Figure 3. ΔF mutant attenuates myeloma cell growth in a cell-autonomous mode.

A subcutaneous plasmacytoma model, in which tumor cells were inoculated subcutaneously in flank of syngeneic naïve mice, was used to determine the role of the bone marrow microenvironment. A. GFP expression of 5TGM1-EV and 5TGM1-ΔF cells analyzed by flow cytometry immediately before inoculation in mice. A single peak for each cell type indicates relative homogeneity of GFP expression in either population. Consistent with this observation, median GFP expression of the gated M1 populations did not differ significantly from the median GFP for all cells. B. Mice were inoculated subcutaneously with 5TGM1-EV cells (EV, n = 5) or 5TGM1-ΔF cells (ΔF, n = 5). Representative mice are shown (left panel); whole-body optical images of tumor-emitting green fluorescence in anesthetized live mice inoculated with above cells taken post-tumor inoculation (right panel). Tumor growth in mice inoculated with 5TGM1-ΔF cells (bottom) was markedly inhibited compared with those inoculated with 5TGM1-EV cells (top). C. Quantitative analysis of tumor volume (days 14 and 21 post-tumor cell inoculation) and excised tumor wet weight (at the end of the experiment; day 23 post-tumor cell inoculation). Days 14, 21 and 23 refer to the number of days after tumor cell inoculation in the flank of mice. Expression of ΔF in 5TGM1 cells almost completely inhibited plasmacytoma growth in vivo. Data represent mean ± SEM; *, P < 0.05. D. Tumor tissue was harvested from 5TGM1-EV- and 5TGM1-ΔF-injected mice, disaggregated, sieved, stained with annexin V-phycoerythrin and 7-AAD, and analyzed by flow cytometry to quantify apoptotic cells (annexin V⁺, 7-AAD⁻; lower-right quadrant). Apoptotic cells in tumor tissue harvested from 5TGM1-ΔF-inoculated mice increased by 10-fold compared with 5TGM1-EV tumors.