Use of DPP4 as NK / t cell lymphoma tumor marker

Abstract

Provided is the use of DPP4 as an NK / T cell lymphoma tumor marker, the use of a DPP4 detection reagent in preparing a kit for screening for NK / T cell lymphoma, and the use of a DPP4 inhibitor in preparing a medicine for treating NK / T cell lymphoma.

Description

Application of DPP4 as a tumor marker for NK / T cell lymphoma Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to the application of DPP4 as a tumor marker for NK / T cell lymphoma. Technical Background

[0002] NK / T-cell lymphoma is a type of non-Hodgkin's lymphoma with a low incidence but rapidly progressive malignancy. Its onset is strongly correlated with Epstein-Barr virus infection, and the vast majority of patients present with lesions invading the nasal cavity or nasopharynx. Clinically, elevated P-glycoprotein expression in NK / T-cell lymphoma tumor cells contributes to resistance to traditional anthracycline-based lymphoma chemotherapy regimens (such as the CHOP regimen), necessitating the use of multidrug combinations incorporating non-anthracycline agents (levoaspartase or pegaspargase) and concurrent / sequential radiotherapy. These discoveries and improvements have significantly improved the clinical efficacy of NK / T-cell lymphoma, with 5-year overall survival rates reaching 70% or higher for early-stage patients. Despite this, patients with advanced, relapsed, and refractory NK / T-cell lymphoma, representing approximately 30%, continue to have a poor prognosis, making treatment options more limited and challenging. In recent years, with the continuous improvement of cancer treatment technology, new targeted and immunotherapy regimens (such as immune checkpoint inhibitors and histone deacetylase inhibitors) have also been tried in the treatment of NK / T cell lymphoma patients and have achieved certain results. However, the efficacy of new treatment regimens is not stable, and the degree of treatment response varies greatly among patients. Therefore, this suggests that NK / T cell lymphoma patients have a high degree of heterogeneity, and there is an urgent need to find new therapeutic targets and design new precision treatment strategies to address this heterogeneity.

[0003] The heterogeneity of NK / T-cell lymphomas is primarily characterized by the following aspects: First, in terms of the type of tumor cell origin, NK / T-cell lymphomas are categorized as either NK-cell or T-cell-derived. T-cell-derived patients account for only a small proportion of patients, and the transformation from normal lymphocytes to NK / T-cell lymphoma tumor cells results in the formation of diverse cell morphologies ranging from small to large. Regarding genetic background, our previous studies have identified sequence alterations in genetic loci located in genes such as HLA-II and IL18RAP. These alterations in genetic loci contribute to susceptibility to NK / T-cell lymphoma, thus enabling genetic stratification of the population into more susceptible and less susceptible groups. At the molecular level, diverse teams, both domestic and international, have analyzed DNA and RNA sequencing data using high-throughput sequencing, identifying specific somatic mutations and transcriptional dysregulations characteristic of NK / T-cell lymphomas. This information can be used to molecularly stratify patients and design appropriate treatment strategies. The above studies have revealed the high heterogeneity among NK / T cell lymphoma patients from different levels. However, these findings are still insufficient to effectively explain the pathogenic mechanism and tumor progression mechanism of NK / T cell lymphoma.

[0004] In addition to tumor cells, tumor tissues also harbor a large number of infiltrating immune cells, which together with tumor cells constitute a heterogeneous tumor microenvironment. In recent years, with the widespread adoption of single-cell transcriptome sequencing technology, numerous studies have explored the heterogeneous immune and stromal cell components within the tumor microenvironment of various cancers. Combined with other approaches, these studies have revealed the associations between these immune microenvironmental components and tumor progression, as well as patient treatment responses. Previous studies have reported the presence of a large number of immune cells infiltrating NK / T cell lymphoma tissues. However, the specific composition of these immune microenvironmental components and their association with NK / T cell lymphoma development and progression remain unknown. Therefore, it is urgent to identify key molecules, key cell populations, and cell-cell interactions that are closely associated with tumor development and progression from the perspective of the tumor microenvironment. These could serve as novel and effective tumor markers and therapeutic targets for NK / T cell lymphoma, ultimately improving patient survival.

[0005] Summary of the Invention

[0006] In view of the defects of the prior art, the purpose of the present invention is to provide a new tumor marker for NK / T cell lymphoma and its application to solve the problems raised in the above background technology.

[0007] The present invention provides the use of DPP4 (dipeptidyl peptidase 4) as a tumor marker for NK / T cell lymphoma.

[0008] On the other hand, the present invention also provides use of a DPP4 detection reagent in preparing a kit for screening NK / T cell lymphoma.

[0009] Preferably, the kit comprises the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0010] On the other hand, the present invention also provides use of a DPP4 inhibitor in the preparation of a medicament for treating NK / T cell lymphoma.

[0011] Preferably, the DPP4 inhibitor includes one or more of linagliptin, alogliptin, anagliptin, begoloizumab, gemagliptin, alogliptin, saxagliptin, sitagliptin, trelagliptin, and vildagliptin.

[0012] The expression level of DPP4 in various cell types in the peripheral blood and tumor tissues of NK / T cell lymphoma patients was detected by single-cell transcriptome sequencing, and it was found that DPP4 was significantly highly expressed in tumor cells. The expression of DPP4 in NK / T cell lymphoma tumor cells can be detected by immunofluorescence co-staining of paraffin-embedded wax blocks of tumor tissue, and it was found that DPP4 is widely and abundantly expressed in tumor cells. The present invention also found that DPP4 expressed by NK / T cell lymphoma tumor cells can effectively degrade chemokines such as CXCL2, CXCL9 and CXCL10, and the expression level of DPP4 in tumor tissue is negatively correlated with the protein content of CXCL2, CXCL9 and CXCL10 chemokines. Further functional experiments confirmed that DPP4 expressed by tumor cells hinders the chemotaxis of normal NK cells, and can be used for the accurate classification and precise treatment of NK / T cell lymphoma patients in clinical practice. Furthermore, DPP4's inhibitory effect on normal NK cell chemotaxis can be suppressed by DPP4 inhibitors. Therefore, DPP4 could serve as an important target for the treatment of NK / T cell lymphomas, and DPP4 inhibitors could be used to treat NK / T cell lymphoma patients. As a tumor marker for NK / T cell lymphomas, DPP4 could be promoted and applied in clinical screening (primary screening) for NK / T cell lymphoma patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG1 shows the expression level and expression ratio of DPP4 in various cell types in NK / T cell lymphoma single-cell transcriptome sequencing data.

[0014] FIG2 shows the results of immunofluorescence co-staining experiments.

[0015] FIG3 shows the results of Western blotting.

[0016] FIG4 shows the strength of the CXCL2 / CXCL9 / CXCL10 / CXCL11-DPP4 interaction pair between tumor cell subsets and myeloid cell subsets obtained by cell communication analysis based on single-cell transcriptome sequencing data of NK / T cell lymphoma.

[0017] FIG5 is a schematic diagram of an in vitro chemotaxis experiment between cell supernatants derived from NK / T cell lymphoma tumor cells and normal NK cells.

[0018] Figure 6 shows the migration rate of NK cells in different treatment groups obtained by in vitro chemotaxis experiments of cell supernatants derived from NK / T cell lymphoma tumor cells and normal NK cells. A: blank control, chemokine, DPP4 protein, DPP4 inhibitor, chemokine + DPP4 protein, chemokine + DPP4 protein + DPP4 inhibitor; B: cell supernatant (YT cells), cell supernatant (YT cells) + chemokine, cell supernatant (YT cells) + chemokine + DPP4 inhibitor, cell supernatant (NK-92 cells), cell supernatant (NK-92 cells) + chemokine, cell supernatant (NK-92 cells) + chemokine, cell supernatant (NK-92 cells) + chemokine + DPP4 inhibitor.

[0019] Figure 7 shows the results of multi-color immunofluorescence co-staining experiments: A: high expression of DPP4; B: low expression of DPP4. DETAILED DESCRIPTION

[0020] To more clearly understand the above-mentioned objects, features, and advantages of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and specific examples. Those skilled in the art should understand that the examples are merely helpful for understanding the present invention and should not be construed as limiting the present invention. Unless otherwise specified, the sequencing and analysis methods used in the following examples are conventional methods.

[0021] Example 1: DPP4 is highly expressed in NK / T cell lymphoma patients

[0022] A total of 10 tumor tissue samples from patients with NK / T cell lymphoma were collected from the Sun Yat-sen University Cancer Center for single-cell transcriptome sequencing. Briefly, the NK / T cell lymphoma tumor tissue samples were enzymatically digested to obtain a single-cell suspension. A commercial single-cell transcriptome library kit (10x Genomics) was then used to load and build libraries from the live cells. Finally, the resulting cDNA library was subjected to high-throughput sequencing to generate sequence files. Analysis of the acquired single-cell transcriptome sequencing data revealed the full range of cell types present in the tumor tissues of NK / T cell lymphoma patients, including normal NK cells, tumor NK cells, T cells, B cells, myeloid cells, and other cells. Figure 1 shows the expression levels and proportions of DPP4 in various cell types in the NK / T cell lymphoma single-cell transcriptome sequencing data. DPP4 was primarily expressed in tumor NK cells and was largely absent in normal NK cells or other cell types, indicating that DPP4 expression is a characteristic of NK / T cell lymphoma tumor cells.

[0023] On the other hand, immunofluorescence co-staining was performed on paraffin-embedded wax blocks of NK / T cell lymphoma tumor tissue, and real-time fluorescence quantitative PCR and Western blotting were performed on NK / T cell lymphoma cell lines. In the immunofluorescence co-staining experiment, a CD56-specific antibody (Zsbio; Cat#ZM-0057) was used to mark tumor cells in tumor tissue wax blocks, and a DPP4-specific antibody (CST; Cat#67138) was used to mark DPP4 protein in tumor tissue. The expression of DPP4 in NK / T cell lymphoma tumor cells was determined by detecting the corresponding fluorescent signals. The primer sequences (SEQ ID NO: 1 and SEQ ID NO: 2) used in the real-time fluorescence quantitative PCR experiment on NK / T cell lymphoma cell lines are shown in Table 1 below to detect the expression level of DPP4 transcription.

[0024] Table 1. Real-time fluorescence quantitative PCR primer sequences

[0025] The oligonucleotide chain sequences of the above primers were synthesized by Guangzhou Paizhen Biotechnology Co., Ltd.

[0026] Western blotting also used the above-mentioned DPP4-specific antibody to detect cell lysate samples and separated supernatants of NK / T cell lymphoma cell lines to clarify the expression and secretion of DPP4 in tumor cells.

[0027] The above method further confirmed the expression of DPP4 in tumor cells of NK / T cell lymphoma, and the presence of cell-secreted DPP4 molecules in the tumor cell supernatant (as shown in Figures 2 and 3). Figure 2 shows the expression of DPP4 in tumor cells in paraffin-embedded wax block sections of NK / T cell lymphoma tumor tissue detected by immunofluorescence co-staining experiments (CD56 and DPP4 double-positive cells). Figure 3 shows the expression and secretion of DPP4 protein in cell lysates and cell supernatants of NK / T cell lymphoma tumor cell lines (YT cells and NK-92 cells) confirmed by Western blotting.

[0028] Example 2: Overexpression of DPP4 in NK / T cell lymphomas can inhibit the chemotactic recruitment of normal NK cells

[0029] CellPhoneDB software was used to perform cell communication analysis on the single-cell transcriptome sequencing data of the above-mentioned NK / T cell lymphoma patients, and significant cell-cell interactions between tumor cell subsets and immune cell subsets were obtained. Figure 4 shows the intensity of the CXCL2 / CXCL9 / CXCL10 / CXCL11-DPP4 interaction between tumor NK cell subsets and myeloid cell subsets. As shown in Figure 4, it was found that all tumor NK cell subsets (NK_C5_ISG15, NK_C6_ITM2A, NK_C7_CCL4, NK_C8_MT1E, NK_C9_CXCL13 and NK_C10_STMN1) can play the role of DPP4 in degrading chemokines through the above-mentioned interactions.

[0030] Furthermore, by conducting in vitro chemotaxis experiments with cell supernatants derived from NK / T cell lymphoma tumor cells and normal NK cells, we observed whether the cell supernatants potentially containing DPP4 secreted by tumor cells had an inhibitory effect on the chemotaxis of normal NK cells, and whether this effect could be further inhibited by DPP4 inhibitors. Figure 5 is a schematic diagram of an in vitro chemotaxis assay of NK / T cell lymphoma cell supernatants and normal NK cells. Cell supernatants from NK / T cell lymphoma cell lines pretreated with the DPP4 small molecule inhibitor linagliptin (CAS No. 668270-12-0; Selleck; Cat# S3031) or a DSMO control (Sigma-Aldrich; Cat# D4540) were used to treat normal NK cells, demonstrating the presence of secreted DPP4 in the supernatants of NK / T cell lymphoma cells, which can inhibit the chemotaxis of normal NK cells by degrading chemokines (CXCL2, CXCL9, and CXCL10). The chemotactic strength was determined by the ratio of NK cells that migrated through the small hole into the lower chamber. A higher migration rate indicates a stronger chemotactic ability of the normal NK cells in the treated group. Figure 6 shows the migration rate of NK cells in different treatment groups obtained from the in vitro chemotaxis experiment of cell supernatants derived from NK / T cell lymphoma tumor cells and normal NK cells (A: blank control, chemokine, DPP4 protein, DPP4 inhibitor, chemokine + DPP4 protein, chemokine + DPP4 protein + DPP4 inhibitor; B: cell supernatant (YT cells), cell supernatant (YT cells) + chemokine, cell supernatant (YT cells) + chemokine + DPP4 inhibitor, cell supernatant (NK-92 cells), cell supernatant (NK-92 cells) + chemokine, cell supernatant (NK-92 cells) + chemokine + DPP4 inhibitor). The chemotaxis of normal NK cells caused by chemokines (CXCL2, CXCL9, CXCL10) is inhibited by DPP4, and this inhibitory effect is inhibited by DPP4 inhibitors.

[0031] The DPP4 inhibitor Linagliptin can specifically target DPP4. Here, Linagliptin is used as an example to illustrate the specific embodiment of the present invention. However, based on the basic principles of the present invention, the DPP4 inhibitor of the present invention includes but is not limited to Linagliptin. Other DPP4 inhibitors can also be applied to the present invention and fall within the scope of protection of the present invention, such as Alogliptin (SYR-322; CAS No.: 850649-61-5), Anagliptin (CAS No.: 739366-20-2), Begelomab (SAND-26; CAS No.: 1403744-56-8), Gemigliptin (LC15-0444; CA S No.: 911637-19-9), Omarigliptin (MK-3102; CAS No.: 1226781-44-7), Saxagliptin (BMS-477118; CAS No.: 361442-04-8), Sitagliptin (MK-0431; CAS No.: 486460-32-6), Trelagliptin (SYR-472; CAS No.: 865759-25-7), Vildagliptin (LAF-237; CAS No.: 274901-16-5).

[0032] Furthermore, multicolor immunofluorescence co-staining was performed on paraffin-embedded sections of NK / T cell lymphoma tissue to examine the correlation between DPP4 and chemokine (CXCL2, CXCL9, and CXCL10) protein expression in tumor tissue. In addition to using the aforementioned CD56 and DPP4-specific antibodies to label tumor cells and DPP4 protein expression, respectively, chemokine-specific antibodies—CXCL2 (Proteintech; Cat#26791-1-AP), CXCL9 (Proteintech; Cat#22355-1-AP), and CXCL10 (Proteintech; Cat#10937-1-AP)—were also used in the immunofluorescence co-staining experiments to clarify the expression of these chemokines in NK / T cell lymphoma tissue.

[0033] Figure 7 shows the expression of a small amount of chemokines (CXCL2, CXCL9, CXCL10) in patients with NK / T cell lymphoma who highly express DPP4 (A), compared to the expression of a large amount of chemokines in patients with low DPP4 expression (B). This result further indicates that NK / T cell lymphoma tumor cells can degrade chemokines by expressing DPP4, thereby hindering the chemotactic recruitment of normal NK cells into tumor tissue.

[0034] These findings suggest that DPP4, expressed on tumor cells, inhibits the chemotaxis of normal NK cells and can be used clinically for the precise classification and treatment of patients with NK / T cell lymphoma. DPP4's inhibitory effect on normal NK cell chemotaxis can be inhibited by DPP4 inhibitors. Therefore, DPP4 could serve as an important target for the treatment of NK / T cell lymphoma, and DPP4 inhibitors could be used to treat patients with NK / T cell lymphoma. As a tumor marker for NK / T cell lymphoma, DPP4 could be promoted and applied clinically for the initial screening of patients with NK / T cell lymphoma.

Claims

1. Application of DPP4 as a tumor marker for NK / T cell lymphoma.

2. Application of DPP4 detection reagent in the preparation of a kit for screening NK / T cell lymphoma.

3. The use according to claim 2, characterized in that: The kit includes the nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:

2.

4. Application of DPP4 inhibitors in the preparation of drugs for the treatment of NK / T cell lymphoma.

5. The use according to claim 4, characterized in that: The DPP4 inhibitor includes one or more of linagliptin, alogliptin, anagliptin, begololimab, gemagliptin, omagliptin, saxagliptin, sitagliptin, trelagliptin, and vildagliptin.

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