-tumor immune response by inhibiting TDO2. Compared with the prior art, the present invention not only reveals the specific mechanism of action of TDO2 in cSCC, but also provides a new targeted treatment strategy, which is expected to significantly improve the treatment effect of cSCC.

[0032] 1. Improve treatment effect:

[0033] By inhibiting TDO2 expression, the present invention can significantly increase the infiltration of CD8+T cells in cSCC tissues, thereby enhancing the anti-tumor immune response, improving the therapeutic effect, and reducing the risk of tumor recurrence and metastasis.

[0034] 2. Identify therapeutic targets:

[0035] The study found that TDO2 is highly expressed in cSCC and plays a key role in immune escape, which provides a clear target for the targeted treatment of cSCC and has important scientific significance and clinical application value.

[0036] 3. Reduce Side Effects:

[0037] Traditional surgery, radiotherapy and chemotherapy often have many side effects. By targeting and inhibiting TDO2, we can reduce damage to normal cells, reduce unnecessary side effects during treatment, and improve the quality of life of patients.

[0038] 4. Innovative treatment strategies:

[0039] The treatment method based on TDO2 inhibition proposed in the present invention is an innovative strategy in the field of immunotherapy and is expected to provide new ideas and methods for the treatment of other types of tumors.

[0040] 5. Combination of scientific research and clinical application:

[0041] The present invention systematically studied the expression and mechanism of TDO2 in cSCC through multiple technical means such as single-cell transcriptome sequencing, in situ hybridization, and immunohistochemistry, achieving a close integration of scientific research and clinical application.

[0042] 6. Economic benefits:

[0043] Improving the treatment effect of cSCC and reducing recurrence and metastasis will greatly reduce medical costs and the economic burden on patients, with significant economic benefits.

[0044] 7. Social benefits:

[0045] By improving the therapeutic effect of cSCC and the quality of life of patients, the present invention helps to reduce the social burden caused by skin cancer, improve the public health level, and has important social benefits.

[0046] In summary, the present invention is not only of great significance in scientific research, but also has significant advantages in clinical application, and is expected to provide a new, efficient and safe method for the treatment of cSCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1Middle (A-D) are diagrams of analyzing TDO2 expression using single-cell transcriptome public data in the present invention;

[0048] Figure 2 This is a diagram of the co-localization of TDO2 and ACTA2 in cutaneous squamous cell carcinoma (cSCC) verified by in situ hybridization in the present invention;

[0049] Figure 3 This is a diagram of the co-localization of TDO2 and α-SMA verified by cell immunofluorescence in the present invention;

[0050] Figure 4 It is a diagram showing that TDO2 is highly expressed in primary cancer-associated fibroblasts (CAFs) verified by Western blotting in the present invention;

[0051] Figure 5 It is a diagram showing the correlation between TDO2+ cells and CD4+T cells, CD8+T cells, FOXP3+ cells and CD206+ cells analyzed by immunohistochemistry in the present invention;

[0052] Figure 6 Representative multiple immunofluorescence (mIHC) images of co-localization of TDO2+ cells and CD8+ T cells in the present invention; Figure 7 The present invention is a diagram of the inhibition of mouse tumor growth by intervening TDO2; wherein (A) is a timeline of mouse model construction and drug intervention; (B) is a representative schematic diagram of the tumor formation process of mice in the control group, TDO2 inhibitor group (TDO2i) and aryl hydrocarbon receptor inhibitor group (AHRi); (C) is a curve chart of the changes in tumor volume of mice in different treatment groups over time; (D) is a statistical chart of the changes in the number of tumors in mice in different treatment groups over time;

[0053] Figure 8 Graphs showing the detection of immune cell infiltration by flow cytometry in the present invention; wherein (A) is a representative scatter plot of flow cytometry of CD4+T cells and CD8+T cells expressed in CD45+CD3+ living cells in mice in the control group and the TDO2 inhibitor group; (B) on the left is a statistical graph of the percentage of CD8+T cells in CD45+ cells, showing that the proportion of CD8+T cells in the TDO2 inhibitor group increased significantly; on the right is a statistical graph of the proportion of CD4+T cells in CD45+ cells, showing no significant difference;

[0054] Fig. 9The figure is a diagram of the effect of TDO2 inhibitor on tumor tissue detected by immunohistochemistry in the present invention; wherein (A) is the result of CD8 immunohistochemistry staining, showing the comparison of the number of CD8+T cells in tumor tissue between the control group and the TDO2 inhibitor group; (B) is the result of Ki67 immunohistochemistry staining, showing the comparison of the number of Ki67+ cells in tumor tissue between the control group and the TDO2 inhibitor group;

[0055] Fig.10 This is the Gene Ontology (GO) functional annotation analysis diagram in the present invention;

[0056] Fig.11 It is the KEGG (Kyoto Encyclopedia of Genes and Genomes) functional annotation analysis diagram in the present invention;

[0057] Fig.12 This is a functional enrichment analysis diagram of the gene set enrichment analysis (GSEA) in the present invention. DETAILED DESCRIPTION

[0058] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0059] Unless otherwise specified, the various chemical reagents and chemicals mentioned in the present invention are all known and commonly used chemical reagents and chemicals in the prior art.

[0060] Example 1

[0061] 1. Expression analysis of TDO2 in cSCC:

[0062] Single-cell RNA sequencing (scRNA-seq): The public dataset GSE193304 was used to analyze the single-cell transcriptome data of cSCC in the Chinese population to determine the expression of TDO2 in various cell subpopulations of cSCC.

[0063] In situ hybridization (ISH): In cSCC tissues, the cellular localization of TDO2 was verified by in situ hybridization to identify the main cell types with high expression.

[0064] Primary cell separation and detection: Primary cell separation was used to isolate cancer-associated fibroblasts (CAFs) from cSCC patient specimens, and the expression of TDO2 was detected by cell immunofluorescence (IF) and Western Blot (WB) experiments.

[0065] 2. Correlation between TDO2 expression and immune cell infiltration in clinical specimens:

[0066] Immunohistochemistry (IHC): cSCC clinical specimens were collected and immunohistochemical staining of TDO2 was performed to analyze the correlation between its expression level and clinical pathological characteristics.

[0067] Multiplex immunofluorescence staining (mIF): further analyze the correlation between TDO2+ cells and immune cells such as CD4+T cells, CD8+T cells, Treg cells, and M2 macrophages, and explore the relationship between TDO2 and immune cell infiltration.

[0068] 3. Experiments of TDO2 inhibitors in cSCC mouse models:

[0069] Ultraviolet-induced cSCC mouse model: A UV-induced cSCC mouse model was established, and the effect of inhibiting TDO2 on tumor formation in mice was observed through TDO2 inhibitor intervention experiments.

[0070] Immune cell infiltration detection: Flow cytometry (FCM) and immunohistochemistry were used to analyze the infiltration of immune cells in mouse cSCC tissues after TDO2 inhibitor intervention, especially the infiltration changes of CD8+T cells.

[0071] 4. Gene expression and pathway analysis:

[0072] RNA sequencing (RNA-seq): RNA sequencing was performed on cSCC tissues of mice treated with TDO2 inhibitors, combined with Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and Gene Set Enrichment Analysis (GSEA) to explore the key genes and pathways of TDO2-induced cSCC immune escape.

[0073] Example 2

[0074] The expression of TDO2 in cSCC was analyzed using public single-cell transcriptome data. Cell clustering and gene expression displayed using the UMAP algorithm: Figure 1 As shown: (A) Cell clustering diagram displayed by the UMAP algorithm, different colors represent different cell subpopulations, and a total of 15 subpopulations were identified; (B) COL1A1 gene expression displayed by the UMAP algorithm, and the color depth represents the relative expression intensity of COL1A1; (C) COL1A2 gene expression displayed by the UMAP algorithm, and the color depth also represents the relative expression intensity of COL1A2; (D) TDO2 gene expression displayed by the UMAP algorithm.

[0075] Example 3

[0076] To further verify the localization of TDO2 in cSCC, the co-localization of TDO2 and ACTA2 in cSCC tissues was verified by in situ hybridization. Figure 2 Shown: This figure shows the co-localization of TDO2 (red) and ACTA2 (green) in cSCC tissue samples. DAPI staining (blue) is used to mark cell nuclei. The merged image reveals the overlap in the spatial distribution of TDO2 and ACTA2; scale bar = 15 μm).

[0077] Example 4

[0078] To verify the co-localization of TDO2 and α-SMA, a cell immunofluorescence experiment was performed. Figure 3 Shown: This figure shows the cell immunofluorescence image of primary CAFs, in which the co-localization of TDO2 (red) and α-SMA (green) further confirmed their common high expression in CAFs, and DAPI staining (blue) was used to mark the cell nucleus; scale bar = 60 μm.

[0079] Example 5

[0080] Western blotting was performed to analyze the expression level of TDO2 in primary cancer-associated fibroblasts (CAFs). Figure 4 As shown: the upper band in the figure represents TDO2 (about 48 kDa), and the lower band represents GAPDH (about 37 kDa).

[0081] Example 6

[0082] To study the correlation between TDO2 and different immune cells, the correlation between TDO2+ cells and CD4+T cells, CD8+T cells, FOXP3+ cells and CD206+ cells was analyzed by immunohistochemistry. Figure 5 Shown: The figure shows the correlation scatter plot and regression analysis between the number of TDO2+ cells and the number of CD4+T cells, CD8+T cells, FOXP3+ cells, and CD206+ cells.

[0083] Example 7

[0084] The co-localization of TDO2+ cells and CD8+ T cells in cSCC tissues was further analyzed by multiplex immunofluorescence (mIHC) experiments. Representative mIHC images of TDO2+ cells and CD8+ T cells, such as Figure 6 Shown: Immunofluorescence staining of TDO2 (green) and CD8 (orange) in cSCC patient tissue samples. The left image represents the TDO2 high expression area, the right image represents the TDO2 low expression area, and DAPI (blue) is used for nuclear staining. Scale bar = 50 μm.

[0085] Example 8

[0086] To verify the inhibitory effect of TDO2 inhibitor on cSCC tumor growth, a mouse model experiment was conducted. Illustration of the inhibition of mouse tumor growth by intervening TDO2, (A) is the timeline of mouse model construction and drug intervention; (B) is a representative schematic diagram of the tumor formation process of mice in the control group, TDO2 inhibitor group (TDO2i) and aryl hydrocarbon receptor inhibitor group (AHRi); (C) is a curve chart of the changes in tumor volume over time in mice in different treatment groups; (D) is a statistical chart of the changes in the number of tumors in mice in different treatment groups over time, such as Figure 7 As shown: (A) Mouse model construction and drug intervention timeline: 6-week-old SKH-1 mice were irradiated with UV light until about 30 weeks of age, then irradiation was stopped and drug intervention was performed. All mice were killed after 4 weeks of observation; (B) Representative schematic diagram of the tumor formation process of mice in the control group, TDO2i group, and AHRi group; (C) Curves of changes in tumor volume over time in mice in different treatment groups; (D) Statistics of changes in the number of tumors in mice in different treatment groups over time.

[0087] Example 9

[0088] To analyze the effect of TDO2 inhibition on immune cell infiltration in mice, flow cytometry was used to detect immune cells in tumor tissues of mice in different treatment groups. Figure 8Shown: (A) is a representative scatter plot of flow cytometry of CD4+T cells and CD8+T cells expressed in CD45+CD3+ living cells in mice in the control group and TDO2 inhibitor group; (B) on the left is a statistical chart of the percentage of CD8+T cells in CD45+ cells.

[0089] Example 10

[0090] The specific effects of TDO2 inhibitors on mouse tumor tissues were analyzed by immunohistochemistry. The effects of TDO2 inhibitors on tumors were detected by immunohistochemistry, such as Fig. 9 Shown are: (A) CD8 immunohistochemical staining results, showing the comparison of the number of CD8+T cells in tumor tissues between the control group and the TDO2 inhibitor group; (B) Ki67 immunohistochemical staining results, showing the comparison of the number of Ki67+ cells in tumor tissues between the control group and the TDO2 inhibitor group.

[0091] Embodiment 11

[0092] To analyze the potential function of TDO2 in cSCC, Gene Ontology (GO) functional annotation analysis was performed. Fig.10 Figure 1 shows the results of GO enrichment analysis. The size of the dots represents the number of genes involved in each process, and the color depth indicates the significance of the P value, from blue (least significant) to red (most significant).

[0093] Example 12

[0094] To further clarify the signaling pathway function of TDO2 in cSCC, Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analysis was performed. The KEGG functional annotation is shown in the figure. Fig.11 Shown: The bar chart shows the pathways with the greatest impact after KEGG pathway enrichment analysis. The length of the bar represents the percentage of genes, and the color represents different biological process categories.

[0095] Embodiment 13

[0096] To explore the correlation between TDO2 and other gene sets in cSCC, gene set enrichment analysis (GSEA) was performed. Fig.12 Shown: These line graphs show the enrichment of Th1 and Th2 cell differentiation and TNF signaling pathways based on GSEA. The normalized enrichment score (NES), P value and false discovery rate (FDR) provide the statistical significance of enrichment for each set.

[0097] In summary, the present invention shows that TDO2, as a potential oncogene, can promote the proliferation, migration and invasion of tumor cells through activation of related signaling pathways and functional enrichment, providing a new direction and molecular target for the targeted treatment of patients with cutaneous squamous cell carcinoma.

[0098] Embodiment 14

[0099] Application of TDO2 inhibitors in the preparation of drugs for treating cutaneous squamous cell carcinoma (cSCC). TDO2 inhibitors include but are not limited to molecules that have an inhibitory effect on TDO2 expression and / or function.

Claims

1. Use of a TDO2 inhibitor in the preparation of a drug for preventing and / or treating cutaneous squamous cell carcinoma.

2. The application according to claim 1, characterized in that: Use of the TDO2 inhibitor in the preparation of targeted drugs for preventing and / or treating skin squamous cell carcinoma.

3. Use of a TDO2 inhibitor in the preparation of drugs for inhibiting the growth and metastasis of cutaneous squamous cell carcinoma.

4. Application of a TDO2 inhibitor in the preparation of anti-tumor immune response drugs.

5. The application according to claim 4, characterized in that: Application of the TDO2 inhibitor in the preparation of an anti-tumor immune response drug for skin squamous cell carcinoma.

6. The use according to any one of claims 1 to 5, characterized in that: The TDO2 inhibitors include agents that inhibit the expression or activity of TDO2.

7. The use according to any one of claims 1 to 5, characterized in that: The medicine also includes single-ingredient preparations or compound preparations.

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