Extracellular vesicle RNA as systemic modulators of the tumor microenvironment in B cell lymphoma
Final Report Abstract
Cancer cells are secreting extracellular vesicles (EV) and thereby modulate the tumor microenvironment in a way that it supports the development and progression of the cancer. As little is known concerning the role of EV in haematological malignancies, we aimed at analysing EV as modulators of the tumor microenvironment in B-cell lymphoma, including chronic lymphocytic leukemia (CLL). In our previous work, we showed that yRNA4 contained within CLL-derived EV activates Tolllike receptor (TLR)-7 in myeloid cells and thereby induces a pro-tumorigenic phenotype in these cells. To enhance our understanding on the role of yRNA4 in tumor-derived EV, we have generated in this project cells and EV that are deficient for yRNA4 as important tools to study the function of this molecule, a topic little is known about. As the generation of these knock-out cells took longer than expected, the functional analyses with these tool organisms will be performed as follow-up work of this project. By using myeloid cells of different mouse lines with deficiencies of various TLR family members and signalling molecules, we provide now evidence for the involvement of further TLR family members as well as other ligand-receptor interactions in inducing a protumorigenic phenotype in myeloid cells. Based on proteome data of EV obtained from patients or mouse models with B-cell lymphoma, we have identified several potential candidate proteins that are currently further investigated. In addition, the proteome data allowed us to identify disease-specific proteins contained in the EV of B-cell lymphoma patients, and we are currently exploring their potential as diagnostic liquid biopsy markers. To gain a better understanding of the EV-induced changes in the tumor microenvironment, we have performed in-depth analyses of immune cells after treatment with EV in vitro and in mouse models. Protein array data of human monocytes treated either with yRNA4 or CLL- derived EV revealed a differential pattern of deregulated proteins, the relevance of which in supporting CLL development is currently followed up on. Interestingly, flow cytometry and transcriptome analyses of T cells that were treated with CLL-derived EV revealed a phenotype of reduced activity. In addition, EV treatment resulted in an activated phenotype of regulatory T cells that are known to contribute to immune suppression. The proteome data of CLL-derived EV suggest that these activities are mediated by inhibitory molecules present in the EV. To finally explore the relevance of EV for the development of CLL, we have generated a mouse line that is deficient for the secretion of EV due to the lack of Rab27a and b expression. Both, by tumor cell transplantation studies and by analysing primary CLL development in this knock-out mouse line, we showed that in the absence of EV, CLL develops much slower. These results clearly show that the production of EV by CLL cells contributes to leukemia development in the mouse model. We further developed a novel risk score for multiple myeloma which is based on the expression of APOBEC family members and inflammation-related genes, which we detected by proteome analyses to be enriched in B-cell lymphoma-derived EV. This score allowed us to identify a subgroup of patients with very good prognosis and an “ultra-high risk” group of patients who do not benefit from high-dose chemotherapy followed by autologous transplantation and maintenance therapy. Altogether, the data of this project provide evidence for a pro-tumoral role of EV in B-cell lymphoma. They further enhance our understanding of the mode-of-action of EV in modulating the tumor microenvironment. The identification of relevant ligand-receptor pairs that are involved in these processes will serve as a basis for the development of novel treatment approaches to avoid EV-mediated tumor-supportive activities. In addition, this project provides a first step towards the identification of liquid biopsy marker proteins for the diagnosis of B-cell lymphoma and for predicting outcome of patients.
Publications
- High-dimensional mass cytometry analysis revealed microenvironment complexity in chronic lymphocytic leukemia. Oncoimmunology, 7(8):e1465167, 2018
Wierz M, Janji B, Berchem G, Moussay E, Paggetti J
(See online at https://doi.org/10.1080/2162402x.2018.1465167) - Purification of Leukemia-Derived Exosomes to Study Microenvironment Modulation. Methods Mol Biol, 1884:231-245, 2019
Wierz M, Pierson S, Gargiulo E, Guerin C, Moussay E, Paggetti J
(See online at https://doi.org/10.1007/978-1-4939-8885-3_16) - Method for the Analysis of the Tumor Microenvironment by Mass Cytometry: Application to Chronic Lymphocytic Leukemia. Front Immunol, 11:578176, 2020
Gonder S, Fernandez Botana I, Wierz M, Pagano G, Gargiulo E, Cosma A, Moussay E, Paggetti J, Largeot A
(See online at https://doi.org/10.3389/fimmu.2020.578176) - Optimized protocol for isolation of small extracellular vesicles from human and murine lymphoid tissues. Int J Mol Sci, Sci., 21(15):5586, 2020
Bordas M, Genard G, Ohl S, Nessling M, Richter K, Roider T, Dietrich S, Maaß KK, Seiffert M
(See online at https://doi.org/10.3390/ijms21155586) - Targeting IRAK4 disrupts inflammatory pathways and delays tumor development in chronic lymphocytic leukemia. Leukemia, 34(1):100-114, 2020
Giménez N, Schulz R, Higashi M, Aymerich M, Villamor N, Delgado J, Juan M, López-Guerra M, Campo E, Rosich L, Seiffert M, Colomer D
(See online at https://doi.org/10.1038/s41375-019-0507-8) - EOMES and IL-10 regulate antitumor activity of T regulatory type 1 CD4+ T-cells in chronic lymphocytic leukemia. Leukemia. 2021 Feb 1
Roessner PM, Llaó Cid L, Lupar E, Roider T, Bordas M, Schifflers C, Arseni L, Gaupel AC, Kilpert F, Krötschel M, Arnold SJ, Sellner L, Colomer D, Stilgenbauer S, Dietrich S, Lichter P, Izcue A, Seiffert M
(See online at https://doi.org/10.1038/s41375-021-01136-1) - Intrinsic Resistance of Chronic Lymphocytic Leukemia Cells to NK Cell-Mediated Lysis Can Be Overcome In Vitro by Pharmacological Inhibition of Cdc42-Induced Actin Cytoskeleton Remodeling. Front Immunol, 12:619069, 2021
Wurzer H, Filali L, Hoffmann C, Krecke M, Biolato AM, Mastio J, De Wilde S, François JH, Largeot A, Berchem G, Paggetti J, Moussay E, Thomas C
(See online at https://doi.org/10.3389/fimmu.2021.619069)
