Application of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis

Abstract

The invention discloses application of fibroblast-MMP11 in pancreatic cancer angiogenesis and tumor cell heterogeneity and metastasis, relates to the technical field of biological medicine, and discloses application of fibroblast-MMP11 in pancreatic cancer angiogenesis and tumor cell heterogeneity and metastasis. Preferably, the drug targets the FbMMP11 or the VEGF and NOTCH signal channels activated by the FbMMP11. According to the application of the fibroblast-MMP11 as the pancreatic cancer diagnosis marker, prognosis and metastasis risks of patients are evaluated by detecting the expression level of the FbMMP11. The invention relates to a pharmaceutical composition for inhibiting angiogenesis and metastasis of pancreatic cancer. The pharmaceutical composition comprises an inhibitor targeting FbMMP11 and a VEGF / NOTCH signaling pathway blocker. In the invention, the molecular basis of the FbMMP11 subgroup for promoting tumor matrix fibrosis through high expression of collagen synthesis genes such as COL1A1 and INHBA is defined; secondly, a new mechanism that FbMMP11 promotes tumor angiogenesis by activating VEGF and NOTCH signal pathways in EnCOL15A1 endothelial cells is disclosed.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal prognosis. Its mortality rate remains high among all cancer types and is projected to become the second leading cause of cancer-related death in the future. This dire situation stems from three key factors: First, PDAC is highly insidious, with most patients already at advanced stages at diagnosis, missing the opportunity for surgical cure; second, existing chemotherapy regimens have limited efficacy, while emerging immunotherapies and targeted therapies have limited response rates in PDAC. This unique tumor microenvironment (TME) of PDAC presents multiple therapeutic barriers.

[0003] In-depth studies have shown that the TME of PDAC is characterized by high fibrosis, high immunosuppression, high heterogeneity, and low vascularization. Among them, cancer-associated fibroblasts (CAFs), as the main component of the TME, secrete a large amount of extracellular matrix (ECM) to form a dense fibrotic barrier, which not only restricts drug penetration but also promotes tumor progression through complex intercellular interactions.

[0004] However, existing research on CAFs still leaves significant gaps in understanding: Firstly, the high heterogeneity of CAFs has resulted in their tumor-promoting mechanisms being incompletely elucidated; secondly, the specific molecular mechanisms by which CAFs regulate angiogenesis and immunosuppression in PDAC remain unclear. The dysplasia of tumor blood vessels in PDAC not only affects the nutrient supply of tumor cells but is also closely associated with immune evasion and therapeutic resistance.

[0005] In recent years, the development of single-cell sequencing technologies has provided new opportunities for understanding the cellular composition and functional heterogeneity of the TME. Previous studies have initially revealed the heterogeneity of CAFs in PDAC using single-cell RNA sequencing, but functional studies of specific CAF subpopulations remain elusive. In this context, further investigation into the mechanisms of action of specific CAF subpopulations in PDAC angiogenesis, tumor heterogeneity, and metastasis is not only of great scientific significance but also provides new insights and targets for overcoming current PDAC treatment bottlenecks.

[0006] Therefore, we proposed the application of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis in order to solve the above problems.

[0007] The above information disclosed in this background technology is only for enhancing understanding of the background technology of the present invention and therefore it may contain information that does not constitute the prior art that is already known to a person of ordinary skill in the art. Summary of the Invention

[0008] The purpose of the present invention is to provide an application of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis, so as to solve the problems in the current market raised by the above background technology.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] Fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity, and metastasis.

[0011] Preferentially, drugs target Fb_MMP11 or its activated VEGF and NOTCH signaling pathways.

[0012] The application of fibroblast-MMP11 as a diagnostic marker for pancreatic cancer is to assess the patient's prognosis and metastasis risk by detecting the expression level of Fb_MMP11.

[0013] A pharmaceutical composition for inhibiting pancreatic cancer angiogenesis and metastasis comprises an inhibitor targeting Fb_MMP11 and a VEGF / NOTCH signaling pathway blocker.

[0014] Preferably, the drug reduces angiogenesis and tumor cell metastasis in the tumor microenvironment by inhibiting the expression or activity of Fb_MMP11.

[0015] Preferably, the VEGF / NOTCH signaling pathway blocker is an anti-VEGF antibody or a NOTCH inhibitor.

[0016] Preferably, the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] In this invention, the molecular basis of how the Fb_MMP11 subpopulation promotes tumor matrix fibrosis by overexpressing collagen synthesis genes such as COL1A1 and INHBA is clarified; secondly, a new mechanism is revealed by which Fb_MMP11 promotes tumor angiogenesis by activating VEGF and NOTCH signaling pathways in En_COL15A1 endothelial cells; at the same time, it is found that Fb_MMP11 drives the formation of epithelial cell heterogeneity by regulating 13 key genes such as ITGB6 and COL1A2, and regulates the differentiation process of tumor cells through transcription factors such as ZNF202 and ZSCAN10, which not only provides a new perspective for understanding the mechanism of pancreatic cancer metastasis, but also provides multiple application values ​​for clinical diagnosis and treatment.

[0019] In the present invention, in terms of diagnosis, the expression level of Fb_MMP11 can be used as a reliable marker for assessing patient prognosis and metastasis risk; in terms of treatment, the combined targeting strategy of Fb_MMP11 and its downstream VEGF / NOTCH signaling pathway has shown significant anti-tumor effects, providing a new approach to improve the treatment effect of pancreatic cancer, a difficult-to-treat tumor.

[0020] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the dimensionality reduction clustering result diagram of the Fb_MMP11 subcluster in the present invention; the characteristics and distribution of the Fb_MMP11 subcluster can be observed.

[0022] Figure 2 This is a heat map of angiogenesis-related gene expression in the En_COL15A1 subpopulation of the present invention; it shows the normalized expression levels of a series of tumor angiogenesis, matrix cell proteins, ECM interaction, collagen-related factors, antigen presentation, and actin remodeling genes in endothelial cell subtypes.

[0023] Figure 3 This is the enrichment of angiogenesis-related genes in the second type of patients in the present invention; it shows that in En_COL15A1, angiogenesis-related genes are significantly enriched in the second type of patients, which is consistent with the role of Fb_MMP11 in promoting angiogenesis.

[0024] Figure 4 It is a scatter plot of the correlation between Fb_MMP11, En_COL15A1 and EP_TFF1 in the present invention; it shows the correlation between Ep_TFF1 and Fb_MMP11, Fb_MMP11 and En_COL15A1, and Ep_TFF1 and En_COL15A1.

[0025] Figure 5 This is a Circos diagram of the interaction between Fb_MMP11 and endothelial cells through the VEGF and NOTCH signaling pathways in the present invention; it shows the interaction between fibroblasts and endothelial cell subpopulations in type 2 patients through the VEGF signaling pathway and the NOTCH signaling pathway.

[0026] Figure 6 This is a scatter plot of the correlation between Fb_MMP11 and epithelial subpopulations in the present invention; it shows the correlation between Fb_MMP11 and various epithelial subpopulations.

[0027] Figure 7 Figure 3 is a Venn diagram of the 13 common genes regulated by Fb_MMP11 in the present invention; it shows the 13 common regulated genes affected by Fb_MMP11 ligand in Ep_COL1A1 and Ep_CP.

[0028] Figure 8 The figure is a histogram of GO functional enrichment analysis of 13 genes in the present invention; it shows the Gene Ontology (GO) functional enrichment analysis of 13 co-regulated genes.

[0029] Figure 9 This is a diagram of transcription factor enrichment and regulatory network analysis in the present invention; it shows the transcription factor enrichment related to the co-regulated genes between Ep_COL1A1 and Ep_CP, gene regulatory network analysis, and identified transcription factor binding motifs. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] Example 1: Sample collection and analysis

[0032] Five pancreatic ductal adenocarcinoma (PDAC) single-cell RNA sequencing datasets, GSE154778, GSE155698, GSE197177, GSE212966, and GSE217847, were downloaded from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo). These datasets cover samples from 24 patients, including 18 primary tumor tissues, 7 adjacent normal tissues, and 9 liver metastasis tissues.

[0033] After integrating the data, cells with a mitochondrial gene ratio of less than 25%, a number of RNA features less than 9000, and an RNA count less than 25000 were filtered out for quality control, and finally 172,476 cells were obtained.

[0034] Principal component analysis (PCA) was used to reduce dimensionality, and the Harmony R package was used to reduce the batch effect between patients. Unsupervised clustering was then performed using uniform manifold approximation and projection to divide the data into 36 clusters. Based on known marker gene annotations, the data were integrated into 15 major cell types, including fibroblasts. Figure 1 shown.

[0035] Example 2: Identification and Analysis of Fibroblast-MMP11 Subpopulations

[0036] By dimensionality reduction clustering, cancer-associated fibroblasts were divided into five subclusters, among which the Fb_MMP11 subcluster showed a typical myofibroblast-like cancer-associated fibroblast phenotype, characterized by high expression of collagen synthesis genes (COL1A1, INHBA, COL5A1), and was composed of Figure 1 As shown in Figure 2, this subcluster promotes matrix fibrosis by oversecreting extracellular matrix (ECM) proteins and remodeling enzymes. Figure 2 shown to promote the progression of pancreatic ductal adenocarcinoma.

[0037] Example 3: Study on the mechanism of fibroblast-MMP11 promoting angiogenesis

[0038] Endothelial cell subpopulation analysis: Endothelial cells were clustered by dimensionality reduction and divided into 8 functional subclusters. Functional analysis showed that the En_COL15A1 subpopulation was closely related to tumor angiogenesis, extracellular matrix formation and microenvironment remodeling, and specifically overexpressed pro-angiogenic genes (including FLT4, PDGFB, NRP2 and TIE1). Figure 2 Correlation analysis showed that En_COL15A1 was significantly correlated with Fb_MMP11 fibroblasts. Figure 4 There was no direct correlation with EP_TFF1 epithelial cells, suggesting that fibroblasts are the main drivers of angiogenesis.

[0039] Signaling pathway activation: Cell-cell communication analysis using single-cell RNA sequencing data and the CellChatDB human database revealed that VEGF and NOTCH signaling pathways were strongly activated in tumor tissues. Figure 5 Compared with patients in group 1, patients in group 2 (characterized by the co-enrichment of invasive EP_TFF1 epithelial cells and pro-angiogenic Fb_MMP11 fibroblasts) were significantly enriched for angiogenesis-related pathways (VEGF and NOTCH), as indicated by the presence of Figure 3 This suggests that Fb_MMP11 activates the VEGF and NOTCH signaling pathways in En_COL15A1, thereby increasing tumor angiogenesis.

[0040] Example 4: Fibroblast-MMP11 regulates tumor cell heterogeneity and metastasis mechanism

[0041] Analysis of epithelial cell differentiation trajectories: Temporal trajectory analysis of epithelial cells revealed that the EP_CENPF subpopulation differentiates through three intermediate states: Ep_SCGB3A1, EP_COL1A1, and Ep_CP, into four terminally differentiated cell types, including Ep_TFF1, which is derived from the attached Figure 6 shown.

[0042] Correlation analysis showed that Fb_MMP11 was strongly correlated with transition cells EP_COL1A1 (p=1.24e-80, R=0.93), Ep_CP (p=1.89e-8, R=0.40) and terminal cells EP_TFF1 (p=9.55e-08, R=0.38). Figure 6 Ligand-target regulatory network analysis showed that Fb_MMP11 synergistically regulated 13 genes (ITGB6, COL1A2, CD44, etc.), driving transition cells to differentiate into EP_TFF1. Figure 7 shown.

[0043] Gene function and regulation analysis: Functional enrichment analysis of these 13 target genes showed that the upregulated genes in category 2 patients were enriched in pathways related to cell adhesion and collagen metabolism. Figure 8 Nine transcription factors that may regulate the expression of these common genes in EP_COL1A1 and Ep_CP were predicted using RcisTarget. Gene regulatory network analysis highlighted that the expression changes of transcription factors ZNF202 and ZSCAN10 were the most significant, indicating that they may play a key role in this regulatory process. Figure 9 shown.

[0044] Example 5: Application of fibroblast-MMP11 in the treatment and diagnosis of pancreatic cancer

[0045] By the attached Figure 5 As shown in the results, fibroblast-MMP11 and its related signaling pathways (VEGF and NOTCH) can be used as potential targets for the treatment of pancreatic cancer, and inhibitors targeting Fb_MMP11 or its activated signaling pathways can be developed to inhibit tumor angiogenesis and tumor cell metastasis.

[0046] By the attached Figure 1 、 4 As shown, detecting the expression level of Fb_MMP11 in patient tumor tissue can be used as a diagnostic marker to assess the prognosis and metastasis risk of pancreatic cancer patients. High expression of Fb_MMP11 may indicate a poor prognosis and a higher risk of metastasis.

[0047] By the attached Figure 2 、 3 As shown in Figures 5 and 5, based on the mechanism of action of fibroblast-MMP11, new treatment methods can be developed, such as the combined use of drugs that inhibit Fb_MMP11 activity and block VEGF and NOTCH signaling pathways to improve the treatment effect of pancreatic cancer.

[0048] In summary, this invention clarifies for the first time the key role of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis, providing a new perspective for a deeper understanding of the pathogenesis of pancreatic cancer.

[0049] Fibroblast-MMP11 and its related signaling pathways can serve as potential targets for pancreatic cancer treatment, and their expression levels can be used as diagnostic markers to help achieve accurate diagnosis and personalized treatment of pancreatic cancer.

[0050] Based on the mechanism of action of fibroblast-MMP11, it is expected that more effective pancreatic cancer treatments can be developed, providing a new way to improve the prognosis of pancreatic cancer patients.

[0051] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0052] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Application of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis.

2. The use of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis according to claim 1, characterized in that: Drugs target Fb_MMP11 or its activated VEGF and NOTCH signaling pathways.

3. Application of fibroblast-MMP11 as a diagnostic marker for pancreatic cancer, characterized in that: The prognosis and metastasis risk of patients were evaluated by detecting the expression level of Fb_MMP11.

4. A pharmaceutical composition for inhibiting angiogenesis and metastasis of pancreatic cancer, characterized in that: Contains inhibitors targeting Fb_MMP11 and VEGF / NOTCH signaling pathway blockers.

5. The use of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis according to claim 1, characterized in that: The drug reduces angiogenesis and tumor cell metastasis in the tumor microenvironment by inhibiting the expression or activity of Fb_MMP11.

6. The pharmaceutical composition according to claim 4, characterized in that: The VEGF / NOTCH signaling pathway blocker is an anti-VEGF antibody or a NOTCH inhibitor.

7. The use of fibroblast-MMP11 in pancreatic cancer angiogenesis, tumor cell heterogeneity and metastasis according to claim 1, characterized in that: Pancreatic cancer is pancreatic ductal adenocarcinoma.

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