3-D Ultrasound Localization Microscopy for Identifying Microvascular Morphology Features of Tumor Angiogenesis at a Resolution Beyond the Diffraction Limit of Conventional Ultrasound

Fanglue Lin, Sarah E Shelton, David Espíndola, Juan D Rojas, Gianmarco Pinton, Paul A Dayton, Fanglue Lin, Sarah E Shelton, David Espíndola, Juan D Rojas, Gianmarco Pinton, Paul A Dayton

Abstract

Angiogenesis has been known as a hallmark of solid tumor cancers for decades, yet ultrasound has been limited in its ability to detect the microvascular changes associated with malignancy. Here, we demonstrate the potential of 'ultrasound localization microscopy' applied volumetrically in combination with quantitative analysis of microvascular morphology, as an approach to overcome this limitation. This pilot study demonstrates our ability to image complex microvascular patterns associated with tumor angiogenesis in-vivo at a resolution of tens of microns - substantially better than the diffraction limit of traditional clinical ultrasound, yet using an 8 MHz clinical ultrasound probe. Furthermore, it is observed that data from healthy and tumor-bearing tissue exhibit significant differences in microvascular pattern and density. Results suggests that with continued development of these novel technologies, ultrasound has the potential to detect biomarkers of cancer based on the microvascular 'fingerprint' of malignant angiogenesis rather than through imaging of blood flow dynamics or the tumor mass itself.

Keywords: Angiogenesis; acoustic angiography; biomarker.; microbubble contrast agent; super-resolution; ultrasound localization microscopy.

Conflict of interest statement

Paul A. Dayton is an inventor on a patent describing the superharmonic acoustic angiography technology mentioned here (although not ULM which this paper is focused on), and a co-founder of SonoVol, Inc., a company which has licensed the patent enabling acoustic angiography.

Figures

Figure 1
Figure 1
Bubble detection process. (A) Acquired beamformed B mode image. (B) Image after using a spatiotemporal filter to remove static and slow-moving signals. (C) Localized microbubbles using hysteresis thresholding. (D) Detected bubble center position superposed on a filtered image.
Figure 2
Figure 2
In vitro: ULM images (A-C) and super harmonic images (D-F) of microtubes with inner diameter of 150 µm (A, D), 75 µm (B, E) and 50 µm (C, F). Bar = 200 µm.
Figure 3
Figure 3
In vivo: ULM images of a 2D slice of a rat FSA tumor and cross-section profiles of three vessels with different size. Bar = 5 mm.
Figure 4
Figure 4
In vivo: Maximum intensity projection through three-dimensional acoustic angiography image data (A, B) and three-dimensional ULM image (C) of a same rat FSA tumor. The resulting two-dimensional images are orientated in the sagittal anatomic plane (A) and coronal anatomic plane (B) for acoustic angiography and in the sagittal anatomic plane (C) for the ULM image. Bar = 4 mm.
Figure 5
Figure 5
In vivo: Maximum intensity projection through three-dimensional ULM image data for four rats. The resulting two-dimensional images are orientated in the sagittal anatomic plane. Vessel density and morphological differences are obvious between the control rats (A, B) and tumor rats (C, D). Bar = 4 mm.
Figure 6
Figure 6
Six extracted vessels and their corresponding tortuosity metric values.
Figure 7
Figure 7
Box plots of the vessel tortuosity (Distance metric) of images from the control rat population and tumor-bearing rat population.

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