SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1183

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Ait-Belkacem et al.
cell death ligand-1 have led the way to a second generation
of immune checkpoint inhibitors.4
Despite all these promising existing and currently developed therapeutic modalities, and because of the immune system's complexity and the diversity of strategies used by tumors
to escape host immunity, challenges to successful immunotherapy remain, with the need for further understanding of the
pathways and biomarkers involved in the tumor immunology.5
Many findings have already indicated that indoleamine-2,3-dioxygenase 1 (IDO1) may be a reliable and promising prognostic indicator for the treatment of human cancers.6-9 IDO1 is a
natural endogenous mechanism of immune suppression acting
through modulation of the Trp degradation pathway. IDO1
catabolizes Trp into Kyn, which could be further metabolized
into Kyn pathway metabolites.10 The fact that IDO1 has a role
in enabling tumors to escape the host immune system, it is
defined as an attractive new target for cancer immunotherapy.
Therefore, IDO1 inhibitors are currently being developed in
immuno-oncology.11
Trp and Kyn are key biomarkers of IDO1 functions, and
the balance between these 2 metabolites drives immune cell
homeostasis.12 The existing and standard quantitation methods are based on the total level of Trp and its metabolites
determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis in plasma, cerebrospinal
fluid, and brain. However, this methodology does not take
into account important parameters such as spatial distribution and tumor heterogeneity.
In this study, we describe the setup, development, and
application of a new quantitative method based on mass
spectrometry imaging (MSI) to detect, localize, and quantify Trp and Kyn in biological tissues. Transfected murine
tumor models expressing different levels of IDO1 were
used as proof-of-concept models for this study. Basically,
MSI is able to simultaneously record the distribution of
hundreds of biomolecules directly from tissue, without
labeling and without prior knowledge.13 MSI based on
matrix-assisted laser desorption/ionization (MALDI)
applied with different tissue preparation procedures can be
used to analyze proteins, peptides, glycans, lipids, metabolites, and drugs.14-18 Often, the large number of molecules
detected within a single experiment often includes isobaric
species that have the same nominal mass but different exact
mass. It has been demonstrated how high-mass-resolution
instruments, such as Fourier transform ion cyclotron resonance (FTICR) and Orbitrap mass spectrometers, are able
to resolve many of the isobaric species, whereas the more
commonly used time-of-flight systems amalgamate the
signals.
Here, we report a quantitative ultra-high-mass-resolution
study, performed using a 7T MALDI-FTICR that allowed us to
highlight key metabolites of the Trp pathway that are responsible for the immunosuppressive tumor microenvironment.

Materials and Methods
Chemicals and Reagents
All chemicals including 1,5-diaminonaphtalene (1,5-DAN),
tryptophan, kynurenine, tryptophan-d5 and kynurenine-d4,
and formic acid were purchased from Sigma-Aldrich (St.
Louis, MO). Methanol, acetonitrile, and Optima LC-MS
water were purchased from Fisher Scientific (Waltham,
MA). Indium tin oxide (ITO)-coated glass slides were purchased from Bruker Daltonics (Bremen, Germany).
Primary rabbit anti-mouse IgG IDO (106134) and GCN2
(137543), secondary goat anti-rabbit IgG H&L (HRP) secondary antibody (205718), and the detection system through
horseradish peroxidase followed by steady DAB/plus (brown
chromogen) were purchased from Abcam (Cambridge, UK).

Sample Collection and Tissue Preparation
IDO1-positive P815 murine mastocytoma clones were generated as described in Uyttenhove et al.19 Two P815-IDO1
clones were selected according to the level of IDO1 expression (one IDO1-low and one IDO1-high P815 clone).19
P815-IDO1 cells were subcutaneously grafted in DBA/2 6to 8-week-old mice (Charles River Laboratories, France).
After 10 to 15 days of tumor growth, mice were sacrificed,
and tumors were sampled and snap frozen in liquid nitrogen
for 15 s. The samples were kept at -80 °C until use. Ten
micrometer thick tissue sections were obtained using a
cryostat microtome (CM-3050S, Leica, Germany) with a
microtome chamber and a specimen holder chilled at -23 °C.
They were thaw mounted onto ITO-coated slides for downstream MALDI imaging and serial sections on SuperFrost
slides for histological and immunohistochemistry (IHC)
analysis. Biological triplicates in duplicate (distant serial
sections) were performed for analytical reproducibility. For
LC-MS/MS analysis, five tissue sections of 10 µm thickness were harvested to perform calibration curves and
quantitation of both Trp and Kyn.
All animal experiments were compliant with the
2010/63/UE European Directive on Laboratory Animal
Welfare and were approved by an Ethical Committee.

LC-MS/MS Analysis
Five tissue sections of 10 µm thickness liver were collected to
perform the calibration curve. Then, two calibration curves
were used in a range of 0 to 500 nM that contain 5 nM of internal standard each (Trp-d5 and Kyn-d4). Afterward, between
0.5 and 2 mg of serial tumor sections was collected in an acidified aqueous extraction solution (H2O 0.1% formic acid =
mobile phase A) that contains 5 nM on internal standards. An
overnight extraction at 4 °C, centrifugation at 3000 × g, for
4 °C/15 min allowed us to recover 500 µL of supernatant from
Table of Contents for the Digital Edition of SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017
