Silencing of CD44 gene expression in human 143-B osteosarcoma cells promotes metastasis of intratibial tumors in SCID mice

Abstract

Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.

Figure 1. shRNA-mediated downregulation of CD44 expression in 143-B OS cells.

(A) Western blot analysis with the panCD44 Hermes3 antibody of total CD44 gene-derived protein products in extracts of 143-B EV (EV), 143-B Ctrl shRNA (Ctrl shRNA) or 143-B shCD44 (shCD44) cells. β-Actin was used as a loading control. (B) Cell immunostaining of CD44 (red) in saponin permeabilized 143-B EV (EV), 143-B Ctrl shRNA (Ctrl shRNA) or 143-B shCD44 (shCD44) cells. Actin filaments (green) and cell nuclei (blue) were visualized with Alexa Fluor 488-labeled phalloidin and DAPI, respectively. Bars, 100 µm.

Figure 2. Effects of CD44 silencing on in-vitro malignant properties of 143-B OS cells.

(A) Adhesion to HA (n = 3), (B) trans-filter migration (n = 6), (C) proliferation (n = 3) and (D) anchorage-independent growth (n = 4) of 143-B EV (EV), 143-B Ctrl shRNA (Ctrl shRNA) or 143-B shCD44 (shCD44) cells. Values represent the mean ± SEM; *, p<0.05.

Figure 3. Effects of CD44 silencing on intratibial primary tumor growth and lung metastasis of 143-B OS cells in SCID mice.

(A) Primary tumor development over time monitored by X-ray or (B) by tumor leg volume measurement at indicated time points in mice intratibially injected with 143-B EV (EV) (n = 9), 143-B Ctrl shRNA (Ctrl shRNA) (n = 6) or 143-B shCD44 (shCD44) (n = 9) cells. (C) Representative images and (D) quantification of X-gal stained (blue) metastases on whole-mounts of lungs collected from mice intratibially injected with 143-B EV (EV) (n = 9), 143-B Ctrl shRNA (Ctrl shRNA) (n = 6) or 143-B shCD44 (shCD44) (n = 9) cells. Values are expressed as mean ± SEM; *, p<0.05.

Figure 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 µm paraffin sections with cell nuclei counterstained with hematoxylin. Bars, 100 µm.

Figure 5. CD44 silencing in 143-B OS cells reduced the density of proliferating cells in intratibial primary tumors and decreased the content of the tumor suppressor merlin in primary tumors and pulmonary metastases to immunohistochemically nearly undetectable levels.

Representative images of 6 µm paraffin sections of primary tumor (PT) (panels A-C and G-I) and of lung tissue with pulmonary metastases (PM) (panels D-F and J-L) stained for the proliferation marker Ki67 (panels A-F) and for merlin (panels G-L). Tissues were collected at sacrifice from SCID mice intratibially injected with 143-B EV (EV), 143-B Ctrl shRNA (Ctrl shRNA) or 143-B shCD44 (shCD44) cells. Bars, 100 µm.