A method for constructing a mouse model to promote formation and maturation of tertiary lymphoid structures

Abstract

The application discloses a method for constructing a mouse model for promoting formation and maturation of a three-level lymph structure, and belongs to the field of biological medicines. The method successfully constructs a mouse model with a large number of three-level lymph structures and high maturation degree by adopting a combination of a PD-1 inhibitor and low-dose radiotherapy. When the low-dose radiotherapy intensity is 1 Gy and the PD-1 inhibitor is combined, the number of B220+ B cells and CD19+ B cells is significantly increased, and the number, density and maturation degree of the three-level lymph structure (TLS) are improved. The application has a positive promoting effect on in-vitro screening of immune-promoting drugs and research on formation of immune tissues.

Description

Technical Field

[0001] This invention relates to a method for constructing a mouse model that promotes the formation and maturation of tertiary lymphoid structures, belonging to the field of biomedicine. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is the most common type of cancer worldwide and a leading cause of cancer-related death. The application of innovative screening technologies has improved the early identification of NSCLC. Despite some progress in radiotherapy, targeted therapy, and immunotherapy for NSCLC, overall survival has only slightly improved. Therefore, exploring new treatment methods for lung cancer is imperative. Recently, tertiary lymphoid structures (TLS) have attracted attention as an important factor related to clinical response.

[0003] Tertiary lymphoid structures (TLS) are vascularized structures that form in non-lymphoid tissues during chronic inflammation (such as autoimmune diseases, chronic infections, and cancer). TLS consist of T-cell regions containing mature dendritic cells (DCs), germinal centers containing follicular dendritic cells and proliferating B cells, and high endothelial venules (HEVs). The presence of TLS is associated with favorable prognoses in various cancers, including lung cancer, colorectal cancer, melanoma, and pancreatic cancer. Recently, numerous reports have shown that TLS can enhance the response to ICB therapy. Inducing TLS formation for antitumor purposes is a promising research area. However, current methods remain limited. Major approaches include stimulating TLS formation in tumors by injecting TFH cells, inducing spontaneous TLS formation in the kidneys, liver, and lungs in adult mice using the standard Notch signaling mediator Rbpj with conditional Notch signal deletion, and inducing TLS formation by intratumoral injection of the B lymphocyte chemokine cxcl13.

[0004] Constructing a tertiary lymph node structural model is of great significance in drug screening, disease research, and immunology. This model helps to understand how lymphocytes exert their anti-tumor effects, providing new insights into cancer treatment. Furthermore, for cancer research, the model can be used to study lymphocytes. In immunology, this model helps to study the complexity of immune responses and the interactions of immune cells in lymph nodes. In addition, the model can be used to assess the potential toxicity of drugs to the lymphatic system and to detect possible side effects in advance. In summary, constructing a tertiary lymph node structural model provides a powerful tool for understanding the biological processes of the lymphatic system, drug development, and disease treatment.

[0005] Tertiary lymphoid structures are highly complex, encompassing diverse cell types and microenvironments. Constructing a model that accurately reflects this diversity and complexity is a challenging task. Clinically, immunotherapy and chemotherapy have been reported to induce TLS formation in cancer patients with favorable prognoses. Intratumoral TLS has been identified as a promising predictive biomarker for anti-PD-1 and / or anti-CTLA-4 antibody responses in various tumor types, and studies have shown increased TLS density following immunotherapy. However, these induction methods are generally unstable and inefficient. The exact mechanisms of TLS development within tumors and the specific contributions of TLS to antitumor immunity remain elusive. Therefore, a stable and accurate method for constructing tertiary lymphoid structures is urgently needed to facilitate researchers' better simulation and exploration of the formation principles and mechanisms of action of tertiary lymph nodes. Summary of the Invention

[0006] Currently, there is a lack of mature and abundant methods for constructing tertiary lymphoid structures. Furthermore, most studies on combination therapy focus on specific types of immune cells within the tumor microenvironment (TME), neglecting changes within the overall microenvironment. Additionally, there are no mature studies on radiotherapy combined with immunotherapy inducing tertiary lymphoid structures. This invention uses a lung cancer mouse model, employing a combination of PD-1 inhibitors and low-dose radiotherapy to construct a mature and abundant mouse model of tertiary lymphoid structures. Building upon this, this invention departs from the conventional approach of studying only a few cells within the TME, treating the TME as a whole. This invention reveals that the TME comprises tertiary lymphoid structures composed of various immune cells.

[0007] A method for constructing a mouse model that promotes the formation and maturation of tertiary lymphoid structures, wherein the method uses PD-1 inhibitors to treat mice.

[0008] In one implementation, mice were treated with a combination of a PD-1 inhibitor and low-dose radiotherapy.

[0009] In one implementation, the mice are lung cancer model mice.

[0010] In one implementation, C57BL / 6JSmoc-Krasem4(LSL-G12D)Smoc mice (purchased from Shanghai Model Organisms Center, Inc.) were anesthetized with isoflurane inhalation and administered via nasal drops, 50 μL per mouse, for 8 weeks.

[0011] In one implementation, the PD-1 inhibitor is pembrolizumab, nivolumab, toripalimab, tislelizumab, camrelizumab, and sintilimab, etc.

[0012] In one embodiment, the PD-1 inhibitor is administered at a concentration of 0.1 to 10 μg / mL.

[0013] In one implementation, the PD-1 inhibitor is administered at 3-day intervals.

[0014] In one implementation, X-rays are used for radiotherapy, with a dose of 1-2 Gy.

[0015] In one implementation, C57BL / 6JSmoc-Krasem4(LSL-G12D)Smoc mice were anesthetized with isoflurane inhalation and administered nasal drops (50 μL / mouse) for 8 weeks (referred to as Kras-LSL-G12D model mice). The mice were administered a PD-1 inhibitor via intraperitoneal injection of 10 mg / kg / 3 days, and simultaneously irradiated daily with 1 Gy of 7.2 Gy / min, 160 KV, 25 mA, 30 cm SSD using a biological X-ray irradiator (RS2000Plus). This resulted in mice with a large number of mature tertiary lymphoid structures.

[0016] The present invention also provides mouse models constructed by any of the above methods.

[0017] The present invention also provides applications of the above-mentioned mouse model, including: analyzing the immunological mechanism of tertiary lymphoid structure formation, analyzing the potential role of tertiary lymphoid structure in immunotherapy, analyzing the dynamic changes of cells or molecules in tertiary lymphoid structure, analyzing the feasibility of mature TLS as a treatment strategy, and screening drugs that promote the formation of tertiary lymphoid structure.

[0018] This invention also provides the use of PD-1 inhibitors in the preparation of drugs that promote the formation of tertiary lymphoid structures.

[0019] In one embodiment, the drug refers to a drug suitable for radiotherapy patients with non-small cell lung cancer, which promotes the maturation of the tertiary lymphoid structure in radiotherapy patients with non-small cell lung cancer.

[0020] In one embodiment, the dose of the radiotherapy is 1-2 Gy.

[0021] In one implementation, a PD-1 inhibitor is combined with low-dose radiotherapy to promote the maturation of tertiary lymphoid structures.

[0022] In one embodiment, the dosage form of the drug is a liquid or a solid.

[0023] Beneficial effects

[0024] This invention successfully constructed a large number of mature mouse models with a tertiary lymphoid structure using a PD-1 inhibitor combined with low-dose radiotherapy. Specifically:

[0025] (1) Low-dose radiotherapy of 1 Gy combined with PD-1 inhibitor was used to treat lung cancer mice, and a mouse model of three-level lymphoid structure was successfully constructed.

[0026] (2) Low-dose radiotherapy of 1 Gy combined with PD-1 inhibitor treatment in lung cancer mice significantly increased the number of B220+B and CD19+B cells and improved the number and density of tertiary lymphoid structures (TLS);

[0027] (3) Low-dose radiotherapy of 1 Gy combined with PD-1 inhibitor was used to treat lung cancer mice to promote TLS maturation. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0029] Figure 1 : Figure 1 A schematic diagram of mouse modeling; Figure 1 B is a CT scan of the lungs of the modeling mouse; Figure 1 C and 1E are staining images of lung tissue; Figure 1 F is the TLS percentage chart;

[0030] Figure 2 : Figure 2 A is a schematic diagram of low-dose radiation; Figure 2 B and 2C are CD19 representation diagrams in TLS; Figure 2 D and 2E are graphs showing the number of TLS and CD3+T; Figure 2 F~I are flow cytometry images;

[0031] Figure 3 : Figure 3 A is a schematic diagram of PD-1 and low-dose radiation; Figure 3 B~3D are flow cytometry results; Figure 3 E is the H&E staining diagram; Figure 3 F reflects the number of TLS in different groups; Figure 3 G reacts to different groups of TLS densities; Figure 3 H represents immunofluorescence images of different cell markers and different stages of TLS; Figure 3 I reflects the different TLS maturity stages of different groups; Figure 3 J reflects the number of E-TLS; Figure 3 K reflects the number of PFL-TLS responses;

[0032] Figure 4 : Figure 4A is a schematic diagram of C57BL / 6J mouse modeling; Figure 4 B is a CT image of the lungs of the modeling mouse; Figure 4 C and 4D are staining images of lung tissue; Figure 4 E represents the number of TLS instances;

[0033] Figure 5 : Figure 5 A is a schematic diagram of low-dose radiation in C57BL / 6J mice; Figure 5 B shows the results of CD19 and CD45 flow cytometry. Figure 5 C represents the expression diagram of CD19;

[0034] Figure 6 : Figure 6 A is a schematic diagram of PD-1 in C57BL / 6J mice and low-dose radiation; Figure 6 B is the expression diagram of CD19. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0036] Example 1: Construction of a mouse model with a three-tiered lymphatic structure

[0037] 1. Constructing a lung cancer model mouse model, Kras-LSL-G12D:

[0038] C57BL / 6JSmoc-Krasem4(LSL-G12D)Smoc mice were purchased from Shanghai Model Organisms Center, Inc. and housed under specific pathogen-free conditions. Orthotopic spontaneous lung cancer model construction: AAV2 / 9-CMV bGI-Cre-EGFP-pA (Shanghai Model Organisms Center, Inc., Shanghai, China) was prepared as a 50 uL / test working solution using PBS; C57BL / 6JSmoc-Krasem4(LSL-G12D)Smoc mice were anesthetized with isoflurane inhalation and then treated with the above working solution via nasal instillation to construct the Kras-LSL-G12D model mouse. The experimental protocol is as follows: Figure 1 As shown in Figure A. Eight weeks later, lung tumor formation was observed using a small animal in vivo Micro-CT imaging system (Quantum GX2, PerkinElmer), and the results are as follows. Figure 1 As shown in B.

[0039] 2. Construct a three-tiered lymphatic structure:

[0040] The model mice were randomly divided into three groups for low-dose radiotherapy: (1) control group (no radiotherapy); (2) 1 Gy group; and (3) 2 Gy group. Radiation was administered through the chest. Mice were anesthetized with isoflurane and irradiated with a biological X-ray irradiator (RS2000Plus) at 7.2 Gy / min, 160 KV, 25 mA, and 30 cm SSD, for 1 and 2 Gy irradiations respectively. A 3 × 3 cm collimator was used to focus the radiation, depending on the location. Seven days later, the mice were euthanized, and whole lung samples were collected. The experimental protocol is as follows: Figure 2 As shown in Figure A.

[0041] After selecting the optimal irradiation dose, the model mice were randomly divided into four groups for modeling: (1) control group (untreated group), (2) 1Gy group, (3) PD-1 inhibitor group (PD-1 inhibitor (InVivoPlus anti-mouse PD-1 (CD279), purchased from BioXcell, USA) intraperitoneal injection 10mg / kg / 3d), and (4) 1Gy + PD-1 combination group (PD-1 inhibitor intraperitoneal injection 10mg / kg / 3d). Seven days later, the mice were euthanized, and whole lung samples were collected. The experimental method was the same as that used in the previous method. Figure 3 As shown in Figure A.

[0042] 3. Detection Experiment

[0043] (1) Immunohistochemistry and multiplex immunofluorescence

[0044] Lung tumor tissue was fixed in 10% formalin, embedded in paraffin, and serially sectioned; 4 μm thick sections were cut from the formalin-fixed paraffin-embedded lung cancer tissue for each staining. Slides were dewaxed, rehydrated, and epitope repair was performed by boiling at 97°C for 20 minutes in sodium citrate (pH=6.0) or EDTA (pH=8.0). Endogenous peroxidase was then blocked by incubation in endogenous peroxidase blocking solution for 10 minutes, followed by protein blocking with 10% goat serum at room temperature for 30 minutes.

[0045] For IHC staining, the primary antibody was diluted and completely coated onto the tissue, then incubated overnight at 4°C. Mouse tissue TLS and T cell markers included CD3 (ab16669, Abcam), CD19 (ab245235, Abcam), B220 (RA3-6B2, eBioscience), CD21 (abs145284, Absin), CXCL13 (ab78237, Abcam), GL7 (14-5902-82, eBioscience), and CD4 (ab183685, Abcam) and CD8 (ab209775, Abcam). For MIF staining, only one antigen was detected per round, including primary antibody incubation, secondary antibody incubation, visualization of tyramine signal amplification (TSA), and then the next antibody was labeled after epitope repair and protein blocking. Antibody information was the same as for IHC. TSA visualization was performed using the XTSA 7-color multiplex IHC kit (AXT37025031, Alpha X), which contains the fluorophore (4', 6-diamidinyl-2-phenylindole (DAPI)), XTSA 520 (B220), XTSA 570 (CD3), XTSA 620 (CD21), XTSA 690 (CD19), XTSA 780 (CXCL13), XTSA signal amplification buffer, and anti-quenching mounting medium. After labeling all antigens on each panel, the slides were microwaved to remove the TSA antibody complexes with EDTA buffer at 97°C for 20 minutes. All IF slides were counterstained with DAPI for 5 minutes and then blocked in anti-quenching mounting medium. The equipment used for image acquisition in this experiment was the ZEISS Axioscan7 whole-slice imaging system (Carl Zeiss Microscopy GmbH Carl-Zeiss-Promenade 1007745 Jena, GERMANY), and the ZEN 3.3 software was used for image reading.

[0046] (2) Evaluation of TLS

[0047] For mouse tumor samples, those observed to have both positive H&E staining for immune cell aggregation and positive IHC B220 or CD19 staining were classified as TLS-positive (TLS+), otherwise as TLS-negative (TLS+). The number of TLSs in each slice was counted and divided by the slice area to calculate the TLS density. CD3 was assessed by counting the number of CD3+ T cells around and inside all TLSs. GL7 was used as a marker for germinal centers (GCs). TLS stage classification and CD8 assessment were performed as described above. Mouse spleens were used as positive controls for all the above staining. The assessment of different TLS stages was based on previous studies. Clusters without follicular dendritic cells (FDCs) were represented as the first stage of TLS development and were called early TLS (E-TLS). TLSs containing FDCs but without GCs were called primary follicular-like TLS (PFL-TLS), and TLSs containing GCs were called secondary follicular-like TLS (SFL-TLS).

[0048] (3) Flow cytometry

[0049] Whole lung tissue containing tumors was cut into small pieces and then suspended in PBS to form a cell suspension. After treatment with a combination of collagenase, deoxyribonuclease I, and hyaluronidase (Sigma, USA), the tumor was digested at 37°C for 50 minutes and then screened through a 70 μm sieve (BD Falcon, USA). Immune cells were isolated using a mouse tumor-infiltrating tissue mononuclear cell isolation kit (SolarBio, China). Immune cells were suspended in PBS, counted, stained with specific flow cytometry antibodies, and incubated on ice for 30 minutes. After washing twice with PBS, the suspended cells were analyzed by flow cytometry. Flow cytometry data were acquired and analyzed on a NovoCyte Penteon flow cytometer (Agilent, NovoExpress 1.6.2).

[0050] 4. Results Analysis

[0051] Figure 1 C and 1E are defined as TLS in KrasG12D mice stained with H&E and IHC and defined as B220, CD19, CD3, and CD21 cells; Figure 1 C and 1E are staining images of lung tissue; 1F is the percentage of positive tertiary lymphoid structures. The results indicate that TLS can be identified in KrasG12D mouse tumors, suggesting successful modeling of tertiary lymphoid structures. However, the cells in TLS are mainly B cells, with very few T cells, and a complete FDC network is not formed. That is, TLS can appear in the KrasG12D model, but only in small numbers as E-TLS.

[0052] Figure 2 B and 2C represent the generation of CD19 in TLS and the generation of CD3 in T cells. Figure 2D and 2E indicate that 1 Gy irradiation is sufficient to increase TLS production and CD3+ T cell count; flow cytometry results are as follows: Figure 2 As shown in Figures F-I, consistent with the above results, TLS production increased, and the number of CD3+ T cells increased. That is, 1 Gy radiotherapy can increase the infiltration of immune cells and the formation of TLS, but TLS is still in a relatively early stage.

[0053] Figure 3 B~3D are the results of flow cytometry, which first demonstrated that the two main types of B cells, B220+B and CD19+B cells, were significantly increased in the 1Gy+PD-1 treatment group. Figure 3 E~3G indicates that the PD-1+1Gy treatment group has a significantly higher number and density of TLS compared to other groups; and Figure 3 H shows the results of MIF staining using B cell markers (B220, CD19), T cell markers (CD3), FDC markers (CD21), GC markers (GL7), and B lymphocyte chemokine (CXCL13), which co-localized TLS and its different maturation stages (E-TLS, PFL-TLS, SFL-TLS). Figure 3 I~3K indicates that the number of TLS at different maturity stages in the PD-1+1Gy dual-processor group is significantly higher than that in other groups.

[0054] Comparative Example 1: Effects of different mouse modeling methods on tertiary lymphoid structures

[0055] C57BL / 6J mice were purchased from GemPharmatech (Nanjing, China) and housed under specific pathogen-free conditions. Mice were intraperitoneally injected with urethane / carbamate (Sigma, USA) 800 mg / kg (8% urethane, 0.1 ml / 10 g body weight prepared with physiological saline), twice weekly for five weeks. Lung tumor formation was observed using a small animal in vivo Micro-CT imaging system at week 20 after the start of the model. Figure 4 (A, 4B), the remaining detection steps and administration methods are the same as in Example 1, and urethane model (abbreviated as: EC model) mice are obtained.

[0056] The results showed that H&E staining and immunohistochemical staining were used to evaluate CD19, the most commonly used biomarker of TLS, in EC model mice. No TLS was found in the urethane model mice. Figure 4 C,4D), with only a small number of tumor-infiltrating lymphocytes (TILs). Compared with the Kras-LSL-G12D model in Example 1, no TLS was found in the EC model mice. Figure 4E). Subsequently, EC model mice were subjected to low-dose radiotherapy of 1-2 Gy. Flow cytometry CD19 cell detection and immunohistochemical CD19 staining showed no formation of TLS (E). Figure 5 (A~C). Following radiotherapy combined with a PD-1 inhibitor, immunohistochemical CD19 staining revealed that although TLS (transferable lesions) were formed, the number was small, and the model was not stable. Figure 6 (A~B).

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of constructing a mouse model to promote formation and maturation of tertiary lymphoid structures, comprising, treatment of mice with a PD-1 inhibitor in combination with low dose radiotherapy; wherein the mice are Kras-LSL-G12D lung cancer model mice; the low dose radiotherapy is performed using X-rays at a dose of 1-2 Gy; and the PD-1 inhibitor is administered at a concentration of 0.1-10 μg / mL.

2. The method of claim 1, wherein, The PD-1 inhibitor is pembrolizumab, nivolumab, tremelimumab, tislelizumab, camrelizumab, and sintilimab.

3. The method of claim 1, wherein, The PD-1 inhibitor is administered at an interval of 3 days.

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