Physiologic upper limit of pore size in the blood-tumor barrier of malignant solid tumors

Abstract

Background: The existence of large pores in the blood-tumor barrier (BTB) of malignant solid tumor microvasculature makes the blood-tumor barrier more permeable to macromolecules than the endothelial barrier of most normal tissue microvasculature. The BTB of malignant solid tumors growing outside the brain, in peripheral tissues, is more permeable than that of similar tumors growing inside the brain. This has been previously attributed to the larger anatomic sizes of the pores within the BTB of peripheral tumors. Since in the physiological state in vivo a fibrous glycocalyx layer coats the pores of the BTB, it is possible that the effective physiologic pore size in the BTB of brain tumors and peripheral tumors is similar. If this were the case, then the higher permeability of the BTB of peripheral tumor would be attributable to the presence of a greater number of pores in the BTB of peripheral tumors. In this study, we probed in vivo the upper limit of pore size in the BTB of rodent malignant gliomas grown inside the brain, the orthotopic site, as well as outside the brain in temporalis skeletal muscle, the ectopic site.

Methods: Generation 5 (G5) through generation 8 (G8) polyamidoamine dendrimers were labeled with gadolinium (Gd)-diethyltriaminepentaacetic acid, an anionic MRI contrast agent. The respective Gd-dendrimer generations were visualized in vitro by scanning transmission electron microscopy. Following intravenous infusion of the respective Gd-dendrimer generations (Gd-G5, N = 6; Gd-G6, N = 6; Gd-G7, N = 5; Gd-G8, N = 5) the blood and tumor tissue pharmacokinetics of the Gd-dendrimer generations were visualized in vivo over 600 to 700 minutes by dynamic contrast-enhanced MRI. One additional animal was imaged in each Gd-dendrimer generation group for 175 minutes under continuous anesthesia for the creation of voxel-by-voxel Gd concentration maps.

Results: The estimated diameters of Gd-G7 dendrimers were 11 +/- 1 nm and those of Gd-G8 dendrimers were 13 +/- 1 nm. The BTB of ectopic RG-2 gliomas was more permeable than the BTB of orthotopic RG-2 gliomas to all Gd-dendrimer generations except for Gd-G8. The BTB of both ectopic RG-2 gliomas and orthotopic RG-2 gliomas was not permeable to Gd-G8 dendrimers.

Conclusion: The physiologic upper limit of pore size in the BTB of malignant solid tumor microvasculature is approximately 12 nanometers. In the physiologic state in vivo the luminal fibrous glycocalyx of the BTB of malignant brain tumor and peripheral tumors is the primary impediment to the effective transvascular transport of particles across the BTB of malignant solid tumor microvasculature independent of tumor host site. The higher permeability of malignant peripheral tumor microvasculature to macromolecules smaller than approximately 12 nm in diameter is attributable to the presence of a greater number of pores underlying the glycocalyx of the BTB of malignant peripheral tumor microvasculature.

Figure 1

Transmission electron microscopy of higher generation Gd-dendrimers. Annular dark-field scanning transmission electron microscopy (ADF STEM) images of unstained Gd-G5, Gd-G6, Gd-G7, and Gd-G8 dendrimers adsorbed onto an ultrathin carbon support film. The diameters of one hundred Gd-G7 and Gd-G8 dendrimers were measured. Scale bar = 20 nm.

Figure 2

Pharmacokinetics of Gd-dendrimer generations in orthotopic RG-2 gliomas and ectopic RG-2 gliomas over 600 to 700 minutes. Respective Gd-dendrimer generation was intravenously infused over 1 minute (0.09 mmol Gd/kg) during the initial 15 minute dynamic contrast-enhanced MRI scan session. Subsequent dynamic scan sessions of re-anesthetized animals were conducted at 30 to 90 minute time intervals. Whole tumor tissue Gd concentrations for the orthotopic and ectopic RG-2 gliomas were calculated for each of the dynamic scan session time points. Shown is the change in the Gd concentration of respective Gd-dendrimer generations in orthotopic RG-2 gliomas and ectopic RG-2 gliomas over 600 to 700 minutes. Superimposed is the best fit curve Gd concentration curve for the respective Gd-dendrimer generations. Panels A through D are orthotopic glioma Gd concentrations over time. Panels E through H are ectopic glioma Gd concentrations over time A. Gd-G5 (Orthotopic, N = 6), B. Gd-G6 (Orthotopic, N = 6), C. Gd-G7 (Orthotopic, N = 5), D. Gd-G8 (Orthotopic, N = 5), E. Gd-G5 (Ectopic, N = 6), F. Gd-G6 (Ectopic, N = 6), G. Gd-G7 (Ectopic, N = 5), H. Gd-G8 (Ectopic, N = 5).

Figure 3

Gd concentration maps of Gd-dendrimer contrast enhancement over 175 minutes. For one additional animal in each Gd-dendrimer generation group the respective Gd-dendrimer generation was intravenously infused over 1 minute (0.09 mmol Gd/kg) while the animal was maintained under anesthesia for the duration of the 175 minute dynamic contrast-enhanced MRI session. Voxel-by-voxel Gd concentration maps were generated. Shown are the voxel-by-voxel Gd concentration maps for the respective Gd-dendrimer generations at the 15 minute time point and then at 30 minute time intervals thereafter. First row, Gd-G5 dendrimer (Orthotopic RG-2 glioma tumor volume, 45 mm³; ectopic RG-2 glioma tumor volume, 113 mm³). Second row, Gd-G6 dendrimer (Orthotopic RG-2 glioma tumor volume, 97 mm³; ectopic RG-2 glioma tumor volume, 184 mm³). Third row, Gd-G7 dendrimer (Orthotopic RG-2 glioma tumor volume, 53 mm³; ectopic RG-2 glioma tumor volume, 135 mm³). Fourth row, Gd-G8 dendrimer (Orthotopic RG-2 glioma tumor volume, 50 mm³; ectopic RG-2 glioma tumor volume, 163 mm³).

Figure 4

Tumor volumes of orthotopic and ectopic RG-2 gliomas of each Gd-dendrimer generation. Whole tumor tissue volumes, in mm³, were determined for the orthotopic and ectopic RG-2 gliomas of each of the Gd-dendrimer generation groups using the T₂ weighted anatomical scans and dynamic contrast-enhanced MRI data sets as described in the Methods section. Shown are the average whole tumor volumes of orthotopic and ectopic RG-2 gliomas of each Gd-dendrimer generation. A. Gd-G5 (Orthotopic, N = 6; Ectopic, N = 6), B. Gd-G6 (Orthotopic, N = 6; Ectopic, N = 6), C. Gd-G7 (Orthotopic, N = 5; Ectopic, N = 5), D. Gd-G8 (Orthotopic, N = 5; Ectopic, N = 5). Error bars represent standard deviation.

Figure 5

Blood pharmacokinetics of Gd-dendrimer generations over 600 to 700 minutes. Five to ten voxels were selected from within the common carotid arteries. For the selected voxels, the average blood Gd concentrations were determined for each of the dynamic scan session time points. Shown is the change in average blood Gd concentration of the respective Gd-dendrimer generations over 600 to 700 minutes. A. Gd-G5 (N = 6), B. Gd-G6 (N = 6), C. Gd-G7 (N = 5), D. Gd-G8 (N = 5).