Progesterone boosts abiraterone-driven target and NK cell therapies against glioblastoma

Abstract

Introduction: Glioblastoma (GBM) poses a significant challenge in oncology, with median survival times barely extending beyond a year due to resistance to standard therapies like temozolomide (TMZ). This study introduces a novel therapeutic strategy combining progesterone (Prog) and abiraterone (Abi) aimed at enhancing GBM treatment efficacy by modulating the tumor microenvironment and augmenting NK cell-mediated immunity.

Methods: We employed in vitro and in vivo GBM models to assess the effects of Prog and Abi on cell viability, proliferation, apoptosis, and the immune microenvironment. Techniques included cell viability assays, Glo-caspase 3/7 apoptosis assays, RNA-seq and qPCR for gene expression, Seahorse analysis for mitochondrial function, HPLC-MS for metabolomics analysis, and immune analysis by flow cytometry to quantify NK cell infiltration.

Results: Prog significantly reduced the IC50 of Abi in TMZ-resistant GBM cell, suggesting the enhanced cytotoxicity. Treatment induced greater apoptosis than either agent alone, suppressed tumor growth, and prolonged survival in mouse models. Notably, there was an increase in CD3^-/CD19^-/CD56⁺/NK1.1⁺ NK cell infiltration in treated tumors, indicating a shift towards an anti-tumor immune microenvironment. The combination therapy also resulted in a reduction of MGMT expression and a suppression of mitochondrial respiration and glycolysis in GBM cells.

Conclusion: The combination of Prog and Abi represents a promising therapeutic approach for GBM, showing potential in suppressing tumor growth, extending survival, and modulating the immune microenvironment. These findings warrant further exploration into the clinical applicability of this strategy to improve outcomes for GBM patients.

Fig. 1

Therapeutic efficacy of Abi combined with Prog on GBM in vitro and in vivo. A After treatment with Abi with or without 40 µM Prog for 72 h, cell viability was estimated using CCK8 assay. IC50 was calculated using the Prism software. Experiments, each containing three replicates, were performed independently three times. B Clinical relevant of PGR expression was analysed using the Gliovis website. C Upper: After transfection with GFP or GFP-PgR for 24 h, Pt#3-R cells were treated with Abi with or without Prog for 72 h, followed by the CCK8 assay. Lower: Medium were collected and analysed using the Glo-caspase 3/7 assay. GFP and GFP-PgR groups were compared statistically using two-way ANOVA. D Upper: After intracranial implantation with U87MG-luc-R, tumor growth was detected using IVIS image. Abi and Prog were injected intraperitoneally on Day 6 (once per three days). Lower: Mouse brain was subjected to paraffin-embedded slides preparation, followed by the HE staining. TMZ: 12 mice; TMZ + Abi: 12 mice, TMZ + Abi + Prog: 11 mice. E-F. Log-rank test was used to performed survival comparison was performed in Scid E receiving U87MG-R-luc implantation and wild type C57BL/6 F mice receiving GL-261-R implantation. For the experiment of C57BL/6 mice. F Six mice were included for each group. Survival extension days by Abi and Abi + Prog was shown

Fig. 2

Effect of Prog on MGMT expression. A, B After treatment with Prog in the absence and presence of Abi, T98G cells were harvested for Western blotting (Left) and RT-qPCR (Right). C Left: After treatment for 48 h, T98G cells were treated with 20 µM cycloheximide (CHX) for the indicated time interval, and cell lysates were collected for Western blotting. Right: Quantitative results. D After treatment with Prog, gDNA of T98G and U87MG-R cells F was purified, and subjected to pyrosequencing analysis. The percentage of methylation in 5 CpG residues in T98G and U87MG-R cells were quantified. Experiments were performed independently three times. (*P < 0.05, **P < 0.01)

Fig. 3

Effect of Abi on mitochondria respiration. After treatment with Abi for 72 h, T98G A and U87MG-R B cells were subjected to Seahorse XF Cell Mito Stress Test. Experiments, each containing two replicates, were performed independently three times. C Upper: Primary and recurrent specimens from GBM patients were homogenized and subjected to RNA-seq. Genes related to Complex I to V in mitochondria were shown in the heatmaps. Lower: RNA extracts from Pt#3 and Pt#3-R were prepared for RT-qPCR. D After treatment with Abi for 72 h, U87MG-R and T98G E cells were harvested for RNA extraction, followed by RT-qPCR. Experiments, each containing two replicates, were performed independently three times. (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

Abi suppresses mitochondria respiration through decreasing CDK1 expression. After treatment with Abi for 72 h, RNA extracts of T98G cells were subjected to RNA-seq. Genes influenced by Abi were shown A and functional clustered by Gene Set Enrichment Analysis (GSEA) B. C Abi-downregulated genes which were involved in G2/M transition and DNA replication were shown. D Western blotting for cyclin A2 and CDK1. E After treatment for 48 h, T98G cells were treated with 20 µM CHX for the indicated time interval, and cell lysates were collected for Western blotting. F Left: After transfection for 24 h, T98G cells were fixed and immunostained using the anti-GFP antibody. Right: After transfection for 24 h, cells were treated with Abi with or without Prog for 72 h. Cell viability was estimated using the CCK8 assay. Experiments, each containing three replicates, were performed independently three times. IC50 was calculated using the Prism software. G After transfection for 24 f and treatment with Abi for the additional 72 h, mitochondria respiration in T98G cells was estimated using Seahorse XF analyser. Experiments, each containing two replicates, were performed independently three times

Fig. 5

Effect of Prog on glycolysis in GBM cells. A After treatment with Prog (40 µM) for 72 h, RNA extracts of T98G cells were subjected to RNS-seq. Genes involved in lipid and glucose metabolism were shown in the heatmaps B. C After treatment with Prog for 72 h, protein lysates of T98G cells were subjected to metabolomics analysis using HPLC-MS. D After treatment for 72 h, glycolytic activity of T98G cells was estimated by the Seahorse Glycolysis Stress Test. Experiments, each containing two replicates, were performed independently three times. E Western blotting for CDK1. F After transfection for 24 h and treatment with Prog for the additional 72 h, RNA extracts were harvested for RT-qPCR targeting ALDOC, PC and IDH3G. Experiments, each containing three replicates, were performed independently three times. (*P < 0.05)

Fig. 6

Effect of Prog on NK cell infiltration in GBM. A Genes related to NK cell activation and suppression were shown in the heatmap. B Abi-influenced NK ligands in the T98G cells. C Gene expression related to NK cell, macrophage and GBM in human GBM specimens. D NK cell infiltration was estimated using the TIMER website. E Correlation of gene expression was analysed using the Gliovis website. F After intracranial implantation with GL261-R cells for 7 days, experimental C57BL/6 mice were intraperitoneally injected with Abi or Abi + Prog twice per week for 3 weeks. Tumor in the brain was excised and subjected to immune-analysis using the indicated antibody by flow cytometry. Before gating for NK cells, cell suspension was gated for CD45⁺ cells. Four mice were included for each group. G Quantitative results for immune cells detected by flow cytometry. One symbol represents one mouse

Fig. 7

Effect of Prog on NK cell therapy against GBM. A GFP-expressed U87MGluc cells were co-cultured with Hoechst-stained NK-92 cells for 24 h. Cells were photographed by laser confocal microscope at the 24th h, and photographed every minute from 24th ~ 26th h B. B After co-culturing for 24 h, cells were monitored using a time-lapse microscope for 42 min. Four images were captured during the final 42 min of observation. a ~ d: from the beginning to the end. C GBM cells, U87MG-luc-R (left), A172 (middle) and T98G (right), were transfected with GFP-PgR and treated with the indicated drug for 48 h. Subsequently, GBM cells were co-incubated with NK-92 cells for 24 h. Cell viability of GBM was estimated using the luciferase assay (top). After co-culture, medium was collected for estimation of caspase 3/7 (middle) and LDH (bottom). Cell number: GBM: NK cells = 1:0.5. Experiments, each containing three replicates, were performed independently three times. (*P < 0.05, **P < 0.01, ***P < 0.001). D After co-culture for 24 h, NK-92 cell suspension was removed, and attached T98G cells were stained with PI, followed by the flow cytometric analysis. E After implantation with U87MG-luc or co-implantation with U87MG-luc and NK-92 cells, Abi and Prog were injected intraperitoneally on Day 6 (once per three days). IVIS images were acquired on the indicated day. Six mice were included for each group. F Brain slides for mice on Day 5 were prepared from the indicated mouse, and subjected to immunostaining. Green: PCNA; Red: NKG2D