gms | German Medical Science

Objective: Given the increasing importance of immunotherapeutic treatment strategies in neuro-oncology, the investigation of immune escape mechanisms, which lead to treatment resistance, is crucial. Therefore, studying tumor heterogeneity to understand the clonal dynamics and emergence of immune escape variants during cancer immunoediting can yield valuable insights into the adaptive processes of treatment resistance.

Methods: We applied a novel multicolor labeling strategy, termed optical barcoding, which makes use of flow-cytometric read-out to compare in-vivo clonality in an immunocompetent versus -deficient environment. The optically barcoded syngeneic GL261 glioma was administered to wildtype and immunodeficient C57BL/6 pfp-/- rag2-/- mice, and tumor growth was monitored. We also analyzed the phenotype of tumor-infiltrating lymphocytes and the gene-expression signatures of explanted tumors using RNAseq.

Results: Ex-vivo analysis of individual tumor clones demonstrated that tumors grown in immunocompetent C57BL/6 mice were composed of a significantly different clonal architecture compared to pfp-/- rag2-/- C57BL/6 (p=0.01). Whereas pfp-/- rag2-/- mice displayed a homogeneous clonal distribution in all tumors, immunoedited tumors in wildtype mice exhibited reduced clonality with emergence of immune escape clones, independent of proliferation rates. Beyond the expected prolonged survival of wildtype mice, immunoediting was further associated with differences in macroscopic growth patterns, vascularity and invasiveness. Tissue staining, time-course microarray analyses and flow-cytometric phenotyping of tumor-infiltrating immune cells highlighted the exemplary role of PD-L1 as mechanism to adapt to interferon-ɣ and infiltration of T-cells. Furthermore, RNAseq profiling of GL261 tumors grown in wt vs immunodeficient mice allowed the identification of an interferon-ɣ-mediated T-cell-shaped gene expression signature and new pathways potentially involved in the immune escape of glioma.

Conclusion: Taken together, using optical barcoding we have demonstrated that the process of cancer immunoediting during tumor evolution not only shapes the gene expression profile and growth pattern, but also has a profound impact on the intratumoral heterogeneity of glioma in the syngeneic GL261 model.