P14 Biomarker Monitoring by Quantitative MALDI Imaging: Application to the Tryptophan-Kynurenine Pathway in Immuno-Oncology

Rima AIT-Belkacem , ImaBiotech, Loos, France
Vanesa Bol , iTeos Therapeutics SA, Gosselies, Belgium
Bruno Gomes , iTeos Therapeutics SA, Gosselies, Belgium
Gregory Hamm , Imabiotech, Loos, France
Stefan Linehan , ImaBiotech Corp, Billerica, MA
Jonathan Stauber , Imabiotech, Loos, France

Tryptophan (Trp) is an amino acid for cell proliferation and survival that is metabolized through different pathways, a major route being the kynurenine (Kyn) pathway. The first, rate-limiting enzyme is the indoleamine-2,3-dioxygenase 1 (IDO1); an endogenous molecular mechanism of immune suppression. Inhibition of T-cell functions, activation of its regulatory, and inhibition of Natural Killer cells are among the important immunosuppressive effects of IDO1. IDO1 is proposed to have a therapeutic potential in immunodeficiency-associated abnormalities, including cancer. The standard quantification methods are based on the total level of Tryptophan and its metabolites determined by LC-MS/MS in plasma and tumors. We describe the setup and application of a MSI method to localize and quantify Tryptophan and Kynurenine in the microenvironment of tumors.


10μm thick tissue sections of P815 tumor xenografts (previously inoculated in BALB/c mice) were thaw mounted onto ITO coated slides. 2,5-dihydroxybenzoic acid (2,5-DHB) and 1,5-diaminonaphthalene (1,5-DAN) matrices were coated using TM-Sprayer (HTX) device. MSI analysis was performed using 7T MALDI FTI-ICR (SolariX XR, Bruker Daltonics,) in positive and negative ion modes (m/z range 50 – 1000) at high-resolution with an on-line calibration. Data acquisition, processing, and visualization were performed using the Flex software suite (ftmsControl 2.1.0, FlexImaging 4.1 and DataAnalysis 4.2). Quantification data analysis done with Multimaging (ImaBiotech, France). Immunohistochemistry anti-IDO and anti-GCN2 (AbCam, UK) with rabbit polyclonal antibodies. Goat anti-rabbit IgG H&L (HRP) secondary antibody was used. Detection system was through horseradish peroxidase followed by Steady DAB/plus (brown chromogen).


This method allows both the study of the sub-tissular localization and the detection/quantitation of metabolites of interest in tumor tissues. In the present study, an experimental tumor model overexpressing IDO1 and its wild-type counterpart were implanted in mice. Then, tissue sections of different tumors were realized and used for mass spectrometry imaging analysis. MALDI FTICR high resolution imaging followed by data analysis enabled an absolute on tissue quantitation. Internal standards of tryptophan (Trp-d5) and kynurenine (Kyn-d4) metabolites were used for normalization. As expected, our results showed an increase of Kyn in parallel to a decrease of Trp amount in IDO1-positive tumors. Following, immunostainings of IDO1 and Trp-depletion sensor (GCN2) pathways were carried. Overlaying images between the immunostainings and the molecular MS images allowed co-localization studies and underlined both the biology and the tumor heterogeneity. This study allowed us to highlight key metabolites of the Trp pathway that are responsible for the immunosuppressive tumor microenvironment. Furthermore, this study illustrated the heterogeneity of tumor immune areas. Because the development of immunotherapies such as IDO1 inhibitors requires a deep understanding of the interplay between the immune system and cancer cells, these immune endogenous metabolites can now be followed by quantitative MALDI imaging and as biomarkers towards enhancing immunotherapies efficiency.