P06.03 Optical barcoding reveals immunoediting of the clonal tumor architecture in a syngeneic glioma model

Since tumors are subject to constant selective pressure by tumor-specific immune cells, cancer immunoediting plays a critical role throughout all stages of tumor evolution. Therefore, investigating the actual impact of the immune system on tumor heterogeneity is crucial to understand the mechanisms of immune escape and tumor recurrence during chemo- and immunotherapy. We compared the in-vivo clonality, as well as cellular and molecular phenotypes of syngeneic mouse gliomas grown in an immunocompetent versus immunodeficient environment.To quantify the impact of the immune system on tumor cell clonality we developed a new clonal labeling strategy, termed optical barcoding. This lentiviral multicolor labeling strategy allows to track individual clones and, in combination with ex-vivo flow cytometry, to quantify the clonal architecture of individual tumors. Optically barcoded mouse GL261 glioma cells were administered through stereotactic intracranial injection in wild-type and immunodeficient pfp -/- rag2 -/- C57BL/6 mice. Clonal composition, survival, tumor growth pattern, vascularization and phenotype of tumor-infiltrating immune cells were analyzed by immunohistochemistry. Gene expression profiling of ex-vivo isolated tumors was performed using RNA sequencing. Phenotype and cytokine production of infiltrating lymphocytes were assessed using multicolor flow cytometry and correlated to PD-L1 expression over a time-course during tumor development. Ex-vivo flow-cytometric analysis of individually labelled tumor clones demonstrated that brain tumors established in immunocompetent C57BL/6 showed a different clonal architecture compared to those in pfp -/- rag2 -/- C57BL/6 mice. Whereas pfp -/- rag2 -/- C57BL/6 mice displayed a homogeneous clonal distribution in all tumors, wildtype mice showed a more heterogeneous clonal mixture with greater variation in clonal composition. As expected, survival of immunodeficient mice was significantly shorter compared to wildtype mice. Interestingly, significant differences in macroscopic growth patterns, defined by tumor expansion morphology, vascularity and invasion were evident. Immunohistochemical stainings and phenotyping of tumor-infiltrating immune cells highlighted a role for PD-L1 in response to interferon-ɣ expression of infiltrating T lymphocytes. Furthermore, RNASeq of GL261 tumors grown in wt vs pfp -/- rag2 -/- mice allowed the identification of new pathways potentially involved in the process of cancer-immune ce