Multimodal Imaging
MULTIMODAL IMAGING OF PHOTOSENSITIZERS
IN 3D TUMOR CELL MODELS
Jennifer-Christin Müller
1
, Lousia Petersen
2
, Dr. Dennis Mulac
2
, Prof. Dr.
Klaus Langer
2
and Prof. Dr. Uwe Karst
1
1
Institute of Inorganic and Analytical Chemistry, University of Münster,
Germany
2
Institute of Pharmaceutical Technology and Biopharmacy,
University of Münster
▪ Introduction
Photodynamic therapy offers an innovative and
alternative cancer treatment. A photosensitive
compound (photosensitizer, PS) is administered
and the tumor is subsequently irradiated. The
activation of the PS leads to a formation of reactive
oxygen species and subsequently to cell
apoptosis. The compound 5,10,15,20-tetrakis(3-
hydroxy-phenyl)-porphyrin (mTHPP), and its
palladium-tagged analogue mTHPP-Pd were
studied in this work (Figure 1). One main challenge
in the development of PS is the hydrophobic
character of the compounds that hinders the tissue
penetration. Additionally, the orally administered
compound needs to pass through the mucus layer
in the gastro-intestinal tract. Thus, the
determination of the penetration depth of those
compounds is of great interest.
The use of 3D tumor spheroids enables in vitro
drug screening, while simulating the tumor
environment better than 2D cell cultures. Here, the
combination of elemental and molecular imaging
by means of laser ablation-inductively coupled
plasma-mass spectrometry (LA-ICP-MS) and
matrix-assisted laser desorption/ionization-mass
spectrometry imaging (MALDI-MSI) was used to
investigate the spatial distribution and
concentration of mTHPP and mTHPP-Pd in 3D
tumor spheroids.
mTHPP-Pd were synthesized according to known
literature[2] and the tumor spheroids were
incubated with the dissolved PS for 24 h with a
final PS concentration of 5 μM.
Sample preparation
For MALDI-MSI, 14 μm thin sections of the
spheroids embedded in Tissue-Tek
®
O.C.T. were
placed on indium tin oxide-coated glass slides.
The cryo-compound was removed using
ammonium acetate buffer and α-cyano-4-
hydroxycinnamic acid (CHCA) was sublimated
onto the tissue using the iMLayer
TM
(20 min,
250 ℃, 5·10¬2 Pa).
For LA-ICP-MS analysis, 5 μm thin tissue sections
of the spheroids treated with mTHPP-Pd were
placed on microscope slides to enable the
detection via ICP-MS. An external calibration with
matrix-matched gelatin standards was applied for
quantification. To do so, 10% gelatin was spiked
with aqueous palladium ICP standard solution in a
concentration range from 0.1 to 100 μg/g. The
standards were heated, homogenized, and cut into
5 μm thin sections, as well.
Instrumentation
For molecular imaging, the iMScope Trio was
utilized. The MALDI source, equipped with a 355
nm Nd:YAG laser, was operated at atmospheric
pressure with a laser pulse frequency of 1000 Hz.
For each pixel, 100 laser shots were accumulated.
The sample voltage was set to 3.5 kV and the
detector voltage was 1.9 kV.
No. SCA-116-004
▪ Material and methods
Tumor spheroids
Stable tumor spheroids based on the human colon
carcinoma cell line HT29 were obtained using the
liquid overlay technique.[1] The PS mTHPP and
