Figure 1.
Representative tumorsphere and attached phenotypes of cultures derived from primary retinoblastoma tumor cells.
Biopsies from human Rb tumor cells were placed in culture medium. Cultures arose either as three-dimensional spherical aggregates of cells called tumorspheres or as an attached monolayer. Typically, tumorspheres arose when Rb cells were placed in neural stem cell-optimized medium without serum, and adherent cultures arose when Rb cells were placed in classical medium with FBS. However, in rare cases tumorspheres and attached cells both arose from Rb cells placed in classical medium with FBS. Scale bar = 100 µm.
Table 1.
RB1 genotypes of primary Rb-derived cultures.
Figure 2.
Expression of pRb and synaptophysin in primary retinoblastoma tumor-derived cultures.
Stains for pRb and synaptophysin of primary Rb tumor-derived cultures were compared to the expression pattern of the original tumor. A. In pRb-negative Rb tumors, the tumor-derived tumorspheres were negative for pRb expression, yet almost all attached cells express pRb (brown). B. Tumorspheres diffusely express high levels of synaptophysin similar to the original tumor (brown), while all adherent cultures are negative for synaptophysin expression except one (Table 2). In one adherent culture, <25% of cells were synaptophysin-positive. Scale bar = 50 µm. C. Total area of the section staining for synaptophysin was quantified for the tumor-derived cultures (n = 8), and tumorsphere cultures were compared to adherent cultures. Synaptophysin expression was seen in all tumorsphere cultures, and rarely in adherent cultures. Results shown as mean ± SD. *** = p<0.001.
Table 2.
Expression of tumor and stromal cell markers in Rb-derived cultures.
Figure 3.
Expression of stromal cell and retinal cell markers in primary retinoblastoma tumor-derived cultures.
Expression of MAP2 (n = 5), GFAP (n = 8), cytokeratin (n = 9), and CD34 (n = 5), which identify photoreceptors/ganglion cells, glial cells, retinal pigment epithelial cells and vascular endothelial cells, respectively, was determined in tumor-derived cultures and compared to the original tumor. A. Representative staining shows the typical result for each marker (brown). While MAP2 labeled 100% of cells in all tumorsphere cultures, no MAP2-positive cells were seen in all adherent cultures except one (Table 2). In one adherent culture, <25% of cells were MAP2-positive. GFAP, cytokeratin and CD34 were not seen in tumorsphere cultures. Very rare, isolated GFAP-positive cells were seen in only 2 of 5 adherent cultures. Cytokeratin-positive cells were seen in all adherent cultures. A significant CD34-positive subpopulation was seen in 3 of 5 cultures. Scale bar = 50 µm. B. Representative sections showing the staining intensity grading (from Table 2) based on approximate number of cells stained. C. Quantification of grading of adherent cultures shows higher levels of cytokeratin-positive cells than CD34-positive cells. Results are shown as median ± interquartile range. CK = cytokeratin. ** = p<0.01. *** = p<0.001.