Wnt signaling regulates MFSD2A-dependent drug delivery through endothelial transcytosis in glioma

Abstract

Background: Systemic delivery of anti-tumor therapeutic agents to brain tumors is thwarted by the blood-brain barrier (BBB), an organotypic specialization of brain endothelial cells (ECs). A failure of pharmacological compounds to cross BBB is one culprit for the dismal prognosis of glioblastoma (GBM) patients. Identification of novel vascular targets to overcome the challenges posed by the BBB in tumors for GBM treatment is urgently needed.

Methods: Temozolomide (TMZ) delivery was investigated in CT2A and PDGFB-driven RCAS/tv-a orthotopic glioma models. Transcriptome analysis was performed on ECs from murine gliomas. Mfsd2a deficient, Cav1 deficient, and Mfsd2a EC-specific inducible mice were developed to study the underlying molecular mechanisms.

Results: We demonstrated that inhibiting Wnt signaling by LGK974 could increase TMZ delivery and sensitize glioma to chemotherapy in both murine glioma models. Transcriptome analysis of ECs from murine gliomas revealed that Wnt signaling inhibition enhanced vascular transcytosis as indicated by the upregulation of PLVAP and downregulation of MFSD2A. Mfsd2a deficiency in mice enhances TMZ delivery in tumors, whereas constitutive expression of Mfsd2a in ECs suppresses the enhanced TMZ delivery induced by Wnt pathway inhibition in murine glioma. In addition, Wnt signaling inhibition enhanced caveolin-1 (Cav1)-positive caveolae-mediated transcytosis in tumor ECs. Moreover, Wnt signaling inhibitor or Mfsd2a deficiency fails to enhance TMZ penetration in tumors from Cav1-deficient mice.

Conclusions: These results demonstrated that Wnt signaling regulates MFSD2A-dependent TMZ delivery through a caveolae-mediated EC transcytosis pathway. Our findings identify Wnt signaling as a promising therapeutic target to improve drug delivery for GBM treatment.

Keywords: Wnt signaling; blood-brain barrier; drug delivery; endothelial cell; glioblastoma.

Figure 1.

Wnt signaling activity is correlated with BBB status in human brain/GBM ECs. (A–B) Co-immunofluorescence staining (A) and quantification (B) of LEF1 and CD31on human non-malignant brain, lower grade glioma and glioblastoma. (C–D) Co-immunofluorescence staining (C) and quantification (D) of β-catenin and CD31 on human non-malignant brain, lower grade glioma and glioblastoma. Endothelial cells with nuclear staining of β-catenin were indicated by white arrow. (E) Expression of core BBB genes (SLC2A1, ABCG2), BBB dysfunction gene (PLVAP) and Wnt targets (MFSD2A, APCDD1) across endothelial cells sorted by SPIN based on a core BBB gene set. Scale bar: 50 μm (A), 20 μm (C).

Figure 2.

Wnt singling pathway inhibition increases TMZ delivery and sensitizes GBM to TMZ therapy. (A) Schematic illustration of the in vitro BBB models used in the study. bEND.3 cells formed a confluent monolayer in the upper part of chamber. TMZ was added to the upper compartment to measure the BBB transport. (B) Concentration of TMZ in the bottom compartment of the chamber. (C) Immunohistochemical staining of CD31 in CT-2A glioma from mice treated with or without LGK974. (D–E) Stereological quantification of vascular area (D) and mean vascular diameters (E) in CT-2A glioma. (F–G) Co-immunofluorescence staining (F) and quantification (G) of LEF1 and CD31 in CT-2A tumors from mice treated with or without LGK974. (H-I) Co-immunofluorescence staining (H) and quantification (I) of β-catenin and CD31 in CT-2A tumors from mice treated with or without LGK974. Endothelial cells with or without nuclear staining of β-catenin were indicated by white arrow or arrowhead respectively. (J) Quantification of TMZ concentration in CT-2A glioma from mice treated with or without LGK974. (K) Immunohistochemical staining of CD31 in RCAS/tv-a glioma from mice treated with or without LGK974. (L–M) Stereological quantification of vascular area (L) and mean vascular diameters (M) in RCAS/tv-a glioma. (N–O) Co-immunofluorescence staining (N) and quantification (O) of LEF1 and CD31 in RCAS/tv-a glioma from mice treated with or without LGK974. (P–Q) Co-immunofluorescence staining (P) and quantification (Q) of β-catenin and CD31 in RCAS/tv-a glioma tumors from mice treated with or without LGK974. Endothelial cells with or without nuclear staining of β-catenin were indicated by white arrow or arrowhead respectively.(R) Quantification of TMZ concentration in RCAS/tv-a glioma from mice treated with or without LGK974. (S–T) Survival curve of CT-2A glioma- (S) or RCAS/tv-a glioma- (T) bearing mice treated with LGK974 and/or TMZ. (U–V) Quantification of paclitaxel concentration in CT-2A glioma (U) or RCAS/tv-a glioma (V) from mice treated with or without LGK974. Scale bar: 100μm (C, K), 50μm (F, N), 20μm (H, P).

Figure 3.

Wnt signaling inhibition enhances nano-particles BBB delivery. (A) Schematic illustration of the in vitro BBB models with CT-2A glioma cells cultured in the bottom part of the chamber. Nano-particles (NPs) was added to the upper compartment to measure the BBB transport. (B) Concentration of NPs in the bottom compartment of the chamber. (C–D) Fluorescence image (C) and quantification (D) of NPs uptake by tumor cells in the bottom compartment of the chamber. (E–F) Fluorescence image (E) and quantification (F) of NPs in CT-2A glioma from mice treated with or without LGK974. Scale bar: 50 μm (C) and 200 μm (E).

Figure 4.

Wnt regulates genes associated with inflammatory and transcytosis in tumor ECs. (A) Schematic overview of the transcriptome study design. (B) Expression level of selected genes in tumor ECs isolated from control or LGK974 treated tumor-bearing mice. (C) GSEA analysis of up- and down-regulated gene sets in tumor ECs isolated from control or LGK974 treated tumor-bearing mice. (D–E) Immunofluorescence (D) and quantification (E) of PLVAP in CT-2A glioma from mice treated with or without LGK974. (F–G) Immuno-staining (F) and quantification (G) of Mfsd2a in CT-2A glioma from mice treated with or without LGK974. (H–I) Immunofluorescence (H) and quantification (I) of Cav1 in CT-2A glioma from mice treated with or without LGK974. Scale bar: 100 μm.

Figure 5.

Enhanced TMZ delivery by Wnt signaling inhibition is dependent on Mfsd2a. (A) Quantification of TMZ concentration in CT-2A glioma from wild type or Mfsd2a-ko mice. (B) Survival curve of CT-2A glioma form wild type or Mfsd2a ko tumor-bearing mice treated with or without LGK974. (C) Concentration of TMZ passing monolayer of control or Mfsd2a overexpressing bEND.3 cells with or without LGK974 treatment. (D) Technical principle for construction of ROSA26^{STOPfloxMfsd2a/TdTomato} mice and schematic overview to generate Mfsd2a-GOF mice. (E–F) Immunofluorescence (E) and quantification (F) of CD31 and Mfsd2a in CT-2A glioma from tamoxifen induced or non-induced Mfsd2a-GOF mice treated with or without LGK974. (G) Quantification of TMZ concentration in CT-2A glioma from tamoxifen induced or non-induced Mfsd2a-GOF mice treated with or without LGK974. (H) Survival curve of CT-2A glioma form tamoxifen induced or non-induced Mfsd2a-GOF tumor-bearing mice treated with or without LGK974. Scale bar: 100 μm.

Figure 6.

Wnt signaling inhibition increases TMZ delivery through enhancing Mfsd2a-mediated EC caveolae transcytosis. (A) Quantification of TMZ concentration in CT-2A glioma from wild type, Mfsd2a^-/-, Cav1^-/- or Mfsd2a^-/-/Cav1^-/- mice. (B) EM analysis with immune-gold labeling Cav1 in CT-2A tumors from mice with or without LGK974 treatment. (C) Quantification of Cav-1 positive vesicles in tumor vessels. n = 3 per group. At least 6 vessels per animal were analyzed. (D) Quantification of TMZ concentration in CT-2A glioma from wild type or Cav1^-/- mice treated with or without LGK974. (E–F) TEM images (E) and quantification (F) of endothelial transcytotic vesicles in CT-2A tumors. (G) Survival curve of CT-2A glioma form wild type or Cav1^-/- tumor-bearing mice treated with or without LGK974. (H) Scheme illustration on the role of Wnt signaling in controlling TMZ delivery. Scale bar: 1 μm.