Immune microenvironment of experimental rat C6 gliomas resembles human glioblastomas

www.nature.com/scientificreports OPEN Received: 7 August 2017 Accepted: 22 November 2017 Published: xx xx xxxx Immune microenvironment of experimental rat C6 gliomas resembles human glioblastomas Anna Gieryng1, Dominika Pszczolkowska1, Katarzyna Bocian1, Michal Dabrowski2, Wenson David Rajan1, Michal Kloss1, Jakub Mieczkowski1 & Bozena Kaminska 1 Glioblastoma (GBM) is the most aggressive primary brain tumor, with ineffective anti-tumor responses and a poor prognosis despite aggressive treatments. GBM immune microenvironment is heterogenous and activation of specific immune populations in GBM is not fully characterized. Reliable animal models are critical for defining mechanisms of anti-tumor immunity. First we analyzed the immune subpopulations present in rat C6 gliomas. Using flow cytometry we determined kinetics of infiltration of myeloid cells and T lymphocytes into glioma-bearing brains. We found significant increases of the amoeboid, pro-tumorigenic microglia/macrophages, T helper (Th) and T regulatory (Treg) cells in tumorbearing brains, and rare infiltrating T cytotoxic (Tc) cells. Transcriptomic analyses of glioma-bearing hemispheres revealed overexpression of invasion and immunosuppression-related genes, reflecting the immunosuppressive microenvironment. Microglia, sorted as CD11b+CD45low cells from gliomas, displayed the pro-invasive and immunosuppressive type of activation. Accumulation of Th and Treg cells combined with the reduced presence of Tc lymphocytes in rat gliomas may result in the lack of effective anti–tumor responses. Transcriptional profiles of CD11b+ cells and composition of immune infiltrates in C6 gliomas indicate that rat C6 gliomas employ similar immune system evasion strategies as human GBMs. Tumor cells and their products are capable of regulating and altering gene expression in non-tumor cells within or infiltrating into the microenvironment, thereby shaping their phenotype. Although various immune effector cells are recruited to the tumor site, their anti-tumor functions are down-regulated, largely in response to tumor-derived signals. Numerous immunosuppressive networks operate in malignant tumors1–4. Reliable animal models are critical for defining tumor immunity mechanisms. Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Due to frequent dysfunctions of tumor suppressors and oncogenes, diffusive growth and resistance to standard therapies, the median survival is approximately 14 months and the treatment remains mainly palliative5–7. Histopathological and flow cytometry analyses of human gliomas revealed infiltration of GBMs with numerous immune cells: resident (microglia) and peripheral macrophages (collectively called glioma-associated microglia/macrophages, GAMs), granulocytes, myeloid-derived suppressor cells (MDSCs) and T lymphocytes (for reviews1,8,9). Intratumoral densities of glioma-associated microglia/macrophages (GAMs) and MDSCs were correlated with the histological grade of gliomas, immunosuppression and patient’s survival10–12. A recent pan-cancer analysis using mRNA sequencing data from The Cancer Genome Atlas for 11 tumor types representing 3485 tumors revealed that macrophage signatures predicted worse survival in GBM13. Although GAMs have a few innate immune functions intact, this is not sufficient to initiate effective immune responses14,15. Moreover, GAMs support tumor invasion16–19 and release immunosuppressive cytokines and chemokines. MDSCs inhibit cytotoxic responses mediated by natural killer cells, and block the activation of tumor-reactive CD4 + T helper (CD4 + Th) cells and CD8 + T cytotoxic (CD8 + Tc) cells12,20. The presence of regulatory T cells may also contribute to the lack of effective immune responses against malignant gliomas21. Tissue macrophages, depending on a stimulus, activate inflammatory responses or cytoprotective, immunosuppressive responses typical for macrophages involved in tissue repair and inflammation resolution22. Studies 1 Laboratory of Molecular Neurobiology, Neurobiology Center, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warszawa, Poland. 2Laboratory of Bioinformatics, Neurobiology Center, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Warszawa, Poland. Correspondence and requests for materials should be addressed to B.K. (email: ) SCIENtIfIC REPOrts | 7: 17556 | DOI:10.1038/s41598-017-17752-w 1 www.nature.com/scientificreports/ attempting to dissect a functional phenotype of human GAMs demonstrated conflicting results indicating either a non-inflammatory, pro-tumorigenic phenotype10, a mixed inflammatory/anti-tumor phenotype23 or M0 phenotype24. Among leukocytes, mostly CD4+ Th, CD4+CD25+FoxP3+ T regulatory (Treg) and CD8+ Tc subsets were detected in GBMs20,21,25–27. Accumulation of CD4+Th and CD4+Treg subsets in GBMs was correlated with the worst prognosis28. Increased expression TGF-β (transforming growth factor β) and IL-10 (interleukin-10) in GBMs may result in inhibition of Tc activation/proliferation and differentiation of naïve T cells into Treg25. Animal models are still instrumental for providing ‘proof of concept” and appropriate rodent glioma models that recapitulate innate and adaptive immune responses occurring in human GBMs would be desirable. Rat C6, 9 L and T9 gliomas were induced by repeated injections of methylnitrosourea to pregnant rats. C6 glioma cells which typically grow in an outbred, not syngeneic Wistar rat and have potential to evoke an alloimmune response29, are commonly used as a glioma model in pharmacological studies. In vivo magnetic resonance imaging and angiography studies showed that similarity of C6 gliomas to human malignant gliomas is better than other models30. In the present study, we analyzed immune heterogeneity in rat C6 gliomas and kinetics of immune cell infiltration of GAMs, MDSCs and T cell subpopulations. We found overexpression of invasion and immunosuppression-related genes in glioma-bearing hemispheres and in tumor infiltrating microglia. Characteristics of immune infiltrates, gene expression profiles and well known histopathological similarities of C6 gliomas to human GBMs, led to conclusion that the rat C6 gliomas employ similar immune evasion strategies as human mesenchymal GBMs. Material and Methods Animals. Male Wistar rats (2–3 months old, body weigh 250–300 g at the beginning of the study) were housed with free access to food and water, on a 12 h/12 h day and night cycle. All efforts have been made to minimize the number of animals and animal suffering. All research protocols conformed to the Guidelines for the Care and Use of Laboratory Animals (European and national regulations 2010/63/UE September 22, 2010 and Dz. Urz. UE L 276/20.10.2010, respectively). Animals were decapitated by a qualified researcher. The First Warsaw Local Ethics Committee for Animal Experimentation approved the study (the protocol 262/2012 from 15/03/2012). C (...truncated)