Figure 2
In vivo growth of MM requires EXT1. L363-TetR or L363-shEXT1a MM cells, transduced to express a GFP-luciferase fusion protein, were injected intracardially into Rag-2−/−γc−/− mice. Subsequently, the mice were divided into 2 (L363-TetR) or 3 (L363-shEXT1a) groups; the first group did not receive doxycycline (no dox), the second group was treated with doxycycline throughout the entire experiment (dox day 1), and the third group (L363-shEXT1a only) received doxycycline after 6 weeks (dox week 6). Bones were collected after 9 weeks, fixed in normal buffered formalin, decalcified, and embedded in paraffin. (A) Bioluminescence images of the ventral side of mice injected with L363-shEXT1a MM cells, taken at week 3 (w3), w6, w7, and w9. Representative mice are shown for each group. (B) Tumor growth in mice injected with L363-TetR or L363-shEXT1a MM cells, as determined by the average photon emission intensity (arbitrary units) measured for the total body. Data are mean ± SD (n = 5 per group). A Mann-Whitney test (2-tailed) was applied to determine significance: *P < .05; **P ≤ .01; ***P ≤ .001. (C) Hematoxylin-stained femur sections of representative mice. Within the femurs of the no dox and dox week 6 mice, myeloma tumors (indicated with asterisks) were found. Bars represent 500 μm and 100 μm for top and bottom panels, respectively. #Necrotic MM cells. *MM cells. (D) To visualize the tumors and their proliferation, anti–human syndecan-1 (clone MI-15, Dako Denmark) and anti–human Ki67 (M7240, Dako Denmark) immunostainings were performed. The no dox and dox week 6 mice had tumors with approximately 87% and approximately 34% Ki67+ MM cells, respectively (data not shown). In the dox week 6 mice, areas of necrosis with weak and diffuse syndecan-1 and Ki67 staining were found. Representative areas are shown. Bars represents 50 μm. (Insets) Isotype controls. IC micrographs were obtained with an Olympus BX51 light microscope, using a 10×/0.30 Plan FI, 20×/0.50 Plan FI, or 40×/0.85 PlanApo objective, connected to an Olympus DP70 camera. Images were captured with Olympus DPController software (Version 1.2.1.108) and further processed with Adobe Photoshop (Version 7.0). (E) Cell-surface HS expression of GFP+ L363-shEXT1a MM cells harvested from the bones of no dox and dox week 6 mice, as determined by FACS. The mean fluorescence intensity (MFI) of the untreated mice was normalized to 100%. N.D. indicates not determined (no tumor).

In vivo growth of MM requires EXT1. L363-TetR or L363-shEXT1a MM cells, transduced to express a GFP-luciferase fusion protein, were injected intracardially into Rag-2−/−γc−/− mice. Subsequently, the mice were divided into 2 (L363-TetR) or 3 (L363-shEXT1a) groups; the first group did not receive doxycycline (no dox), the second group was treated with doxycycline throughout the entire experiment (dox day 1), and the third group (L363-shEXT1a only) received doxycycline after 6 weeks (dox week 6). Bones were collected after 9 weeks, fixed in normal buffered formalin, decalcified, and embedded in paraffin. (A) Bioluminescence images of the ventral side of mice injected with L363-shEXT1a MM cells, taken at week 3 (w3), w6, w7, and w9. Representative mice are shown for each group. (B) Tumor growth in mice injected with L363-TetR or L363-shEXT1a MM cells, as determined by the average photon emission intensity (arbitrary units) measured for the total body. Data are mean ± SD (n = 5 per group). A Mann-Whitney test (2-tailed) was applied to determine significance: *P < .05; **P ≤ .01; ***P ≤ .001. (C) Hematoxylin-stained femur sections of representative mice. Within the femurs of the no dox and dox week 6 mice, myeloma tumors (indicated with asterisks) were found. Bars represent 500 μm and 100 μm for top and bottom panels, respectively. #Necrotic MM cells. *MM cells. (D) To visualize the tumors and their proliferation, anti–human syndecan-1 (clone MI-15, Dako Denmark) and anti–human Ki67 (M7240, Dako Denmark) immunostainings were performed. The no dox and dox week 6 mice had tumors with approximately 87% and approximately 34% Ki67+ MM cells, respectively (data not shown). In the dox week 6 mice, areas of necrosis with weak and diffuse syndecan-1 and Ki67 staining were found. Representative areas are shown. Bars represents 50 μm. (Insets) Isotype controls. IC micrographs were obtained with an Olympus BX51 light microscope, using a 10×/0.30 Plan FI, 20×/0.50 Plan FI, or 40×/0.85 PlanApo objective, connected to an Olympus DP70 camera. Images were captured with Olympus DPController software (Version 1.2.1.108) and further processed with Adobe Photoshop (Version 7.0). (E) Cell-surface HS expression of GFP+ L363-shEXT1a MM cells harvested from the bones of no dox and dox week 6 mice, as determined by FACS. The mean fluorescence intensity (MFI) of the untreated mice was normalized to 100%. N.D. indicates not determined (no tumor).

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