Modulation of blood-tumor barrier transcriptional programs improves intra-tumoral drug delivery and potentiates chemotherapy in GBM

Abstract

Glioblastoma (GBM) is the most common malignant primary brain tumor. GBM has an extremely poor prognosis and new treatments are badly needed. Efficient drug delivery to GBM is a major obstacle as the blood-brain barrier (BBB) prevents passage of the majority of cancer drugs into the brain. It is also recognized that the blood-brain tumor barrier (BTB) in the growing tumor represents a challenge. The BTB is heterogeneous and poorly characterized, but similar to the BBB it can prevent therapeutics from reaching effective intra-tumoral doses, dramatically hindering their potential. Here, we identified a 12-gene signature associated with the BTB, with functions related to vasculature development, morphogenesis and cell migration. We identified CDH5 as a core molecule in this set and confirmed its over-expression in GBM vasculature using spatial transcriptomics of GBM patient specimens. We found that the indirubin-derivative, 6-bromoindirubin acetoxime (BIA), could downregulate CDH5 and other BTB signature genes, causing endothelial barrier disruption in endothelial monolayers and BBB 3D spheroids in vitro. Treatment of tumor-bearing mice with BIA enabled increased intra-tumoral accumulation of the BBB non-penetrant chemotherapeutic drug cisplatin and potentiated cisplatin-mediated DNA damage by targeting DNA repair pathways. Finally, using an injectable BIA nanoparticle formulation, PPRX-1701, we significantly improved the efficacy of cisplatin in patient-derived GBM xenograms and prolonged their survival. Overall, our work reveals potential targets at the BTB for improved chemotherapy delivery and the bifunctional properties of BIA as a BTB modulator and potentiator of chemotherapy, supporting its further development.

Figure. 1.. Identification of tumor-endothelium associated (BTB-genes) using bulk and spatial RNA-sequencing datasets from GBM clinical samples.

(A) Workflow of identification and filtering of genes associated with tumoral vasculature in GBM. Using a gene expression correlation tool, cBIO, top-50 genes that co-expressed with CD31, VWF, CD34 and CLEC14A were selected, and their expression in tumor above normal brain examined in the Rembrandt dataset using the GlioVis visualization tool, and evaluated regional expression using the IVY GAP atlas resource. (B) Gene expression of 12 tumor endothelium-associated genes (BTB-genes) identified in the cBio Portal following the workflow shown in (A), *** p-value<0.001 by Tukey’s Honest Significant Difference test. Individual values are colored by GBM subtype Classical, Mesenchymal or Proneural, NA indicates unknown sample information. (C) Regional expression of the 12 BTB-genes in GBM using the IVY GAP resource, *** p-value<0.001 by Tukey’s Honest Significant Difference test. (D) Gene ontology analysis (GO Biological Process 2023) of the 12 BTB-genes using EnrichR soaware. Biological processes are ranked by p-values, which are indicated next to the GO designation. (E) STRING network analysis on the 13 identified BTB-henes. 12 nodes, 17 edges, average node degree 2.83. PPI enrichment p-value 1ê−16.

Figure 2.. Spatial transcriptomic analysis of GBM patient samples confirms CDH5 upregulation in tumor associated endothelium.

(A) Surface plots of CDH5 expression from spatial transcriptomic performed on GBM tumors and non-tumorigenic cortex from cortex and tumor issues. (B) Spatial UMAP plot of CDH5 expression in cortex (lea) and tumor (right) showing specific clusters per sample. (C) Violin plots indicating endothelial cell marker expression and CDH5 from tumor issues and clustered according to spatial clustering from (B). (D) Gene Ontology (GO) indicating associated pathways with CDH5 gene expression in cluster 3 of sample UKF_248 and (F) clusters 2 and 5 of UKF_334. GO biological processes highlighted in red indicate vasculature development and vascular processes of the circulatory system as pathways enriched for CDH5. Top 20 gene lists for highlighted GO pathways are shown. Data was obtained and re-analyzed from Ravi et al., (2022) using the SPATA2 package from R-studio.

Figure 3.. BIA modulates BTB-associated genes and cell mobility, vascular development, angiogenesis and L-serine metabolism in brain endothelial cells.

(A) Heat-map generated from the top-15 upregulated and downregulated genes (Log2FC) from Bulk-RNA sequencing analysis performed on HCMEC/D3 cells treated with BIA (1 μM, 24hrs). (B) Gene ontology analysis of >1.5 (Log2FC) significantly upregulated and downregulated genes by BIA in brain endothelial cells from (A). Biological processes are ranked by p-values, which are shown next to the GO designation. Analysis performed using the EnrichR soaware. (C) Volcano plot analysis from all the upregulated and downregulated genes by BIA. Labels on genes related to angiogenesis, TGF-β and WNT pathways are highlighted. (D) Gene expression fold-change (Log2) levels of dysregulated genes by BIA related to the TGF-β and WNT pathways, angiogenesis and the tumor vascular associated genes (BTB-genes, highlighted). (E) Spatial expression of 7 of the 12 BTB-genes regulated by BIA vitro highlighted in (D) for non-tumor (lea) and tumor (right) issues from sample UKF_248. (F) Clustered gene-expression of UKF_248 showing the BTB-genes from (E) in non-tumor (lea) and tumor issue (right).

Figure 4.. BIA prevents barrier forma-on by brain endothelial cells in vitro and increases dextran uptake in a three-dimensional BBB spheroid model.

(A) Immunofluorescence staining of CDH5 (red) and nuclei (blue) in HCMEC/D3 cells treated with BIA (1 μM, 24hrs). Scale bar=20 μm. (B) TEER values of HCMEC/D3 treated with BIA upon monolayer confluence. Time-point of BIA addition is indicated. (C) Immunofluorescence images of BBB spheroids treated with indicated doses of BIA for 72 hours. Staining of F-actin (green), CDH5 (red) and nuclei (blue). Maximal projection intensity is shown from z-stack images (50 μm depth, 20 layers). Scale bar=100 μm. (D) FITC-conjugated dextran (70kDa) permeability assay in BBB spheroids. Dextran (gray) and nuclei (purple) are shown. Scale bar=100 μm. Mean fluorescence quantification of (E) CDH5, (F) Phalloidin and (G) FITC-dextran from images in (C) and (D) using the ImageJ soaware. Data shows mean and standard deviation, n=4–5. Ordinary one-way ANOVA test. ** p=0.0028, *** p=0.008, **** p<0.0001. (H) Human phospho-kinase array of HCMEC/D3 cells exposed to 1 μM BIA for 24 hours. Highlighted wells related to indicated pathways. Samples were analyzed in duplicates. (I) Quantification of signal by ImageJ of dot-blot shown in (H). Mean and standard deviation of duplicates are shown. Two-way ANOVA analysis was performed. ** p=0.0015, *** p=0.005, **** p<0.0001.

Figure 5.. Systemic administration of BIA increases the selective uptake of sodium fluorescein and platinum chemotherapy in murine GBM tumors.

(A) Workflow schematic of BIA administration and subsequent injection of sodium fluorescein for BTB permeability assessment. (B) In vivo imaging system (IVIS) pictures of G30-tumor bearing brains from mice injected with BIA and sodium fluorescein as shown in (A). (C) Quantification of image intensity was performed with ImageJ. Mean and standard deviation are shown, n=7–8. Unpaired t-test for statistical significance, ** p=0.0024. (D) Platinum quantification via ICP-MS of brain and tumor issue from tumor-bearing mice injected with Cisplatin in G9-PCDH, (E) G34-PCDH and (F) GL261 murine models. Cisplatin (5 mg/kg) was administered 24 hours aaer BIA injec0on. Mean and standard deviation are shown, n=3–5/group. Two-way ANOVA test was performed, * p<0.05. (G) Platinum quantification via ICP-MS of tumor and brain issue of a G9-PCDH tumor-bearing xenograa model administered with increasing BIA doses. Mean and standard deviation are shown, n=3/group. Two-way ANOVA test, * p=0.0177, ** p=0.0016. (H) Immunofluorescence imaging from frozen and sectioned brain issue from G9-PCDH and G34-PCDH xenograa murine models, 24 hours aaer injection with 20 mg/kg of BIA. Tumor (green), CDH5 (red) and CD31 (blue) are shown. Scale bars at 100 μm. (I) CDH5 fluorescence quantification from experiment in (E) using ImageJ. Mean and standard deviation are shown. Unpaired t-test (n=3/group). ** p=0.0013, * p=0.0334.

Figure 6.. BIA poten-ates pla-num-based cytotoxicity by targe-ng DNA-repair pathways in pa-ent-derived GBM cells.

(A) GBM cell viability of BIA and cisplatin combination treatment. Cisplatin doses are indicated in x-axis, BIA remained at a constant concentra0on of 1 μM (5 days exposure). Mean and standard deviation are shown, n=3/group. (B) Dose-response matrix showing inhibi0on percentage of BIA and Cisplatin combinations at various concentra0ons for 5 days using SynergyFinder 3.0. G9-PCDH cells were treated, and viability analyzed as indicated in the cell viability assay section (see Materials and Methods). (C) ZIP method synergy score of BIA and Cisplatin combinations. The overall average δ-score is indicated on top of the chart. The dose combinations showing an increased likelihood of synergy are highlighted. (D) Immunofluorescence staining of γH2AX (green) and nuclei (blue) in G9-PCDH cells treated with 1 μM of Cisplatin and/or BIA, for 72 hours. Representative image of 3 pictures per condition. Pictures taken at 40x, scale bar=20 μm. (E) Quantification of γH2AX foci from (D) using ImageJ. (F) Western blot of G9-PCDH cells treated with 1 μM of Cisplatin and/or BIA, for 72 hours, probing for the p-CHK1 (Ser345), total CHK1 and H2AX (Ser139) proteins. GAPDH was used as loading control. Representative image from triplicate experiments.

Figure 7.. Systemic administra-on of BIA in combina-on with cispla-n treatment shows enhanced pre-clinical efficacy in murine GBM models.

(A) and (C) Diagrams of the experimental design for G34-PCDH and G9-PCDH xenograa efficacy studies using BIA/PPRX-1701 and Cisplatin combinations. (B) Efficacy studies of G34-PCDH xenograa using BIA and (D) PPRX-1701 in combination with Cisplatin. For PPRX-1701 studies, vehicle formulation was used as control and in combination with Cisplatin. n=8/group. Log-rank test analysis for statistical significance. (E) Confocal immunofluorescence imaging of γH2AX (Alexa Fluor 647, red) nuclear foci from tumor issue collected from study (D). Nuclei were stained with Hoechst 33342 (blue). Representative pictures taken at 20x. Scale bar=50 μm. (F) Quantification of γH2AX foci from (E) using Image J, n=6/group. Ordinary One-way ANOVA was performed for statistical evalua0on. * p=0.01, ** p=0,0086. (G) Schematic of proposed model of BIA/PPRX-1701 mechanism of action and its effects in GBM tumor drug delivery and efficacy.