Feasibility of a novel image guided injection tool for non-surgicalimplantation of orthotopic tumors
TM Kierski1, JD Rojas1, N Beaumont1, K Young2, TE Charity2, RD Marek2, G Palmer2, RC Gessner1, TJ Czernuszewicz1(1) Revvity, Inc., (2) Duke University. tomasz.czernuszewicz@revvity.com

NeedleNeedleExternal
Transducer

Liver

Prostate

Pancreas

NeedlePositioner

Prostate

BladderHeart

InjectionsiteInjection
location

Figure 1: Schematic of the image-guided injection system (left) and positioning of the animal for different organ
injections (right). Transducer was connected to Vega imaging engine (Revvity), and ultrasound stream visualized inSonoEQ software (Revvity). In the positioning diagrams, the green box indicates the approximate location of theultrasound transducer, and the red ‘X’ indicates the entry point of the needle.

Study Design

Male athymic nude mice (n=20) were split evenly into two groups:••

Group 1 received intracardiac injections (left ventricle).
Group 2 mice received injections in the pancreas, prostate, and liver.

Injection solutions were prepared as follows:••Pancreas

SpleenUltrasoundfield of viewGroup 1 – 250 µL IVISBrite HepG2 Red F-luc cells (Revvity, 2.5e5 cells/mL in PBS), 50 µL luciferin (15 mg/mL),
30 µL VesselVue ultrasound contrast agent (Revvity)
Group 2 – 250 µL IVISBrite HepG2 Red F-luc cells (2.5e5 cells/mL in 50% Matrigel, 50% PBS), 50 µL luciferin (15mg/mL), 5 µL VesselVue ultrasound contrast agent

After
Injection

BeforeInjection

10/10

Prostate

Heated Animal
Bed3

Injectionsite

Injection
site

Imaging Methods

After
Injection

BeforeInjection

Liver

GOAL: Evaluate the feasibility of a newly-designed image guided injection platform bymeasuring hit-rate in the prostate, pancreas, liver, and left ventricle of the heart.

2NeedleInjection
site
BeforeInjection

AfterInjection

9/10
Pancreas

Injectionsite

Injectionsite

Figure 3: Representative ultrasound images pre- and post-injection. Left column: images of the target organs before
the needle has been inserted into the tissue. Center column: images of the needle at the injection site. Right column:
images acquired post-injection show clear enhancement from the microbubbles, confirming that cells were delivered tothe target organs.

5Results: Intra-Cardiac Injections

10/10

For each injection, animals were positioned on the heated platform and translated such that the target
site was in the line-of-sight of the ultrasound beam and the needle (Figure 1). Using the ultrasoundlivestream as a visual aid, the needle was guided to each target organ.
••Figure 5: Ex vivo IVIS images collected post-injection for Group 2 (all displayed on logarithmic scale with 3x3
smoothing). Clear BLI signal was seen in 10/10 liver images, 9/10 prostate images, and 10/10 pancreas images. In
some cases, the liver images were relatively dim, and repeat images were collected in various orientations to ensurethat the signals were genuine. It was noted during these injections that the density of the liver tissue sometimes
resulted in cells leaking along the needle path rather than remaining at the site of injection. Additionally, the liver is ahighly vascular organ and the cells could be seeing going into a large blood vessel and being carried away from theinjection site. Both of these factors may have contributed to the brightness of these data.

Group 1 – 100 µL injection volume
Group 2 – 20 µL injection volume

Hit-rate verification:••

Group 1 – In addition to the enhancement on the ultrasound stream, the animals were euthanized and the injectedorgans dissected, placed in a six-well plate, and imaged ex vivo using an IVIS Kinetic (Revvity).
Group 2 – The mice were imaged in vivo using an IVIS Kinetic to verify that the bioluminescent signal wasdetected throughout the mouse.
BeforeInjection
RightAtrium

Aorta
NeedleLeft Ventricle©2024, Revvity, Inc. All rights reserved.

AfterInjection

Figure 2: Representativeultrasound images pre- andpost-injection for Group 1(cardiac). Left: image of theleft ventricle before theneedle has been inserted.
Center: image of the needle
at the injection site. Right:
image acquired postinjection show clear
enhancement from themicrobubbles, confirmingthat cells were delivered intocirculation.

Results: Liver, Prostate, & Pancreas Injections

6LiverLiver• Orthotopic tumor models are becoming more widely adopted in preclinical cancer research asthese models more faithfully recapitulate the original tumor microenviroment and exhibit greater
clinic translatability.
• Traditionally, orthotopic tumors are instantiated either via open surgery or blind free-hand injection.
The former requires specific surgical skill, is time consuming, low-throughput, and highly invasive,
while the latter may be prone to error, technician variability, and unintended animal harm.
• Ultrasound-guided percutaneous injections offer a minimally invasive approach to target an organof interest with high accuracy and repeatability.

Results: Representative Ultrasound Images

Prostate

4

Pancreas

Background

17

9/10
Figure 4: IVIS images collected post-injection for Group 1. Nine out of ten animals show full-body signal with hot spotsat the brain and kidneys. One animal (top row, right) showed signal only in the lungs and was considered a failure. Itshould be noted that bright-red blood was seen in the hub of the needle after this injection, and microbubbles weredetected in the liver and kidneys using ultrasound imaging. This suggests that the injection was indeed in the left
ventricle, but it remains unclear why the cells were only deposited in the lungs. One hypothesis is that the cells wereclumping, which may affect their distribution or the right ventricle was pierced causing dissemination directly to thelung.
For research use only. Not for use in diagnostic procedures.

Conclusions• Device achieved a 95% hit rate across 4 different anatomical targets• Time to perform 3 injections in the same mouse = 36 minutes• Time to perform a cardiac injection = 6 min• Future studies will explore design improvements to further increase speed oforgan localization and injection, and test additional injection site accuracy(including: kidney, spleen, bladder, ovary, thymus, and others).
Funding sources: NIH R44CA239830

Revvity, 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.revvity.com