Remodeling the Vascular Microenvironment of Glioblastoma with α-Particles

Abstract

Tumors escape antiangiogenic therapy by activation of proangiogenic signaling pathways. Bevacizumab is approved for the treatment of recurrent glioblastoma, but patients inevitably develop resistance to this angiogenic inhibitor. We previously investigated targeted α-particle therapy with ²²⁵Ac-E4G10 as an antivascular approach and showed increased survival and tumor control in a high-grade transgenic orthotopic glioblastoma model. Here, we investigated changes in tumor vascular morphology and functionality caused by ²²⁵Ac-E4G10.

Methods: We investigated remodeling of the tumor microenvironment in transgenic Ntva glioblastoma mice using a therapeutic 7.4-kBq dose of ²²⁵Ac-E4G10. Immunofluorescence and immunohistochemical analyses imaged morphologic changes in the tumor blood-brain barrier microenvironment. Multicolor flow cytometry quantified the endothelial progenitor cell population in the bone marrow. Diffusion-weighted MR imaged functional changes in the tumor vascular network.

Results: The mechanism of drug action is a combination of remodeling of the glioblastoma vascular microenvironment, relief of edema, and depletion of regulatory T and endothelial progenitor cells. The primary remodeling event is the reduction of both endothelial and perivascular cell populations. Tumor-associated edema and necrosis were lessened, resulting in increased perfusion and reduced diffusion. Pharmacologic uptake of dasatinib into tumor was enhanced after α-particle therapy.

Conclusion: Targeted antivascular α-particle radiation remodels the glioblastoma vascular microenvironment via a multimodal mechanism of action and provides insight into the vascular architecture of platelet-derived growth factor-driven glioblastoma.

Keywords: 225Ac; actinium-225; glioblastoma; pericytes; radioimmunotherapy; vascular endothelium.

FIGURE 1.

Histologic changes in glioblastoma microenvironment 10 d after ²²⁵Ac-E4G10 treatment. (A) Confocal immunofluorescence imaging of tumor sections co-stained for anti-CD31, anti-desmin, and anti–collagen IV. Overlay of all stains is presented, with colocalization of endothelial cells (red) and pericytes (green) in yellow and counterstaining with DAPI (blue). Scale bars are 20 μm. (B and C) Quantification of pericyte density (desmin normalized by DAPI) (B) and pericyte coverage of blood vessels (desmin normalized by CD31) (C). (D) Immunohistochemical staining for regulatory T cells. Arrows indicate FoxP3-positive cells. (E) Quantification of FoxP3-positive regulatory T cells. Scale bars are 100 μm (full panels) and 20 μm (inset panels). Representative images of treatment and control groups are shown in A and D; positively stained area was counted and calculated as percentage of whole-tumor section for quantifications shown in B, C, and E. Data are mean ± SEM. HSA = human serum albumin.

FIGURE 2.

Diffusion-weighted MRI study of vessel functionality after ²²⁵Ac-E4G10 treatment. (A) Representative MR images on day 10 after treatment: axial T2-weighted fast spin echo (left); corresponding echoplanar at b = 50 (predominantly perfusion) (middle); and echoplanar at b = 600 (predominantly diffusion) (right). Scale bars indicate relative intensities; dotted outline, tumor margin; solid arrow, tumor; and dashed arrows, water-filled phantom (measurement control). (B) Representative logarithmic plot of signal intensities of whole-tumor ROIs—plotted normalized to intensity at b = 0 and as natural logarithm (ln(S_b/S_b=0))—vs. measured b-values. Biexponential decay fitting of data (intravoxel incoherent motion, straight line) results in 2 slopes, D* (red line) and D (green line). ADC is derived by monoexponential decay fitting of data (dashed line). (C–E) Quantification of coefficients: ADC (C), D (D), and D* (E). Data are mean ± SEM.

FIGURE 3.

Dasatinib penetration of glioblastomas 10 d after ²²⁵Ac-E4G10 treatment. Representative axial whole-brain sections stained with hematoxylin and eosin are shown, with matching autoradiography of ¹⁴C-labeled dasatinib from mice treated with vehicle (n = 5) or ²²⁵Ac-E4G10 (n = 6). Arrows indicate tumor margins. Scale bar indicates relative intensities. Autoradiography data are quantified on right. Data are mean ± SEM of mean intensities of whole-tumor ROIs. ***P < 0.001.

FIGURE 4.

(A) Flow cytometry gating scheme to identify bone marrow EPCs in Ntva mice. After gating out debris (panel 1) and cell doublets based on forward scatter (panel 2), population of live (DAPI-negative) TER 119–negative and CD11b-negative cells is selected (third panel) and identified as VEGF receptor 2–positive, c-kit–positive EPCs (red square, panel 4). (B and C) Pretreatment (B) and posttreatment (C) comparisons of EPC population between Ntva mice and controls. Data are mean ± SEM. FSC = forward scatter; SSC = side scatter.