High-frequency Ultrasound
Guided Injection andMonitoring of Orthotopic
Murine Models

FACTSHEETIntroduction

Key Advantages

Orthotopic tumor mouse models provide several distinct
advantages compared to their subcutaneous counterparts dueto their organ-specific tumor microenvironment (TME) andincreased clinical relevance and predictivity. However, orthotopic
implantation of cell lines can be time consuming, technicallychallenging and inefficient due to the laboratory and surgicalprocedures required to establish these tumor models, also
necessitating careful consideration of animal welfare due toincreased risks to the species used for study. Furthermore, theluc-tagged cell lines used to create orthotopic animal models for
BLI studies are limited in number and have been shown to create
additional hurdles when evaluating certain immuno-oncology
therapeutics.

The utilization of HF ultrasound for orthotopic tumor guidance
and monitoring offers many benefits. These include:

Crown Bioscience has evaluated the use of high-frequency (HF)
ultrasound technology as a preclinical tool to overcome many of
the challenges associated with orthotopic tumor mouse models.
The benefits of this technology include efficient, precise andhighly reproducible tumor implantation, non-invasive real-time
tumor tracking, orthotopic use of non-luc-tagged cell lines, andimproved animal welfare.

• Satisfies the 3 R’s of Replace, Reduce, Refine• Efficient, precise and highly reproducible tumor implantation• High-resolution imaging and rapid tumor volume measurement• No BLI loss-of-signal due to necrotic tumor tissue• Applicability to many different types of cancer models, such aslung, liver, colon, brain, and pancreatic cancers• Orthotopic imaging of non-luc-tagged cell lines• Improved animal welfare

Comparison of HF Ultrasound to Surgical Implantation andTraditional Monitoring

HF ultrasound in orthotopic mouse models is a promising toolthat provides accurate, reproducible and translational insights fortoday's therapeutic development.
HF Ultrasound Technology Overview

HF ultrasound guided implantation offers higher throughput
when compared to surgical implantation due to the shorter
procedure time, need for fewer technicians, and faster animal
recovery time. The end result is a shortened study duration whencompared to surgical implantation. Results of a comparison
study of ultrasound guided implantation (USGI) versus surgicalimplantation conducted with liver and pancreas tumors is shownin Figure 1 below.

Crown Bioscience has selected the Vevo 3100 HF ultrasound (Fuji
Film Visual Sonics). This is a micro-ultrasound technology thatoffers high frequency to produce higher resolution. The frequency
range of 12 – 71 MHz and 30-micron resolution are well-suited
for small animal studies. The high resolution of the Vevo 3100 HF
ultrasound allows for precise and less invasive orthotopic tumor
implantation using ultrasound guidance, as well as accurate
tumor detection and sizing in 2D and 3D imaging.
Figure 1: HF ultrasound compared with surgical
USGI vs. Surgical Implantation and Recovery Time
60
50

Minutes

40
30
20
100

Procedure Time Liver Procedure Time Pancreas
USGI

Recovery Time Liver

Recovery Time Pancreas

Surgical2

Crown Bioscience recently completed a study that assessed the benefits of HF ultrasound in orthotopic tumor implantation and tumor
monitoring. A summary of the study method and results is provided below.
HF Ultrasound Guided Orthotopic Implantation and Tumor Monitoring: Hep3B-luc Liver Model
Study Goal

Study Results

Assess the benefits of replacing traditional invasive surgicalorthotopic implantation of hepatic tumor cell lines with a minimallyinvasive ultrasound guided approach. Evaluate tumor monitoring
using HF ultrasound as compared to bioluminescence imaging.

In-life tumor volumes were assessed by bioluminescent imaging(BLI) using the Spectrum BL (PerkinElmer, US) and high frequency
ultrasound (HFUS) using the Vevo 3100 system (Visualsonics,Canada). HFUS images were analyzed using Vevolab® software(Visualsonics, Canada). 100% of Hep3B-luc tumors implanted
orthotopically using HFUS demonstrated progressive tumor
growth, as measured by both tumor associated bioluminescence(TABL) (Figure 3) and HFUS (Figure 4). No significant weight loss instudy animals due to the presence of these tumors was observed(Figure 3).

Study Methods• Hep3B cells were transduced to express firefly luciferase shorttandem repeat (STR) profile match to parental cell line.
• 1x106 Hep3B-luc cells in 20 µL of Matrigel were injected
orthotopically into the liver via ultrasound guided injection.
• In-life growth and terminal ex vivo assessed by bioluminescenceimaging (BLI) using Spectrum BL (PerkinElmer, US) and HF
ultrasound imaging using Vevo 3100 system (Visualsonics,Canada).

HFUS imaging acquisition enabled the tumor location within theorgan of interest to be accurately pinpointed. The HFUS images
were then analyzed using Vevolab® software (Visualsonics,Canada), enabling 3D reconstruction of these tumors, giving atumor volume reading (Figure 4). HFUS imaging analysis confirmed
the location of these tumors within the liver, and this was further
validated by ex vivo BLI of the livers upon termination (Figure 4).

Figure 2: Relative body weight change after orthotopic
Hep3B-luc tumor inoculation and In-life tumor growth as assessed by BLI.
Relative Body Weight (% Day 0)

Tumor Associated Bioluminescence (TABL)

2x1011

Total Flux (photons/sec)

Relative Body Weight(%first recorded)

120

110

100

900510152025

1.5x1011

1x10115x10100300510Study Day

110Vehicle (0mg/kg)
Sorafenib (50mg/kg)

1x10115x101005101520

Study Day

202530Study Day

Relative Body Weight(% of 1st day of dosing)Total Flux (photons/second)

1.5x1011

15253035Vehicle (0mg/kg)
Sorafenib (50mg/kg)

105

100

95

010152025

Study Day

30353

Figure 3: Tumor volumes as assessed by high frequency ultrasound andex vivo BLI confirmation of tumor location within the liver

Mean Ultrasound Tumor Volumes

Individual Ultrasound Tumor Volumes
1000Tumor volume (mm3)Tumor volume (mm3)8006004002000*600

4002000051015202530

Study Day

*8000102030

Study Day

* In- life/ex vivo BLI and Ultrasound3D tumor measurements

Day 13Day 6V = 4.6 mm3Day 20V = 17.5 mm3

Day 25V = 220.7 mm

3

V = 647.9 mm3* Example images taken from the same animal

Left: Final in-life BLI image. Right: (top to bottom) photograph,ex vivo BLI and final 3D ultrasound image confirming
tumor location.

Orthotopic Hep3B-luc model: SoC Validation
Mice were implanted orthotopically with Hep3B-luc cells using HF ultrasound guided injection. After 7 days of tumor growth mice wererandomized and treated with 50 mg/kg daily Sorafenib or vehicle control for 21 days. Treatment with Sorafenib resulted in a statisticallysignificant inhibition in tumor growth of HF ultrasound injected orthotopic Hep3B-luc (p≤0.0001).

4

1.5x1011

110

Vehicle (0mg/kg)
Sorafenib (50mg/kg)

Relative Body Weight(% of 1st day of dosing)Total Flux (Photons/second)

Figure 4: Effects of SOC therapy on orthotopically implanted Hep3B-luc tumors as assessed by BLI and bodyweight change

1x10115x101005101520253035Vehicle (0mg/kg)
Sorafenib (50mg/kg)

105

10095010Study Day

1520253035Study Day

Study Conclusions• The time taken for HF ultrasound guided implantation compared to traditional surgical methods is significantly reduced, enabling ahigher throughput of animals on study.
• HF ultrasound guided implantation is less traumatic than traditional surgical methods, resulting in faster recovery times and fewer
surgery related complications.
• Tumor tracking and measurement by HF ultrasound aligns with BLI imaging.
• HF ultrasound is a promising tool for oncology researchers seeking accurate, reproducible and translational insights for today's
therapeutic development.
Summary
HF ultrasound guided implantation of orthotopic tumors is a minimally invasive technique that reduces study time, enables accuratemonitoring of tumor localization and growth, and adheres to the principles of the 3R’s (reduce, refine, replace).
HF ultrasound guided orthotopic models enable tumor implantation without the need for luciferase tagging of tumor cells, resulting inshortened study timelines. HF ultrasound guided injection takes less time than traditional surgical implantation meaning that a larger
number of animals can be inoculated in a shorter period of time. The use of HF ultrasound guided injection also reduces the risk ofsurgery related complications and potential tissue trauma and inflammation.
The use of HF ultrasound imaging of orthotopic models to track tumor progression has several advantages over traditional imaging
modalities used to track bioluminescently-tagged tumors, including higher resolution and rapid, precise tumor volume measurements.
Additionally, with HF ultrasound imaging, bioluminescently-tagged tumor models are not required, eliminating the potential of loss-ofsignal due to necrotic tumor tissue. Studies have found that the signal for using bioluminescence for tracking tumors in vivo can drop out
after a certain period of time due to necrotic tumor tissue. HF ultrasound methods can continue to track even necrotic tumor masses inlive animals1,2.
HF ultrasound in orthotopic mouse models is a promising tool that provides accurate, reproducible and translational insights for today's
therapeutic development.
References
1Badr CE. Bioluminescence imaging: basics and practical limitations. Methods Mol Biol. 2014;1098:1-18. doi: 10.1007/978-1-62703-718-1_1. PMID: 24166364.

2

Shen YT, Asthana R, Peeters C, Allen C, DeAngelis C, Piquette-Miller M. Potential Limitations of Bioluminescent Xenograft Mouse Models: A Systematic Review. J Pharm Pharm
Sci. 2020;23:177-199. doi: 10.18433/jpps30870. PMID: 32407285.

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