Fig. 5.

Limiting dilution cell transplantation confirms that the FN-RMS mesenchymal-enriched subfraction has tumor propagating potential in vivo. a. Representative images of NSG mice engrafted with CD44+/CD90+ mesenchymal-enriched or CD44−/CD90− MAST139 PDX RMS cells (100 and 1,000 cells/mouse). Mice imaged at specific days post-transplantation as noted. Dashed lines delineate tumor. Representative image from n=3 independently engrafted mice shown for each dilution with similar results. b. Latency of tumor regrowth following engraftment into NSG mice. Day 0 is the day of initial engraftment. c. Barbell plot showing differences in the percentage of tumor propagating cells (TPCs) determined by limiting dilution cell transplant of FN-MAST139 and FN-MSK74711 (n=3 animals engrafted per log10 fold dilution). * p=0.02, ** p=0.007, *** p=0.0004. d. Flow analysis of tumors generated from sorted cell populations. Representative FACS plot with mean±SEM noted for analysis of three independently engrafted animals, *** p=0.0005. e. Histopathological analysis of tumors engrafted from RMS sorted cell subpopulations. Representative images of hematoxylin and eosin stain (H&E), immunohistochemistry analysis for Desmin, immunofluorescence for proliferation marker Ki67 (*** p=0.0006) and differentiated muscle markers TNNT3 (*** p=0.0005) and MF20 (*** p=0.001). Quantitation is mean±SEM from analysis of three independently engrafted animals (average obtained from four randomly imaged fields for each tumor). Statistics provided for Extreme Limiting Dilution Analysis ⁶⁰ (c) and two-sided Student’s t-test (d,e). Not significant (ns). Scale bar equal 50 microns (e).