Constitutive integrin activation on tumor cells contributes to progression of leptomeningeal metastases

Abstract

Leptomeningeal metastases are a serious neurological complication in cancer patients and associated with a dismal prognosis. Tumor cells that enter the subarachnoid space adhere to the leptomeninges and form tumor deposits. It is largely unknown which adhesion molecules mediate tumor cell adhesion to leptomeninges. We studied the role of integrin expression and activation in the progression of leptomeningeal metastases. For this study, we used a mouse acute lymphocytic leukemic cell line that was grown in suspension (L1210-S cell line) to develop an adherent L1210 cell line (L1210-A) by selectively culturing the few adherent cells in the cell culture. beta1, beta2, and beta3 integrins were in a constitutively high active state on L1210-A cells and in a low, but inducible, active state on L1210-S cells. Expression levels of these integrins were comparable in the two cell lines. Static adhesion levels of L1210-A cells on a leptomeningeal cell layer were significantly higher than those of L1210-S cells. All mice that were injected intrathecally with L1210-A cells died rapidly of leptomeningeal leukemia. In contrast, 45% long-term survival was seen after intrathecal injection of mice with L1210-S cells. Our data indicate that constitutive integrin activation on leukemic cells promotes progression of leptomeningeal leukemia by increased tumor cell adhesion to the leptomeninges. We argue that an aberrantly regulated inside-out signaling pathway underlies constitutive integrin activation on the adherent leukemic cell population.

Fig. 1

Morphological and proliferation characteristics of L1210-S and L1210-A cells. A and B. Light-microscopic pictures of L1210-S and L1210-A cells. Panel A shows the L1210 acute lymphocytic leukemic suspension cell line (L1210-S cell line). A few cells (<1%) strongly adhere and spread on the bottom of noncoated culture flasks. The adherent leukemic cells were selectively cultured by aspirating the suspended L1210 cells every day for two weeks, resulting in the adherent L1210 cell line (L1210-A), shown in panel B. These leukemic cells form extensive filopodia and lamellipodia on the bottom of the noncoated culture flask. C. Proliferation of L1210-S and L1210-A cells in culture medium. Leukemic cells were seeded at a density of 2 × 10⁴ cells on noncoated wells of a 48-well plate in culture medium (RPMI, 10% fetal calf serum, 60 μM β-mercaptoethanol). At 24, 48, and 72 h, the number of leukemic cells was counted with a cell counter. The mean number of leukemic cells of six wells (± SEM) was plotted. One representative experiment out of six is shown. D. Proliferation of L1210-S and L1210-A cells in CSF. L1210-A and L1210-S cells were seeded at a density of 2 × 10⁴ cells on non-coated wells of a 48-well plate in CSF supplemented with 60 μM β-mercaptoethanol. At 24, 48, and 72 h, the number of leukemic cells in three wells was counted with a cell counter. The mean number of leukemic cells (± SEM) is plotted. One representative experiment out of three is shown.

Fig. 2

L1210-A cells adhere and spread more efficiently to a leptomeningeal cell layer than do L1210-S cells. Adhesion assays of fluorescently labeled leukemic cells to determine adhesion to a leptomeningeal cell layer were performed at 37°C for 30 or 60 min, whereafter nonadherent cells were washed away and adhered cells were fixed with 2% paraformaldehyde. A. The mean number of leukemic cells (± SEM) that adhered to the leptomeningeal cell layer after 30 and 60 min of static adhesion is plotted. Two independent experiments were performed in quadruplicate. B and C. Light-microscopic pictures of L1210-S cells (B) and L1210-A cells (C) that adhered to a leptomeningeal cell layer after 60 min of static adhesion. 40×.

Fig. 3

Significant difference in survival of mice with L1210-A and mice with L1210-S leptomeningeal leukemia. Kaplan-Meier survival curves of mice intrathecally injected with L1210-A cells (solid line; n = 22) or L1210-S cells (dashed line; n = 22). Leukemic cells (2 × 10⁵) were injected into the cisterna magna of the mice, and survival was recorded.

Fig. 4

β₁, β₂, and β₃ integrins are constitutively active on L1210-A cells. Static adhesion assays of leukemic cells were performed on collagen (A), mouse ICAM-1 (B), and vitronectin (C). Assays were done without extracellular stimulation (DMEM); in the presence of Mg²⁺ (5 mM), Mn²⁺ (0.5 mM), or EDTA (10 mM); or after pretreatment of leukemic cells with PMA (100 ng/ml), integrin-blocking or isotype control MoAbs (10 μg/ml), or dRGD-w peptide (100 μM) for 30 min at 37°C. The percentage of adhered cells after 30 min of static adhesion and three washing steps is plotted on the y-axis. White and black bars represent the mean percentage of adhered L1210-A and L1210-S cells (± SEM), respectively. Data were obtained from more than three independent experiments performed in triplicate. ^*Significantly different compared to the mean percentage adhered L1210-A cells in DMEM. ^#Significantly different compared to the mean percentage adhered L1210-S cells in DMEM.

Fig. 5

Mn²⁺ induces L1210-S cell adhesion to a leptomeningeal cell layer and L1210-A cell adhesion is β₃ integrin dependent. A. Static adhesion assays of leukemic cells were performed on confluent mouse leptomeningeal cell layers without extracellular stimulation (DMEM); in the presence of Mg²⁺ (5 mM) or Mn²⁺ (0.5 mM); or after pretreatment of leukemic cells with PMA (100 ng/ml), β₁, β₂, β₃ integrin-blocking or isotype control MoAbs (10 μg/ml), or dRGD-w peptide (100 μM) for 30 min at 37°C. B. Static adhesion assays of leukemic cells on confluent mouse leptomeningeal cell layers were performed after pretreatment of leukemic cells with MoAbs against the single β₁, β₂, or β₃ integrin subunits (10 μg/ml) or all three β integrin chains, and results were compared to results for pretreatment with the isotype control MoAbs (10 μg/ml) for 30 min at 37°C. The percentage of adhered cells after 30 min of static adhesion and three washing steps is plotted on the y-axis. White and black bars represent the mean percentage of adhered L1210-A and L1210-S cells (± SEM), respectively. Data were obtained from at least three independent experiments performed in triplicate. ^*Significantly different compared to the mean percentage adhered L1210-A cells in DMEM or after pretreatment with isotype control MoAbs. ^#Significantly different compared to the mean percentage adhered L1210-S cells in DMEM.