American Society of Hematology

Figure 2

$Figure 2. In vivo and in vitro characterization of bone marrow–derived cells in TRAMP prostate tumors. (A) FACS analysis of spontaneous prostate tumors in TRAMP mice (24-30 weeks of age). Representative plots of GFP+ cells retrieved from tumors costained with the indicated antibodies is shown. (B) Quantitative analysis of hematopoietic/myeloid cells in TRAMP prostate tumors (n = 3). Approximately 99% of all BMDC are CD45+ hematopoietic cells. (C) Sections of well-differentiated prostate tumors were stained with GFP and CD11b antibodies (i). Yellow cells indicate the merged coexpression. Nuclei were counterstained with DAPI (blue). Scale bar represents 100 μm. (D) After bone marrow ablation/engraftment, collagenase-digested prostate tumors were plated into tissue-culture dishes. Numerous GFP+ colonies appeared after approximately 1 week in culture from which spindle-shaped GFP+ cells emerged (i). Scale bar represents 0.5 mm. After 2 weeks, cells were treated with trypsin, and tightly adhered, trypsin-resistant cells TD-CFUs were analyzed by qRT-PCR. Similar to PBM cells, TD-CFUs were enriched in expression of CD45, CD11b, F4-80, and GR-1 but not VE-Cadherin (ii). Also by qRT-PCR, MMP2 is predominately expressed by TECs, while PBM cells and TD-CFUs express both MMP9 and MMP2 (iii). (E) TECs formed branching, cord-like structures when plated at low densities and admixed with freshly isolated PBM cells on thin matrigel layers. PBM cells, which appear as small, refractive cells in the high-powered magnification, closely aligned with these branching structures along the TEC surface (i). At right, the same field is shown where TECs fluoresce in red and PBM cells from GFP+ donor mice fluoresce in green (ii). Scale bars represent 50 μm. (F) PBM cells addition increased TEC branching when cells were plated in low serum and at low density on matrigel. The addition of TECs only resulted in few branching structures (i) while TECs cocultured with PBM cells resulted in TEC branching and tube formation (ii). PBM cells addition increased TEC branching 10-fold when averaged from 3 independent observations (iii). Data were evaluated by Student t test and the P value is indicated on the graph. Scale bars represent 0.5 mm.$

In vivo and in vitro characterization of bone marrow–derived cells in TRAMP prostate tumors. (A) FACS analysis of spontaneous prostate tumors in TRAMP mice (24-30 weeks of age). Representative plots of GFP⁺ cells retrieved from tumors costained with the indicated antibodies is shown. (B) Quantitative analysis of hematopoietic/myeloid cells in TRAMP prostate tumors (n = 3). Approximately 99% of all BMDC are CD45⁺ hematopoietic cells. (C) Sections of well-differentiated prostate tumors were stained with GFP and CD11b antibodies (i). Yellow cells indicate the merged coexpression. Nuclei were counterstained with DAPI (blue). Scale bar represents 100 μm. (D) After bone marrow ablation/engraftment, collagenase-digested prostate tumors were plated into tissue-culture dishes. Numerous GFP⁺ colonies appeared after approximately 1 week in culture from which spindle-shaped GFP⁺ cells emerged (i). Scale bar represents 0.5 mm. After 2 weeks, cells were treated with trypsin, and tightly adhered, trypsin-resistant cells TD-CFUs were analyzed by qRT-PCR. Similar to PBM cells, TD-CFUs were enriched in expression of CD45, CD11b, F4-80, and GR-1 but not VE-Cadherin (ii). Also by qRT-PCR, MMP2 is predominately expressed by TECs, while PBM cells and TD-CFUs express both MMP9 and MMP2 (iii). (E) TECs formed branching, cord-like structures when plated at low densities and admixed with freshly isolated PBM cells on thin matrigel layers. PBM cells, which appear as small, refractive cells in the high-powered magnification, closely aligned with these branching structures along the TEC surface (i). At right, the same field is shown where TECs fluoresce in red and PBM cells from GFP⁺ donor mice fluoresce in green (ii). Scale bars represent 50 μm. (F) PBM cells addition increased TEC branching when cells were plated in low serum and at low density on matrigel. The addition of TECs only resulted in few branching structures (i) while TECs cocultured with PBM cells resulted in TEC branching and tube formation (ii). PBM cells addition increased TEC branching 10-fold when averaged from 3 independent observations (iii). Data were evaluated by Student t test and the P value is indicated on the graph. Scale bars represent 0.5 mm.

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