Fig. 3: Bilateral, early-onset retinoblastomas accumulate CD45⁺ immune cells, unlike control or late-onset tumors which don’t.

A Retinae were harvested from P7, P15, P30 and P60 Vsx2-Cre⁺; Rb1^lox/lox; p107^−/−; Pten^lox/lox mice and stained by IHC for CD45⁺ cells. B Fifteen-day old control and Vsx2-Cre⁺; Rb1^lox/lox; p107^−/−; Pten^lox/lox tumor samples were isolated, trypsinized to single cells, processed for flow cytometry to quantify CD45⁺ cell numbers, and graphed as percent of total cells. C Cells (P15) from 3B were further analyzed for GFP by flow cytometry to detect Cre:GFP⁺/CD45⁺ cells native to retina, or Cre:GFP⁻/CD45⁺ cells external to the retina, and graphed as the percent of total cells. D Retinae were harvested from P15 control, Vsx2-Cre⁺; Rb1^lox/lox; p107^−/− and Vsx2-Cre⁺; Rb1^lox/lox; p107^−/−; Pten^lox/lox and stained by H&E, or stained by IHC to detect apoptotic cells by TUNEL, proliferation by Ki67, and CD45⁺ immune cells. E The numbers of CD45⁺ cells detected by IHC were counted and graphed as the % CD45⁺ cells compared to total. *p < 0.05; **p < 0.01; ***p < 0.001.