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To investigate whether this self renewing subpopulation is more tumorigenic than the adherent population, we injected different numbers of RD cells and their corresponding sphere cultures (106, 105 and 104) intramuscularly (i.m).
To demonstrate that all xenograft tumors were indeed RMS tumors, we collected tumor samples and constructed a xenograft tissue microarray (TMA) with adherent RD cells and corresponding sphere cultures as controls.
In Figure 3A, a heat map of all samples is shown which revealed that RD and Rh36 cells cluster with their corresponding sphere cells indicating that both cell lines are more different from each other than their different passages.
Different amounts of adherent cells and their corresponding sphere cultures were injected intra muscularly into the right leg of NOD.CB17-Prkdcscid/J (NOD/Scid) and NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (The Jackson Laboratory) and tumor size was determined every 2 to 3 days by measuring two diameters (d1 and d2) in right angles of both legs with a calliper.
The CCS values of individual subunits were used to simulate the radius of the corresponding sphere model as described in the Methods section.
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Six spherical VOIs, having the same diameters of the spheres of NEMA phantom, were drawn on the corresponding spheres on PET/CT images.
In this phantom study, the reciprocal of body weight per injected dose was 9000 Bq/g so that background SUV was 1. Regarding calculation of SUVs, 10-, 17-, 17-, 28-, 28-, and 37-mm circular ROIs were drawn exactly on the corresponding spheres in the central slice by following the CT boundaries of the fused SPECT/CT images.
To characterize sphere cultures in further detail and to identify marker proteins specifically up- or downregulated, a gene expression profiling was performed with a human exonmicroarray (HuEx-1_0-st-v2) for both RD and Rh36 cells and three different passages (early, intermediate and late) of their corresponding spheres.
Open image in new window Figure 12 The preparation processes of the composite spheres and their corresponding hollow spheres (Wang et al. 2009b ).
Let be the corresponding unit sphere.
The material aspect of the experienced object as experienced, although arising in dependence upon the activity of the corresponding sense sphere, at the same time obstructs and limits it: a wall limits movement, night restricts vision, etc.
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