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Background  Single rare cell characterization represents a new scientific front in personalized therapy. Imaging mass cytometry (IMC) may be able to address all these questions by combining the power of MS-CyTOF and microscopy.    Methods  We have investigated this IMC method using < 100 to up to 1000 cells from human sarcoma tumor cell lines by incorporating bioinformatics-based t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis of highly multiplexed IMC imaging data. We tested this process on osteosarcoma cell lines TC71, OHS as well as osteosarcoma patient-derived xenograft (PDX) cell lines M31, M36, and M60. We also validated our analysis using sarcoma patient-derived CTCs.    Results  We successfully identified heterogeneity within individual tumor cell lines, the same PDX cells, and the CTCs from the same patient by detecting multiple protein targets and protein localization. Overall, these data reveal that our t-SNE-based approach can not only identify rare cells within the same cell line or cell population, but also discriminate amongst varied groups to detect similarities and differences.    Conclusions  This method helps us make greater inroads towards generating patient-specific CTC fingerprinting that could provide an accurate tumor status from a minimally-invasive liquid biopsy.

Rare osteosarcoma cell subpopulation protein array and profiling using imaging mass cytometry and bioinformatics analysis