Figure 1.
ZAP70 expression is associated with CNS infiltration in vivo. A: Efficiency of ZAP70 knockdown by shRNA, as determined by qRT-PCR. RQ=2^-DDCT. B: Semi-quantitative measurement of CNS infiltration as described in Methods. Hematoxylin and Eosin staining of a negative (−, left), an intermediate (+, middle), and a high (++, right) sample as examples for the scoring method, 200× magnification. * signifies leukemic infiltration. C: Mice were xenografted with 697 (n=10 per group), REH (n=8 per group) and JURKAT (n=10 per group) cells bearing an shRNA against ZAP70 (shZAP70) or a control (shGFP). Mice were sacrificed upon appearance of hind limb paralysis, and semi-quantitative scoring of the xenograft CNS is shown in the table (Fisher´s exact test, one-sided P-values. *P is significant). In the 697-shZAP70 group, 1 cage containing 3 mice was accidentally eliminated from the experiment on day +2. These animals were not included in the statistical analysis. D: Number of leukemic blasts recovered from the CNS of mice xenografted with either JURKAT-shGFP (n=4) or JURKAT-shZAP70 (n=3) (unpaired t-test, one-sided *P-value 0.041). E: 1×105 REH-shGFP or REH-shZAP70 cells/mouse were xenografted into 10 NOD/SCID mice/group by tail vein injection, xenograft survival (Kaplan-Meier log-rank test, **P=0.0021). F: 10 primary samples of pediatric BCP-ALL patients chosen according to ZAP70 expression (5 ZAP70lo and 5 ZAP70hi) were xenografted into duplicate NSG-mice. Mice were sacrificed when leukemic symptoms were visible and semi-quantitative scoring of the CNS was performed. Patient and xenograft characteristics are depicted in the Online Supplementary Table S1. *P<0.05; **P<0.01. CSF: cerebrospinal fluid; DM: dura mater; CNS: central nervous system; BCP-ALL: B-cell precursor acute lymphoblastic leukemia; Xeno: xenograft; shGFP: shRNA against GFP(green fluorescent protein); shZAP70: shRNA targeting ZAP70 (zeta-chain-associated protein kinase 70).