Ds, EOL-1 cells displayed only a small subpopulation of sialyl Lewis a (CA19-9) positive cells (3.7 ), but were highly positive for sialyl Lewis 6 (CD15s),E- and P-Selectin Essential in Leukemia XenograftFigure 2. Human EOL-1 cells and K562 cells cells show tethering and adhesion to E- and P-selectin in laminar flow assays. Interactions of human CEL and CML cells from culture under laminar flow (0.25 dyn/cm2) in selectin coated channels. The cell lines EOL-1 and K562 were tested for adherence (A) or tethering (B). Given are the numbers and corresponding standard deviations of adhering and tethering cells, respectively. Channels were coated with human E-selectin (E), human P-selectin (P), murine E-selectin (mE) or murine P-selectin (mP). No adhering or tethering cells were observed in Fc control coated channels. All experiments were done in triplicates. doi:10.1371/journal.pone.0070139.gwhereas K562 cells were negative for both (Figure 6). Both cells lines were positive for CD162 (PSGL-1).Antibodies against sialyl Lewis x (CD15s, Figure 7A) and sialyl Lewis a (CA19-9, Figure 7B) did not inhibit E- and P-selectinE- and P-Selectin Essential in Leukemia XenograftFigure 3. Human CEL and CML cells can be clearly detected by immunohistochemistry in the tissues of the scid mice. A: Tissue section of an EOL-1 chloroma of selectin wt scid mouse. Immunohistochemical 86168-78-7 supplier staining for human HLA-DR in red. B: Tissue section of a K562 chloroma of a wt scid mouse. Immunohistochemical staining for human mitochondria in red. doi:10.1371/journal.pone.0070139.gbinding to EOL-1 and K562 cells although anti-CA19-9 (and not anti-CD15s) did completely block E-selectin binding to PaCacells as previously described [34]. Even the monoclonal antibody HECA-452 which has a broader specificity (CA19-9 and CD15sE- and P-Selectin Essential in Leukemia XenograftFigure 4. Xenograft model of CEL with the human cell line EOL-1 in wild-type and E- and P-selectin knockout scid mice. Selectin deficiency order ��-Sitosterol ��-D-glucoside dramatically increases survival of the animals after injection of 26106 EOL-1 cells and decreases the number of leukemia cells in the blood. A: Kaplan-Meyer survival curve for wild-type (wt, selectin competent, 8 animals, grey curve) and selectin knockout (k.o., E-and P-selectin deficient, 10 animals, black curve). The percentage of surviving animals on a given day is shown. The experiment was terminated after 53 days. Median survival after transplantation was: wt 32 days, k.o. not reached. The curves were significantly different, ***: P,0.0001 (Log-rank test). B: Human EOL-1 cells in the animals’ blood at the time of death as determined by quantitative real-time PCR. Selectin competent (wt, 8 animals) are compared with E- and Pselectin deficient mice (k.o., 9 animals). Given in the box plot are the median (line), highest and lowest number of EOL-1 cells per ml of the animals’ blood (whiskers) and upper and lower quartile (box). Median cell number per ml blood was 32950 for the wt group and 7.8 for the k.o. group. The difference between the groups was significantly different, ***: P = 0.0002 (Mann Whitney test). doi:10.1371/journal.pone.0070139.gamong other carbohydrate moieties) had no inhibitory effect on Eand P-selectin binding to the CEL and CML cells, but blocked Eselectin binding to PaCa 5061 cells (not shown). An antibody against CD162 (PSGL-1) blocked P-selectin binding to EOL-1, butnot to K562 (Figure 7A), whereas E-selectin binding to both cell lines was not influenced b.Ds, EOL-1 cells displayed only a small subpopulation of sialyl Lewis a (CA19-9) positive cells (3.7 ), but were highly positive for sialyl Lewis 6 (CD15s),E- and P-Selectin Essential in Leukemia XenograftFigure 2. Human EOL-1 cells and K562 cells cells show tethering and adhesion to E- and P-selectin in laminar flow assays. Interactions of human CEL and CML cells from culture under laminar flow (0.25 dyn/cm2) in selectin coated channels. The cell lines EOL-1 and K562 were tested for adherence (A) or tethering (B). Given are the numbers and corresponding standard deviations of adhering and tethering cells, respectively. Channels were coated with human E-selectin (E), human P-selectin (P), murine E-selectin (mE) or murine P-selectin (mP). No adhering or tethering cells were observed in Fc control coated channels. All experiments were done in triplicates. doi:10.1371/journal.pone.0070139.gwhereas K562 cells were negative for both (Figure 6). Both cells lines were positive for CD162 (PSGL-1).Antibodies against sialyl Lewis x (CD15s, Figure 7A) and sialyl Lewis a (CA19-9, Figure 7B) did not inhibit E- and P-selectinE- and P-Selectin Essential in Leukemia XenograftFigure 3. Human CEL and CML cells can be clearly detected by immunohistochemistry in the tissues of the scid mice. A: Tissue section of an EOL-1 chloroma of selectin wt scid mouse. Immunohistochemical staining for human HLA-DR in red. B: Tissue section of a K562 chloroma of a wt scid mouse. Immunohistochemical staining for human mitochondria in red. doi:10.1371/journal.pone.0070139.gbinding to EOL-1 and K562 cells although anti-CA19-9 (and not anti-CD15s) did completely block E-selectin binding to PaCacells as previously described [34]. Even the monoclonal antibody HECA-452 which has a broader specificity (CA19-9 and CD15sE- and P-Selectin Essential in Leukemia XenograftFigure 4. Xenograft model of CEL with the human cell line EOL-1 in wild-type and E- and P-selectin knockout scid mice. Selectin deficiency dramatically increases survival of the animals after injection of 26106 EOL-1 cells and decreases the number of leukemia cells in the blood. A: Kaplan-Meyer survival curve for wild-type (wt, selectin competent, 8 animals, grey curve) and selectin knockout (k.o., E-and P-selectin deficient, 10 animals, black curve). The percentage of surviving animals on a given day is shown. The experiment was terminated after 53 days. Median survival after transplantation was: wt 32 days, k.o. not reached. The curves were significantly different, ***: P,0.0001 (Log-rank test). B: Human EOL-1 cells in the animals’ blood at the time of death as determined by quantitative real-time PCR. Selectin competent (wt, 8 animals) are compared with E- and Pselectin deficient mice (k.o., 9 animals). Given in the box plot are the median (line), highest and lowest number of EOL-1 cells per ml of the animals’ blood (whiskers) and upper and lower quartile (box). Median cell number per ml blood was 32950 for the wt group and 7.8 for the k.o. group. The difference between the groups was significantly different, ***: P = 0.0002 (Mann Whitney test). doi:10.1371/journal.pone.0070139.gamong other carbohydrate moieties) had no inhibitory effect on Eand P-selectin binding to the CEL and CML cells, but blocked Eselectin binding to PaCa 5061 cells (not shown). An antibody against CD162 (PSGL-1) blocked P-selectin binding to EOL-1, butnot to K562 (Figure 7A), whereas E-selectin binding to both cell lines was not influenced b.
