Visualizing the acute effects of vascular-targeted therapy in vivo using intravital microscopy and magnetic resonance imaging: correlation with endothelial apoptosis, cytokine induction, and treatment outcome

Abstract

The acute effects of the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) were investigated in vivo using intravital microscopy (IVM) and magnetic resonance imaging (MRI). Changes in vascular permeability and blood flow of syngeneic CT-26 murine colon adenocarcinomas were assessed at 4 and 24 hours after DMXAA treatment (30 mg/kg, i.p.) and correlated with induction of tumor necrosis factor-alpha (TNF-alpha), endothelial damage [CD31/terminal deoxynucleotidyl transferase (TdT)], and treatment outcome. Intravital imaging revealed a marked increase in vascular permeability 4 hours after treatment, consistent with increases in intratumoral mRNA and protein levels of TNF-alpha. Parallel contrast-enhanced MRI studies showed a approximately 4-fold increase in longitudinal relaxation rates (DeltaR(1)), indicative of increased contrast agent accumulation within the tumor. Dual immunostained tumor sections (CD31/TdT) revealed evidence of endothelial apoptosis at this time point. Twenty-four hours after treatment, extensive hemorrhage and complete disruption of vascular architecture were observed with IVM, along with a significant reduction in DeltaR(1); and virtual absence of CD31 immunostaining. DMXAA-induced tumor vascular damage resulted in significant long-term (60-day) cures compared to untreated controls. Multimodality imaging approaches are useful in visualizing the effects of antivascular therapy in vivo. Such approaches allow cross validation and correlation of findings with underlying molecular changes contributing to treatment outcome.

Trial registration: ClinicalTrials.gov NCT00119275.

Figures

Figure 1

Intravital microscopic images of normal and tumor-associated vasculature in a BALB/c mouse dorsal skin window chamber. Representative images of normal host cutaneous vasculature (control) and vasculature associated with a growing CT-26 tumor within dorsal skinfold window chambers implanted in BALB/c mice. Serial intravital images were acquired following the injection of CT-26 tumor cells to monitor changes in vessel geometry and architecture with tumor growth. Arrows indicate corresponding areas in the images acquired at different times (days 1, 4, and 6) after tumor implantation that showed significant host vessel dilation and increased tortuosity.

Figure 2

IVM, contrast-enhanced MRI, and immunohistochemical assessment of CT-26 tumor response to DMXAA. Intravital (upper panel) and contrast-enhanced MR (middle panel) images of CT-26 colon adenocarcinomas acquired before treatment (preRx), 4 hours after treatment with DMXAA (4 h), and 24 hours after treatment with DMXAA (24 h). Imaging-based changes in vascular function correlated with immunohistochemical analysis (CD31/TdT) of tumor sections (lower panel). Four hours after treatment, endothelial apoptosis (arrows) was visible in DMXAA-treated tumors. Ghost outlines of vessels were seen at 24 hours after treatment (rectangles). Representative images of individual mice from each methodology have been shown.

Figure 3

Temporal change in the longitudinal MR relaxation rate (ΔR1 tumor/blood) of control and DMXAA-treated CT-26 tumors implanted in BALB/c mice. Graph shows the change in T1 relaxation rates (ΔR1) over time of untreated control tumors (squares) and tumors treated with 30 mg/kg DMXAA following administration of the macromolecular contrast agent. Significant differences in ΔR1 values (**P < .01; two-tailed t-test) were seen 4 hours after DMXAA treatment (n = 3) compared to untreated controls (n = 3).

Figure 4

Induction of TNF-α at different times after DMXAA treatment. BALB/c mice bearing subcutaneous CT-26 tumors were injected with DMXAA and, at different times after treatment, tumors were excised for the determination of mRNA (A) and protein levels of TNF-α (B) using PCR and ELISA, respectively. Statistical analyses (two-tailed t-test) revealed significant differences between control and treatment groups at 2 and 4 hours after treatment (***P

Figure 5

Long-term treatment outcome following antivascular therapy. BALB/c mice bearing subcutaneous CT-26 tumors were injected with 30 mg/kg DMXAA, and tumor growth was monitored for 60 days after treatment. Kaplan-Meier survival curves of untreated controls (n = 10) and DMXAA-treated animals (n = 6). A significant difference in survival was seen between control and treatment groups (**P