Florian Barré (Presenter)
Maastricht University, M4I
Authorship: F.P.Y. Barré(1), C. Côme(2), F. Dewez(1), K. Grønbæk(2), A.H. Lund(3), R.M.A. Heeren(1), B. Cillero-Pastor(1)
(1) The Maastricht Multimodal Molecular Imaging Institute (M4I), Division of Imaging Mass Spectrometry, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands. (2) Epigenomlaboratoriet, Rigshospitalet Dept. 3733, Bartholin Institute, Copenhagen Biocenter, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark. (3) Biotech Research and Innovation Centre, Ole Maaloes vej 5, 4., 2200 Copenhagen N, Denmark.
| Short Abstract Diffuse large B-cell lymphoma (DLBCL) is a biologically aggressive disease and up to one-third of patients will ultimately become refractory to initial therapy or relapse after treatment and display poor survival outcome. The high mortality rate in patients with relapsed or refractory DLBCL highlights the urgent need for novel therapeutic approaches based upon selective molecular targets. We propose to combine in vivo luminescent/fluorescent DLBCL xenograft models with mass spectrometry imaging (MSI) analysis to study the tumor characteristics during R-CHOP treatment. The aim is to investigate changes in the chemical composition during tumoral development, treatment response and identify yet uncharacterized targets that could become alternative targets for therapy. |
Long Abstract
Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin’s lymphoma (NHL) throughout the world, comprising 30–35% of all NHLs, with approximately 71 000 new cases and 19 000 deaths estimated for 2014 (1). Currently, R-CHOP, a combination of immunotherapy (Rituximab, targeting the cell surface protein CD20 expressed by B cell lymphoma) and chemotherapy (Cyclophosphamide, doxorubicin, vincristine and prednisone), remains the most commonly used regimens for newly diagnosed advanced DLBCLs (2–4). DLBCL is a biologically aggressive disease and up to one-third of patients will ultimately become refractory to initial therapy or relapse after treatment (5) and display poor survival outcome (6,7). The standard therapy for patients with relapsed or primary refractory DLBCL is high dose chemotherapy with autologous stem cell transplantation (ASCT), but the long-term outcome is poor (8,9). The high mortality rate in patients with relapsed or refractory DLBCL highlights the urgent need for novel therapeutic approaches based upon selective molecular targets. DLBCL is a heterogeneous disease comprising at least two distinct subtypes; germinal center B-cell (GCB) and the activated B-cell (ABC) type that respond differently to standard treatments (10). Further subtypes were revealed by genetic analysis of DLBCL tumor samples and include a diverse range of somatic mutations and aberrant intracellular signaling pathways (11,12). Still, knowledge of the molecular characteristics of the relapsed/refractory cases is sparse. However, our previous studies have shown that the most aggressive primary DLBCLs carry inactivation of components of central tumor suppressor pathways including TP53, ARF, CDKN2A, ATM and miR34A (13–16). Moreover, the last decade has underlined the importance of the tumor microenvironment (TME) in tumor resistance, especially B-cell lymphomas. Indeed, these tumors derive from B-cells, impacted by their interaction with various cell types such as T-cells, dendritic cells, macrophages, stromal cells, etc… And these TME cells appear more and more to be key components of the resistance mechanisms to therapy (17–20). Therefore, it is of high importance to enhance our knowledge of the various mechanisms leading to tumor resistance/relapse in GCB and ABC DLBCL in order to develop efficient therapies against relapsing tumors. Our hypothesis is that, not only tumor cells but also TME cells are evolving to become resistant to therapy. Our aim is to investigate changes in the chemical composition during tumoral development, treatment response and identify yet uncharacterized targets that could become alternative targets for therapy.
For this purpose, we combine in vivo luminescent/fluorescent DLBCL xenograft models with “imaging mass spectrometry” analysis. The in vivo imaging approach allows us to precisely quantify tumoral development and response to therapy, as well as to differentiate tumoral cells from the tumoral micro-environment. On the other hand, MSI technique provides information regarding analyte composition at an almost cellular level. Therefore, we can identify, localize the molecules, proteins, drugs or metabolites in the therapy resistant and sensitive areas of each tumor.
Luminescent and fluorescent cancer cell lines were generated in BRIC institute constitutively expressing a cassette containing luciferase-2 coding sequence fused to a fluorescent protein (GFP or TdTomato), based on lentiviral vectors protocol. 10 million cells of a U2932 lymphoma cell line were xenografted into 20 athymic nude immuno-deficient mice. Tumoral growth was repeatedly quantified in a non-invasive manner based on tumors’ luminescent signal using the in vivo imaging system (IVIS) Lumina II. Consequently, R-CHOP treatment was applied to 10 mice after primary tumoral growth. 3 types of samples are generated: i) before treatment, ii) during response treatment (shrinking of the tumor), iii) after tumor relapse.
Matrix assisted laser/desorption ionization mass spectrometry imaging (MALDI-MSI)(21-22) is then used to analyze and compare the chemical and biological profiles of DLBCL xenografts at these three stages of tumoral growth. (MSI) is a relatively new biomolecular tool. Compared to conventional imaging techniques, such as MRI, PET or autoradiography, MSI offers several important advantages. First, it does not require the labeling of molecules and can produce high spatial resolution images. Moreover, MSI allows for the simultaneous detection of thousands of different molecules that can be detected from histological tissues sections in a single experiment. Each mass spectrum reflects the local molecular composition at a given pair of x and y coordinates. All mass spectra acquired from the tissue constitute an image dataset analogous to pixels in a digital photograph. Images show the distribution of the selected compound within the tissue section according to the specific m/z value which is extracted from all the collected spectra and the relative abundance of that ion in each pixel can be visualized by a color intensity in scale in a two-dimensional (2D) map. Subsequent developments of imaging computer algorithms, that allowed instrument control as well as data acquisition and processing, provide the tools for obtaining images of compound distributions within thin tissue sections.
The growth of the tumors was followed by IVIS before sacrificing the animals in order to study the effect of the treatment and the relapse on the mice. Different methods for sample preparation have been tested for lipid, metabolite and protein analysis. The protocols have been optimized using different matrices, also different devices for matrix application (TM-Sprayer from HTX-technologies and Suncollect from SunChrom) have been used as well as several mass spectrometers such as the Synapt G2-Si (Waters), the RapifleX and the Solarix (Bruker). HDImaging, FlexImaging, BioMap and SCiLS lab software have been used for the data analysis.
The heat stabilization of the tumor prior to snap freezing with the Denator device showed an improvement in the intensity of metabolites and some proteins. The tumors at different stages of response to R-CHOP therapy have been analyzed and compared from lipidomics, metabolomics and proteomics point of view by MSI showing different molecular profiles. For example, multivariate analysis such as principal component analysis have revealed a specific protein profile of relapsed tumor and tumor before treatment . In addition to this, different metabolites have been identified by high resolution mass spectrometry (FT-ICR) and imaged by MSI to look at their biodistribution within the tissue sections.
Combining IVIS and MSI allow us for a better understanding of the disease and the treatment effects. We optimized protocols using different devices (Denator, different sprayers) and mass spectrometers to reveal lipidomics, metabolomics or proteomics signatures between the different stages of DLBCL response to R-CHOP treatment. We show the potential of combining these techniques in order to identify new candidates for alternative therapies.
References & Acknowledgements:
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I would to thanks Ron Heeren and Berta Cillero to have let me developed this collaboration.
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