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both calibration curves of Trp/Kyn and all the IDO1-low and
high P815 sections that were used for the LC-MS/MS analysis.
A total of 5 µL was injected into the LC-MS/MS system. No
additional filtration step was necessary. The LC-MS/MS
system consisted of an ultra-high-performance liquid chromatography-focused+ LC system composed of RS column compartment, RS pump, and RS autosampler (Dionex Ultimate
3000, Thermo Fisher Scientific) that contains a Thermo
Scientific column (Hypersil Gold C18 50 × 21, particle size =
1.9 µ) coupled to a TSQ Quantiva Thermo Scientific (Thermo
Fisher Scientific). Data acquisition and processing were carried out using TSQ Quantiva software version 2.0 1292 and
Xcalibur 3.0 software.

Quantitative MALDI Analyses
Quantitative MALDI analyses were performed using our
internal newly developed software Multimaging 1.2 software (ImaBiotech SAS, France). This multimodal imaging
platform combines quantitative mass spectrometry imaging
(qMSI) and microscopy platform with statistical analysis
for the understanding of the Omics information at cellular
levels. Regarding the quantitative part, internal standards
optimal concentrations were first determined using deuterated compounds (Trp-d5 and Kyn-d4). Using isotopic modified compounds should allow getting the same suppression
effect if present within the tissue regarding the endogenous
molecules of interest. These compounds were sprayed over
tissues of interest (IDO1-low and -high P815 tumors) to calculate the ratio between internal molecule and deuterated
molecule intensities that should not exceed 10 (5 µM of
Trp-d5 and 10 µM of Kyn-d4 were chosen for following
experiments). Afterward, calibration curves were measured
following two steps (large and restricted ranges). Following
these, low limit of quantitation, upper limit of quantitation,
and limit of linearity parameters were defined for each calibration curve that has been performed using at least 10
spiked concentrations on 10 µm thickness liver tissue sections. The concentration range was 125 to 0.5 pmol for both
Trp and Kyn, which has been validated as a linear regression model. On the same big ITO slide, calibration curves
and tissue sections of interest (IDO1-high and -low P815 in
triplicates and duplicates) were deposited and matrix containing internal standards sprayed over. Then, using
Multimaging software, normalized Trp and Kyn intensities
over calibration curves and tissues of interest were performed. Finally, using the calculated equations, different
quantities in µg/mm2, then converted to µg/g of tissue, were
obtained in selected regions of interest (ROIs; triplicates of
IDO1-high and -low P815 tumors analyzed in duplicates).

MALDI-FTICR Imaging
For MALDI MSI of metabolites, a uniform layer of filtrated
1,5-diaminonaphtalene (1,5-DAN) prepared at 10 mg/mL

SLAS Discovery 22(10)
with 50/50 ACN/H2O matrix was deposited onto the tumor
tissue sections using an HTX-TM sprayer device (HTX
Technologies, LLC, Carrboro, NC) with a nozzle heated to
60 °C and high of 40 mm from the sample slide. Ten passes
at 0.1 mL/min flow rate and 1200 mm/min velocity with 3
mm track spacing and a pressure of 10 psi and 2 L/min of
gas flow rate. Then, MALDI MSI analysis was performed
using 7T MALDI-FTICR (SolariX XR, Bruker Daltonics,
Bremen, Germany) with a SmartBeam II laser. MSI data
were recorded in negative ion mode (m/z range 100-1000)
and at 120 µm and then 30 µm of spatial resolutions with an
online calibration.
Data acquisition was performed using the Flex software
suite (FtmsControl 2.1.0 and FlexImaging 4.1x64) from
Bruker Daltonics and data processing and visualization
using Multimaging 1.2 software (ImaBiotech SAS, France).
This multimodal imaging platform combines qMSI and
microscopy platform with statistical analysis for understanding the Omics information at cellular levels.
MSI data were acquired from each tissue section as well
as matrix control areas adjacent to the tissue sections to
check for analyte dispersion/delocalization during sample
preparation.

IHC Analyses, Digital Scan Image, and HighDefinition Overlay
Sections were stained for IDO1 and GCN2 using rabbit polyclonal antibodies purchased from Abcam (Cambridge, UK)
and adapted to fresh frozen tissue sections. Sections were first
exposed to 0.5% Triton-X for 15 min at room temperature and
washed with phosphate-buffered saline prior the addition of
the primary anti-IDO1 and anti-GCN2 antibody (1:50 and
1:100 dilutions, respectively) and processed with goat antirabbit IgG H&L (HRP) secondary antibody (1:2000). The
detection system was through horseradish peroxidase followed
by steady DAB/plus (brown chromogen).
After MSI data acquisition, any residual matrix was
removed with a 70% ethanol wash, and the tissue samples
were then stained with hematoxylin and eosin (H&E)
solution.
High-resolution histological images of H&E stain or
IHC were then recorded using a digital slide scanner (3D
Histech Pannoramic) then loaded in Multimaging technology to perform the high definition overlays with convoluted
molecular images.

Results
MALDI MSI of Tryptophan Pathway: An Insight
in the Tumor Metabolism
As expected, we detected decreased Trp in IDO1-high compared with IDO1-low P815 tumors. This low Trp level in
the IDO1-high tumors was consistent with the detected high

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