1225 Non-invasive Imaging of Irradiation Induced Tumor-Infiltrating Macrophages.

Rao V. L Papineni*#, Parmesh Diagaradjane, William Mclaughlin#, Sean Orton# and Sunil Krishnan
#Carestream Health, Inc., New Haven, CT and Department of Radiation Oncology, MD Anderson Cancer Center, Houston, TX, USA

*E.Mail: rao.papineni1@carestreamhealth.com

Macrophage infiltration occurs in cancer and other inflammatory

pathways. We recently demonstrated that both the endogenous
peritoneal macrophages and the exogenous murine cells that were
transplanted into peritoneal cavity migrate to the site of
inflammation. This was obtained using the Thermal injury mouse
model. Likewise, tumor irradiation enhances the expression of
inflammatory mediators and modulators. In this work, we
determined if this irradiation induced changes in the
microenvironment, influences the recruitment of peritoneal
macrophages. We utilized colorectal cancer xenograft model in nude
mice using HCT 116 xenograft in nude mice and treated with 8 Gy of
g-radiation. A commercially available imaging system was used to
obtain X-ray and near infrared fluorescence (NIRF) images at
Steady increase in infiltration of macrophages at site of thermal burn injury
Quantitation of the NIR signals from X-SIGHT Nanospheres 650 and X-SIGHT Nanospheres 761 at the thermal burn injury are
provided (Fig.1). We earlier1 reported that a non lethal scald injury induced to the left hind limb of mice resulted in infiltration
of endogenous X-SIGHT Nanosphere laden peritoneal macrophages at the site of inflammation. Further, in a different set of
experiments, the cultured macrophages labeled1,2 with X-SIGHT Nanospheres that were introduced by i.p.trafficked to thermal
burn injury site.
Infiltration of X-Sight 650 Nanosphere Laden
Macrophages
90000000
ROI :Thermal Injury Paw

ROI :Contralateral Paw

different time intervals. We demonstrate that upon irradiation, the 80000000

Net Intensity (a.u.)

peritoneal macrophages migrate to tumor region, as determined by 40000000
non-invasive NIR fluorescence imaging (figures below). This
infiltration property of peritoneal macrophages to the irradiated tumor 30000000

has immense potential in development of platforms for drug Figure 1. X-SIGHT 650 and X-SIGHT 761
20000000
discovery, drug delivery, and real-time evaluation of radiation Nanospheres sequestered at the injury site.
therapy progression in mice. The role of clinically relevant doses of 10000000
Measurements of the NIR signals were determined
irradiation in the infiltration of compartmentalized macrophages to from the Time-Lapse images (Representative
0
the tumor microenvironment, and the prospects of developing novel 20 40 60 80 100 1065 Image- Inset) obtained with KODAK in-Vivo
Does focused radiation therapy lead to transfer of Time (min)
Multispectral FX system. NIRF imaging: EX:630
therapeutic strategies utilizing these cell trafficking pathways will be
discussed. peritoneal cavity transplanted payload to the tumor Infiltration of X-Sight 761 Nanosphere Laden Macrophages EM:700 and EX:730 EM:790; 10 sec images. No
17000000

ROI :Thermal Injury Paw

binning. ROI were drawn around the injury site
15000000
ROI :Contralateral Paw and the control ROI’s were drawn at several non-
target sites, including the contralateral paw.

Net Intensity (a.u.)

8 Gy -radiation

8000000

6000000
Experimental Protocol 0.5 nmoles X-SIGHT –
761 Nanospheres.

4000000

Colorectal Cancer Xenograft Model:

Cell Culture:HCT 116 cells were cultured in DMEM supplemented
with 10% FBS and 1% Penicillin-streptomycin (Invitrogen, Grand
Island, NY).
Xenograft implantation of HCT 116 cells: Athymic nu/nu mice (4
weeks old) were obtained from the breeding colony of the
Department of Experimental Radiation Oncology at The University of
Texas M. D. Anderson Cancer Center. The animals were housed
five/cage in standard mouse plexiglass cages in a room maintained at
constant temperature and humidity under 12-h light and dark cycles
and fed with regular autoclaved chow diet with water ad libitum.
None of the mice exhibited any lesions, and all were tested pathogen
free. Before initiating the experiment, we acclimatized all mice to a
pulverized diet for 3 days. Experimental protocol was reviewed and
approved by the Institutional Animal Care and Use Committee at MD
Anderson Cancer Center.
Xenograft implantation of HCT 116 cells were harvested from sub-
confluent cultures after a brief exposure to 0.25% trypsin and 0.2%
EDTA. Trypsinization was stopped with medium containing 10% fetal
bovine serum. The cells were washed once in serum-free medium and
resuspended in phosphate-buffered saline (PBS). Only suspensions
consisting of single cells with >90% viability were used for the
injections. Mice were anesthetized with ketamine-xylazine solution,
and 2 x 106 cells were injected subcutaneously into the right leg of
each mouse using a 27-gauge needle and a calibrated push button-
controlled dispensing device (Hamilton Syringe Company, Reno,
NV).
Near IR Nanosphere injection and Irradiation: One week after
implantation, mice were randomized based on the tumor volume
measured by Vernier calipers prior to for X-Sight Nanosphere 761
injection (i.p.). 500 pmoles of the X-SIGHT Nanosphere 761 was
injected into the peritoneal cavity and immediately subjected to
Irradiation protocol. For irradiation, animals were anesthetized and
immobilized in the treatment position with their right legs extended.
Radiation was delivered at a dose of 8 Gy through a single posterior to
anterior collimated 3-cm cobalt beam with a 5-mm bolus placed over
the tumor. Near IR imaging (NIRF) was performed after 17 hrs.
2000000
Conclusions
Subcutaneous colorectal cancer
xenograft implanted mice
0
20 40 60 80 100 1065 • Focused radiation therapy potentially
Time (min)
trigger inflammatory response and
Figure 2. Cartoon putatively promotes infiltration of
describing the tumor peritoneal macrophages.
bearing mouse. i.p.
administration of the • Like the near IR dye loaded X-SIGHT-
761 Nanospheres, drug payloads carrying
Near IR nanoparticles is
prior to the irradiation nanostructures can be compartmentalized
in the peritoneal cavity for delivery at
irradiated site. This will likely minimize
the toxic side-effects at the healthy tissues.
•Thus the recruitment of peritoneal
macrophages to the irradiated tumor has
A B
immense potential in the development of
platforms for targeted delivery of drugs3,
vectors, nanoparticles, and other payloads
directly and in high concentrations to the
Figure 4. Anatomical co registration of tumor using a radiation beam physically
the Near-IR fluorescence signal. X-Ray collimated to conform to the edges of the
and NIR fluorescence images were tumor, minimizing unnecessary exposure of
obtained using KODAK in-Vivo other parts of the body to these agents.
Multispectral FX system . X-ray 1. Papineni et. al. Multiplex Multimodal Imaging of
imaging: 60 sec exp. f-stop 2.8, no Infiltrating Cells in vivo- Mouse Burn Wound Model.
binning. Fluorescence imaging: 60 sec Presented at World Molecular Imaging Congress,
exp, f-stop 2.8, EX 730 EM 790. The Nice, France. 2008.
fluorescence signal was contrasted for 2. Driving Discovery form inside out. Genetic
overlay. Engineering & Biotechnology News. Feb-1, 2008.
3. Patent in process for Dr. Rao Papineni
Figure 3. NIRF imaging of tumor infiltration. The different view (Panels A and
B and fig.4) show the accumulation of near IR fluorescence at the tumor bearing
region after 17 hrs post-irradiation. The fluorescence images were obtained Carestream and X-SIGHT are trademarks of Carestream Health, Inc. The Kodak trademark is used under license from
using KODAK In-Vivo Imaging System FX no binning, EX: 730 EM 790; f- Kodak. Carestream Molecular Imaging is a division of Carestream Health, Inc. Although the Kodak In-Vivo
Multispectral Imaging System FX can be used for in vivo and in vitro molecular imaging of materials, researchers
stop 2.8. should be aware that the methods of preparing and viewing the materials for molecular imaging may be subject to
various patent rights.

"Molecular Imaging - Wisdom To See For Maladies To Flee"

Dr. Rao V. L. Papineni