Local Delivery of Minocycline and Vorinostat Targets the Tumor Microenvironment to Inhibit the Recurrence of Glioma

Abstract

Background: Postoperative recurrence is the main reason for poor clinical outcomes in glioma patients, so preventing tumor recurrence is crucial in the management of gliomas.

Methods: In this study, the expression of matrix metalloproteinases (MMPs) in normal tissues was detected via RNA-seq analysis. Glioma cases from the public databases (The Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA)) were included in this study. The hydrogel contains minocycline (Mino) and vorinostat (Vor) (G/Mino+Vor) was formed under 365 nm when the photoinitiator was added. High-performance liquid chromatography (HPLC) was used to assess the release of drugs in the G/Mino+Vor hydrogel. An MTT assay was used to explore the biosecurity of GelMA. Immunohistochemistry, ELISA, and TUNEL assays were used to demonstrate the antitumor effect of the G/Mino+Vor hydrogel.

Results: We successfully developed a G/Mino+Vor hydrogel. The experiments in vitro and in vivo confirmed the MMPs-responsive delivery of minocycline and vorinostat in hydrogel and the anti-glioma effect on an incomplete tumor operation model, which indicated that the G/Mino+Vor hydrogel effectively inhibited the recurrence of glioma after surgery.

Conclusion: In summary, the G/Mino+Vor hydrogel could continuously release drugs and improve the therapy effects against recurrent glioma.

Keywords: hydrogel; minocycline; recurrent glioma; tumor microenvironmennt; vorinostat.

Figure 1

Association of MMP2 and MMP9 expression with patient survival. (A) Glioma tissue samples from patients were stained to visualize MMP2 and MMP9 (brown). Scale bar, 50 µm. (B and C) The difference in MMP2 expression among different histological types of anaplastic gliomas patients and normal people, one-way ANOVA. (D and E) Kaplan–Meier survival curves for the glioma patients with low MMP2 expression (blue) compared to those with high MMP2 expression (red), Mantel-Cox Log rank test. (F and G) Expression of MMP2 protein level in different grade glioma, one-way ANOVA.

Figure 2

M1 and M2 macrophages expression in GBM. (A) Proportion of immune cells in normal and tumor tissues (1–4, normal; 5–12, GBM). (B) Expression of M1 and M2 level in normal and GBM, t-test.

Figure 3

Preparation and characterization of G/Mino+Vor hydrogels. (A) Gelation of G/Mino+Vor hydrogel before and after UV irradiation. (B) SEM images of G/Mino+Vor hydrogel. Scale bar, 2 µm. (C) Schematic illustration of G/Mino+Vor hydrogel degradable process under MMPs conditions. (D) The drug release profile of G/Mino+Vor hydrogels under different conditions. (E and F) Toxicity detection of GelMA on GL261 and HA1800 cell lines.

Figure 4

Reduced MMPs expression in response to G/Mino+Vor treatments. (A) Immunohistochemistry staining of representative tumor sections using MMP2 and MMP9 antibodies on the 28thday after tumor implantation. Scale bar, 50 μm. (B and C) Immunohistochemical slides were analyzed quantitatively using integral optical density (IOD) analysis.

Figure 5

Repolarization of M2 macrophages caused by G/Mino+Vor treatments. (A–D) Levels of TNF-α, IL-4, IL-6 and IL-10 in glioma tissue by ELISA on the 21th day after tumor implantation. One-way ANOVA.

Figure 6

Therapeutic efficacy of G/Mino+Vor hydrogel in the GL261 glioma tumor model. (A) Schematic outline shows the in vivo experimental design for treatments. (B) Representative micrographs of H&E stained tumors in orthotopic models. (C) Kaplan–Meier survival curves for the mice (n =6), one-way ANOVA. (D) Body weight change. Data are expressed as the mean ± SEM (n =6). (E) TUNEL staining of coronal sections from mouse brains with orthotopic tumors. Scale bar, 50 µm.

Figure 7

Toxicity analysis of G/Mino+Vor treatments. H&E staining of heart, liver, spleen, lung and kidney from mice treated from different groups. Scale bar, 200 µm.

Scheme 1

The structure of G/Mino+Vor hydrogel and its effects in inhibiting the recurrence of glioma. (A) The G/Mino+Vor hydrogel is formed under UV light and corresponds to MMPs. (B) Minocycline and vorinostat released from G/Mino+Vor hydrogel under post-operative environment, which could repolarize M2 to M1 and reduce expression of MMPs.