Research ArticleExperimental Studies
Open Access

Humanized PD-1 Knock-in Mice Reveal Nivolumab’s Inhibitory Effects on Glioblastoma Tumor Progression In Vivo

CHUN-YU CHIANG, MENG-CHU HUANG, SHIH-CHONG TSAI, FEI-TING HSU, TSAI-LAN LIAO, JEI-HWA YU, TZU-HSIANG LIN, HUA-HSIH HUANG and PEN-AN LIAO
In Vivo September 2023, 37 (5) 1991-2000; DOI: https://doi.org/10.21873/invivo.13296

Abstract

Background/Aim: Immunotherapy has been considered a promising approach for brain tumor treatment since the discovery of the brain lymphatic system. Glioblastoma (GBM), the most aggressive type of brain tumor, is associated with poor prognosis and a lack of effective treatment options. Materials and Methods: To test the efficacy of human anti-PD-1, we used a humanized PD-1 knock-in mouse to establish an orthotopic GBM-bearing model. Results: Nivolumab, a human anti-PD-1, effectively inhibited tumor growth, increased the survival rate of mice, enhanced the accumulation and function of cytotoxic T cells, reduced the accumulation and function of immunosuppressive cells and their related factors, and did not induce tissue damage or biochemical changes. The treatment also induced the accumulation and activation of CD8+ cytotoxic T cells, while reducing the accumulation and activation of myeloid-derived suppressor cells, regulatory T cells, and tumor-associated macrophages in the immune microenvironment. Conclusion: Nivolumab has the potential to be a treatment for GBM.

Key Words:

Glioblastoma (GBM) is a prevalent malignant tumor of the central nervous system that frequently occurs in both adults and children. Despite the benefits of resection, radiotherapy, and temozolomide chemotherapy in improving the prognosis, glioblastoma patients continue to face a significant mortality risk. The improvement of prognosis in glioblastoma is a critical area of research that necessitates the development of effective innovative approaches (1-3).

Vascular endothelial growth factor (VEGF) is an angiogenic protein required for tumor progression. Glioblastoma employs the expression of VEGF to induce angiogenesis and increase blood-brain barrier (BBB) permeability. Consequently, this process results in the development of peritumoral edema (4, 5). Tumor-associated edema is frequently treated with corticosteroid therapy (6). Bevacizumab is a humanized anti-VEGF monoclonal antibody that was approved by the FDA in 2009 for the treatment of recurrent glioblastoma. By targeting the VEGF pathway, it does not only inhibit angiogenesis but also alleviates BBB permeability and peritumoral edema (4, 7, 8).

The synergy between the innate and adaptive immune systems is crucial for achieving antitumor immunity, which involves the combined action of both immune responses to effectively suppress tumors (9). CD8+ T cells, also known as cytotoxic T lymphocytes (CTLs), play a vital role in the immune response against tumors by eliminating tumor cells through two main pathways of apoptosis: the perforin/granzyme B pathway and the Fas/Fas ligand (FasL) pathway (10). The programmed death receptor 1 (PD-1)/programmed death ligand 1 pathway is an immune checkpoint signaling pathway that inhibits the anti-cancer function of CD8+ T cells, thereby promoting tumor immune evasion (11, 12).

Nivolumab, an anti-PD-1 monoclonal antibody, has demonstrated potential therapeutic benefits in recurrent glioblastoma patients who do not use baseline corticosteroids. In terms of median overall survival, nivolumab exhibits equivalent efficacy to bevacizumab in patients with recurrent glioblastoma (13). Nivolumab may offer an effective approach for the treatment of glioblastoma, and further evaluation of its anti-glioblastoma mechanism is warranted. Therefore, the main goal of the present study was to investigate the impact of nivolumab on anti-tumor immunity in glioblastoma in vivo.

Materials and Methods

Culture of GL261 cells. The GL261 cells were obtained from the Leibniz Institute DSMZ in Braunschweig, Germany. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (10,000 U/ml). The cells were maintained in a 5% CO2 and 37°C incubator. All culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

GL261 bearing animal model. PD-1 humanized mice (humanized PD-1 knock-in) were generously provided by Dr. Lan at the National Yang Ming Chiao Tung University in Taipei, Taiwan. For the intracerebral injections, we used 10,000 GL261 cells resuspended in 3 μl of phosphate-buffered saline (PBS) and administered with a 26G Hamilton syringe. The injection site was precisely targeted using a stereotaxic apparatus, positioned 3 mm lateral to the right and 1 mm posterior to the bregma, and a depth of 3 mm. The cells were injected at a rate of 1 μl per min, and the Hamilton syringe was held in place for 15 min before being removed at a rate of 1 mm per min (14). This allowed for the cells to be effectively delivered to the targeted location and minimized the potential for backflow or leakage. On day 10 following the initial injection of cells, the mice were anesthetized for magnetic resonance imaging (MRI) scanning, which was designated as day 0 for treatment initiation.

Animal grouping and experimental procedures. The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University, Taichung, Taiwan (ID: CMUIACUC-2021-396). The mice were housed in an appropriate environment under specific pathogen-free conditions throughout the experiment. Once the tumor volume reached 10 mm3, the mice were randomly assigned to one of two groups: a control group or a group receiving nivolumab treatment at a dose of 5 mg/kg. Nivolumab was administered by intravenous injection twice per week for a total of six treatments (on days 1, 4, 7, 10, 13, and 16). The control group was also injected intravenously with 100 μl PBS containing 0.1% DMSO. Tumor progression was monitored using MRI scans, as described in the following section. The Kaplan–Meier method was employed for survival analysis.

Magnetic resonance imaging (MRI) scanning of mouse brain. Tumor progression was monitored using 2D TurboRARE sequences acquired with a Brucker 9.4T PharmaScan scanner on days 0, 14, 21, and 28. Tumor volume was quantified using the PMOD Biomedical Image Quantification Software VOI toolbox (PMOD Technologies LLC, Zürich, Switzerland). To evaluate tumor progression, we performed T2-weighted RARE sequence scans on mice bearing GL261 tumors. A 2D TurboRARE MRI scan with fat suppression was performed using a rapid acquisition with relaxation enhancement sequence, with the following parameters: field of view (FOV) of 20×20 mm2, a matrix size of 256×256, a repetition time (TR) of 2,500 ms, two excitations (NEX), an effective echo time (15) of 33 ms, and 16 slices with a thickness of 0.5 mm (14).

Flow cytometry analysis of cells isolated from mouse organs. We isolated the spleen (SP), bone marrow (BM), and tumor-draining lymph nodes (TDLN) from both the control and nivolumab-treated mice on day 30. Single cells were filtered through a 40 μm strainer twice, and red blood cells were lysed using ACK buffer (Thermo Fisher Scientific). Afterward, the cells were neutralized using Hank’s Balanced Salt Solution buffer and stained using specific markers, namely CD8, CD11b, and Gr-1. Intracellular markers, such as IFN-γ and IL-2, were stained after the cells were fixed and permeabilized using BD fixation and permeabilization solution (BD Biosciences, Franklin Lakes, NJ, USA) as per the manufacturer’s protocol. The fluorescence intensity from each group was recorded using a NovoCyte flow cytometer and quantified using NovoExpress® software (Agilent Technologies Inc., Santa Clara, CA, USA) (16, 17). The markers used in the flow cytometry analysis are listed in Table I.

View this table:
Table I.

Antibodies used for flow cytometry.

Hematoxylin and Eosin (H&E) staining. On day 29, we isolated the heart, liver, kidney, and intestine from both control and nivolumab-treated mice. The tissues were embedded in paraffin and sliced to 5 μm thickness for further staining. The H&E staining has been previously described (18).

Biochemistry analysis. On day 29, we collected and isolated serum from each group of mice and stored it at −80°C. The serums were sent to Bio-Check Laboratories (New Taipei City, Taiwan, ROC) for evaluating Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), and gamma-glutamyl transferase (γGT) values using standardized tests (19).

Immunohistochemistry (IHC) staining. On day 29, we isolated tumors from both the control and nivolumab-treated mice, embedded them in paraffin, and sliced them to a thickness of 5 μm for further staining. We followed the IHC staining protocol described in previous studies (20) and used the primary antibodies listed in Table II.

View this table:
Table II.

Antibodies used for immunohistochemistry.

Statistical analysis. The significant difference between the control and nivolumab-treated groups was calculated using the Student t-test, and the specific p-value is presented in each figure.

Results

Nivolumab demonstrates efficacy in inhibiting GL261 tumor progression in in vivo model. To validate the efficacy of human anti-PD-1, we established a GL261 orthotopic mouse model using humanized PD-1 knock-in mice. To assess tumor progression, GL261 cells (1×105 cells) were injected into the right cerebral hemisphere of mice. Mice with tumors were then randomized into a control group and a nivolumab (5 mg/kg) treatment group. The tumor progression was monitored by performing MRI scans on days 0, 14, 21, and 28 (Figure 1A). The MRI images of three mice from the control group and four mice from the nivolumab group were obtained throughout the treatment process (Figure 1B). Our results show that nivolumab treatment inhibited tumor growth compared to the control group, as evidenced by the reduced tumor size observed on MRI scans (p=0.0402) (Figure 1C). Furthermore, mice in the control group began to die on day 14, while those in the nivolumab group survived longer (log-rank p=0.0143) (Figure 1D). Overall, our findings suggest that nivolumab treatment may be effective in treating GL261 tumors on humanized PD-1 knock-in mice.

Figure 1.
Figure 1.

Treatment efficacy of nivolumab was validated using orthotopic GBM bearing model. (A) The experimental flowchart for efficacy and survival analysis of nivolumab is shown. (B) The representative magnetic resonance images from both the control and nivolumab groups of mice are presented. (C) The tumor volume of mice was quantified and displayed using PMOD software. (D) The survival outcome of both control and nivolumab groups of mice measured by Kaplan–Meier analysis is presented.

Nivolumab does not induce tissue damage, biochemical changes, or changes in the body weight of mice. To assess the safety profile of nivolumab, we evaluated tissue pathology staining, biochemical changes in the serum, and changes in body weight during treatment. As shown in Figure 2A, only the control group exhibited weight loss, which may be attributed to tumor progression. In contrast, nivolumab-treated mice maintained their weight throughout the treatment process. Furthermore, we examined liver function by measuring the levels of AST and ALT in the serum, and found no significant differences between the control and nivolumab-treated groups (Figure 2B). We also measured the level γGT in the serum before and after treatment (Table III). The level of γGT remained stable after nivolumab treatment, indicating that nivolumab did not induce liver damage or dysfunction. Additionally, we performed H&E staining on the heart, liver, kidney, and intestine of mice in both groups to assess whether nivolumab treatment altered tissue pathology. Importantly, no significant pathological alterations were observed in the nivolumab group compared to the control group (Figure 2C). Following the administration of nivolumab treatment, the changes in pathology were thoroughly assessed using Shackelford’s (2002) four-scale method to record the severity scores of each treatment group (20). These severity scores were used to evaluate the extent and severity of any alterations in the observed pathology, providing a comprehensive and detailed analysis of the treatment’s effects on the specific condition under investigation. However, no obvious change was observed (Table IV). Our findings demonstrate that nivolumab is well-tolerated in mice and suggest its potential for use in GBM treatment.

Figure 2.
Figure 2.

The general toxicity of nivolumab was evaluated in vivo using body weight measurements, biochemical analyses, and H&E staining. (A) The body weight of mice was recorded every three days during the treatment. (B) The biochemistry analysis of mouse serum, including AST and ALT, is displayed. (C) The pathology of the heart, liver, kidney, and intestine of mice evaluated by H&E staining is displayed. ns: No significant difference. Scale bar=100 μm.

View this table:
Table III.

The serum level of gamma-glutamyl transferase (γGT).

View this table:
Table IV.

Severity scores for pathological alterations after Nivolumab treatment (19).

Nivolumab effectively induces the accumulation and activation of CD8+ cytotoxicity T cells in vivo. To investigate the effects of nivolumab on the immune microenvironment in mice, we conducted flow cytometry analysis and IHC staining. We isolated tumor draining lymph nodes (TDLN) and splenocytes (SP) from mice and stained them with different immune cell markers as described in the Materials and Methods section. Figure 3A shows the population of CD8+ T cells and the expression levels of IFN-γ and IL-2 in TDLN. We found that nivolumab effectively induced the accumulation of CD8+ T cells in TDLN compared to the control group (p=0.0219) (Figure 3B). Moreover, nivolumab treatment also increased the levels of CD8+/IFN-γ and CD8+/IL-2, which are markers of T cell function, in TDLN. We also observed an increase in the accumulation and activation of CD8+ cytotoxic T cells in both TDLN and SP (Figure 3C and D).

Figure 3.
Figure 3.

The positive immune regulation of nivolumab was validated using flow cytometry. (A) The pattern of CTL accumulation and activation in TDLN is presented. (B) Quantification of CD8+ T cells, CD8+IFN-γ+ T cells, and CD8+IL-2+ T cells in TDLNs from both control and nivolumab-treated mice is displayed. (C) The pattern of CTL accumulation and activation in SP is presented. (D) Quantification of CD8+ T cells, CD8+IFN-γ+ T cells, and CD8+IL-2+ T cells in SP from both control and nivolumab-treated mice is displayed. (E) The pattern of MDSC accumulation and activation in BM and SP are presented. (F) Quantification of CD11b+Gr-1+ MDSC cells in BM and SP from both control and nivolumab-treated mice is displayed.

We further investigated the expression levels of CD8 and IFN-γ in the tumor area using IHC staining (Figure 4A). The protein expression levels of CD8 and IFN-γ were found to be increased in the nivolumab group. In contrast, the expression level of IDO-1 was decreased following nivolumab treatment. These findings suggest that nivolumab positively regulates the immune response by activating cytotoxic T cells (CTL) and their associated factors. The activation of CTL may facilitate the treatment efficacy of nivolumab against GBM.

Figure 4.
Figure 4.

The inhibitory effect of nivolumab on the immune system validated using IHC staining. (A) The protein expression of CD8, IFN-γ and IDO-1 in tumor tissues was validated using IHC staining and is presented. (B) The protein expression of VEGF, FOXP3, and Gr-1 in tumor tissues was validated using IHC staining and is presented. (C) The protein expression of CD86, and CD206 in tumor tissues was validated using IHC staining and is presented.

Nivolumab effectively reduces the accumulation and activation of immunosuppressive cells. To investigate the impact of nivolumab on the immune microenvironment, we performed flow cytometry analysis and IHC staining. Specifically, we isolated bone marrow (BM) and splenocytes (SP) from mice and analyzed their immune cell populations using various markers. As shown in Figure 3E-F, nivolumab effectively reduced the accumulation and activation of immunosuppressive cells, MDSC, in BM and SP. Furthermore, we observed a decrease in the protein expression level of Gr-1 in tumor tissue in the nivolumab group (Figure 4B).

Additionally, the protein levels of VEGF, FOXP3, and CD206, which play a crucial role in the recruitment of Treg and tumor-associated macrophages, were found to be reduced in the nivolumab group compared to the control group (Figure 4B and C). However, the inflammatory macrophage marker CD86 was found to be induced in the tumor area after nivolumab treatment (Figure 4C). These results suggest that nivolumab not only suppresses the accumulation of immunosuppressive cells and their related factors but also triggers the accumulation of immunosupportive cells and the expression of their factors, leading to a more favorable immune microenvironment for GBM treatment.

Discussion

The interaction between PD-L1 expressed on tumor cells and PD-1 on activated CD8+ T cells leads to the inactivation of CD8+ T cells and this interaction can be prevented using monoclonal antibodies targeting PD-L1 and PD-1 (21, 22). High expression of PD-L1 is an unfavorable prognostic factor for glioblastoma, whereas increased infiltration of CD8+ T cells within tumors is associated with improved prognoses among glioblastoma patients (23, 24). Our data demonstrated that nivolumab effectively promoted CD8+ T cells activation and the accumulation of tumor-infiltrating CD8+ T cells, while also inhibiting the growth of orthotopic glioblastoma (Figure 3A-D and Figure 4A). Apart from the presence of CD8+ T cells, M1-like macrophages contribute to anti-tumor immunity by triggering cytotoxic effects against cancer cells (25, 26). Additionally, we also discovered that nivolumab significantly upregulates the expression of CD86, a critical marker of M1-like macrophages (Figure 4C).

In addition to tumor cells, immunosuppressive cells, including MDSCs, Tregs, and M2-like macrophages, contribute to the attenuation of the anti-tumor immune response and the facilitation of tumor immune evasion (27-29). MDSC cells express PD-L1 to inactivate CD8+ T cells and induce conversion of T helper type 1 (Th1) cells into Tregs. Furthermore, Tregs also express PD-L1 (30-32). The increase of MDSCs has been reported to correlate with poor prognosis of glioblastoma (33). The results demonstrated that nivolumab exerted a significant inhibitory effect on the population of MDSCs in the spleen and bone marrow. Moreover, it demonstrated effective reduction in the protein levels of Gr-1 and FOXP3, which are crucial markers associated with MDSCs and Tregs, respectively (Figure 4B).

The proportion of M2-like macrophages in gliomas is known to increase in parallel with higher tumor grades (34). M2-like macrophages are conducive to tumor progression by secreting protein growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor beta (TGF-β). In addition, M2-like macrophages also participate in the inactivation of CD8+ T cells (35, 36). The immunosuppressive proteins IDO-1 and VEGF, which are expressed by tumors, suppress the anticancer function of CD8+ T cells and promote the recruitment of Tregs and MDSCs (37, 38). Our research revealed that the administration of nivolumab effectively eliminated both IDO-1 and CD206, a marker of M2-like macrophages (Figure 4).

In conclusion, our study offers compelling evidence of the effectiveness of nivolumab in treating glioblastoma and provides insights into its impact on anti-tumor immunity. Based on our data, we suggest that the reduction of immunosuppressive factors, including IDO-1, VEGF, and immunosuppressive cell populations such as MDSCs, Tregs, and M2-like macrophages, may contribute to the enhanced anti-glioblastoma efficacy of CD8+ T cells in response to nivolumab treatment.

Acknowledgements

The Medical Research Core Facilities Center, Office of Research & Development at China Medical University, Taichung, Taiwan, ROC, was utilized to conduct experiments and analyze data in part.

Footnotes

  • Authors’ Contributions

    CYC, MCH, and FTH conducted all the experiments, THL carried out the statistical analysis, and summarized the data. FTH, HHH, and PAL drafted the initial version of the paper. HHH and PAL conceptualized the presented idea, supervised the findings of this work, and performed the literature review. TLL, SCT, JHY, and PAL prepared the final version of the paper.

  • Conflicts of Interest

    The Authors state that they have no financial interests that could be perceived as conflicting with the results or conclusions presented in this study.

  • Funding

    The study received support from the Show Chwan Memorial Hospital, Changhua, Taiwan (ID: SRD-111017) and Cathay General Hospital, Taipei, Taiwan, R.O.C (ID: CGH-MR-A11138).

  • Received June 20, 2023.
  • Revision received July 20, 2023.
  • Accepted July 21, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Humanized PD-1 Knock-in Mice Reveal Nivolumab’s Inhibitory Effects on Glioblastoma Tumor Progression In Vivo
CHUN-YU CHIANG, MENG-CHU HUANG, SHIH-CHONG TSAI, FEI-TING HSU, TSAI-LAN LIAO, JEI-HWA YU, TZU-HSIANG LIN, HUA-HSIH HUANG, PEN-AN LIAO
In Vivo Sep 2023, 37 (5) 1991-2000; DOI: 10.21873/invivo.13296
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