Stromal cells sustain the activation of PI3-K/Akt pathway. Sorted CD19⁺ CLL cells were incubated in suspension and in coculture for 2 days either untreated or treated with LY294002 or wortmannin. As shown in panel A, a weak staining for Akt-pS473 is observed in suspension cultures, and a further decrease occurred after exposure to LY294002 and wortmannin (Ai,vi,xi). An intense staining for Akt pSer-473 is shown in the nonadherent and adherent compartments in the coculture (ii-iii), which was diminished by exposure to the inhibitors LY294002 (vii-viii) and wortmannin (xii-xiii). Red fluorescence shows the fibronectin matrix that is produced by bone marrow fibroblasts (iv,ix,xiv). Islands of adherent CLL cells could be seen above or in close contact with BMSCs and fibronectin matrix (v). Cell adhesion was decreased after exposure to the inhibitors (x,xv). Western blotting confirmed the effect of coculture and PI3-K inhibitors on Akt phosphorylation (B; S indicates suspension; NAd, nonadherent cells; Ad, adherent cells). Panel C shows weak staining for PIP3 in CLL cells in suspension cultures (i) that was absent after LY294002 and wortmannin treatment (ii-iii). A bright fluorescence for PIP3 was seen in CLL cells from cocultures (iv) that was reduced after LY294002 and wortmannin treatment (v-vi). The pattern of expression of the main components of the PI3-K pathway (PI3-K–p85, PDK1, Akt, and PTEN) is shown in sorted (CD19⁺) CLL cells from 2 patients before and after exposure to LY294002 (1μM) under both culture conditions (D). As shown by Western blotting, the amounts of PI3-K p85, PDK1, pPDK1, pAkt were relatively lower in suspension cultures compared with cocultures and were further decreased after exposure to LY294002 under both culture conditions.