Use of tlqp-21 in the treatment of pancreatic cancer or pancreatic cancer-depression comorbidity

Abstract

The application relates to the field of biotechnology, and provides application of TLQP-21 in treatment of pancreatic cancer or pancreatic cancer-depression comorbidity. It is disclosed that TLQP-21 has the effect of inhibiting the progression of pancreatic cancer, can improve the depression-like behavior and pain symptoms, provides a new target for drug development of pancreatic cancer and pancreatic cancer-depression comorbidity, and provides a new treatment strategy, is expected to be clinically used, provides an effective treatment method, improves the life quality of pancreatic cancer patients and prolongs the survival time, and provides a new research direction for the treatment of pancreatic cancer and pancreatic cancer-depression comorbidity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, more particularly, to the application of TLQP-21 in treating pancreatic cancer or pancreatic cancer-depression comorbidity. BACKGROUND

[0002] The 5-year survival rate of pancreatic cancer is only 11%, which is one of the malignant tumors with the worst prognosis. Depression is often comorbid with cancer and is associated with poor prognosis, reduced treatment adherence, and increased cancer mortality. Pancreatic cancer is the tumor disease with the highest incidence of depression among digestive system tumors. Surveys have shown that more than 50% of pancreatic cancer patients have mental symptoms that appear 43 months earlier than somatic symptoms. Despite the significant comorbidity of pancreatic cancer and depression, little is known about the comorbidity mechanisms.

[0003] For pancreatic cancer, there are currently two chemotherapy methods, gewcitbinemab-paclitaxel and FOLFIRINO (irinotecan. oxaliplatin. S-fluororacil and leucovorin), however, these two chemotherapy methods are only effective for 30% of patients. Although surgical resection of the tumor is a potential cure, less than 20% of cases are resectable at the time of pancreatic cancer diagnosis, and most patients who undergo surgery still die due to local recurrence and / or metastasis. And for patients with advanced pancreatic cancer, there is still no clear cure.

[0004] Recent studies have found that nerves are an important part of the tumor microenvironment and play an important role in cancer progression. Neurotrophic factors and neuropeptides are important mediators of cancer cell and nerve interactions. VGF is a secreted protein and neuropeptide precursor. There are relatively few reports on the molecular mechanisms by which VGF affects tumor progression. Existing literature suggests that in lung cancer, high expression of VGF can reduce cancer cell apoptosis, while silencing VGF expression reduces tumor growth by enhancing cancer cell apoptosis. In addition, VGF can promote the migration and invasion of lung cancer cell lines. In pancreatic endocrine tumors, VGF promotes the proliferation and metastasis of cancer cells. In glioblastoma, glioblastoma stem cells promote the growth and self-renewal of glioblastoma stem cells by autocrine VGF, while maintaining the survival of differentiated glioblastoma cells through paracrine effects, promoting tumor growth in vivo. However, the role of VGF and its derived peptides in pancreatic cancer has not been reported.

[0005] Vitamin B complex (VGF) is expressed in the central and peripheral nervous systems and pancreatic islets, participating in and regulating energy balance and metabolism, and possessing antidepressant functions. VGF exerts different physiological functions by breaking down into various peptides, such as TLQP-21, TLQP-62, NERP-1, and NERP-2. Among these, TLQP-21 and its receptor are enriched in the central nervous system and pancreas, playing a crucial role in maintaining pancreatic B cell survival, stimulating insulin secretion, and regulating energy metabolism. Researching the impact of TLQP-21 on pancreatic cancer progression and its regulatory mechanisms is beneficial in finding a breakthrough to break the comorbid cycle between pancreatic cancer and depression, which is essential for improving the quality of life and survival time of pancreatic cancer patients. Summary of the Invention

[0006] The present invention aims to overcome at least one of the defects (deficiencies) of the prior art and provide an application of TLQP-21 in the treatment of pancreatic cancer or pancreatic cancer-depression comorbidity, thus providing a new treatment strategy for pancreatic cancer and pancreatic cancer-depression comorbidity.

[0007] One object of this invention is to provide the use of TLQP-21 in the preparation of a medicament for treating pancreatic cancer or pancreatic cancer-depression comorbidity. In one or more embodiments of this invention, the effect of the VGF-derived peptide TLQP-21 on the occurrence and development of pancreatic cancer was investigated for the first time. It was found that, unlike the previously known properties of VGF promoting lung cancer and glioma, the VGF-derived peptide has an inhibitory effect on pancreatic cancer progression, while simultaneously improving its depressive-like behavior and pain symptoms. The amino acid sequence of the TLQP-21 polypeptide is: TLQPPASSRRRHFHHALPPAR (SEQ ID NO.1).

[0008] Furthermore, when TLQP-21 is used in the preparation of a drug for treating pancreatic cancer-depression comorbidity, TLQP-21 alleviates activation of the insular cortex. In one or more embodiments of the present invention, after administration of TLQP-21 to pancreatic cancer model mice, the results showed that activation of the insular cortex was alleviated, and the pain level of the mice was positively correlated with the activity of the insular cortex, indicating that TLQP-21 improves pancreatic cancer-depression comorbidity by acting on pain information mediated by the insular cortex.

[0009] Furthermore, when TLQP-21 is used in the preparation of a drug for treating pancreatic cancer-depression comorbidity, TLQP-21 alleviates astrocyte-mediated neuroinflammatory responses in brain regions including the dorsal vagus nucleus, the area reticularis, and the nucleus tractus solitarius. In one or more embodiments of the present invention, GFAP immunofluorescence staining analysis was performed on 15 brain regions related to mood and pain. The results showed that among the 15 brain regions included in the analysis, GFAP in the area reticularis and the dorsal vagus nucleus was significantly increased in pancreatic cancer model mice; and after injection of TLQP-21 into pancreatic cancer model mice, GFAP expression in the area reticularis, the dorsal vagus nucleus, and the nucleus tractus solitarius was significantly reduced, the inflammatory response of astrocytes was alleviated, thereby reducing susceptibility to depression.

[0010] Furthermore, when TLQP-21 is used in the preparation of drugs for treating pancreatic cancer, TLQP-21 downregulates the expression of differentially expressed genes in pancreatic tumors, including pro-inflammatory cytokine-related genes, genes related to promoting M2 macrophage polarization, angiogenesis-related genes, and genes related to immune infiltration. Further, the pro-inflammatory cytokine-related genes include A2m, Ccl2, Plek, Tsc22d3, Zc3h12a, and Cxcl1; the genes related to promoting M2 macrophage polarization include Per1, Fgl2, Adgre1, Socs3, Cc120, Rel, Herpud1, and Ccl2; the angiogenesis-related genes include Ccl2, E2f2, Icam1, Tlr2, and Cxcl1; and the immune infiltration-related genes include Fgl2, Cdl4, Nlrp3, Syk, and Cxcl1.

[0011] This invention analyzed the cell types in which DEGs were enriched and expressed in the pancreas of a mouse model of pancreatic cancer. It found that the screened DEGs were expressed in various cell types, including ductal cells, metaplastic cells, endocrine cells, endothelial cells, immune cells, pericytes, and fibroblasts, with the highest enrichment level in macrophages. Furthermore, this invention used qPCR to validate the expression of some DEGs, finding that compared to the control group, the mRNA expression levels of Gpr141, Per1, Birc3, Slamf7, Icam1, Adrb2, Ccl20, and A2m in the tumor tissue of the TLQP-21 intervention group were significantly reduced.

[0012] Another object of the present invention is to provide the application of TLQP-21 in the preparation of a drug for inhibiting the migration and growth of pancreatic cancer cells. Further, TLQP-21 inhibits the migration of pancreatic cancer cells by acting on the C1QBP receptor or by acting on TAM cells. In one or more embodiments of the present invention, a co-culture experiment of tumor cell-associated macrophages and pancreatic tumor cells revealed that TLQP-21 can inhibit the growth of pancreatic tumor cells by regulating unknown factors secreted by TAM cells.

[0013] Another object of the present invention is to provide a pharmaceutical composition for treating pancreatic cancer or depression comorbid with pancreatic cancer, comprising TLQP-21. Further, the dosage form of the pharmaceutical composition is an injection.

[0014] Another object of the present invention is to provide the application of TLQP-21 in the preparation of angiogenesis inhibitors, wherein the angiogenesis inhibitor is a drug that inhibits angiogenesis at pancreatic cancer lesions.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention reveals that TLQP-21 has the effect of inhibiting the progression of pancreatic cancer, while improving its depressive-like behavior and pain symptoms. It provides a novel target for the development of drugs for pancreatic cancer and pancreatic cancer-depression comorbidity, and also provides a new treatment strategy. It is expected to be used in clinical practice, providing an effective treatment method, improving the quality of life and prolonging the survival time of pancreatic cancer patients, and also providing a new research direction for the treatment of pancreatic cancer and pancreatic cancer-depression comorbidity. Attached Figure Description

[0016] Figure 1Display: The relationship between changes in VGF mRNA expression levels in pancreatic cancer tumor samples and patient survival time. (AD) Kaplan-Meier survival analysis plot of four neurotrophic factors VGF(A), NGF(B), BDNF(C), and GDNF(D) genes in the GEPIA database and overall survival (OS) in human pancreatic cancer (PDAC) patients (statistical analysis used log-rank test). (EF) Kaplan-Meier survival analysis plot of neurotrophic factor VGF gene and OS in human lung adenocarcinoma (LUAD, E) and glioblastoma multiforme (GBM, F) patients in the GEPIA database (statistical analysis used log-rank test). (G) Expression of neurotrophic factor VGF gene in tumor tissue and adjacent normal tissue of human PDAC patients in the GEPIA database. (H) Distribution of VGF protein expression in human tissues. The data comes from the human proteomics database PreteomicsDB.

[0017] Figure 2 Display: TLQP-21 inhibits tumor growth in orthotopic pancreatic cancer model mice. (A) Schematic diagram of the experimental procedure for the effect of TLQP-21 on tumor progression in orthotopic pancreatic cancer model mice. (B) Quantitative analysis of tumor volume in orthotopic pancreatic cancer model mice after 14 consecutive days of intraperitoneal injection of saline or TLQP-21. (C) Representative IVIS plots of orthotopic pancreatic cancer model mice after 3, 7, and 14 days of intraperitoneal injection of saline or TLQP-21. (D) Average fluorescence quantum number plots at different days measured by IVIS. (E) Total fluorescence quantum number plots at different days measured by IVIS. All data are expressed as mean ± SEM and analyzed by Student's t tests. *p<0.05.

[0018] Figure 3Display: TLQP-21 alleviates pain and depressive behavior in pancreatic cancer model mice. (A) Schematic diagram of the experimental procedure for TLQP-21 affecting the emotional behavior and pain of orthotopically implanted pancreatic cancer model mice; Sham: Mice that underwent sham surgery but did not receive tumor implantation, injected with saline solution daily via intraperitoneal injection; KPC+Saline: Tumor-implanted mice, injected with saline solution daily via intraperitoneal injection; KPC+TLQP-21: Tumor-implanted mice, injected with TLQP-21 (5ug / g / d) daily via intraperitoneal injection; OF: Open field test; SIT: Social interaction test; VFT: Von frey test; TST: Tail suspension test. (B) Changes in body weight of mice every three days after tumor implantation. (C) Tumor volume of the KPC+Saline and KPC+TLQP-21 groups 20 days after tumor implantation, analyzed using Student's t tests, n=9-12. (D) Trajectory representation of the three groups of mice in OF. (E) Total distance traveled by the three groups of mice in OF, analyzed using One-way Anova, n = 10⁻¹². (F) Changes in pain threshold in VFT before and after modeling in the three groups of mice, analyzed using One-way Anova, with multiple comparison correction using Tukey's test, α = 5–6. (G) Immobility time of the three groups of mice in TST, analyzed using One-way Anova, with multiple comparison correction using Kruskal-Wallis test w / uncorrected Dunn's test, n = 10⁻¹². (H) Trajectory representation heatmap of the three groups of mice in SIT. (I) Total exploration time and differentiation index (DI%) of the three groups of mice in SIT, analyzed using One-way Anova, with multiple comparison correction using Fisher's LED test, n = 10⁻¹². All data are expressed as mean = SEM.

[0019] Figure 4The image shows that TLQP-21 downregulates the expression level of c-FOS, an active biomarker in specific brain regions of a pancreatic cancer model mouse. (A) Mouse brain atlases related to 15 brain regions included in the whole-brain c-FOS analysis. In the figure, red represents brain regions where c-FOS expression was increased in the KPC+Saline group compared to the Sham group, and decreased after TLQP-21 administration (KPC+TLQP-21 group). Gray represents brain regions with no significant changes. (B) c-FOS positive cell density (number of c-FOS positive cells / area of ​​target region), fold change of the mean in the three groups (all data are expressed as mean ± SEML and analyzed by Student's ttests. IL: n = 9, 10, 12; PL: n = 8, 10, 12; IC: n = 9, 10, 12; LS: n = 9, 9, 12; BNST: n = 9, 9, 12; MPO: n = 9, 9, 11; RE: n = 9, 10, 12; PVN: n = 9, 10, 12; MH: n = 9, 10, 12; LH: n = 9, 10, 12; dHC: n = 9, 10, 12; ARC: n = 9, 10, 12; AP: n = 9, 10, 12; DMV: n = 9, 10, 12; NTS: n = 9, 9, 12). (C) Representative immunofluorescence staining images of c-FOS expression in the IC brain region of three groups of mice, scale bar = 300 μm. (D) Correlation analysis of pain threshold changes in mice during the VFT experiment and the density of c-FOS-positive neurons in the IC brain region (linear regression and Pearson correlation analysis), n = 15. *p < 0.05***p < 0.0001.

[0020] Figure 5The image shows that TLQP-21 downregulates GFAP expression levels in astrocytes of specific brain regions in pancreatic cancer model mice. (A) Mouse brain atlases related to 15 brain regions included in the whole-brain GFAP analysis. In the figure, red represents brain regions where GFAP expression was increased in the KPC+Saline group compared to the Sham group, and decreased after TLQP-21 administration (KPC+TLQP-21 group). Gray represents brain regions with no significant changes. (B) The proportion of GFAP fluorescence intensity in the target brain region (GFAP fluorescence intensity / target region area), and the fold change of the mean values ​​of the three groups (all data are expressed as mean ± SEML and analyzed by Student's t tests. IL: n = 9, 10, 12; PL: n = 9, 9, 12; IC: n = 10, 9, 11; LS: n = 10, 9, 12; BNST: n = 10, 9, 12; MPO: n = 10, 9, 12; RE: n = 10, 8, 10; PVN: n = 10, 10, 12; MH: n = 10, 8, 11; LH: n = 10, 10, 12; dHC: n = 10, 10, 12; ARC: n = 10, 10, 12; AP: n = 10, 10, 12; DMV: n = 9, 10, 12; NTS: n = 10, 10, 12). (C) Percentage of GFAP fluorescence coverage (GFAP fluorescence coverage area / target area * 100), fold change of the three mean values ​​(all data are expressed as mean ± SEM value, and are verified by Student's...). The tests were analyzed. IL: n = 9, 10, 12; PL: n = 9, 9, 12; IC: n = 10, 9, 11; LS: n = 10, 9, 12; BNST: n = 10, 9, 12; MPO: n = 10, 9, 12; RE: n = 10, 10, 12; PVN: n = 10, 10, 12; MH: n = 10, 8, 11; LH: n = 10, 10, 12; dHC: n = 10, 10, 12; ARC: n = 10, 10, 12; AP: n = 10, 10, 12; DMV: n = 9, 10, 12; NTS: n = 10, 10, 12. (D) Representative immunofluorescence staining images of GFAP expression in the AP, DMV, and NTS brain regions of the three groups of mice, scale bar = 300 μm. *p<0.05**p<0.001***p<0.0001.

[0021] Figure 6The diagram shows the inhibition of pancreatic cancer cell migration by TLQP-21. (A) Schematic diagram of the proliferation (CCK8 assay) and migration (Transwell assay) of tumor cells after TLQP-21 treatment. (B) Effect of TLQP-21 treatment for 48 h on KPC cell proliferation using the CCK8 assay. (C) Effect of TLQP-21 treatment for 48 h on Pan02 cell proliferation using the CCK8 assay. (D) Representative diagram of the effect of TLQP-21 treatment for 18 h on KPC cell migration using the Transwell assay, scale bar = 300 μm. (E) Effect of TLQP-21 treatment for 18 h on KPC cell migration using the Transwell assay. (F) Representative diagram of the effect of TLQP-21 treatment for 18 h on Pan02 cell migration using the Transwell assay, scale bar = 300 μm. (G) Effect of TLQP-21 treatment for 18 h on Pan02 cell migration using the Transwell assay. All data are expressed as mean ± SEML and analyzed using Oue-way ANOVA.

[0022] Figure 7The results show that TLQP-21 inhibits pancreatic cancer cell migration by binding to the C1QBP receptor. (A) Expression of the neurotrophic factor receptor C1QBP gene in tumor tissues (Tumor) and adjacent normal tissues (Normal) of human PDAC patients in the GEPIA database. (B) Expression of the neurotrophic factor receptor C3AR1 gene in tumor tissues (Tumor) and adjacent normal tissues (Normal) of human PDAC patients in the GEPIA database. (C) Correlation analysis plot (linear regression and Pearson correlation analysis) between the expression levels of neurotrophic factor VGF and its receptor C1QBP gene in the GEPIA database. (D) qPCR verification that the receptor C1qbp of TLQP-21 is expressed on tumor cells, with BV2 microglia as a positive control. (E) UMAP plot showing the major cell types in the pancreas of PtfIa-CreER, LSL-Kras-GI2D, LSL-tdTomato (PRT) mice 17 days, 6 weeks, 3 months, 5 months, 9 months, and 15 months after injection of tamoxifen. Data are from a single-cell transcriptome analysis of preinvasive pancreatic lesions and cancer in mice published in 2020 by Yehuda Schlesinger et al. (Schlesinger Y, Yosefov-Levi O, Kolodkin-Gal D, et al. Single-cell transcriptomes of pancreatic preinvasive lesions and Nat Commun, 2020, 11(1):4516). (F) Distribution of C1qbpmRNA in mouse tissues. (G) Distribution of C3ar1 mRNA in mouse tissues. (H) Representative graph of the effect of Transwell migration assay on KPC cell migration after competitive inhibition of 0.1 μM TLQP-21 by 0.1 μM C1qbp mAb, scale bar = 300 μm. (I) Effect of Transwell migration assay on KPC cell migration ability after competitive inhibition of 0.1 μM TLQP-21 by 0.1 μM C1qbp mAb - gray bars represent the Ctrl group. (J) Representative plot of the effect of C1qbppmAb competitive inhibition of TLQP-21 on Pan02 cell migration using Transwell migration assay, scale bar = 300 μm. (K) Effect of C1qbp mAb competitive inhibition of TLQP-21 on Pan02 cell migration ability using Transwell migration assay - gray bars represent the Ctrl group. All data are expressed as mean ± SEML and analyzed by one-way ANOVA. *p < 0.05.

[0023] Figure 8 Display: TLQP-21 regulates gene expression profiles and functional pathways in pancreatic tumor tissue. (A) Schematic diagram of experimental and analytical workflow. An orthotopic pancreatic cancer model was established in mice. Starting from the second day after tumor implantation, mice were continuously injected with TLQP-21 via intraperitoneal injection (IP) (control group injected with saline). Saline: tumor-implanted mice, injected with saline via IP daily; TLQP-21: tumor-implanted mice, injected with TLQP-21 (5ug / g / d) daily. On days 7 and 14 after tumor implantation, mice underwent IVIS to observe the growth of the tumor in vivo. After confirming that TLQP-21 slowed the growth of pancreatic cancer tumors, fresh tumor tissue was harvested on day 15 after tumor implantation, and RNA was extracted from the tumor tissue immediately. (B) Cluster heatmap of differentially expressed genes (DGEs) (DGE screening criteria: p value < 0.01). Scale bar (right): log2 (CPM). (C) DGE volcano plot: fold change and p value of the TLQP-21 group compared to the Saline group. Red: Upregulated expression; Blue: Downregulated expression. (D) UMAP plot showing the major cell types in the pancreas of Ptf1a-CreER, LSL-Kras-G12D, LSL-tdTomato (PRT) mice 17 days, 6 weeks, 3 months, 5 months, 9 months, and 15 months after tamoxifen injection. Data are from a 2020 article by Yehuda Schlesinger et al. on single-cell transcriptomic analysis of pre-invasive lesions and cancers in the mouse pancreas. (E) Distribution of mRNA of all DGEs in mouse tissues. (F) qPCR validation of the fold change in expression of selected DGEs relative to the mean in the Saline group (all data are expressed as mean ± SEM and analyzed by Student's t tests). (G) Gene Set Enrichment Analysis (GSEA), enrichment selection criteria: absolute enrichment score (NES) > 0; false discovery rate (FDR) < 0.25. GO-BP enrichment analysis, GO-CC enrichment analysis, GO-MF enrichment analysis, KEGG enrichment analysis. Red: entries enriched in the Saline group; blue: entries enriched in the TLQP-21 group. Scale: dots represent -log. 10 FDRq value. Color representation: NES.

[0024] Figure 9The diagram shows that TLQP-21 indirectly inhibits pancreatic cancer cell growth by acting on TAM cells. (A) TAM cell sorting flowchart. TAM cells were sorted using FACS, and the sorted TAM cells were then co-cultured with KPC cells using a non-contact Transwell system. (B) TAM cell sorting strategy (CD45) + CD11b + F4 / 80 + (C) Schematic diagram of TAM cells co-cultured with KPCs for 48 h using a non-contact Transwell system (0.4 μm pore size). (D) Representative diagram of KPC cell growth in the lower chamber after 48 h of TAM cell co-culture with KPCs, scale bar = 100 μm. (E) Statistical graph of the number of KPC cells in the lower chamber after 48 h of TAM cell co-culture with KPCs. (F) Schematic diagram of KPC co-cultured with KPCs (control experiment) for 48 h using a non-contact Transwell system (0.4 μm pore size). (G) Representative diagram of KPC cell growth in the lower chamber after 48 h of KPC co-culture with KPCs, scale bar = 100 μm. (H) Statistical graph of the number of KPC cells in the lower chamber after 48 h of KPC co-culture with KPCs. All data are expressed as mean ± SEM and analyzed by Student's t-test. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following (if the specific experimental conditions are not specified in the embodiments, they are usually performed according to conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments can be obtained commercially unless otherwise specified).

[0028] Example 1

[0029] I. Relationship between the expression levels of different neurotrophic factors and survival time of cancer patients

[0030] To explore the potential impact of different neurotrophic factors on pancreatic cancer survival, this embodiment first used the GEPIA database to analyze whether there was an association between the expression of four neurotrophic factors, including VGF, NGF, BDNF, and GDNF, in pancreatic cancer tumor samples and the overall survival of human pancreatic cancer patients. Figure 1 A-1D results showed that patients with high VGF mRNA expression in tumor samples had significantly longer overall survival than patients with low VGF mRNA expression. Figure 1 A), while the mRNA expression levels of NGF, BDNF, and GDNF were not significantly associated with overall survival in human pancreatic cancer patients. Figure 1 B-1D). Previous studies have reported that VGF plays a role in promoting tumorigenesis and development in lung cancer (Hwang W, Chiu YF, Kuo MH, et al. Expression of Neuroendocrine Factor VGF in Lung Cancer Cells Confers Resistance to ECFR Kinase Inhibitors and Triggers Epithelial-to Mesenchymal Transition[J]. Cancer Res, 2017, 77(11):3013-26) and glioma (Wang X, Prager BC, Wu O, et al. Reciprocal Signaling between Glioblastoma stem Cells and Differentiated Tumor Cells Promotes Malignant Progression[J]. Cell Stem Cell, 2018 22(4):514-28e5). To verify the reliability of the GEPIA data, the inventors also queried whether there was a correlation between the expression level of neurotrophic factor VGF mRNA and the overall survival time of human lung adenocarcinoma and glioma patients ( Figure 1 E-1F). Results showed that high expression of VGF mRNA in tumor samples significantly reduced the risk of lung adenocarcinoma in humans (E-1F). Figure 1 E) and glioma ( Figure 1 F) Overall survival time of patients. Notably, there was no significant difference in VGF mRNA expression levels between tumor tissue and adjacent normal tissue in human pancreatic cancer patients. Figure 1G), this embodiment further used the human proteomics database ProteowicsDB to analyze the distribution of VGF protein in human tissues. The results showed that under normal physiological conditions, the expression level of VGF protein was highest in the pancreatic islets. Figure 1 H). This suggests that VGF may play an important physiological role in the pancreas under normal physiological conditions.

[0031] Overall, the data from this study indicate that high VGF expression significantly reduced overall survival in human lung adenocarcinoma and glioma tumor samples, while high VGF expression significantly increased survival in human pancreatic cancer tumor samples.

[0032] II. TLQP-21 inhibits tumor progression in a mouse model of pancreatic cancer.

[0033] To investigate the effect of TLQP-21 on tumor growth, an orthotopic pancreatic cancer mouse model was established in this embodiment. Starting from the second day after tumor implantation, mice were continuously injected intraperitoneally with TLQP-21 (the control group was injected with physiological saline). In vivo imaging (IVIS) was performed on days 3, 7, and 14 after tumor implantation to observe tumor growth. On day 15, mice were sacrificed to measure tumor volume. Figure 2 A). The results showed that, compared with the control solvent injection group, administration of TLQP-21 to pancreatic cancer model mice significantly reduced tumor volume (A). Figure 2 B). IVIS results showed that, compared with the control group, the tumor size in the TLQP-21 intervention group mice did not change significantly on days 3 and 7 after tumor implantation, but on day 14 after tumor implantation, the mean fluorescence quantum number of the tumor in the TLQP-21 intervention group mice ( Figure 2 D) and total fluorescence quantum number ( Figure 2 E) significantly reduced.

[0034] Overall, the above data suggest that the VGF-derived peptide TLQP-21 can inhibit the growth of pancreatic cancer.

[0035] III. TLQP-21 can alleviate depressive-like behavior and pain in pancreatic cancer model mice.

[0036] To investigate the effects of VGF-derived peptide TLQP-21 on emotion-like behavior and pain in a mouse model of pancreatic cancer, this study used an orthotopic implantation model of pancreatic cancer and divided the animals into three groups:

[0037] (1) Animals in the sham surgery group underwent sham surgery but were not implanted with tumors, and received intraperitoneal injections (IP) of saline once a day starting from the second day after surgery.

[0038] (2) Animals in the tumor control (KPC+Saline) group received daily injections of saline starting from the second day after tumor implantation;

[0039] (3) Animals in the experimental (KPC+TLQP-21) group received daily injections of TLQP-21 (5ug / g / d) starting the day after tumor implantation. The sequence of TLQP-21 is as follows:

[0040] Starting from day 14 post-surgery, this embodiment performed a series of behavioral tests on the mice to assess their depressive-like and pain-like behaviors. Mice were perfused and harvested 72 hours after the completion of the behavioral tests. Figure 3 A). The results showed that the body weight of mice in all three groups steadily increased after tumor implantation, with no significant difference between the groups. Figure 3 B). Compared with the KPC+Saline group, administration of TLQP-21 to pancreatic cancer model mice significantly reduced tumor volume (B). Figure 3 C). In the OF test, there was no significant difference in the overall movement distance among the three groups of mice, suggesting that the mice's motor ability was not affected. Figure 3 D and Figure 3 E). In the VFT test, compared with the Sham group, the mechanical threshold of the KPC-Saline group was significantly decreased, indicating that the pancreatic cancer model mice exhibited pain sensitization; while administration of TLQP-21 to the pancreatic cancer model mice significantly increased the mechanical pain threshold and improved the pain sensitization phenomenon in the pancreatic pain model mice. Figure 3 F). In the tail suspension test (TST), compared with the Sham group, the immobility time of animals in the KPC+Saline group was significantly increased, while administration of TLQP-21 significantly reduced the immobility time of pancreatic cancer model mice. Figure 3 G). In the social interaction test (SIT), compared with the Sham group, the KPC-Saline group showed a significantly reduced exploration time to unfamiliar mice, while administration of TLQP-21 significantly increased the exploration time to unfamiliar mice in the pancreatic pain model mice. Figure 3 H and Figure 3 I). There was no significant difference in the total exploration time among the three groups of mice in the SIT test, suggesting that the mice's motor and exploration abilities remained unchanged. Figure 3 H and Figure 4 I).

[0041] Overall, the above experimental results indicate that pancreatic cancer model mice exhibit depressive-like behaviors such as despair and social impairment, and are sensitive to pain. Intraperitoneal injection of TLQP-21 can significantly alleviate depressive-like behaviors and pain in pancreatic cancer model mice.

[0042] IV. Effects of TLQP-21 on the expression level of c-FOS, a marker of functional activity in the brain regions of pancreatic cancer model mice.

[0043] To further explore the neural mechanism by which the VGF-derived peptide TLQP-21 improves pancreatic cancer-depression comorbidity, in this embodiment, 72 hours after the completion of behavioral studies, perfusion tissue was collected from three groups of mice. Frozen sections of mouse brain tissue were prepared, and c-FOS immunofluorescence staining analysis was performed on 15 brain regions related to emotion and pain to screen for brain regions with altered activity. Figure 4 A). c-FOS is a protein encoded by an immediate-early gene and is commonly used as an indicator of neuronal activity. Increased expression levels indicate increased brain region activity (Guzowski JF, Timlin J A, Roysam B, et al. Mapping behaviorally relevant neural circuits with immediate-early gene expression[J]. Curr Opin Neurobiol, 2005, 15(5): 599-606). The results showed that among the 15 brain regions included in the analysis, compared with the un-sham group mice, the density of c-FOS-positive cells in the PL and PL brain regions of the pancreatic KPC cell-implanted and saline-injected (KPC+Saline) group mice was significantly increased (). Compared with the sham group, the density of c-FOS-positive cells in the PL, LS, PVN, LH and dHC brain regions of the pancreatic cancer model mice injected with TLQP-21 (KPC-TLQP21) group mice was significantly increased (). Figure 4 B). Notably, compared to the Sham group, the KPC+Saline group showed a significant increase in c-FOS-positive cell density in the insular cortex (IC), a condition that could be significantly alleviated by TLQP-21 intervention. Figure 4 B and Figure 4 C). According to literature reports, the insular cortex (IC) has functions in regulating mood, pain, motivation, and cognition, and is also the main cortex for receiving and processing visceral sensory information (Gogolla N. The insular cortex D. Curr Biol, 2017, 27(12): R580-R6). Further correlation analysis showed that the change in pain threshold in VFT in pancreatic cancer model mice was significantly negatively correlated with the density of c-FOS positive cells in the IC brain region, suggesting that the higher the pain sensitivity of pancreatic cancer model mice, the stronger the activity of the IC brain region (C). Figure 5 D).

[0044] Overall, the results show that the activation of the IC brain region in pancreatic cancer model mice was alleviated after administration of TLQP-21. Furthermore, the pain level in the mice was positively correlated with the activity of the IC brain region, suggesting that pain information mediated by the IC brain region may be one of the neural mechanisms by which TLQP21 improves the comorbidity of pancreatic cancer and depression.

[0045] V. Effect of TLQP-21 on the expression level of GFAP, an inflammatory marker, in the brain region of astrocytes in a mouse model of pancreatic cancer.

[0046] To further explore the neural mechanism by which the VGF-derived peptide TLQP-21 improves the comorbidity of pancreatic cancer and depression, this study analyzed GFAP immunofluorescence staining in 15 brain regions related to mood and pain. Figure 5 A). GFAP is a biomarker of inflamed astrocytes, and its increased expression often indicates astrocyte-mediated neuroinflammation (Sofironiew MV. Molecular dissection of reactive astrogliosis and glial scarformation[J]Trends Neurosci.2009.32(12):638-47). The results showed that among the 15 brain regions included in the analysis, compared with the Sham group, only the last brainstem region (AP) and DMV brain region showed a significant increase in GFAP (including fluorescence intensity and percentage of fluorescence coverage) in pancreatic cancer model mice (KPC+Saline group); administration of TLQP-21 to pancreatic cancer model mice significantly inhibited the expression of GFAP in the AP and DMV brain regions ( Figure 5 B-5D). Compared with the Sham group, the expression level of GFAP in the nucleus of the solitary tract (NTS) brain region of pancreatic cancer model mice did not change significantly, but administration of TLQP-21 to pancreatic cancer model mice could significantly reduce the expression of GFAP in this brain region. Figure 6 B-5D).

[0047] Overall, the above results indicate that astrocytes in the DVC (DMV, AP, and NTS collectively referred to as the DVC brain region) of pancreatic cancer model mice exhibited an inflammatory response. Administering TLQP-21 to pancreatic cancer model mice could alleviate the inflammatory response of DVC astrocytes, suggesting that inflammatory activation of astrocytes in the DVC brain region may be one of the neural mechanisms by which TLQP-21 improves pancreatic cancer-depression comorbidity.

[0048] VI. Direct effects of TLQP-21 on pancreatic tumor cells

[0049] To investigate the direct effects of TLQP-21 on pancreatic cancer cells, this study treated KPC cells with different concentrations of TLQP-21. The effect of TLQP-21 treatment on the proliferation of pancreatic cancer cells was detected using the CCK8 assay; the effect of TLQP-21 treatment on the migration ability of pancreatic cancer cells was detected using the Transwell assay. Figure 6 A). CCK8 cell proliferation assay results showed that treatment with 0.1-1 uM TLQP-21 did not affect the proliferation ability of KPCs. Figure 6 B). To further verify the above results, this embodiment also examined the effect of TLQP-21 on the proliferation of another pancreatic cancer cell line, Pan02, such as... Figure 6 As shown in Figure C, treatment with 0.1–1 μM TLQP-21 did not affect the proliferation ability of PanO2. Transwell migration experiments showed that the KPC (…) group treated with 0.1–1 μM TLQP-21… Figure 6 D and 6E) or Pan02 ( Figure 7 The number of F and 6G cells passing through the transwell was significantly reduced compared to the control group.

[0050] Overall, the experimental results above indicate that TLQP-21 does not directly inhibit the growth of pancreatic cancer cells, but it can inhibit the migration of pancreatic cancer cells.

[0051] VII. The inhibition of pancreatic cancer cell migration by TLQP-21 depends on its receptor C1QBP.

[0052] TLQP21 mainly has two receptors, C1QBP and C3AR1 (Sahu BS, Nguyen ME, Rodriguez P, et al. The molecular identity of the TLQP-21peptide receptor[J]Cell Mol Life Sci.2021,78(23):7133-44). In this example, the C1QBP in tumor tissue and surrounding normal tissue of human pancreatic cancer patients was first analyzed using the GEPIA database. Figure 7 A) and C3AR1 Figure 7 B) Changes in mRNA expression levels revealed that the mRNA expression levels of both receptors in human pancreatic tumor tissues were significantly higher than in normal tissues; and the expression level of C1QBP was approximately twice that of C3AR1. Correlation analysis of VGF and the expression levels of the two receptors showed a significant positive correlation between C1QBP and VGF expression levels. Figure 7C), while the expression levels of C3AR1 and VGF showed no significant correlation (not shown in the figure). Furthermore, this embodiment analyzed the expression of the above two receptors in mouse pancreatic cancer cells and animal models. The inventors used single-cell transcriptome data on the progression of precancerous lesions and pancreatic cancer in mice published by Schlesiger et al. in 2020 (Schlesinger Y, Yosefov-Levi O, Kolodkin-Gal D, et al. Single-cell transcriptomes pancreatic preinvasive lesions and cancer reveal acinar and plastic cells' heterogeneity D Nat Commun, 2020, 11(1):4516) to analyze the cell types expressing C1qbp and C3ar1 mRNA in the pancreas of mouse pancreatic cancer model mice ( Figure 7 E-7G). UMAP clustering showed that mouse pancreatic cancer tissue contained various cell types, including ductal cells, metaplastic cells, acinar cells, endocrine cells, endothelial cells, immune cells, pericytes, and fibroblasts. C3ar1 mRNA was mainly expressed in macrophages, while C1qbp mRNA was expressed in multiple cell types, including metaplastic cells, suggesting that the target of TLQP-21 for pancreatic cancer cells may be C1QBP (E-7G). Figure 7 To verify the above hypothesis, we first used qPCR to detect whether C1qbp mRNA was expressed in KPC and Pan02 cells, with BV2 microglia as a positive control. The results showed that C1qbp mRNA was expressed in both KPC and Pan02 cells. Figure 7 (D) Then, in this embodiment, a monoclonal neutralizing antibody against the C1QBP receptor was used to block C1QBP signaling to evaluate the role of C1QBP in TLQP-21's inhibition of pancreatic cancer cell migration. Transwell migration assays showed that TLQP21 significantly reduced the migration ability of KPC and Pan02 cells, while blocking C1QBP restored the number of cells crossing the Transwell to the control group level.

[0053] Overall, the above data indicate that TLQP-21 exerts its inhibitory effect on cancer cell migration by acting on the C1QBP receptor on the pancreatic cancer cell membrane. Figure 8 H-7K).

[0054] VIII. Effects of TLQP-21 on gene expression profiles in mouse pancreatic tumors

[0055] To investigate the molecular mechanism by which TLQP-21 slows the progression of pancreatic cancer, this study performed whole-transcriptome sequencing (RNA-Seq) analysis on pancreatic cancer tumors in the TLQP-21 intervention group and the control group. In this study, an orthotopic pancreatic cancer model mouse was established. Starting from the second day after tumor implantation, mice were continuously injected intraperitoneally with TLQP-21 (5ug / g / d), while the control group received saline. On day 15 after tumor implantation, pancreatic tumor RNA was extracted. After RNA quality checks (RNA integrity number, RIN value >7), library construction and RNA-Seq were performed on 3-4 independent samples from each group (1-2 animals were pooled into one independent sample). The RNA-Seq results were then analyzed for differential gene expression and gene set enrichment analysis (GSEA). Figure 8 A). In this embodiment, the DESeq2 package in R language was used to perform DEG analysis on the RNA-Seq results. The screening condition was p-value < 0.01, resulting in 194 differentially expressed genes (DEGs), of which 47 genes were upregulated and 147 genes were downregulated. The inventors used the pheatmap, ggplot2, and ggpubr packages in R language to visualize the DEG analysis results as a clustering heatmap. Figure 8 B) and volcano map ( Figure 8C). Notably, the significantly downregulated DEGs include: pro-inflammatory cytokine-related genes such as A2m, Ccl2, Plek, Tsc22d3, Zc3h12a, and Cxcl1; genes related to promoting M2 type macrocytic polarization such as Per1, Fgl2, Adgre1, Socs3, Cc120, Rel, Herpud1, and Ccl2; angiogenesis-related genes such as Ccl2, E2f2, Icam1, Tlr2, and Cxcl1; and genes related to immune infiltration such as Fgl2, Cdl4, Nlrp3, Syk, and Cxcl1. The inventors used single-cell transcriptome data from a 2020 paper by Schlesinger et al. on the progression of precancerous lesions and cancer in the pancreas of mice (Schlesinger Y, Yosefov-Levi O, Kolodkin-Gal D, et al. Single-cell transcriptomes pancreatic preinvasive lesions and cancer reveal acinar metaplastic cells' heterogeneity[J]Nat Commun,2020,11(1):4516) to analyze the cell types in which DEGs were enriched in the pancreas of a mouse model of pancreatic cancer. Figure 8 D). The results showed that the screened DEGs were expressed in various cell types, including ductal cells, metaplastic cells, endocrine cells, endothelial cells, immune cells, pericytes, and fibroblasts, but were most enriched in macrophages. Figure 8 D and 8E).

[0056] This embodiment further validated the expression of some DEGs using qPCR, and found that compared with the control group, the mRNA expression levels of Gpr141, Per1, Birc3, Slamf7, Icam1, Adr2, Ce120 and 42m in the tumor were significantly reduced in the TLQP-21 intervention group. Figure 9 F).

[0057] In this embodiment, GSEA was used to perform gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the gene expression profiles of the TLQP-21 intervention group and the control group. The GO database standardized the description of gene products from the perspectives of molecular function (GO-MF), biological pathway (GO-BP), and cellular localization (GO-CC) (Reimand, Isserlin R, Voisin V, et al. Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap. Nat Protoc, 2019 14(2):482-517.). KEGG is a database for systematically analyzing gene products in cellular signaling pathways (Reimand J, Isserlin R, Voisin V, et al. Pathway enrichment analysis and visualization of omics data using g:Profiler,GSEA,Cytoscape andEnrichmentMap[J].Nat Protoc,2019 14(2)482-517.). The results showed that after administering TLQP-21 to pancreatic cancer model mice, the enriched entries of genes expressed by pancreatic tumors were mainly related to gene expression regulation processes such as ribosome structure, activation of RNA polymerase and RNA methyltransferase, and RNA metabolism; while the entries with downregulated enrichment in pancreatic cancer model mice were mainly related to immune processes such as neutrophil migration, myeloid leukocyte activation, cellular response to biological stimuli, interaction between cytokines and their receptors, hematopoietic cell lineage, and cell adhesion.

[0058] 9. TLQP-21 indirectly inhibits the growth of pancreatic cancer cells by acting on TAM.

[0059] To further explore the molecular mechanism by which TLQP-21 slows the progression of pancreatic cancer, this embodiment used fluorescence-activated cell sorting (FACS) to sort TAM cells (CD45) from the tumor cells of orthotopically implanted pancreatic cancer model mice (implanted with KPC-GFP-luc cells). + CD11b + F4 / 80 + ()( Figure 9 (A and 9B) The sorted TAM cells were co-cultured with KPC cells. In this example, a non-contact Transwell system was used to co-culture TAM cells with pancreatic cancer cells (KPC) for 48 hours. 1 μM TLQP-21 or control solution was added to both the upper and lower chambers. The number of KPC cells after co-culture was detected using a cell counter. Figure 9 C). The results showed that, compared with the control solution treatment group, the number of KPC cells decreased significantly after TLQP-21 treatment of the TAM-KPC co-culture system for 48 h. Figure 9 (D and 9E), suggesting that TLQP-21 can inhibit KPC cell growth by affecting factors secreted by TAM cells. This example also used a Transwell system to observe the direct effect of TLQP-21 on pancreatic cancer cells. KPC cells were cultured in both the upper and lower chambers, and 1 μM TLQP-21 or control solution was added. After 48 h of culture, the number of KPC cells in the lower chamber was measured (D and 9E). Figure 9 F). The results showed that, compared with the control solution treatment group, there was no significant difference in the number of KPC cells after TLQP-21 treatment of the KPC-KPC co-culture system for 48 h. Figure 6 G and 9H). This result is consistent with... ​ The results from the CCK8 experiment were consistent, suggesting that TLQP-21 does not directly inhibit the growth of KPC cells.

[0060] Overall, the above data indicate that TLQP-21 indirectly inhibits the growth of pancreatic cancer cells by acting on TAM.

[0061] Overall, through analysis of experimental data, we found that TLQP-21 inhibits pancreatic cancer tumor growth and simultaneously improves pain and depression-like symptoms in pancreatic cancer model mice. We also revealed that TLQP-21 inhibits cancer cell migration by acting on the C1QBP receptor on the pancreatic cancer cell membrane and inhibits pancreatic tumor cell growth by acting on TAM cells. Furthermore, TLQP-21 improves pancreatic cancer-depression comorbidity by alleviating activation in the IC brain region and reducing inflammatory responses in astrocytes in the DVC brain region. We also disclosed that TLQP-21 can downregulate some differentially expressed genes. This provides a novel target for drug development in pancreatic cancer and pancreatic cancer-depression comorbidity, and offers new treatment strategies with the potential for clinical use, providing effective treatment methods, improving the quality of life and prolonging survival for pancreatic cancer patients, and providing a new research direction for the treatment of pancreatic cancer and pancreatic cancer-depression comorbidity.

[0062] The TLQP-21 sequence used in the above experiments was: TLQPPASSRRRHFHHALPPAR (SEQ ID NO.1).

[0063] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. Use of TLQP-21 in the preparation of a medicament for treating pancreatic cancer or pancreatic cancer-depression comorbidity, wherein the amino acid sequence of the TLQP-21 is TLQPPASSRRRHFHHALPPAR (SEQ ID NO. 1).

2. Use according to claim 1, characterized in that, When the TLQP-21 is used in the preparation of a medicament for treating pancreatic cancer-depression comorbidity, the TLQP-21 alleviates activation of the insular cortex; when the TLQP-21 is used in the preparation of a medicament for treating pancreatic cancer-depression comorbidity, the TLQP-21 alleviates astrocyte-mediated neuroinflammatory responses in brain regions including the dorsal vagal complex, the area postrema, and the nucleus tractus solitarius.

3. Use according to claim 1, characterized in that, When the TLQP-21 is used in the preparation of a medicament for treating pancreatic cancer, the TLQP-21 down-regulates differentially expressed genes in pancreatic tumors, including pro-inflammatory cytokine-related genes, genes related to promoting M2 macrophage polarization, angiogenesis-related genes, and genes related to immune infiltration.

4. Use according to claim 3, characterized in that, The pro-inflammatory cytokine-related genes include A2m, Ccl2, Plek, Tsc22d3, Zc3h12a, and Cxcl1; the genes related to promoting M2 macrophage polarization include Per1, Fgl2, Adgre1, Socs3, Cc120, Rel, Herpud1, and Ccl2; the angiogenesis-related genes include Ccl2, E2f2, Icam1, Tlr2, and Cxcl1; and the genes related to immune infiltration include Fgl2, Cdl4, Nlrp3, Syk, and Cxcl1.

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