Abstract
Abstract Immunotherapeutic strategies are increasingly important in neuro-oncology, and the elucidation of escape mechanisms which lead to treatment resistance is crucial. Understanding heterogeneous clonal dynamics and emergence of immune escape variants during cancer immunoediting can yield valuable insights into the adaptive processes governing treatment resistance. We applied a recently developed multicolor labeling strategy termed optical barcoding to compare in vivo clonality in an immunocompetent vs immunodeficient environment. Optically barcoded GL261 glioma cells were implanted in parallel into the brains of i) wildtype (wt) C57BL/6 mice, ii) immunodeficient Pfp-/- Rag2-/- mice or iii) PD1-/- mice and tumor growth was monitored. Ex vivo analysis of individual tumor clones revealed that tumors in immunocompetent mice had a profoundly different clonal architecture compared to immunodeficient mice. Whereas tumors in pfp-/- rag2-/- mice exhibited a highly polyclonal composition, immunoedited tumors in wt and PD1-/- mice showed reduced clonality with emergence of immune escape clones, independent of in vitro proliferation rates. Survival of wt mice was significantly longer than of Pfp-/- Rag2-/- mice and was even more prolonged in PD1-/- mice. Immunoediting in immunocompetent mice was further associated with striking differences in vascularity and invasiveness. Time-course microarray analyses, flow-cytometric phenotyping of tumor-infiltrating immune cells and immunostaining highlighted the role of PD-L1 and other immunosuppressive mediators as mechanisms to adapt to T-cell infiltration and IFN-ɣ production. Furthermore, RNAseq profiling of FACS-separated tumor cells, T cells and macrophages/microglia allowed the identification of IFN-ɣ-mediated T-cell-shaped gene expression signatures and of new pathways potentially involved in the immune escape of gliomas. To conclude, the process of cancer immunoediting during tumor evolution not only shapes gene expression profiles and growth patterns, but also has a profound impact on the intratumoral clonal heterogeneity in malignant gliomas.
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