Fig. 6

Zebrafish embryos facilitate rapid detection of an aggressive stem-like subpopulation of OSCC cells and of the effects of ICG-001 inhibition. a HSC-3 and CAL27 cells were transduced with a CellTracker™, and 100–200 cells in ~ 1 nL were injected directly into the periviteline space of the embryo. After 24 h, CAL27 cells formed tumors that did not metastasize, whereas HSC-3 cells produced larger tumors that metastasized to the tail and to the craniofacial region. b Immunofluorescence imaging of CellTracker™-labeled CAL27 and HSC-3 cells revealed that treatment with ICG-001 inhibited CAL27 cell-driven tumor growth, as well as growth and metastasis of HSC-3-derived tumors. c Percent of injected fish exhibiting metastasis was quantified and displayed as the mean (± S.D.) per cell type and per treatment group. Majority of vehicle-treated HSC-3-driven tumors (n = 86) metastasized relative to vehicle-treated CAL27-driven tumors (n = 68). ICG-001 treatment prior to injection resulted in a significant reduction in percent fish displaying HSC-3-driven metastasis (n = 104). d Identification of subpopulations of RFP-transduced HSC-3 cells with primitive antigenic cell markers CD44⁺CD24^highCD29⁺ by flow cytometry. e Subpopulations of CD44⁺CD24^highCD29⁺ and CD44⁺CD24^lowCD29⁺ cells were isolated by FACS, grown in culture for 12 h followed by treatment with either DMSO (vehicle) control or ICG-001 for 24 h. Unsorted HSC-3 cells were used as control. Immunofluorescence imaging of tumor xenografts derived from unsorted RFP HSC-3 cells (n = 3), RFP CD44⁺CD24^lowCD29⁺ cells (n = 3), or CD44⁺CD24^highCD29⁺ cells (n = 3) revealed that only CD44⁺CD24^highCD29⁺ cells formed tumors that metastasized rapidly within 2 h to the tail and craniofacial regions (left panel). After 24 h, metastases were detected from tumors derived from CD44⁺CD24^highCD29⁺ (n = 5) as well as unsorted cells (n = 6), and to a much lesser extent, from CD44⁺CD24^lowCD29⁺ cells (n = 6; middle panel). We note that a substantial population of zebrafish injected with CD44⁺CD24^highCD29⁺ cells died within the 24 h (middle panel). Treatment of CD44⁺CD24^highCD29⁺ cells (n = 11) as well as unsorted cells (n = 6) with ICG-001 inhibited tumor growth and significantly abrogated metastases (right panel). In each case, metastasis was quantified as number of metastasis per fish (see “Methods”), and displayed as the mean (± S.D.). *P < 0.01; **P < 0.001; ***P < 0.0001; unpaired t test