Glioblastoma (GBM) is a deadly brain cancer that suppresses cellular immunity through the production of anti-inflammatory cytokines/catabolites, expression of immune checkpoint molecules, and induction of tolerogenic immune cell subsets. While preclinical models have shown each of these mechanisms to negatively impact survival outcome, it remains unclear how these distinct processes manifest themselves within the context of a coordinated immune system and whether additional immunosuppressive mechanisms exist. To address these questions, we have developed an extensible in vivo screening platform to holistically determine how GBM affects cellular immune organization. The method allows for temporal assessment of leukocyte population dynamics in response to tumor progression. Leukocytes derived from lymphoid tissues of tumor-bearing mice are isolated and immunolabeled with a 12-color optimized immunofluorescence panel (OMIP) targeting several major leukocyte lineages. Single-cell data is acquired by flow cytometry and is clustered in an unsupervised manner based on cell-surface protein expression. A vector-based classification system is then used to map phenotypically distinct clusters onto known immune cell lineages. The platform allows for the extraction of immune signatures whose divergence from tumor-naïve standards may be quantified and statistically analyzed; such signatures may also be correlated with response/resistance to therapy. The approach has been validated on orthotopic GL261 glioma in syngeneic C57BL6/J mice, yet it is readily amenable to the study of diverse model systems, including de novo tumors driven by clinically-relevant oncogenes. Alternative flow cytometry antibody panels may easily be substituted to probe more specific immune cell subsets of interest. Systems biology approaches applied to the field of tumor-induced immunosuppression hold promise in identifying novel network-level immune signatures, facilitating a better understanding of immunotherapeutic drug action, and driving next generation high-dimensional biomarker discovery.

Citation Format: Gregory J. Baker, P.S. Thiagarajan, Sucheendra K. Palaniappan, Stephanie H. Davis, Jodene K. Moore, Peter K. Sorger. A flow-based immunoprofiling strategy for interrogating system-wide leukocyte composition in response to glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1678. doi:10.1158/1538-7445.AM2017-1678