- Title
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Vangl2/RhoA Signaling Pathway Regulates Stem Cell Self-Renewal Programs and Growth in Rhabdomyosarcoma
- Authors
- Hayes, M.N., McCarthy, K., Jin, A., Oliveira, M.L., Iyer, S., Garcia, S.P., Sindiri, S., Gryder, B., Motala, Z., Nielsen, G.P., Borg, J.P., van de Rijn, M., Malkin, D., Khan, J., Ignatius, M.S., Langenau, D.M.
- Source
- Full text @ Cell Stem Cell
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Related to Figure 5. Transgenic expression of vangl2 does not affect tumor latency, incidence, or size in the primary zebrafish ERMS. (A-D) Zebrafish ERMS generated by injection of rag2:kRASG12D (A,B) or rag2:kRASG12D + rag2:vangl2 (C,D). rag2:GFP was co-injected to fluorescently label tumor cells. Representative images of tumor histology (B,D). (E) Tumor incidence assessed over time (n=175 animals for kRASG12D and n=214 animals for kRASG12D+Vangl2, p=0.57 log-rank statistic). (F) Tumor size measured at 30 days based on GFP intensity multiplied by 2D pixel area. Each datum point represents tumor size in a single fish. Not significant by Student’s t-test calculation (n.s.). |
Related to Figure 6. Vangl2 transcripts are highly expressed in zebrafish TPCs, sort purity for experiments shown in Figure 6 and establishing long-term selfrenewal potential of myf5:GFP+/vangl2:mCherry+ TPCs. (A) Animal shown following engraftment of bulk myf5:GFP/mylz2:mCherry tumor cells. (B) FACS plot of isolated myf5:GFP+/mylz2:mCherry- (green), myf5:GFP+/mylz2:mCherry+ (yellow), myf5:GFP- /mylz2:mCherry+ (red) and myf5:GFP-/mylz2:mCherry- (black) ERMS cells. Percentage of cells in each gated sub-population are noted. (C) Quantitative RT-PCR analysis for vangl2 expression in sorted myf5:GFP+/mylz2:mCherry- (+/-, green), myf5:GFP+/mylz2:mCherry+ (+/+, yellow), myf5:GFP-/mylz2:mCherry+ (-/+, red) and myf5:GFP-/mylz2:mCherry- (-/-, grey) cells. Sort purity was >85% and >95% cell viability. ***p<0.001, Student’s t-test. Error bars represent standard deviation. Graph is representative of n=2 independent ERMS. (D) FACS plots showing sort purity for tumor cells engrafted into recipient animals depicted in Figure 6FJ. (E-H) Establishing long-term self-renewal potential of myf5:GFP+/vangl2:mCherry+ TPCs. Sort purity of cells isolated from fish engrafted with myf5:GFP+/vangl2:mCherry+ TPCs (E) and then engrafted into recipient fish F-H. Whole animal fluorescent image (F), histology (G), and quantification of engraftment at varying cell doses (H). myf5:GFP+/vangl2:mCherry+ (G+R+, yellow), myf5:GFP+/vangl2:mCherry- (G+, green) cells. (I) Relative expression of fgfr1a, fgfr3 and cd133/prominin1a (prom1a) in sorted myf5:GFP+/vangl2:mCherry- (G+), myf5:GFP+/vangl2:mCherry+ (G+R+), myf5:GFP-/vangl2:mCherry+ (R+), myf5:GFP- /vangl2:mCherry- (NEG) cells and compared with whole zebrafish embryos at 6 and 24 hours post fertilization (hpf). prom1a is not detected in zebrafish ERMS. Graph is representative of n=2 independent ERMS. |
Related to Figure 6. vangl2:mCherry labels self-renewing TPCs in zebrafish ERMS. (A,B) Transgenic zebrafish with primary ERMS expressing vangl2(-5kb):mCherry along with rag2:GFP + rag2:kRASG12D. (C) Whole animal and (D) histology of syngeneic zebrafish engrafted with fluorescently-labeled ERMS imaged at 30 days post transplantation (1x105 unsorted tumor cells/animal). (E) FACS of rag2:GFP/vangl2:mCherry ERMS cells isolated from the transplant fish shown in C. (F) Sort purity of cells following FACS. (G-K) Sorted cell fractions were introduced into recipient fish at limiting dilution. Whole animal (G), histology (H) and FACS analysis (I) of fish engrafted with rag2:GFP+/vangl2:mCherry+ sorted cells. Sort purity of engrafted cells shown in lower left panel G. (J) Quantification of engraftment into syngeneic recipient animals (combined results from analysis of three independent ERMS, n=118 recipient fish total). rag2:GFP+/vangl2:mCherry+ (G+R+, yellow), rag2:GFP+/vangl2:mCherry- (G+, green) and rag2:GFP-/vangl2:mCherry+ (R+, red) tumor cells. (K) Graphical analysis of Extreme Limiting Dilution Analysis (ELDA) denoting overall engraftment rates at various cell dilutions. Dotted lines represent 95% confidence intervals. Estimated tumor propagating cell number for each cell fraction is indicated. ***p<0.001. (L) Sort purity of fluorescent-labeled ERMS cells isolated from tertiary recipient fish following two rounds of FACS. (M) Fluorescent image of fish engrafted with rag2:GFP+/vangl2:mCherry+ ERMS cells, establishing long-term and serial repopulating ability of Vangl2+ TPCs. Sort purity of engrafted cells is indicated in lower left panel M (viability >96%). (N) Histology of fish shown in M. (O) Quantification of tumor engraftment by 90 days post transplantation with sorted ERMS cells, rag2:GFP+/vangl2:mCherry+ (G+R+, yellow) and rag2:GFP+/vangl2:mCherry- (G+, green) cells. |