Cellular Oncology: Single-cell RNA profiling identifies diverse cellular responses to EWSR1/FLI1 down regulation in Ewing sarcoma cells (Houghton, Chen, Kitagawa, Ignatius)

Roxane Khoogar,
Fuyang Li,
Yidong Chen,
Myron Ignatius,
Elizabeth R. Lawlor,
Katsumi Kitagawa,
Tim H.-M. Huang,
Doris A. Phelps &
Peter J. Houghton

Background

The EWSR1/FLI1 gene fusion is the most common rearrangement leading to cell transformation in Ewing sarcoma (ES). Previous studies have indicated that expression at the cellular level is heterogeneous and that levels of expression may oscillate, conferring different cellular characteristics. In ES the role of EWSR1/FLI1 in regulating subpopulation dynamics is currently unknown.

Methods

We used siRNA to transiently suppress EWSR1/FLI1 expression and followed population dynamics using both single-cell expression profiling, CyTOF, and functional assays to define characteristics of exponentially growing ES cells and of ES cells in which EWSR1/FLI1 had been downregulated. Novel transcriptional states with distinct features were assigned using random forest feature selection in combination with machine learning. Cells isolated from ES xenografts in immune-deficient mice were interrogated to determine whether characteristics of specific subpopulations of cells in vitro could be identified. Stem-like characteristics were assessed by the primary and secondary spheroid formation in vitro, and invasion/motility was determined for each identified subpopulation. Autophagy was determined by expression profiling, cell sorting, and immunohistochemical staining.

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