DLL3 as a therapeutic target for cervical neuroendocrine carcinoma and its application

Abstract

This invention discloses DLL3 as a therapeutic target for cervical neuroendocrine carcinoma and its application, belonging to the field of biotechnology. This invention discloses the specific expression of the DLL3 gene in cervical neuroendocrine carcinoma (NECC) and its application as a therapeutic target. Single-cell sequencing and immunohistochemistry revealed activation of the DLL3-NOTCH1 / 2 signaling axis in NECC, and functional experiments confirmed that it drives malignant progression by promoting tumor proliferation and inducing T cell exhaustion. Furthermore, this invention constructed an NECC organoid-TIL co-culture model to verify the synergistic efficacy of the DLL3-targeting drug AMG757 combined with EP chemotherapy. This invention provides new target selection and theoretical basis for the precision diagnosis and treatment of NECC, and has significant clinical translational value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to DLL3 as a therapeutic target for cervical neuroendocrine carcinoma and its application. Background Technology

[0002] Despite widespread HPV vaccination and cytology screening, cervical cancer (CC) remains the fourth most common cancer and the fourth leading cause of cancer death among women. Neuroendocrine carcinoma of the cervix (NECC) is a rare but highly aggressive histological subtype, accounting for 0.9%–1.5% of invasive cervical cancers. Due to the tendency for early lymph node / vascular metastasis, most patients are diagnosed at an advanced stage during hysterectomy. Currently, multidisciplinary treatment options for NECC, including surgery, chemotherapy, and radiotherapy, are ineffective, primarily due to squamous cell carcinoma / adenocarcinoma (SCC / ADC) or neuroendocrine carcinomas in other sites such as the lungs and digestive tract. Despite aggressive treatment, the 5-year survival rate for NECC remains low (25%–39% across all stages), with a significantly reduced survival rate in advanced stages. The ongoing clinical challenges and unmet treatment needs surrounding NECC underscore the importance of systematically studying its biological behavior to elucidate its oncogenic mechanisms and inform therapeutic innovation.

[0003] The tumor immune microenvironment (TIME) plays a crucial role in tumor development and prognosis. It is composed of various cellular / extracellular matrix components and signaling molecules that drive tumor growth and treatment resistance. Elucidating these complex interactions is essential for understanding cancer progression and overcoming therapeutic challenges. However, the TIME mechanisms of NECC remain underdeveloped, and its oncogenic drivers are not fully understood. Therefore, elucidating the molecular mechanisms underlying NECC immune escape and tumorigenesis is of great significance for guiding further precision immunotherapy strategies. Summary of the Invention

[0004] The purpose of this invention is to provide DLL3 as a therapeutic target for cervical neuroendocrine carcinoma and its application, thereby addressing the problems existing in the prior art. Addressing the limitations of existing research models for cervical neuroendocrine carcinoma (NECC) and the lack of therapeutic targets, this invention reveals for the first time the significant value of DLL3 as a specific therapeutic target for NECC, providing important evidence for a deeper understanding of the pathogenesis of NECC and optimization of clinical treatment plans.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of the DLL3 gene or its encoded protein in the preparation of diagnostic markers or therapeutic targets for cervical neuroendocrine carcinoma, wherein the nucleotide sequence of the DLL3 gene is shown in SEQ ID NO.1.

[0007] The present invention also provides the use of a substance that inhibits DLL3 activity in the preparation of a drug for treating cervical neuroendocrine carcinoma, said substance comprising an antibody, siRNA or a small molecule inhibitor, wherein the nucleotide sequence of said DLL3 gene is shown in SEQ ID NO.1.

[0008] Furthermore, the small molecule inhibitor is AMG757.

[0009] The present invention also provides a medicament for preparing a treatment for cervical neuroendocrine carcinoma, wherein the active ingredient of the medicament includes a DLL3 inhibitor, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] Furthermore, the DLL3 inhibitor includes one or more of antibodies, siRNAs, or small molecule inhibitors.

[0011] Furthermore, the small molecule inhibitor is AMG757.

[0012] The present invention also provides the use of a substance for detecting DLL3 in the preparation of diagnostic or prognostic reagents for cervical neuroendocrine carcinoma, wherein the nucleotide sequence of DLL3 is shown in SEQ ID NO.1.

[0013] The present invention also provides the application of DLL3 as a detection target in the development of cervical neuroendocrine diagnostic reagents or prognostic reagents, wherein the nucleotide sequence of DLL3 is shown in SEQ ID NO.1.

[0014] The present invention discloses the following technical effects:

[0015] This invention is the first to explicitly identify SYN / INSM1-positive NPCs as the malignant origin of NECC, deepening our understanding of the disease's biological characteristics and revealing the DLL3-NOTCH1 / 2 signaling axis as a specific molecular feature of NECC, providing new evidence for precise diagnosis. Through multi-omics analysis and functional experiments, it confirms that DLL3 has a dual role in NECC: directly driving tumor progression (proliferation / invasion) and shaping an immunosuppressive microenvironment, making it a highly promising therapeutic target. Furthermore, a biomimetic NECC organoid-TIL co-culture system was successfully constructed, overcoming the limitations of traditional immortalized cell lines and animal models, providing a more clinically relevant in vitro model for NECC research. For the first time, the efficacy of the DLL3-targeting drug (AMG757) was validated in an organoid model, demonstrating its synergistic effect with EP chemotherapy, providing experimental evidence for combination therapy strategies for NECC. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a UMAP diagram of single-cell transcriptome data of three types of cervical cancer (NECC, SCC, and ADC) in an embodiment of the present invention; where A represents 24 cell populations; B represents 12 cell types; C represents 3 subtypes; D represents the expression of cell type-specific marker genes among the 12 cell types; and E represents the compositional distribution of NECC, SCC, and ADC.

[0018] Figure 2 In the diagram, A represents the differential expression analysis of the DLL3-NOTCH1 / 2 and CD38-PECAM1 interaction networks in NECC and ADC / SCC tissues; B represents the expression of NPCs and CD38-PECAM1 interactions in NECC tissues based on multicolor immunohistochemistry (mIHC). 8+ Verification of spatial colocalization of the DLL3-NOTCH1 / 2 signaling axis among T cells; C is verification of spatial colocalization of the DLL3-NOTCH1 / 2 signaling axis between NPCs and B cells in NECC tissue based on multicolor immunohistochemistry (mIHC);

[0019] Figure 3 The results of the functional validation experiment of DLL3 in NECC are shown below. A shows the cell proliferation capacity analysis: (left) comparison between the HeLa cell DLL3 overexpression group (DLL3 OE) and the control group; (right) comparison between the NCI-H82 cell DLL3 knockdown group (DLL3 KD) and the control group. B shows the cell invasion capacity detection: (left) comparison between the HeLa cell DLL3 overexpression group (DLL3 OE) and the control group; (right) comparison between the NCI-H82 cell DLL3 knockdown group (DLL3 KD) and the control group.

[0020] Figure 4 The results are from the functional verification experiment of DLL3 in NECC; where A represents the flow cytometry detection CD. 8+ Changes in the expression of immune checkpoints (PD-1, TIM-3) and effector molecules (GZMB, perforin, CD107a) after T cells were co-cultured with HelaNC / Hela-DLL3 OE; B shows the expression of CD107a by flow cytometry. 8+ Expression levels of PD-1, TIM-3, GZMB, perforin, and CD107a in T cells co-cultured with NCI-H82 NC / NCI-H82 KD;

[0021] Figure 5 A schematic diagram illustrating the establishment of the NECC organoid-TILs co-culture system;

[0022] Figure 6 This section presents the comprehensive characterization results of NECC tissue and NECC organoid models. A represents the three-dimensional morphological observation of NECC organoids; B represents the H&E staining and histological structure analysis of NECC tissue and NECC organoids; C represents the immunohistochemical detection of neuroendocrine markers (SYN / INSM1) in NECC tissue and organoids; D represents the whole-exome sequencing (WES) analysis of NECC tissue and organoids; and E represents the batch RNA sequencing analysis of NECC tissue and organoids.

[0023] Figure 7 The therapeutic effects of different treatment regimens on NECC are shown in Figure A; where A represents the cytotoxic effects of different treatment regimens on NECC organoids; and B represents the CD4+ levels in the NECC organoid-TIL co-culture system detected by flow cytometry after treatment with different regimens. 8+ The expression level of the T-cell effector molecule PERFORIN; C represents the expression level of CD4+ in the NECC organoid-TIL co-culture system after different treatment regimens, detected by flow cytometry. 8+ The expression level of the T-cell effector molecule CD137; D represents the expression level of CD137 in the NECC organoid-TIL co-culture system after different treatment regimens, detected by flow cytometry. 8+ Expression level of T cell effector molecule CD107a. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Technical solution

[0030] 1. Target screening and validation

[0031] Single-cell transcriptome sequencing and immunohistochemical analysis of two cases of NECC, two cases of squamous cell carcinoma (SCC), and two cases of adenocarcinoma (ADC) revealed that DLL3 was specifically highly expressed in neuroendocrine-like cells (NPCs), but not significantly expressed in SCC / ADC. Further multicolor immunohistochemistry (mIHC) and cell communication analysis confirmed the presence of a unique DLL3-NOTCH1 / 2 signaling axis in NECC, which was completely absent in SCC / ADC, suggesting that DLL3 may serve as a specific molecular marker for NECC.

[0032] 2. Functional Mechanism Research

[0033] Gene knockdown (KD) and overexpression (OE) experiments confirmed that DLL3 not only significantly promoted tumor cell proliferation and invasion (p<0.05), but also induced the formation of an immunosuppressive microenvironment by upregulating immune checkpoint molecules (such as PD-L1 and LAG-3). Co-culture experiments further revealed that DLL3 can promote T cell exhaustion and inhibit its activation, indicating that DLL3 is a key molecular hub coordinating the malignant progression of NECC and immune escape.

[0034] 3. Validation of treatment strategies

[0035] An organoid-tIL immune co-culture model of NECC was established, and its high preservation of the pathological and molecular characteristics of the primary tumor was verified by histological (HE / IHC) and multi-omics (WES / RNA-seq) methods. The efficacy of DLL3-targeted therapy (AMG757) as monotherapy and in combination with first-line chemotherapy (EP regimen) was evaluated using this model, demonstrating that DLL3-targeted therapy significantly enhances the antitumor effect of standard chemotherapy.

[0036] Example 1

[0037] This invention was approved by the Ethics Committee of the Obstetrics and Gynecology Hospital Affiliated to Fudan University (2022-105). Six tumor samples were collected from two patients with NECC, two with ADC, and two with SCC for scRNA-seq and TCR-seq. None of the patients had received chemotherapy, radiotherapy, or other anti-tumor treatments prior to surgery. Blood samples were collected from a healthy donor to obtain T cells for co-culture with organoids. Formalin-fixed samples were also collected from the tissue bank of the Obstetrics and Gynecology Hospital Affiliated to Fudan University (2020-22). All tissues were obtained with informed consent and after approval by the hospital's ethics committee.

[0038] Molecular profiling was performed on two cases of NECC, two cases of cervical adenocarcinoma (ADC), and two cases of cervical squamous cell carcinoma (SCC) using single-cell transcriptome sequencing (scRNA-seq) combined with T-cell receptor sequencing (TCR-seq). Single-cell transcriptome sequencing and immunohistochemical analysis were also performed on the two cases of SCC and two cases of ADC.

[0039] Surgically removed tumor tissue was preserved in MACS storage solution, washed with PBS, and cut into 1mm pieces on ice. 3 After digestion, cells were enzymatically digested with type II collagenase at 37°C for 30 minutes, filtered through a 70 μm filter, centrifuged at 300 g for 5 minutes, treated with erythrocyte lysis buffer, washed with 0.04% BSA-PBS, and filtered through a 35 μm filter. Cell viability was assessed by Countstar after AO / PI staining. After staining, cells were allocated for scRNA-seq.

[0040] ScRNA-Seq and TCR-seq: ScRNA-Seq and V(D)J libraries were constructed using a 10X Genomics ChromiumController instrument, a Chromium Single Cell 5' library and gel bead kit, and a V(D)Jenrichment kit (10X Genomics, Pleasanton, CA). Cells were concentrated to 1000 cells / μL and loaded into each channel to create single-cell gel bead emulsions (GEMs), with an expected yield of 5000 single-cell mRNA barcodes per sample. Following a reverse transcription (RT) step, the GEMs were destroyed, and the barcoded cDNA was purified and amplified. The amplified barcoded cDNA was used to construct 5' gene expression libraries and TCR enrichment libraries. For 5' library construction, the amplified barcoded cDNA was fragmented, A-tailed, ligated with adapters, and subjected to indexed PCR amplification. For V(D)J libraries, human T-cell V(D)J sequences were enriched from amplified cDNA, followed by fragmentation, A-tailing, adapter ligation, and indexed PCR amplification. The final libraries were quantified using the Qubit high-sensitivity DNA assay kit (Thermo Fisher Scientific), and library size distribution was determined using a high-sensitivity DNA microarray on a Bioanalyzer 2200 (Agilent). All libraries were sequenced at 150 bp paired ends on an Illumina sequencer (Illumina, San Diego, CA).

[0041] Raw data were processed using the NovelBrain cloud platform. After quality control filtering using FastP, reads were aligned to the GRCh38 human reference genome using CellRanger v5.0.1 software. Seurat v3.1.4 was used for data quality control (retaining cells expressing >200 genes / cell and with a mitochondrial UMI percentage <20%), SCTransform normalization, and cell cycle effect regression. PCA dimensionality reduction analysis was performed based on the top 2000 hypervariable genes (selecting the top 10 principal components for t-SNE / UMAP visualization). After clustering using the shared nearest neighbor (SNN) graph, the Wilcoxon rank-sum test (screening criteria: lnFC>0.25, p<0.05, min.pct>0.1) was used to identify differentially expressed genes in each cluster.

[0042] After multiple rounds of subpopulation re-clustering analysis, 23 cell subpopulations with significant transcriptional heterogeneity were finally identified. Figure 1 Based on expression profile analysis of known cell type marker genes, these subgroups can be further classified into 12 major cell types (A). Figure 1Comparative analysis showed significant differences in cell composition among samples from three different pathological subtypes: NECC, ADC, and SCC. Figure 1 (C, where the color depth of the dot plot represents gene expression level). Notably, cell population composition analysis revealed that neural progenitor cells (NPCs) are specifically enriched in NECC tissues (C). Figure 1 This discovery (D) provides important clues for elucidating the origin of tumor cells in NECC.

[0043] In summary, this invention, through single-cell transcriptome sequencing and immunohistochemical analysis of 2 cases of NECC, 2 cases of squamous cell carcinoma (SCC), and 2 cases of adenocarcinoma (ADC), found that DLL3 is specifically highly expressed in neuroendocrine-like cells (NPCs), while no significant expression was observed in SCC / ADC.

[0044] Example 2

[0045] Multicolor immunohistochemistry (mIHC) and cell communication analysis

[0046] (1) Multicolor immunohistochemistry (mIHC)

[0047] To validate the multicellular ecosystem and spatial distribution of different cell types in NECC TIME, mIHC was used to target the potential NECC target DLL3. Formalin-fixed paraffin-embedded (FFPE) tissue sections from NECC, ADC, and SCC samples were stained using mIHC. 4 μm thick FFPE tissue sections were stained sequentially with primary antibodies against CD8 (CST#70306S, CST), CD38 (ab108403, Abcam), SYN (ab32127, Abcam), PECAM1 (ab108403, Abcam), DLL3 (ab229902, Abcam), Notch1 (ab52627, Abcam), and Notch2 (5732T, CST) and a TSA 6-color kit (abs50015-100T, Absinbio, Shanghai). The sections were then reverse-stained with DAP (DAPI; Absinbio, product number abs47047616) to observe cell nuclei. The staining process was illustrated using PECAM1 as an example: First, the deparaffinized sections were incubated with anti-PECAM1 antibody for 30 minutes, followed by incubation for 10 minutes with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (abs50015-02, Absinbio). Following the manufacturer's instructions, the PECAM1 signal was developed using a TSA520 for 10 minutes. The sections were then washed with triple-buffered saline containing TBST and heated in a preheated citrate solution (90°C) in a microwave oven at 20% maximum power for 15 minutes. The sections were cooled to room temperature in the same solution. Between all steps, the sections were washed with Tris buffer. After air-drying, the slides were imaged using a Pannoramic MIDI II scanner (3DHISTECH). Images were analyzed using Indica Halo software.

[0048] (2) Cell communication analysis

[0049] The Cell Phone DB public database was used as the source of ligand-receptor interaction information to systematically conduct intercellular communication analysis. Specifically, firstly, membrane proteins, secretory proteins, and peripheral proteins expressed in cell clusters at different time points were comprehensively annotated; then, based on known ligand-receptor interaction relationships and combined with single-cell expression matrices obtained through Seurat normalization, the mean of significant interactions between ligand-receptor pairs was calculated, and their statistical significance was evaluated (the significance threshold was set at p<0.05).

[0050] This invention, through multicolor immunohistochemistry (mIHC) and cell communication analysis, confirms the existence of a unique DLL3-NOTCH1 / 2 signaling axis in NECC, which is completely absent in SCC / ADC, suggesting its potential role as a specific molecular marker for NECC. Specifically, multicolor immunohistochemistry (mIHC) revealed a unique DLL3-NOTCH1 / 2 interaction network in NECC tissue, see [link to mIHC]. Figure 2 : Figure 2 The results in A showed that, compared with ADC / SCC, there was a significant DLL3-NOTCH1 / 2 interaction signal in NECC tissue, and differential expression patterns of CD38-PECAM1 were also observed. Figure 2 Results B showed that, through mIHC staining, the spatial co-localization characteristics of DLL3-positive tumor cells and NOTCH2 / SYN double-positive tumor cells in NECC tissue were clearly displayed. Figure 2 Further evidence from cell C confirms that DLL3 / SYN double-positive tumor cells and NOTCH1 / 2 / CD38 triple-positive B cells form a unique tumor-immune microenvironment interaction structure. These findings collectively reveal the crucial role of the DLL3-NOTCH signaling axis in the development and progression of NECC. This series of evidence jointly establishes the diagnostic value of DLL3 as a specific molecular marker for NECC.

[0051] Example 3

[0052] 1. Gene knockdown (KD) and overexpression (OE) experiments

[0053] Cell culture: HeLa and NCI-H82 cells (ATCC) were cultured in DMEM (Gibco, 11965118) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Experiments were performed using cells from passages 10-20, validated by STR profiling analysis. Routine mycoplasma detection was performed using the MycoAlert assay kit (Lonza, LT07-218).

[0054] Cell transfection: For cell transfection experiments, DLL3 overexpression vectors, DLL3-targeting knockdown vectors, and their corresponding control vectors (Genomeditech, Shanghai, China) were transfected into HeLa / NCI-H82 cells in serum-free Opti-MEM medium (51985-034, Gibco, Gaithersburg, MD, USA) using Lipofectamine 2000 (11668-019, Invitrogen, Carlsbad, CA, USA).

[0055] HeLa / NCI-H82 cell lines in good growth condition were passaged into culture dishes at a ratio of 1:3 to 1:5. Transfection experiments were performed when cells reached 70%-80% confluence. Two hours before transfection, the medium was replaced with fresh complete medium (containing 10% FBS). In the 24-well plate transfection system, 0.4 μg of transfection-grade plasmid DNA was first dissolved in 50 μL of Opti-MEM serum-free medium and vortexed to mix. Then, 1.0 μL of HG-Trans293™ transfection reagent was added, gently pipetted to mix, and allowed to stand at room temperature for 30 minutes to form a stable nucleic acid-transfection reagent complex. The complex was then added dropwise to the wells containing cells and complete medium, and the plate was gently shaken in a cross-hatching motion to ensure even distribution (this transfection reagent maintains high transfection efficiency even under serum-containing conditions, with cell viability >95%). Transfected cells were incubated at 37°C in a 5% CO2 incubator, and the medium was replaced with fresh complete medium after 4-6 hours. After culturing for 18-48 hours, the transfection efficiency was detected by Western blotting and qPCR, and subsequent functional experiments were conducted.

[0056] The nucleotide sequence of the DLL3 gene is as follows:

[0057]

[0058] The amino acid sequence of the protein encoded by the DLL3 gene is as follows:

[0059] MVSPRMSGLLSQTVILALIFLPQTRPAGVFELQIHSFGPGPGPGAPRSPCSARLPCRLFFR (SEQ ID NO.2);

[0060] The sequences of the forward and reverse primers used to amplify the DLL3 gene are shown below:

[0061] Forward Primer1:GACCCTCAGCGCTACCTTTT(SEQ ID NO.3);

[0062] Reverse Primer1: GAACCCTCCTGCGTCCTTAG (SEQ ID NO. 4).

[0063] Western blotting and qPCR were used to detect transfection efficiency:

[0064] RNA extraction, reverse transcription, and qRT-PCR analysis: Total RNA was extracted using Trizol reagent (Invitrogen, #15596026), and reverse transcription was performed using first-strand cDNA synthesis premix (YEASEN, #11141ES60) (42℃ for 15 min, 85℃ for 5 s). qPCR was performed on the Step One Plus real-time PCR system using SYBR green mix (YEASEN, #11203ES08) (95℃ for 5 min pre-denaturation, 95℃ for 10 s / 60℃ for 30 s, 40 cycles), with β-actin as an internal control. The relative expression level of the DLL3 gene was calculated using the 2-ΔΔCt method, with three technical replicates.

[0065] Western blotting: Proteins were extracted using Western / IP lysis buffer or RIPA lysis buffer containing protease / phosphatase inhibitors. Protein concentration was determined by the BCA method. Samples were separated by SDS-PAGE and transferred to a PVDF membrane (0.45 μm) using the Bio-Rad system. After blocking with 5% BSA for 1 hour, the membrane was incubated with primary antibody (4°C, overnight) and then with secondary antibody (room temperature, 1 hour). Protein bands were visualized by ECL detection (ImageQuant LAS 4000) and quantified using ImageJ.

[0066] Figure 3 The results of the functional validation experiment of DLL3 in NECC are shown in Figure A. Figure A shows that DLL3 gene overexpression significantly promotes the proliferation activity of HeLa cells (p<0.01); conversely, DLL3 gene knockout effectively inhibits the proliferation ability of NCI-H82 cells (p<0.05). Figure B shows that DLL3 knockout significantly reduces the invasive and metastatic potential of NCI-H82 cells (p<0.01). In summary, this invention, through gene knockdown (KD) and overexpression (OE) experiments, confirms that DLL3 not only significantly promotes tumor cell proliferation and invasion (p<0.05), but also induces the formation of an immunosuppressive microenvironment by upregulating immune checkpoint molecules (such as PD-L1 and LAG-3).

[0067] 2. Co-culture experiment

[0068] Preparation of tumor-infiltrating lymphocytes (TILs): Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation. The specific steps for in vitro expansion and culture were as follows: Day 1: PBMCs were co-incubated with CD3 / CD28 T cell activators (1:1 ratio) and seeded in DMEM complete medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin antibiotics, and 100 U / mL recombinant human IL-2. The cell density was adjusted to 1 × 10⁶ cells / mL. 6 Cells / mL, incubated at 37℃ in a 5% CO2 incubator, with fresh medium replaced every 2-3 days. Day 10: CD8 + T cells were subjected to secondary stimulation, with CD3 / CD28 activators added again to enhance T cell proliferation. Day 14: Expanded T cells were collected at a rate of 2 × 10⁻⁶. 5 The cells were resuspended in fresh culture medium at a concentration of cells / mL, and then seeded at 50 μL / well (containing 10,000 effector T cells) into 96-well U-shaped plates pre-coated with tumor cells, with an effector cell to target cell (E:T) ratio of 10:1. After co-culturing for 72 hours, the expression levels of T cell activation markers (GZMB, PERFORIN, CD107a) and immunosuppressive markers (PDCD1, TIM3) were detected by flow cytometry to assess the cytotoxic efficacy and activation status of T cells.

[0069] Figure 4 The results of the functional validation experiments of DLL3 in NECC are as follows: Regarding immune regulation, experiments in groups A and B confirmed that when co-cultured with tumor-infiltrating lymphocytes (TILs), DLL3-overexpressing cells induced T cell exhaustion by upregulating PDCD1 and TIM3 expression, while downregulating GZMB and CD107a to inhibit T cell activation. DLL3 knockout significantly restored T cell function, manifested by upregulation of perforin and CD107a expression and downregulation of TIM3 expression. These results systematically confirm the dual role of DLL3 in promoting tumor proliferation and mediating immune escape. In summary, this invention, through co-culture experiments, further reveals that DLL3 can promote T cell exhaustion and inhibit its activation, indicating that DLL3 is a key molecular hub coordinating the malignant progression and immune escape of NECC.

[0070] Example 4

[0071] 1. Establish an NECC organoid-TILs immune co-culture model

[0072] Cervical cancer organoid culture: Freshly extracted cervical cancer tissue specimens were immediately placed in pre-cooled DMEM / F12 medium (containing 1% penicillin / streptomycin) at 4°C for preservation. The tissue was mechanically minced to approximately 1 mm using ophthalmic scissors. 3 After sizing, the cells were transferred to a digestion solution containing 1 mg / mL collagenase I (Type IV) and digested at 37°C with shaking (200 rpm) for 30 minutes. The digested tissue suspension was filtered through a 70 μm cell sieve and centrifuged at 300 × g for 5 minutes to collect the cell pellet. After washing the cells three times with PBS, the cells were treated with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 5 minutes to remove erythrocytes. The final cell pellet was resuspended in organoid complete culture medium containing B27 (1:50), N2 (1:100), 10 mM HEPES, 1 mM N-acetylcysteine, 10 ng / mL EGF, and 50 ng / mL FGF2, and incubated at 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 / mL in 24-well plates pre-coated with Matrigel (Corning, 356231), and incubated at 37°C for 30 minutes to allow cell attachment. After removing unattached cells, fresh medium was added, and the medium was changed every other day.

[0073] 2. Preparation of tumor-infiltrating lymphocytes (TILs)

[0074] PBMCs were isolated from peripheral blood using Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation. Lymphocyte culture was prepared using RPMI 1640 supplemented with 2 mM L-glutamine, 100 U / mL penicillin-streptomycin, and 10% heat-inactivated serum from males (type AB). Resuscitated PBMCs were treated with preheated T-cell medium (containing 25 U / mL DNase I (Benzonase) and 10% FBS) at 37°C for 15 minutes, followed by washing and adjustment of the concentration to 2-3 × 10⁻³. 6 Cells / mL, after adding 150 IU / mL of recombinant human IL-2 (PeproTech), were activated and cultured at 37°C and 5% CO2 for 24 hours.

[0075] 3. Establishment of a co-cultivation system

[0076] Organoids were pretreated with 200 ng / mL IFN-γ (PeproTech) for 16 hours, then dissociated into single-cell suspensions using TrypLE Express enzymes. After counting, the cells were sputtered at 5 × 10⁻⁶ cells per cell. 4Cells / mL were resuspended in TILs medium. 24-well plates pre-coated with 5 μg / mL anti-CD28 antibody (BioLegend) were incubated overnight at 4°C. Cells were then resuspended at an effector cell:target cell ratio of 20:1 (TILs 1×10⁻⁶). 6 Cells / wells were co-cultured, with the medium supplemented with 150 IU / mL IL-2. The culture system was maintained at 37°C in a 5% CO2 incubator, with half the medium replaced every 48 hours. A schematic diagram of the NECC organoid-TILs co-culture system is shown below. Figure 5 .

[0077] 4. HE staining identification of constructed human cervical cancer organoids

[0078] Experimental procedure:

[0079] 1) Sample fixation: Collect intact BME droplets containing organoids from the culture plate and incubate them in 5 mL of cell recovery solution placed on ice for 30 min; fix with 4% paraformaldehyde (PFA) for 10 min, wash with distilled water for 2 min; replace with fresh distilled water and wash for another 2 min.

[0080] 2) HE staining: Stain with hematoxylin for 8 min, rinse with tap water to remove excess staining solution for about 10 min, then wash again with distilled water; add differentiation solution for differentiation for about 5 s, rinse with tap water for 10 min; stain with eosin for 1 min.

[0081] 3) Dehydration, clearing, and mounting: 70% ethanol for 10 seconds, 80% ethanol for 10 seconds, 90% ethanol for 10 seconds, and anhydrous ethanol for 10 seconds. Clear with xylene for 5 minutes; replace with fresh xylene and clear again for 5 minutes; mount with neutral resin.

[0082] HE staining results showed that the constructed cervical cancer organoids successfully simulated the pathological features of cervical cancer tissue.

[0083] 5. Differences in mRNA between cervical cancer organoids and primitive tissues

[0084] Experimental procedure:

[0085] (1) RNA extraction: RNA was extracted from cervical cancer organoids and primitive tissue samples;

[0086] (2) RNA quality testing: The extracted RNA was tested using an ultraviolet absorption spectrometer to ensure the integrity, purity and concentration of the RNA;

[0087] (3) RNA library preparation: RNA is converted into sequencing libraries using PCR amplification library preparation methods;

[0088] (4) Quality control: Perform quality control on the library, including total library volume, fragment length and purity, to ensure that the library meets sequencing requirements;

[0089] (5) Sequencing: RNA-Seq technology is used to perform high-throughput sequencing on RNA samples to obtain the sequence information and relative expression levels of transcripts;

[0090] (6) Data analysis: Preliminary processing, quality control and comparison of sequencing data, and application of bioinformatics methods for data analysis to detect differential gene expression.

[0091] 6. Gene mutation map of cervical cancer organoids and primitive tissues

[0092] Experimental procedure:

[0093] (1) DNA extraction: DNA was extracted from cervical cancer organoids and primitive tissue samples by using methods such as cell disruption, protein digestion and DNA purification, and the quality and purity of the extracted DNA were tested.

[0094] (2) DNA library construction: The extracted DNA is converted into a sequencing library. DNA library construction includes the following steps: DNA double-strand breakage, end repair, sequencing primer ligation, aptamer ligation, and PCR amplification, etc.

[0095] (3) Quality control: The total amount, fragment length and purity of the DNA library are tested to ensure that the quality of the library meets the sequencing requirements;

[0096] (4) Sequencing operation: Whole genome sequencing was performed using Illumina HiSeq high-throughput sequencing technology;

[0097] (5) Data analysis: Preliminary processing, quality control and alignment of sequencing data, including removing low-quality sequences, filtering adapter sequences, and aligning sequencing data with the reference genome; followed by bioinformatics analysis such as variant detection, annotation, structural variant analysis and genome assembly.

[0098] (6) Data analysis: Based on the whole genome sequencing results, statistical and biological analyses are performed, including the detection of single nucleotide variants (SNVs), insertion / deletion mutations and copy number variations.

[0099] Whole-genome sequencing analysis showed that the constructed cervical cancer immunogenic organoids were highly similar in genomic characteristics to cervical cancer tissue. Figure 6This paper presents comprehensive characterization results of the NECC organoid model constructed in this invention, including three-dimensional morphological observation of the organoids, HE staining tissue structure analysis, immunohistochemical detection of neuroendocrine markers (SYN / INSM1), whole-exome sequencing (WES), and batch RNA sequencing analysis. Experimental data confirm that the model highly reproduces the cellular morphological characteristics, neuroendocrine differentiation phenotype, and genomic / transcriptomic features of the primary tumor, successfully establishing an in vitro research platform capable of simulating the pathobiological characteristics of NECC.

[0100] In summary, this invention establishes an NECC organoid-TILs immune co-culture model and verifies its high preservation of the pathological and molecular characteristics of the primary tumor through histological (HE / IHC) and multi-omics (WES / RNA-seq) methods.

[0101] Example 5

[0102] The above model was used to evaluate the efficacy of DLL3-targeted drug (AMG757) as monotherapy and in combination with first-line chemotherapy (EP regimen), to verify the efficacy of DLL3-targeted drug (AMG757), and to demonstrate its synergistic effect with EP chemotherapy. Specifically, EP drug, DLL3-targeted drug (AMG757), and combination drugs were added to an organoid T cell co-culture system (see the co-culture system establishment in Example 4) to verify the efficacy.

[0103] The therapeutic effects of different treatment regimens on NECC are shown in the table below. Figure 7 . Figure 7 Figure A in the figure shows that the first-line treatment of NECC, the EP regimen (etoposide + cisplatin) combined with the DLL3-targeting drug AMG757, produces the strongest tumor cell killing effect. Figure 7 B- Figure 7 Further analysis revealed that, compared to the monotherapy group, the EP+AMG757 combination therapy group significantly enhanced co-stimulatory activation signals (upregulation of CD137 expression) and cytotoxic granule release (increased perforin and CD107a expression, P<0.05) in CD8+ T cells. These data indicate that the EP+AMG757 combination therapy not only effectively induces immunogenic tumor cell death but also significantly activates T cell function, providing a novel therapeutic strategy with transformative potential for DLL3-positive NECC patients.

[0104] In summary, this invention used this model to evaluate the efficacy of DLL3-targeted drug (AMG757) as a monotherapy and in combination with first-line chemotherapy (EP regimen), demonstrating that DLL3-targeted therapy can significantly enhance the anti-tumor effect of standard chemotherapy.

[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of a substance that inhibits DLL3 activity for the manufacture of a medicament for the treatment of cervical neuroendocrine cancer, characterized in that, The substance is a small molecule inhibitor, and the nucleotide sequence of the DLL3 gene is shown as SEQ ID NO. 1; The small molecule inhibitor is AMG757.

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