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Photoacoustic endoscopic imaging

study of melanoma tumor growth in a

rat colorectum in vivo

Chiye Li, Joon-Mo Yang, Ruimin Chen, Yu Zhang,

Younan Xia, et al.

Chiye Li, Joon-Mo Yang, Ruimin Chen, Yu Zhang, Younan Xia, Qifa Zhou,

K. Kirk Shung, Lihong V. Wang, "Photoacoustic endoscopic imaging study of

melanoma tumor growth in a rat colorectum in vivo," Proc. SPIE 8581,

Photons Plus Ultrasound: Imaging and Sensing 2013, 85810D (4 March

2013); doi: 10.1117/12.2005470

Event: SPIE BiOS, 2013, San Francisco, California, United States

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Photoacoustic endoscopic imaging st

udy of melanoma tumor growth

in a rat colorectum

in vivo

Chiye Li

1,4

, Joon-Mo Yang

1,4

, Ruimin Chen

, Yu Zhang

, Younan Xia

, Qifa Zhou

, K. Kirk

Shung

, and Lihong V. Wang

Optical Imaging Laboratory, Depart

ment of Biomedical Engineeri

ng, Washington University in St.

Louis, One Brookings Drive, Campus

Box 1097, St. Louis, Missouri, 63130, USA

Ultrasonic Transducer Resource Ce

nter, Department of Biomedical

Engineering, University of

Southern California, 1042 Downey Way, Univer

sity Park, DRB 130, Los Angeles, CA 90089, USA

The Wallace H. Coulter Department of Biomedi

cal Engineering, Georgia Institute of Technology

and Emory University, Molecular Science and Engi

neering Building, 901 Atla

ntic Drive, Atlanta,

GA 30332, USA

ABSTRACT

We performed a photoacoustic endoscopic imaging study of melanoma tumor growth in a nude rat

in vivo

. After

inducing the tumor at the colorectal wall of the animal, we monitored the tumor development

in situ

by using a

photoacoustic endoscopic system. This paper introduces our experimental method for tumor inoculation and presents

imaging results showing the morphological changes of the blood vasculature near the tumor region according to the

tumor progress. Our study could provide insights for future studies on tumor development in small animals.

Keywords

: Photoacoustic endoscopy, melanoma tumor, nude rat, colorectum.

1. INTRODUCTION

Because of its strong spectroscopic and angiographi

c imaging ability, photoaco

ustic endoscopy (PAE)

1-4

could be a

powerful tool for studying tumor development. Importantly, PAE can provide plenty of functional information and

blood vasculature information without using any contrast agent

5-16

Although many

in vivo

photoacoustic (PA) imaging

studies based on tumor models induced in mouse or rats have been reported

8, 17-21

, to our knowledge, none of them were

implemented in the endoscopic form.

In this study, we investigated such capability of PAE by performing a melanoma tumor imaging experiment with a nude

rat. Like colonic carcinoma, melanoma tumor is a well-k

nown epithelial tumor. By injecting the tumor cells (B16

melanoma cell line), we produced a xenograft melanoma tumor m

odel at the colorectal wall of an athymic nude mutant

rat (Hsd:RH-

Foxn1

rnu

Foxn1

). Then, we monitored its development

in situ

using a PA endoscopic system

and could

observe vasculature variation as the tumor developed. Here we describe the experimental methods for tumor induction

and the endoscopic imaging procedure, and present the imaging results.

MATERIALS AND METHODS

2.1.

Photoacoustic endoscopic system

These authors contributed equally to this work.

Corresponding author:

lhwang@biomed.wustl.edu

Photons Plus Ultrasound: Imaging and Sensing 2013, edited by Alexander A. Oraevsky, Lihong V. Wang,

Proc. of SPIE Vol. 8581 85810D-1

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Cornputer

DAQ card

Display

Amplifier

Signal wire

Laser

Delay

generator

Optical

fiber

Micromotor

driver circuit

I- Endoscope

Motorized

pullback stage

For this study, we utilized the 3.8-mm diameter probe based endoscopic system reported in

Nature Medicine

Figure 1

shows the endoscope and its peripheral systems, composed of a micromotor driver circuit, a delay generator, a laser

system, an ultrasonic (US)

pulser-receiver including an amplifier, a data

acquisition (DAQ) card, and a computer for

recording signals and displaying images.

Figure 1.

Block diagram showing the endoscopic

probe and its peripheral systems.

For PA imaging, laser pulses (578 nm, ~10 ns pulse width, ~0.3 mJ/pulse) from a tunable dye laser (Cobra HRR, Sirah),

pumped by a solid-state, diode-pumped Nd:YLF laser (INNOSLAB IS811-E, EdgeWave), are guided by a multimode

optical fiber (BFL22-365, Thorlabs) and emitted through the

central hole of a single element focused US transducer

(LiNbO

, ~36 MHz, 65% fractional bandwidth), which is coaxially

aligned with the optical fiber. After exiting the fiber,

the laser beams are further directed to

the target tissue by a scanning mirror, and finally generate PA waves once

absorbed by the target tissue. The PA wa

ves that propagate to the scanning mirror are reflected by the same mirror, sent

to the US transducer, converted into electrical signals, am

plified by the US pulser-recei

ver (5072PR, Panametrics), and

digitally recorded by the D

AQ card (NI PCI-5124, National Instruments).

With the endoscopic system, we acquired

volumetric PA images with a B-scan frame rate of ~4 Hz. Mo

re information on the endoscope’s structure is available in

our previous report

With the laser fluences used in these experiments, the endoscope’s maximal radial imaging depth was ~7 mm from the

endoscope’s surface, and th

e angular field-of-view (FOV) wa

s limited to approximately 270°

, due to partial blocking by

the probe housing. Experimentally measured highest PA and

US resolutions in the focal z

one of the transducer were

respectively ~55 μm and ~30 μm in the axial direction, and

~80 μm and ~60 μm in the transverse direction, but the

transverse resolution varied with target distance.

2.2.

Tumor inducement

Because of the long rigid distal secti

on (~38 mm) of the employed endoscope,

we chose an athymic nude mutant rat

(Hsd:RH-

Foxn1

rnu

/Foxn1

, Harlan Laboratories) for the host of melanoma tumor.

During the cell culture, we maintained B16 melanoma cells in Dulbecco’s Modified Eagle Medium with 10% fetal

bovine serum and 1% penicillin/streptomycin supplement. The cells were incubated in 37 C°, 5% CO

and divided

every 72 hours. For subculture,

they were seeded at 2–4 ×10

cells/cm

after being dispersed in 0.25% EDTA-trypsin.

For injection, cells were resuspended in phosphate buffered

saline after trypsinization. Then

, we locally injected a 200-

μl tumor cell solution, containing ~10

tumor cells, to the colorectal wall of a nude rat (~16 weeks old) via anus by

using a 30 gauge needle. To dilate the anus, we utilized a plastic catheter which had a small hole at the tip for the needle

introduction. With this injection method, we were able to induce melanoma tumor while minimizing the anatomical

disturbance of animal which frequently occurs in conventional surgical injection methods.

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(a)

Day 1

+120° +60° MD -60°-120°+120° +60° MD

-60°-120°

(b)

Day 2

+120° +60° MD -60°-120°+120° +60° MD

-60°-120°

PA amplitude

2.3.

Endoscopic imaging of the tumor

We targeted monitoring the tumor development with a one

-week interval. When we appr

eciated that an endoscopic

imaging experiment was needed, we fasted the rat for ~20 hr before the day of the experiment to increase the likelihood

of an empty colon for endoscopic imaging.

In each imaging experiment, we anesthe

tized the rat with ~4% isoflurane for

induction, and administered a cocktail of 87 mg/kg ketamine and 13 mg/kg xylazine (IP) to provide time to prepare and

mount the animal. Once the animal was

properly positioned, medical ultrasound gel was inserted into the colon via a

small plastic tube. The ultrasound gel provided acoustic coupling between the tissue and US transducer and lubricated

the probe during colon insertion through the anus. Then we

inserted the endoscopic probe into the colon ~6 cm deep

from the animal’s anus and performed pullback volumetric scans over a ~2–3 cm range during constant pullback

translation of the probe at a

mechanically governed speed of

~160 μm/s (~1 cm/min). We recorded multiple volumetric

data sets, and each volumetric scan required a scanning

time of ~2–3 min. About 500–800 B-scan slices with

longitudinal spacings of ~40 μm were acquired for each im

aging session. While imaging, we maintained anesthesia

with 1.5–2.0% isoflurane supplied through a nose cone and monitored its vital signs during the experiment. Once we

observed that the tumor grew up too big to perform endoscopic imaging, we euthanized the animal by a pentobarbital

overdose (150 mg/kg, IP) and surgically validated.

All procedures in the experiment followed the protocol approved by the Institutional Animal Care and Use Committee

at Washington University in St. Louis.

Figure 2.

Serially-acquired PA-RMAP images from the rat colorectum with a melanoma tumor (views from the

inside of the colon). In each image, the horizontal

-axis corresponds to the angular FOV covering 270°, and the

vertical

-axis corresponds to the pullback length of 2–3 cm.

The approximate mid-dorsal (MD) position and angular

measures from the MD are marked along the horizontal

-axis, where the positive and

negative values correspond to

the right and left sides of the animal. The scale bars represent 5 mm for the vertical direction only.

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PA amplitude

RESULTS AND DISCUSSION

In general, tumor growth speed shows a large variation depending on the health condition of the animal and the

environment of the tumor injection site. So, we frequently checked the tumor progress status and decided the starting

moment of endoscopic imaging experiment. In this study, we performed the first endoscopic imaging after two weeks

from the tumor cell injection, and

could acquire endoscopic image data

twice with a one-week interval.

Figure 2

, we present the serial PA radial-maximum amplit

ude projection (RMAP) images acquired from the rat

using a 578 nm laser wavelength. To show the image reproducibility, we present two sets of image data in parallel for

each experiment day. As shown in the images, the tumor re

gion was clearly visualized

in PA images because the

melanoma tumor tissue includes a lot of highly light-absorbing melanin. Also, one can see the increase of the tumor size

and adjacent vasculature change in the colorectal wall

clearly between the first and second experiment days.

To show the melanoma tumor’s configuration in a three-dimensional space, we produced a volume-rendered PA image

using the data presented in

Figure 2(b)

, and present it with a photograph image in

Figure 3

. As shown in

Figure 3(a)

the melanoma tumor location and its adjacent vascul

ature were clearly mapped by the PA imaging.

Figure 3.

(

) Three-dimensionally rendered PA structural image

obtained with a 578 nm laser excitation wavelength.

The down side of this image is cl

oser to anus, and the negative

-axis corresponds to the dorsal direction of the

animal, respectively. (

) A photo showing the melanoma tumor in the rat

colorectum. The

scale bars represent 5 mm.

In this study, we investigated PA image features

of melanoma tumor in the colorectum of a nude rat

in situ

to show

PAE’s clinical potential. Many types of information related to

tumor, such as its site, size, developing rate, vascular

structure and growth pattern would be important, because they could be used for evaluating degree of maturity,

distinguishing adenomas and carcinomas, and determining a prognosis. As we presented here, PAE was able to provide

information on the tumor location and adjacent blood vasculature clearly without using any contrast agent. The

experimental results show PAE’s potential to be used as a new clinical tool in diagnosing many gastrointestinal

diseases, such as cancer.

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For future studies, we plan to use multi-colored laser light to noninvasively provide clinically relevant information like

total hemoglobin concentration, blood oxygenation, blood

flow, and temperature, and we will also try to perform a

colorectal tumor imaging study using nude rats.

CONCLUSION

We performed the first

in vivo

PA endoscopic imaging study of melanoma tumor growth in the colorectum of a nude

rat. PAE could provide the melanoma tumor region and its adjacent blood vasculature clearly without using any contrast

agent. Our study could lead to a

useful methodology for studying tumo

r development in small animals.

ACKNOWLEDGEMENT

We thank Seema Dahlheimer for her attentive reading of the manuscript. This work was sponsored in part by National

Institutes of Health grants R01 CA157277, R01 NS46214

(BRP), R01 EB000712, R01 EB008085, and U54 CA136398

(Network for Translational Research). L.W.

has a financial interest in Microphotoacoustics, Inc. and Endra, Inc., which,

however, did not support this work.

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