Figure 2. Eµ-Myc/c-rel−/− tumours grow equally well in wild type and c-rel−/− mice and are more sensitive to apoptotic stimuli.
(A & B) Reimplanted Eµ-Myc/c-rel−/− tumours grow equally well in wild type and c-rel−/− mice. Lymph node tumours derived from 3 different Eµ-Myc/c-rel−/− mice were re-implanted in parallel into either 3 wild type (C57Bl/6) or 3 c-rel−/− host mice. Four weeks after implantation, the mice were sacrificed and tumour sizes at different sites were assessed. Shown here is representative data from the spleen (A) and cervical lymph nodes (B) Data representing mean ± SEM and p values were calculated using Student’s unpaired t-tests.
(C) Tumour burden in lymphoid organs (weight of organ/bodyweight of animal (g)) following reimplantation of either Eµ-Myc or Eµ-Myc crel−/− tumours cells into either C57Bl/6 or crel−/− mice. Data shown is the mean of 3 independent tumours each implanted into 3 mice ± SEM. * indicates p<0.05 in an unpaired Student’s t-test but otherwise there were no significant differences between tumour burden in WT or c-rel ko animals at any of the sites assessed.
(D) Cell viability of Eµ-Myc and Eµ-Myc/c-rel−/− tumour cells grown ex vivo. Cell viability was measured using the trypan blue exclusion assay over a 4-hour period after freeze-thawing.
(E) Eµ-Myc/c-rel−/− tumour cells are more sensitive to apoptotic stimuli. Freshly isolated Eµ-Myc or Eµ-Myc/c-rel−/− lymph node tumour cells (5×105 per well) were seeded into 96-well plates. Increasing concentrations of the chemotherapeutic agents, doxorubicin (Sigma) or vincristine (Sigma) or solvent controls were added to three replicate wells. After 96 hours, viability was quantified using the CellTiter96© AQueous One Solution Cell Proliferation Assay (MTS) (Promega), according to manufacturer’s instructions.
Single cell suspensions were prepared from tumour-bearing organs of Eµ-Myc and Eµ-Myc/crel−/− mice upon necropsy. These were then used for downstream analyses or frozen in 90% FBS/10% DMSO for long-term storage and transplantation. For reciprocal microenvironment, 2×106 Eµ-Myc/c-rel−/− lymph node tumour cells from male mice were transplanted intra-venously via the lateral tail vein into 8-week old male C57BL/6 or c-rel−/− recipients. Mice were necropsied when they became moribund and the tumour burden assessed. C57BL/6 mice used for reimplantation studies were purchased from Charles River, UK.