Figure 2. In vivo discovery of tumor-associated genes in TA-MEFs.

(A) BALB/c mice were subcutaneously injected with MEFs in combination with FB61 tumor cells. Injection of FB61 tumor cells or MEFs alone served as control. Tumor volumes were monitored over time as indicated. Mean ± SEM, ^**P< 0.01, two-way ANOVA, n = 6 per group; data are representative of three independent experiments. (B) Schema for the functional development of TA-MEFs from shotgun transduced MEFs (retro-MEFs) via primary tumor passage. (C) BALB/c mice were subcutaneously injected with MEFs, retro-MEFs or TA-MEFs in combination with FB61 tumor cells. Injection of FB61 tumor cells or larger number of TA-MEFs (5×10⁶) alone served as control. Tumor volumes were monitored over time as indicated. Mean ± SEM, ^**P< 0.01, two-way ANOVA, n=6 per group; data are representative of three independent experiments. (D) FB61 cells were mixed with TA-MEFs at different ratios as indicated before co-injection into BALB/c mice. Tumor volumes were monitored over time as indicated. Mean ± SEM, ^*P< 0.05, ^**P< 0.01, two-way ANOVA, n=5 per group; data are representative of two independent experiments. (E) TA-MEFs isolated from tumors established by co-injection of retro-MEFs and FB61 tumor cells were stained for FSP-1, α-SMA/vimentin or ER-TR7 and assessed by confocal microscopy. Nuclei were counterstained with DAPI. Scale bars, 20 μm; data are representative of two independent experiments. (F, G) LAM-PCR was performed for retro-MEFs and TA-MEFs isolated from FB61 (F) and TS/A (G) tumors. TA-1 and TA-2 were two different batches of TA-MEFs isolated from two different tumors. Data are representative of three independent experiments.