Contractile forces contribute to increased glycosylphosphatidylinositol-anchored receptor CD24-facilitated cancer cell invasion

Abstract

The malignancy of a tumor depends on the capability of cancer cells to metastasize. The process of metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The expression of the glycosylphosphatidylinositol-anchored receptor CD24 is increased in several tumor types and is consistently associated with increased metastasis formation in patients. Furthermore, the localization of β1-integrins in lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) mainly through β1 heterodimeric integrin receptors. Here, we studied the invasion of A125 human lung cancer cells with different CD24 expression levels in three-dimensional ECMs. We hypothesized that CD24 expression increases cancer cell invasion through increased contractile forces. To analyze this, A125 cells (CD24 negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24(high) and CD24(low) transfectants was determined in three-dimensional ECMs. The percentage of invasive cells and their invasion depth was increased in CD24(high) cells compared with CD24(low) cells. Knockdown of CD24 and of the β1-integrin subunit in CD24(high) cells decreased their invasiveness, indicating that the increased invasiveness is CD24- and β1-integrin subunit-dependent. Fourier transform traction microscopy revealed that the CD24(high) cells generated 5-fold higher contractile forces compared with CD24(low) cells. To analyze whether contractile forces are essential for CD24-facilitated cell invasion, we performed invasion assays in the presence of myosin light chain kinase inhibitor ML-7 as well as Rho kinase inhibitor Y27632. Cell invasiveness was reduced after addition of ML-7 and Y27632 in CD24(high) cells but not in CD24(neg) cells. Moreover, after addition of lysophosphatidic acid or calyculin A, an increase in pre-stress in CD24(neg) cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase or STAT3 strongly reduced the invasiveness of CD24(high) cells, slightly reduced that of CD24(low) cells, and did not alter the invasiveness of CD24(neg) cells. Taken together, these results suggest that CD24 enhances cell invasion through increased generation or transmission of contractile forces.

FIGURE 1.

High CD24 expression increased human lung carcinoma cell invasion. A, flow cytometric analysis ((R)-PE-labeled-labeled antibodies) of CD24 cell-surface expression on CD24^neg, CD24^low, and CD24^high cells. B, data (MFI) of CD24 expression on CD24^neg, CD24^low, and CD24^high cells are presented as means ± S.D. (n = 3). C, schematic image of the invasion assay. D, modulation contrast images of representative invasive lung carcinoma cells wild type (left), stably transfected with CD24 and sorted for low expression (middle), and high expression (right) that invaded into three-dimensional ECMs. Scale bars, 20 μm. E, percentage of invasive cells and invasion profiles (F) of CD24^neg, CD24^low, and CD24^high cells are shown that invaded into three-dimensional ECMs after 3 days of culture. G, flow cytometric analysis (Cy₂-labeled antibodies) of CD24 cell-surface expression on CD24^high cells transfected with control siRNA (left), CD24-specific (siCD24-1) siRNA (middle), or CD24-specific (siCD24-2) siRNA (right). One representative experiment of three is shown. H, MFI (mean ± S.D., n = 3) of CD24 expression on CD24^high cells treated with control siRNA, siCD24-1, or siCD24-2. I, percentage of invasive cells and invasion profiles (J) of CD24^high cells that were treated with control siRNA, CD24-specific siRNA (siCD24-1 and siCD24-2), or β1-integrin subunit-specific siRNA that invaded into three-dimensional ECMs after 3 days of culture. K, flow cytometric analysis of the β1-integrin subunit cell-surface expression on CD24^high cells transfected with control siRNA (left) or β1-integrin-specific (siβ1) siRNA (right). One representative experiment of three is shown. L, MFI (mean ± S.D., n = 3) of β1-integrin expression on CD24^high cells treated with control siRNA or siCD24-1 or siβ1. (*, p < 0.05; **, p < 0.01; ***, p < 0.001.)

FIGURE 2.

Knockdown of the α1- and α2-integrin subunits in CD24-facilitated invasiveness of cancer cells and effect of serum on cell invasion. A, percentage of invasive cells and invasion profiles of CD24^neg, CD24^low, and CD24^high cells cultured in serum-free medium (SF) (C), serum-free and LPA-stimulated (SF + LPA) (D), and in medium with serum (B) that invaded into three-dimensional ECMs after 3 days of culture. E, flow cytometric analysis of α1 cell-surface CD24^high cells transfected with control siRNA (left) or α1-specific siRNA (siα1, right panel). One representative experiment of three is shown. F, MFI (mean ± S.D., n = 3) of α1-integrin expression on CD24^high cells treated with control siRNA or siα1. G, flow cytometric analysis of α2 cell-surface CD24^high cells transfected with control siRNA (left panel) or α2-specific siRNA (siα2, right panel). One representative experiment of three is shown. H, MFI (mean ± S.D., n = 3) of α2-integrin expression on CD24^high cells treated with control siRNA or siα2. I, percentage of invasive cells and invasion profiles of CD24^high cells (J) treated with control siRNA, α1-integrin subunit-specific siRNA (siα1) and α2-integrin subunit-specific siRNA (siα2) that invaded into three-dimensional ECMs after 3 days of culture. (*, p < 0.05, **, p < 0.05; ***, p < 0.001.)

FIGURE 3.

MT1-MMP expression of CD24^neg, CD24^low, and CD24^high cells and effect of proteinase inhibition on three-dimensional ECM invasion. A, left panel, flow cytometric analysis of the MT1-MMP receptor expression on the cell surface of CD24^neg, CD24^low, and CD24^high cells. One representative experiment of three is shown. Right panel, MFI (mean ± S.D., n = 3) of MT1-MMP receptor expression on CD24^neg, CD24^low, and CD24^high cells. B, percentage of invasive cells in the presence of an protease inhibitor mixture (PI), GM6001 inhibitor, or DMSO buffer control and invasion profiles of CD24^neg, CD24^low, and CD24^high cells cultured in the presence of PI (D), GM6001 inhibitor (E), or DMSO buffer control (C) that invaded into three-dimensional ECMs after 3 days of culture. (*, p < 0.05; **, p < 0.05; ***, p < 0.001.)

FIGURE 4.

Effect of CD24 expression on cell stiffness and cytoskeletal remodeling dynamics. A, representative fluorescence images of CD24^neg (left panel), CD24^low (middle panel), and CD24^high cells (right panel) stained for actin with Alexa-Fluor546-conjugated phalloidin showed equal stress fiber formation on fibronectin-coated glass after 16 h. B, stiffness of CD24^neg, CD24^low, and CD24^high cells as well as of CD24^high cells transfected with control siRNA (control) and two CD24-specific (siCD24-1 and siCD24-2) siRNAs was measured after force application to fibronectin-coated beads using magnetic tweezers. C, flow cytometric analysis (MFI as mean ± S.D., n = 3) of CD24 expression on the cell surface of CD24^high cells transfected with control siRNA (left panel) or CD24-specific siRNA (siCD24-1 and siCD24-2, respectively). D, creep exponent b (cell fluidity and cytoskeletal remodeling dynamics) of CD24^neg, CD24^low, and CD24^high cells as well as of CD24^high cells transfected with control siRNA (control) and two CD24-specific (siCD24-1 and siCD24-2) siRNAs was also determined using magnetic tweezers. The values are expressed as mean ± S.D. 27–60 cells were measured for each condition. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

FIGURE 5.

Increased contractile force generation of CD24^high cells and inhibition of contractile force-mediated cell invasion. A, strain energy per cell (mean ± S.D.) of CD24^high cells (n = 11) was 3-fold increased compared with CD24^low cells (n = 5). B, percentage of invasive cells (mean ± S.D.) of CD24^neg (top panel), CD24^low (middle panel), and CD24^high cells (bottom panel) determined after 3 days in the presence of myosin contraction inhibitors (15 μm ML-7 or 100 μm Y27632), the actin polymerization inhibitor (2 μm latrunculin B), the myosin phosphatase inhibitor (1 nm calyculin A), or DMSO as control. Invasion profiles of CD24^neg, CD24^low, and CD24^high cells treated with DMSO control (C), ML-7 (D), Y26732 (E), latrunculin B (Lat B) (F), or calyculin A (Cal A) (G). (**, p < 0.05; ***, p < 0.001.)

FIGURE 6.

Inhibition of Src kinase and STAT3 of CD24-mediated cell invasion. A–C, percentage of invasive cells (mean ± S.D.) of CD24^neg (A), CD24^low (B), and CD24^high cells (C) determined after 3 days in the presence of an Src inhibitory peptide (Src inh 30 μm), STAT3 inhibitor (STAT3 inh 30 μm), the tyrosine kinase inhibitor herbimycin A (HerbA, 100 μm), or DMSO as control. Invasion profiles of CD24^neg, CD24^low, and CD24^high cells treated with DMSO control (D), Src inhibitory (E), (*, p < 0.05; (F), or herbimycin A (G). (*, p < 0.05; **, p < 0.05; ***, p < 0.001).

FIGURE 7.

High endogenous CD24 expression increased human breast carcinoma cell invasion. A and B, flow cytometric analysis ((R)-PE-labeled-labeled antibodies) of CD24 cell-surface expression on 231endCD24^low (A) and 231endCD24^high subcell lines (B). C, data (MFI) of CD24 expression on 231endCD24^low and 231endCD24^high cells are presented as means ± S.D. (n = 3). D, percentage of invasive cells and invasion profiles (E) of 231endCD24^low and 231endCD24^high cells are shown that invaded into three-dimensional ECMs after 3 days of culture. F and G, flow cytometric analysis (Cy₂-labeled antibodies) of CD24 cell-surface expression on 231endCD24^high cells transfected with control siRNA (F) and CD24-specific (siCD24-2) siRNA (G). H, MFI (mean ± S.D., n = 3) of CD24 expression on 231endCD24^high cells treated with control siRNA or siCD24-2. I, percentage of invasive cells and invasion profiles (J) of 231endCD24^high cells that were treated with control siRNA and CD24-specific siRNA (siCD24-2) that invaded into three-dimensional ECMs after 3 days of culture. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).