Er tumor cell injection, similar osteolytic lesions, characteristic for 143-B cell-derived tumors, were recognized by X-ray in all three Nafarelin cost groups of mice (Figure 3A). Somehow unexpectedly, caliper measurements of the volume of the tumor legs over time revealed growth of intratibial primary tumors to a significantly (p,0.001) larger final size at sacrifice on experimental day 20 in mice injected with 143-B shCD44 cells (108 6 14 mm3, n = 9) than in animals that received 143-B EV cells (39 6 6 mm3, n = 9) (Figure 3B). The mean size of tumors developing in mice injected with 143-B Ctrl shRNA cells (65 6 25 mm3, n = 6) was intermediate, but not significantly different from that in mice injected with 143-B shCD44 or with 143-B EV cells, most probably due to the fact that Ctrl shRNA group consisted of only 6 animals that exhibited greater heterogeneity in tumor size than the other experimental groups. Two of the 6 mice of 143-B Ctrl group, in particular, developed remarkably larger primary tumors thanthe other 4 mice of this group and all mice in the 143-B EV group that were similar in size. Interestingly, beside the described larger size of 143-B shCD44 compared to 143-B EV and 143-B Ctrl shRNA cell-derived primary tumors, the mean number of metastatic lesions on lung surfaces at sacrifice, detected by X-gal staining, were also found significantly increased 2-fold (p,0.05) and 2.4-fold (p,0.05) in mice with 143-B shCD44 cell-derived tumors compared the numbers counted on the lungs of animals injected with 143-B EV or 143-B Ctrl shRNA cells, respectively (Figure 3C, D). In view of the discrepant malignant properties of 143-B shCD44 cells in vitro and in vivo, we assessed the expression of immunoreactive CD44 by immunohistochemistry with Hermes3 antibodies in primary tumor tissue and lung metastases derived from 143-B shCD44 cells and compared it with 143-B EV and 143-B Ctrl shRNA cell-derived tumors. This analysis demonstrated thatCD44 Pentagastrin supplier silencing Promotes Osteosarcoma MetastasisFigure 4. Ex-vivo immunohistochemical analysis of shRNA mediated CD44 silencing in 143-B-lacZ OS cell-derived intratibial primary tumors and pulmonary metastases. Robust expression of immunoreactive CD44 observed in primary tumor (PT) tissue and pulmonary metastases (PM) derived from 143-B EV (EV) (A,G) and 143-B Ctrl shRNA (Ctrl shRNA) cells (B,H) was found suppressed in tumor tissue (C) and lung metastases (I) derived from 143-B shCD44 (shCD44) cells. Immunohistochemically detectable HA in PT (panels D-F) was not affected by CD44 silencing. The figure shows images of representative 6 mm paraffin sections with cell nuclei counterstained with hematoxylin. Bars, 100 mm. doi:10.1371/journal.pone.0060329.gCD44 silencing was maintained in vivo in 143-B shCD44 cells (Figure 4C and I). The content of HA in the extracellular matrix of primary tumors, on the other hand, was indistinguishable in 143-B shCD44, -EV and -Ctrl shRNA cell-derived tumors, indicating that the levels of expression of CD44 gene products in 143-B cells did not affect extracellular HA deposition (Figure 4D-F). Interestingly, nuclear Ki67 immunostaining of proliferating tumor cells on primary tumor and lung paraffin sections showed a specific structure of primary tumors and lung metastases in mice bearing tumors depleted of CD44. 143-B shCD44 cells appeared to be in much looser contact in primary tumors and metastases than 143-B EV and 143-B Ctrl shRNA cells that formed remarkably denser malignant tissue than.Er tumor cell injection, similar osteolytic lesions, characteristic for 143-B cell-derived tumors, were recognized by X-ray in all three groups of mice (Figure 3A). Somehow unexpectedly, caliper measurements of the volume of the tumor legs over time revealed growth of intratibial primary tumors to a significantly (p,0.001) larger final size at sacrifice on experimental day 20 in mice injected with 143-B shCD44 cells (108 6 14 mm3, n = 9) than in animals that received 143-B EV cells (39 6 6 mm3, n = 9) (Figure 3B). The mean size of tumors developing in mice injected with 143-B Ctrl shRNA cells (65 6 25 mm3, n = 6) was intermediate, but not significantly different from that in mice injected with 143-B shCD44 or with 143-B EV cells, most probably due to the fact that Ctrl shRNA group consisted of only 6 animals that exhibited greater heterogeneity in tumor size than the other experimental groups. Two of the 6 mice of 143-B Ctrl group, in particular, developed remarkably larger primary tumors thanthe other 4 mice of this group and all mice in the 143-B EV group that were similar in size. Interestingly, beside the described larger size of 143-B shCD44 compared to 143-B EV and 143-B Ctrl shRNA cell-derived primary tumors, the mean number of metastatic lesions on lung surfaces at sacrifice, detected by X-gal staining, were also found significantly increased 2-fold (p,0.05) and 2.4-fold (p,0.05) in mice with 143-B shCD44 cell-derived tumors compared the numbers counted on the lungs of animals injected with 143-B EV or 143-B Ctrl shRNA cells, respectively (Figure 3C, D). In view of the discrepant malignant properties of 143-B shCD44 cells in vitro and in vivo, we assessed the expression of immunoreactive CD44 by immunohistochemistry with Hermes3 antibodies in primary tumor tissue and lung metastases derived from 143-B shCD44 cells and compared it with 143-B EV and 143-B Ctrl shRNA cell-derived tumors. This analysis demonstrated thatCD44 Silencing Promotes Osteosarcoma MetastasisFigure 4. Ex-vivo immunohistochemical analysis of shRNA mediated CD44 silencing in 143-B-lacZ OS cell-derived intratibial primary tumors and pulmonary metastases. Robust expression of immunoreactive CD44 observed in primary tumor (PT) tissue and pulmonary metastases (PM) derived from 143-B EV (EV) (A,G) and 143-B Ctrl shRNA (Ctrl shRNA) cells (B,H) was found suppressed in tumor tissue (C) and lung metastases (I) derived from 143-B shCD44 (shCD44) cells. Immunohistochemically detectable HA in PT (panels D-F) was not affected by CD44 silencing. The figure shows images of representative 6 mm paraffin sections with cell nuclei counterstained with hematoxylin. Bars, 100 mm. doi:10.1371/journal.pone.0060329.gCD44 silencing was maintained in vivo in 143-B shCD44 cells (Figure 4C and I). The content of HA in the extracellular matrix of primary tumors, on the other hand, was indistinguishable in 143-B shCD44, -EV and -Ctrl shRNA cell-derived tumors, indicating that the levels of expression of CD44 gene products in 143-B cells did not affect extracellular HA deposition (Figure 4D-F). Interestingly, nuclear Ki67 immunostaining of proliferating tumor cells on primary tumor and lung paraffin sections showed a specific structure of primary tumors and lung metastases in mice bearing tumors depleted of CD44. 143-B shCD44 cells appeared to be in much looser contact in primary tumors and metastases than 143-B EV and 143-B Ctrl shRNA cells that formed remarkably denser malignant tissue than.