Noninvasive measurement of murine glioblastoma in vivo with abenchtop ultrasound instrument
TM Kierski1, JD Rojas1, PG Durham2, PA Dayton2, M Thang3, SD Hingtgen3, TJ Czernuszewicz1, RC Gessner1(1) Revvity, Inc., (2) Joint Department of Biomedical Engineering, UNC-Chapel Hill and NC State University, (3) Eshelman
School of Pharmacy, UNC-Chapel Hill

1

Background4

Representative Images

Limits of Detectability

7• Glioblastoma (GBM) is the most common primary brain tumor with <5% ofpatients surviving >5 years post diagnosis.
Tumor Size(diameter) 0-1 mm(volume) 0.5 mm3• Murine models play a vital role in researching new treatment options forGBM.
• Small animal MRI is the most common and sensitive method fornoninvasively tracking GBM tumors in rodent models, however it is costly
and time consuming, which makes large cohort studies challenging.

Detect Rate

• Ultrasound imaging offers a cost-effective and high-throughput
alternative, but historically struggles as a brain imaging modality due topoor skull penetration and low contrast

58%
7 / 1275%
9 / 12100%
6/64-5 mm
65.4 mm3100%
3/3

5-6 mm113.1 mm3100%1/1

Figure 5: Detection rate versus tumor size. For illustrative purposes, edges ofhistogram bins are defined as incremental spheroid diameters with tumor volume
cut-points computed as 4Τ3 𝜋𝑟 3 . US detected 72.2% (26 out of 36) of the tumorsthat were seen in MRI. The mean volume, as measured by MRI, of tumors thatwere missed by US was 2.3 mm3, while the mean volume of tumors that weredetected by US was (8.0 mm3). Smaller tumors were more easily detected in USimages in areas of the brain with lower baseline AA signal (suggesting higher
difference in vascular density between diseased and healthy brain tissue).

GOAL: To evaluate a new 3D contrast-enhanced ultrasoundimaging approach for rapidly measuring tumor volumes in vivo inrodent models for GBM.

20%
0/2

1-2 mm
4.1 mm3

2-3 mm14.1 mm3

3-4 mm33.5 mm3

Imaging Methods

AOutlier Examples

9Figure 8: US (top) and MRI (bottom) images of a brain where one axial location
contained a tumor (left) and the other a cyst (right). The tumor and cyst appear
similar on MRI, while the tumor and cyst are hyperechoic and hypoechoic,
respectively, in the US image. Cysts were excluded from the analysis.

Figure 2: Representative tumor growth images of US (top row) and MRI (third row).
The 3D rendering of the segmentations are shown in rows 2 and 4 for US and MRI,respectively. Tumor location is indicated by the dotted circles.

5
B

Longitudinal Growth Curves

DA

B

KidneysHeartLiverC
Figure 6: US (top) and MRI (bottom) images of the smallest (left) and largest(right) tumors that were detected by US.

Figure 1: Schematic overview of image acquisition approach.
A: How the bottom-up approach captures widefield images of rodents.
B: Photo of Vega imaging system.
C: Illustration of VesselVue microbubble ultrasound contrast agent.
D: Screenshots illustrating in vivo 3D Acoustic Angiography data in a mouse
liver (left) and kidneys (right).

3

Figure 3: Tumor volume over time for US (A) and MRI (B). All tumors from round 2
were detected by MRI 7 days after implantation, except in one case where themouse did not grow a tumor. Only 3 mice from round 1 grew tumors and weredetected by MRI 19, 22, and 29 days after implantation. Furthermore, the growth
curves measured by US and MRI are similar for tumors that US detected in multiple
timepoints.

Study Design

6-week old female athymic nude mice were implanted with U87 GMB cells
in the brain.
Implantation. Cells (3 μL total injection volume) were implanted on the left
side of the brain approximately 0.6 mm anterior from bregma, 2 mm lateralfrom center, and 3 mm from the surface of the brain using a stereotaxicstage, motorized micromanipulator, and microinjection syringe pump.
• Round 1- 6 mice were implanted with 1 x 105 cells.
• Round 2- 8 mice were implanted with 1.5 x 105 cells.
Imaging. Mice were imaged with both US and MRI twice per week for 4
weeks after implantation, starting 1 week after implantation, with thefollowing imaging parameters:• US: Acoustic Angiography images were acquired using the Densitypreset in SonoEQ with an additional 20 sec delay between repetitions. A
1:1 mixture of VesselVue and saline was injected via continuous infusion
pump (30 μL bolus followed by 15 μL/min after).
• MRI: 9.4T T2 images were acquired using a 100 μm and 250 μm in-planeand out-of-plane resolution, respectively, and 6 repetitions.
©2024, Revvity, Inc. All rights reserved.

6A

8Tumor Growth Location
10Conclusions

ACorrelation Analysis
BB

Figure 4: Correlation (A) and Bland-Altman (B) plots of US vs MRI volume
measurements. r-Pearson correlation coefficient, R2-coefficient of determination, ρSpearman correlation coefficient, CV-coefficient of variation, LOA-limits ofagreement.

Figure 9: US (top) and MRI (bottom) images of a tumor that was not detectedwith US until the last imaging timepoint. US detected several tumors that weresmaller than 5 mm3 but failed to detect some that were twice that size.

Figure 7: Tumor center
locations overlaid on MRI
images of a healthy brain in thecoronal (A) and axial (B)orientations. Center locations oftumors that were detected by
US are depicted by yellow
circles, while the locations ofcysts and tumors not detectedby US are shown as teal andblack X’s, respectively. Theaxial location is 0.6 mm caudal
from Bregma and the coronalplane is 3 mm from the top ofthe brain. No pattern was foundto explain why some of thetumors were not detected byUS.

These studies demonstrate that the ultrasound (US)technique of Acoustic Angiography has the potential to be
used for tracking glioblastoma development in mice. While
US was not able to detect as many tumors as MRI, thevolume measurements obtained on detected tumors had astrong correlation (R2 = 0.92) between US and MRI.
Future work will focus on optimizing US imaging
parameters to improve sensitivity and enhance detection.
Furthermore, histology will be performed to explore whysome tumors were not detected by US.
Funding sources: NIH R44CA239830

Corresponding Author: Tomek Czernuszewicz, Ph.D. tomasz.czernuszewicz@revvity.com
Revvity, 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.revvity.com