Abstract 4552: BMX-Seq as a new resource for deciphering the transcriptomic hallmarks of tumor plasticity and stromal interactions in brain metastasis
Authors: Wingrove Emily , Liu Zongzhi Z. , Nguyen Don X. , Arnal-Estape Anna , Patel Kiran D. , Melnick Mary-Ann , Politi Katerina , Valiente Manuel , Kluger Harriet M. , Chiang Veronica L. 
Published: August 2019

Purpose: Brain metastasis remains a major site of relapse for several cancer types. However, the molecular mechanisms of brain metastasis are largely unknown given the complex relationship between tumor cells and the surrounding brain tumor microenvironment (TME). To better characterize the tumor-stromal relationship in the context of brain metastasis, we have developed a Brain Metastatic RNA-Sequencing (BMX-Seq) approach, which leverages xenograft models and can distinguish between the transcriptomes of human tumor and mouse stroma gene expression in vivo. This resource provides an extensive molecular portrait of the co-adaptation of brain metastasis with the brain TME.

Methods: Following intra-arterial injection of the human, lung adenocarcinoma (LUAD), H2030-BrM3 cell model into athymic mice, metastatic brain tumors were macrodisasected, flash frozen and RNA extracted. Healthy brain regions, subcutaneous tumors and H2030-BrM3 cells grown in monolayer were harvested in parallel. Our BMX-Seq pipeline was engineered to analyze bulk xenograft tissue, which includes tumor lesions as well as surrounding stromal tissue. Species-specific Taqman primers and immunofluorescent (IF) staining were used to validate our BMX-Seq results. Models of melanoma and breast cancer brain metastasis were also included in this study.

Summary: In comparing the transcriptomic profiles of tumors grown in the brain as compared to other sites, we identify shifts in epithelial and neuronal-like gene expression programs in malignant cells and show such trends are reversible once tumor cells are removed from the brain TME. Genes involved in WNT signaling and cell projection were also upregulated across melanoma, lung and breast cancer types when cells were grown in the brain versus other sites. In describing the neuroinflammatory response of stromal regions directly surrounding brain lesions, our BMX-Seq analysis revealed increased expression of astrocyte and microglial enriched transcripts. We then utilized IF to confirm the elevated density of both of these cell types in areas directly surrounding and infiltrating tumor regions. We also detect significantly induced expression of stromal TIM3 in areas surrounding tumor lesions and in a model system devoid of T-Cells. Follow-up work revealed TIM3 is largely expressed and induced on tumor-associated macrophages (including microglia) within the tumor region, and we further validate such results in a syngeneic model system as well as in human, brain tissue.

Conclusions: We have developed a highly sensitive RNA-sequencing based approach that can accurately map the transcriptomic adaptation of brain metastatic tumor cells and the surrounding brain TME. We believe these data may serve as an invaluable resource to guide future discovery in CNS metastases.

Citation Format: Emily Wingrove, Zongzhi Z. Liu, Don X. Nguyen, Anna Arnal-Estape, Kiran D. Patel, Mary-Ann Melnick, Katerina Politi, Manuel Valiente, Harriet M. Kluger, Veronica L. Chiang. BMX-Seq as a new resource for deciphering the transcriptomic hallmarks of tumor plasticity and stromal interactions in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4552.