Feasibility of establishing a nude mouse model of thymoma-associated myasthenia gravis

Introduction

Myasthenia gravis (MG) is an acquired autoimmune disease mediated by autoantibodies and characterized by the inhibition of synaptic transmission at neuromuscular junctions (NMJ) in skeletal muscles. T lymphocytes and complement have a key role in the pathogenesis of MG (1,2). Recent comprehensive reviews highlight the heterogeneity of MG subtypes and their distinct immunological mechanisms (3). MG has been reported in ~30% of all patients with thymoma, of which >50% could be classified as type B2 according to World Health Organization (WHO) classification (4). Clinical studies have reported that MG symptoms alleviate or disappear following thymectomy in ~70% of patients with thymoma-associated myasthenia gravis (TAMG) (1,4). Nevertheless, in some patients, symptoms may not improve or may worsen following thymectomy, and therefore immunosuppressive therapy may be required for long periods of time (5). A number of cases may result in mortalities due to respiratory failure. Furthermore, in certain patients with thymoma without MG, symptoms appear following thymectomy. Therefore, these conditions seriously affect the clinical curative effect and prognosis of patients with thymoma.

A large number of studies have confirmed that the pathogenesis of TAMG is associated with thymoma, and autoreactive T cells arise from thymoma (6,7). In contrast to other cancers, >95% of all thymomas (with the exception of type A) generate polyclonal cluster of differentiation CD4⁺ and CD8⁺ thymocytes from bone marrow progenitors (4). This is closely associated with the occurrence of myasthenia gravis (MG) in thymoma patients (7); Regretfully, the specific mechanism remains unclear. Therefore, the pathogenesis of TAMG must be investigated to provide data for clinical treatments and prognosis. At the same time, it would also provide a novel theoretical basis and evidence for research into other subtypes of MG. However, further research on TAMG is hindered by the lack of mature and stable animal models. Therefore, the present study aimed to establish an animal model of TAMG by transplanting thymoma tissues from TAMG patients to the kidney membrane of BALB/C nude mice. The developed animal model mimics some of the clinical pathological characteristics of TAMG and may provide a novel experimental and theoretical basis for further study on the pathogenesis of TAMG. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1125/rc).

Methods

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the ethics committee of Tianjin Medical University (No. KY2018K018). All subjects were volunteers, and informed consent was obtained.

Patient samples

A total of six patients with TAMG received thymectomy. The subjects included 4 males and 2 females aged between 29 and 66 years, with a median age of 63 years. The patients were categorized based on the Ossermann MG classification (8): Type I in 1 case, type IIa in 2 cases, type IIb in 2 cases and type III in 1 case. All of the postoperative thymoma samples were diagnosed as thymoma via pathological examination according to the WHO classification (9) (type B1 in 1 case, type B2 in 1 case, mixed B2 and B3 in 4 cases).

Animal experiments

Based on the quantity of thymoma tissue obtained from surgical patients, healthy female BALB/c nude mice (n=36; 3 to 4 weeks old; 14 to 16 g) were bred in specific pathogen-free (SPF) conditions, and the thymoma tissues were numbered. Water, food and air were sterilized by ultraviolet light and the mice had free access to food and water. The humidity was maintained at 40% to 60%, and the temperature was kept at 26 to 28 ℃. Mats and cages were replaced 2 to 3 times per week.

The mice were anesthetized with 2% chloral hydrate through intraperitoneal injection. Thymoma tissue blocks (1 mm × 1 mm × 2 mm) were transplanted into the left renal subcapsules of the mice. The transplantations were completed within 6 h of thymectomy. The thymoma tissue of one TAMG patient was transplanted into 5 to 6 mice. All the Mice were randomly assigned to transplantation using a computer-generated sequence.

After 2 h, the mice recovered and were able to eat independently. Penicillin (80,000 units, Tianjin Huajin Pharmaceutical Co., Ltd., Tianjin, China) was injected intraperitoneally within 1 week to prevent infection. The mice were fed and observed in SPF conditions for 90 days. Venous blood (0.1 mL) was collected from each mouse. Heparin (Baxter International Inc., Deerfield, USA) was added to the blood samples for anticoagulation, and the samples were subsequently preserved at room temperature. The nude mice were subsequently sacrificed, and the left kidney was extracted, which was made into paraffin-embedded tissue, hematoxylin and eosin staining (H&E) was performed. The results were confirmed by three pathologists. Pathologists analyzing IHC and H&E stains were blinded to transplantation assignments. Experiments were performed under a project license (No. AE2021113) granted by the Ethics Committee of Tianjin Medical University, in compliance with Chinese national or institutional guidelines for the care and use of animals. The protocol (including the research question, key design features, and analysis plan) was prepared and registered before the study.

Flow cytometry

T lymphocytes and subsets in mouse peripheral blood were detected using flow cytometry prior to and following transplantation. 0.1 ml tail vein blood of each nude mice (anticoagulant: ethylenediaminetetraacetic acid) were collected, stored at room temperature. Cells were then stained with fluorescence-labeled antibody (1 µL/1×10⁶ cells) that was specific for the following markers (BD Pharmingen, San Diego, USA): PE-labeled mouse anti-mouse CD8a, PerCP-labeled mouse anti-mouse CD3e, and FITC-labeled mouse anti-mouse CD4, and then 100 µL of tail venous blood of nude mice was added to set the vortex. Mix and incubated in a refrigerator at 4 ℃ for 30 minutes. Red blood cells were lysed by adding 1 mL hemolysin (Optilyse C, BD Pharmingen), mixed and then placed at room temperature for 10 minutes. Centrifuge at 1,500 rpm/min for 5 min and discard the supernatant; add 1 mL phosphate buffer solution, centrifuge at 1,500 rpm/min for 5 min, wash twice. Cells were washed with 300 µL phosphate buffer solution and read on an FACS canto II. Data analysis was performed using FACS Diva II software (Becton, Dickinson and Company, BD). The Experiment was repeated three times pre-transplantation and post-transplantation.

Enzyme-linked immunosorbent assay (ELISA)

Serum levels of total complement cells in mouse peripheral blood were determined using ELISA [Serum total complement CH50 ELISA kit, QiYi Biological technology (Shanghai) Co., Ltd., Shanghai, China] prior to and following transplantation, which performed according to the manufacture’s instructions.

Immunohistochemistry (IHC)

Specimens were cut into 4µm sections, fixed with 10% neutral-buffered formalin and embedded in paraffin. The H&E were used to stain the tissues. A streptavidin-peroxidase (SP) IHC method was applied. The SP IHC kit, Cytokeratin (CKs, mouse anti-human polyclonal antibody), T-cell surface glycoprotein CD5 (rabbit anti-human Monoclonal antibody) and terminal deoxynucleotidyl transferase (TdT, rabbit anti-human polyclonal antibody) were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). All the antibodies were diluted by distilled water. We observed the staining intensity and distribution of positive cells at high magnification; no staining was scored as 0, weak staining but significantly stronger than the negative control was scored as 1, a clear stain is scored as 2, and a strong stain is scored as 3. No positive control cell is scored as 0. The required number of positive cells, i.e., <30%, 30–60%, and >60%, were scored as 1, 2, and 3, respectively. Based on the total scores, the samples were assessed for CKs, CD5, TdT expression. A score of ≤2 was classified as negative, a score of 3 was weakly positive, a score ranging from 4 to 5 was positive, and a score of 6 was strongly positive. The results were confirmed by three pathologists.

Statistical analysis

Data are presented as the mean ± standard deviation (the number of experimental replicates: n=20). Data normality was confirmed using Shapiro-Wilk tests. Data were analyzed using paired t-test using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA) statistical software. P<0.05 was considered to be statistically significant.

Results

Transplantation

The success rate of the operation was 92%. Out of a total of 36 mice, there was a single mortality due to an anesthetic accident, and transplantation failed in 2 cases due to destruction of the renal capsule. A total of 3 months following transplantation, the mouse model did not present with symptoms of MG. A number of measures were performed to increase the survival rates, such as the specimens were stored in cell culture medium, the transplantations was finished within 6h after the specimens were resected, the improved surgical skills and the using of antibiotics.

The thymoma tissue located at the renal subcapsule presented no evident growth, and the boundary between the renal tissue and the transplant was clear (Figure 1). The boundary between the renal parenchyma and the transplant was also clear. Furthermore, the internal structure was clear and staining indicated no ischemia, vacuolization or necrosis (Figure 2). These findings indicated that transplantations were successful in 20 mice, with a success rate of 61%. Further detection performed by IHC confirmed the results (Figure 3). Diffuse CK positivity, scattered weak CD5 expression, and focal dot-like TdT staining were observed, which confirms that the transplants were thymoma tissues. Whereas, the transplant had not significantly increased in size 3 months post-transplantation.

Figure 1 Patient thymoma tissue located at the renal subcapsule.

Figure 2 Hematoxylin and eosin staining of thymoma transplants under the renal subcapsule in mouse. (A) Thymoma tissue; (B) renal tissue. The number of replicates, n=20; the images are representative of all samples taken (×50).

Figure 3 Immunohistochemical analysis (×50): diffuse cytokeratin (A) positivity, scattered weak CD5 expression (B), and focal dot-like TdT staining (C) were observed. TdT, terminal deoxynucleotidyl transferase. The arrow points to the transplanted thymoma tissue.

T lymphocytes and subsets prior to and following transplantation

The peripheral blood of BALB/c nude mice was collected prior to and following transplantation, and changes in T lymphocytes and subsets were analyzed. Flow cytometric analysis in Figure 4 indicated that the percentage of CD3⁺ T and CD4⁺ T cells following transplantation significantly increased compared with the percentage prior to transplantation, whereas the percentage of CD8⁺ T cells decreased (P<0.05; Table 1). However, the proportion of the T lymphocyte in the entire peripheral blood cells remained low.

Figure 4 Flow cytometric analysis of T lymphocyte subsets in mouse peripheral blood prior to and following transplantation. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Detection of complement prior to and following transplantation

Analysis of the number of total complement cells in the mouse peripheral blood by ELISA demonstrated a significant decrease following transplantation compared with pre-transplantation (P<0.05; Table 2).

Discussion

MG is an acquired autoimmune disease mediated by autoantibodies and characterized by the inhibition of synaptic transmission at NMJ in skeletal muscles. MG classification is diverse and complex. MG can be divided into early onset, late-onset and TAMG (6) based on age at onset, gender and genetic differences, antigen specificity and accompanying changes in thymus pathology.

Several studies have confirmed that the pathogenesis of TAMG is associated with thymoma, however the specific underlying mechanism remains unclear (6). Emerging evidence suggests that thymoma-derived autoreactive T cells disrupt immune tolerance through dysregulated cytokine signaling and aberrant antigen presentation (10). Research on TAMG is considerably hindered by the lack of mature and stable animal models. Since the first animal models of MG using rats and guinea pigs were established by Lennon et al. (11) in the 1970s, three different types of experimental autoimmune MG (EAMG) were formed (12). These models included active immunization, where acetylcholine receptor (AChR) was injected into an animal to induce MG, and passive immunization, which involved the injection of anti-AChR antibody refined from patients or animals into another animal. A third model involved the injection of lymphocytes from peripheral blood of patients with MG into an animal (12). However, these models were considered MG alone, rather than the roles of thymoma in the pathogenesis.

Schönbeck et al. (13) demonstrated that lymphoid follicular hyperplasia of the thymus has a key role in the pathogenesis of MG. A model of MG was established by transplanting thymus tissue into mice with severe combined immunodeficiency. The model induced increasing anti-AChR antibody titers, but without clinical myasthenia symptoms. The current animal models of EAMG were established by injecting exogenous antigen or antibody. Several researchers developed animal models by transplanting hyperplastic thymus tissues. However, no MG symptoms were induced, and the specimens lacked the immune characteristics of thymoma. Therefore, the pathogenesis of thymoma has not been investigated in detail (13). An animal model established through the transplant of thymoma tissues, which is able to induce symptoms of MG may be useful to further investigate the pathogenesis of thymoma.

In the present study, an animal model of TAMG was established by transplanting human thymoma tissues into the left renal subcapsules of BALB/c nude mice. The mice were observed for 90 days following transplantation and dissected for removal of the left kidney. No evident growth was present in the thymoma tissues located at the renal subcapsules, and the boundary between the kidney and the transplant was clear. These findings indicated that the success rate of the transplants was 61%.

Thymoma is a benign tumor. The growth rate of thymoma is slow, and thymoma exhibits decreased invasiveness compared with other malignant tumors, such as lung cancer, gastric cancer; therefore, the volume of the transplants may not significantly increase, and the survival rate of transplants may be reduced compared with malignant tumors. To increase the survival rate, a number of measures were performed in the present study. These measures included storing tissue specimens in cell culture medium and completing the transplantation within 6 h to maintain the activity of the sample. In addition, surgical skills were improved to increase the accuracy of the transplant and reduce damage to the surrounding renal tissues. Antibiotics were used to reduce postoperative inflammatory reaction.

The peripheral blood of BALB/c nude mice was collected prior to and following transplantation, and changes in T lymphocytes and subsets were analyzed. The percentage of CD3⁺ T cells following transplantation significantly increased compared with the percentage prior to transplantation. This finding indicates that the transplant affected the peripheral T lymphocytes of the nude mice, and it may partly function as the thymus and reconstruct T-cell immunity. Given that the transplants were type B thymoma, the authors of the present study hypothesize that the cells of type B thymoma were able to promote proliferation, differentiation, maturity, and migration of native T lymphocytes to peripheral blood.

In our study, the percentage of CD3⁺ T and CD4⁺ T cells following transplantation significantly increased compared with the percentage prior to transplantation, whereas the percentage of CD8⁺ T cells decreased. The percentage of CD4⁺/CD8⁺ T cells was imbalanced(high) in T cell homeostasis, and the T lymphocyte in the peripheral blood of the mouse model was disordered. Such imbalances may reflect a broader dysregulation of T helper cell subsets, as observed in other autoimmune models (10). These results are in accord with the T cell changes in patients with TAMG. The results of the present study also suggest that the microenvironment of thymoma was disordered, which may lead to atypical peripheral outputs of reactive T lymphocytes and abnormal immune cell ratios (7). Taking into consideration that the effector cells of CD4⁺ T lymphocytes are T-helper cells, further research will be conducted to study the subsets of T-helper cells in the peripheral blood of the mouse model, which may enable further study of the pathogenesis of TAMG.

The pathological features of MG include the deposition of immunoglobulin G (IgG) and complement components at the NMJ (14,15), which confirms the antibody-mediated pathological mechanism of MG. Notably, specific IgG1 subclass antibodies targeting AChR have been identified as dominant contributors to autoimmune responses in MG, suggesting potential therapeutic targets (16). AChR antibody-induced MG is mainly caused by complement-mediated lysis of the post-synaptic cell membrane, thereby promoting AChR degradation and directly inhibiting the binding between acetylcholine and AChR (17). Researchers have reported the accumulation of anti-AChR-IgG and complement cells in an EAMG mouse model, which causes formation of a membrane attack complex, and results in the destruction of muscle membrane and a reduction in the number of functional AChRs. Other studies, which utilized IHC and electron microscopy, revealed that the complements that accumulated in the postsynaptic membrane at the NMJ were complement component 3 (C3) and C9 fragments. Further studies reported that serum C3 and C4 levels were decreased and AChR antibody titer levels were increased in patients with MG. This finding indicates an excessive consumption of complement components, although a decreased serum level of C3 or C4 was not associated with the severity of MG (17).

It has been reported that susceptibility to MG symptoms is significantly decreased in EAMG mouse models of knockouts of complement components. These mouse models did not show characteristic symptoms of MG, which suggests that the integrity of complement components is necessary for the pathogenesis of AChR antibody-associated MG. Therefore, in the present study, the total serum complement cells in nude mice prior to and following transplantation was analyzed. It was demonstrated that the total complement in the peripheral blood significantly decreased following transplantation. This finding suggests the activation and consumption of complement cells in the mice following transplantation. The complement cells were involved in the incidence of complement-dependent autoimmune disease.

In the present study, thymoma tissues were obtained from patients with TAMG and transplanted into the left kidney capsules of BALB/c nude mice. Despite successful thymoma engraftment and observed immunological changes (T-cell imbalance and complement consumption), no clinical signs of MG (e.g., muscle weakness, fatigability) were observed in the mice during the 90-day study period. The authors of the present study have hypothesized that this finding may be due to a number of reasons.

One possible reason may be the small size of specimens used for the present study. The size of the transplanted tumor tissue blocks was 1 mm × 1 mm × 2 mm. Although the tissues survived, they had not significantly increased in size 3 months post-transplantation. The percentage of peripheral blood T lymphocytes increased following transplantation, but the proportion in the entire peripheral blood cells remained low. All of these findings may be due to the small size of the transplant tissue blocks or the low amount of functional tissues. Another possible reason is that the observation time was too short. Thymoma, particularly B type thymoma, is an indolent tumor and exhibits reduced growth and invasion rates compared with malignant tumors (18). Thirdly, the absence of human B cells or autoantibody-producing plasma cells in the nude mouse model, which are critical for NMJ damage in MG.

To address these limitations, subsequent studies should: (I) test for serum anti-AChR antibodies (AChR-Ab) via radioimmunoprecipitation assay to confirm B-cell activation; (II) perform electrophysiological studies (repetitive nerve stimulation/single-fiber) to detect subclinical NMJ dysfunction; (III) extend observation periods (>6 months) or use humanized mice (e.g., NSG mice engrafted with patient PBMCs to enhance human immune reconstitution; (IV) co-transplant thymoma tissues with autologous PBMCs to promote autoreactive B-cell activation and autoantibody production (16,19).

Conclusions

The results of the present study confirmed that transplanted human thymoma is able to survive for extended lengths of time in BALB/c nude mice. This proof-of-concept study demonstrates the feasibility of transplanting human thymoma tissues into BALB/c nude mice is able to maintain the histological and immunological characteristics of human thymoma. While the model recapitulates key immune alterations (e.g., T-cell dysregulation), the absence of clinical MG symptoms highlights the need for further optimization. In a word, several issues and challenges in relation to the establishment of a TAMG model must be resolved and investigated in future research.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1125/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1125/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1125/prf

Funding: This project was financially supported by the Youth Foundation of The Second Hospital of Tianjin Medical University (No. 2017ydey09).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1125/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the ethics committee of Tianjin Medical University (No. KY2018K018). All subjects were volunteers, and informed consent was obtained. Experiments were performed under a project license (No. AE2021113) granted by the Ethics Committee of Tianjin Medical University, in compliance with Chinese national or institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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