Figure 5.
In vitro myeloid support for T-ALL in the CNS is dependent on integrin-mediated interactions and downstream FAK/PYK2 signaling. (A) Quantification of viable T-ALL cells cultured with myeloid cells from the CNS in the presence of the indicated blocking antibodies to ICAM-1 and/or VCAM-1 or isotype control antibody. Bars show the mean + SEM of cumulative data from 3 experiments; symbols represent distinct primary color-encoded T-ALL stocks. (B-C) Quantification of relative ICAM-1 (B) and VCAM-1 protein levels (C) on the indicated myeloid subsets from the CNS of healthy (black) and LMO2 T-ALL-engrafted (red) mice. (D-F) Quantification of viable T-ALL cells cultured in the absence or presence of myeloid cells with the indicated concentrations of PF562271 ("iFAK/PYK”) to inhibit FAK/PYK2 activity or a DMSO vehicle control. Bars depict the mean + SEM of cumulative data from 6 experiments, each with a color-coded unique T-ALL stock. Data from panel D are also stratified into Hhexhigh (E) and Hhexlow (F) T-ALL subtypes. (G) IF imaging of sections (original magnification ×40) of the spinal cord and surrounding meninges show T-ALL (DAPI+ [4′,6-diamidino-2-phenylindole positive], blue) cells and ICAM-1+ (green) cells in the meninges. (H) LY6C (green), F4/80 (blue), and ICAM-1 (red) immunostaining (right) reveals myeloid cells within the meninges; inset images show dual expression of ICAM-1+LY6C+ (top) and ICAM-1+F4/80+ (bottom) by monocytes (white arrowhead, black outline) and macrophages (black arrowhead, white outline), respectively. In panels G-H, scale bars represent 100 μm. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 (using the repeated measures 1-way analysis of variance with Bonferroni correction in panels A-F). DMSO, dimethyl sulfoxide; iFAK/PYK, inhibitor of FAK/PYK; RFI, relative fluorescence intensity.

In vitro myeloid support for T-ALL in the CNS is dependent on integrin-mediated interactions and downstream FAK/PYK2 signaling. (A) Quantification of viable T-ALL cells cultured with myeloid cells from the CNS in the presence of the indicated blocking antibodies to ICAM-1 and/or VCAM-1 or isotype control antibody. Bars show the mean + SEM of cumulative data from 3 experiments; symbols represent distinct primary color-encoded T-ALL stocks. (B-C) Quantification of relative ICAM-1 (B) and VCAM-1 protein levels (C) on the indicated myeloid subsets from the CNS of healthy (black) and LMO2 T-ALL-engrafted (red) mice. (D-F) Quantification of viable T-ALL cells cultured in the absence or presence of myeloid cells with the indicated concentrations of PF562271 ("iFAK/PYK”) to inhibit FAK/PYK2 activity or a DMSO vehicle control. Bars depict the mean + SEM of cumulative data from 6 experiments, each with a color-coded unique T-ALL stock. Data from panel D are also stratified into Hhexhigh (E) and Hhexlow (F) T-ALL subtypes. (G) IF imaging of sections (original magnification ×40) of the spinal cord and surrounding meninges show T-ALL (DAPI+ [4′,6-diamidino-2-phenylindole positive], blue) cells and ICAM-1+ (green) cells in the meninges. (H) LY6C (green), F4/80 (blue), and ICAM-1 (red) immunostaining (right) reveals myeloid cells within the meninges; inset images show dual expression of ICAM-1+LY6C+ (top) and ICAM-1+F4/80+ (bottom) by monocytes (white arrowhead, black outline) and macrophages (black arrowhead, white outline), respectively. In panels G-H, scale bars represent 100 μm. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 (using the repeated measures 1-way analysis of variance with Bonferroni correction in panels A-F). DMSO, dimethyl sulfoxide; iFAK/PYK, inhibitor of FAK/PYK; RFI, relative fluorescence intensity.

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