Figure 1.
The CLL proliferation center is a hypoxic environment. (A) Immunohistochemistry of a representative (n = 7) CLL LN sample stained with anti–HIF-1α and anti -CAIX antibodies, with more intense signal localized in proliferation centers (black arrows). Original magnification ×10 and ×40. (B) The same antibodies were used to stain reactive LN samples (n = 3), showing dim positivity in correspondence of lymphocytes. Original magnification ×10 and ×40. (C) Spleen samples (n = 6) obtained from CLL xenografts in NSG mice stained with anti–human CD20 to show CLL cells (left panel). The middle panels show HIF-1α and CAIX staining of xenografted mice spleen sections with strong positivity in the areas colonized by human cells. NSG mice xenografted with human CLL cells were injected with 100 mg/kg pimonidazole (PIM). Spleen specimens, obtained 2 hours after injection, were fixed and stained with an anti-PIM antibody (right panel). Original magnification ×4 and ×20. (D) Human cells recovered from the spleen of mice bearing CLL xenografts and injected with PIM were stained with anti–human CD45, CD20 and anti-PIM antibodies before flow cytometry and confocal microscopy analysis (original magnification ×63). (E) RT-PCR analysis of HIF-1α (HIF1A) expression in PB, BM, or LN CLL cells obtained concurrently from the same patient shows higher expression in LN than in the PB or BM (n = 12). The Wilcoxon signed rank test was used for comparative analyses. Panel on the right shows western blot analysis for HIF-1α expression in lysates obtained from the PB, BM, and LN of paired samples. Lysates were obtained by pooling cells obtained from 5 samples. The last lane on the right shows a positive control obtained after culturing purified CLL cells for 48 hours under hypoxia. Immunohistochemical samples were analyzed using an AXIO Laboratory.A1 microscope (Zeiss, Milan, Italy) equipped with a Canon EOS600D and images acquired with the ZoomBrowserEx software (Canon). Immunofluorescence was analyzed using a TCS SP5 laser scanning confocal microscope with an oil immersion 63×/1.4 objective lens (Leica Microsystems, Milan, Italy). Images were acquired using the LAS AF version Lite 2.4 software (Leica Microsystems) and processed with Adobe Photoshop (Adobe Systems). IB, immunoblot.

The CLL proliferation center is a hypoxic environment. (A) Immunohistochemistry of a representative (n = 7) CLL LN sample stained with anti–HIF-1α and anti -CAIX antibodies, with more intense signal localized in proliferation centers (black arrows). Original magnification ×10 and ×40. (B) The same antibodies were used to stain reactive LN samples (n = 3), showing dim positivity in correspondence of lymphocytes. Original magnification ×10 and ×40. (C) Spleen samples (n = 6) obtained from CLL xenografts in NSG mice stained with anti–human CD20 to show CLL cells (left panel). The middle panels show HIF-1α and CAIX staining of xenografted mice spleen sections with strong positivity in the areas colonized by human cells. NSG mice xenografted with human CLL cells were injected with 100 mg/kg pimonidazole (PIM). Spleen specimens, obtained 2 hours after injection, were fixed and stained with an anti-PIM antibody (right panel). Original magnification ×4 and ×20. (D) Human cells recovered from the spleen of mice bearing CLL xenografts and injected with PIM were stained with anti–human CD45, CD20 and anti-PIM antibodies before flow cytometry and confocal microscopy analysis (original magnification ×63). (E) RT-PCR analysis of HIF-1α (HIF1A) expression in PB, BM, or LN CLL cells obtained concurrently from the same patient shows higher expression in LN than in the PB or BM (n = 12). The Wilcoxon signed rank test was used for comparative analyses. Panel on the right shows western blot analysis for HIF-1α expression in lysates obtained from the PB, BM, and LN of paired samples. Lysates were obtained by pooling cells obtained from 5 samples. The last lane on the right shows a positive control obtained after culturing purified CLL cells for 48 hours under hypoxia. Immunohistochemical samples were analyzed using an AXIO Laboratory.A1 microscope (Zeiss, Milan, Italy) equipped with a Canon EOS600D and images acquired with the ZoomBrowserEx software (Canon). Immunofluorescence was analyzed using a TCS SP5 laser scanning confocal microscope with an oil immersion 63×/1.4 objective lens (Leica Microsystems, Milan, Italy). Images were acquired using the LAS AF version Lite 2.4 software (Leica Microsystems) and processed with Adobe Photoshop (Adobe Systems). IB, immunoblot.

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