Figure 1.
ROR1 mRNA expression in pediatric ALL.
(A) Log2 median-centered gene expression profiling data for ROR1 mRNA isoform 1 from a previously published dataset representing 132 pediatric patients with newly diagnosed ALL (www.stjuderesearch.org/data/ALL1) [21] revealed uniform expression in the E2A-PBX1 group (p<0.0001, one-way ANOVA test) and variable expression in all other groups. ROR1 mRNA isoform 1 expression was also detected in normal tissues. (B) ROR1 mRNA isoform 1 expression was significantly higher in the E2A-PBX1 group compared to all other groups combined (p = 0.0001, Mann Whitney test).
Figure 2.
Cell surface ROR1 expression in ALL cell lines.
(A) Flow cytometry profile of pre-B-ALL cell line 697 stained with mouse anti-human ROR1 mAb 2A2 (gray) or mouse IgG1 as isotype control (white) followed by DyLight 649-conjugated goat anti-mouse IgG pAbs. (B) Using the same method, 14 ALL cell lines that represent the immunophenotypic and genotypic heterogeneity in ALL were analyzed for cell surface ROR1 expression. The ΔMFI was calculated as the difference of MFI values obtained for mAb 2A2 and isotype control. Six ALL cell lines revealed cell surface ROR1 expression. (C) Protein lysates (10 µg/lane) from the 5 positive pre-B-ALL cell lines, but not from negative cell lines and normal B cells, revealed a ∼120 kDa band by Western blotting using polyclonal goat anti-human ROR1 pAbs followed by HRP-conjugated donkey anti-goat IgG pAbs. The membrane was stripped and probed with rabbit anti-human GAPDH (∼36 kDa) pAbs followed by HRP-conjugated goat anti-rabbit IgG pAbs to confirm uniform loading of protein lysates. (D) Immunohistochemical analysis of FFPE slides of pre-B-ALL cell lines 697 (left) and REH (right) probed with goat anti-human ROR1 pAbs. (Magnification 20×).
Figure 3.
Cell surface ROR1 expression on primary ALL blasts.
(A) Flow cytometry profiles of primary ALL blasts from B-ALL patients showing the various intensities of cell surface ROR1 expression. (A) Representative ROR1-negative sample. (B), (C), and (D) ROR1+ samples with MFI scores “+”, “++”, and “+++”, respectively. (MFI score: 0, ΔMFI <2; +, ΔMFI >2 and <5; ++, ΔMFI >5 and <10; +++, ΔMFI >10). Four-color flow cytometry was done by first staining with mouse anti-human ROR1 mAb 2A2 (gray) or mouse IgG (white) as isotype control followed by DyLight 649-conjugated goat anti-mouse IgG pAbs, addition of propidium iodide to exclude dead cells from the analysis, and gating with an FITC-conjugated anti-human CD19 mAb and a PE-conjugated anti-human CD10 mAb. Primary ALL blasts in the CD19+ CD10+ gate (left) uniformly expressed cell surface ROR1 (right).
Figure 4.
Cell surface ROR1 expression on primary ALL blasts.
(A) Immunohistochemical analysis of FFPE slides of bone marrow from a pre-B-ALL patient with hyperdiploid genotype (ID 35; Table 1) probed with normal goat pAbs (control; middle panel) and goat anti-human ROR1 pAbs (ROR1; right panel). The nuclei of ALL blasts were identified by hematoxylin and eosin staining (H&E; left panel). (B) Proportion of ROR1+ cases by genotype based on 56 pediatric ALL patients (Table 1).
Table 1.
Origin and characteristics of primary ALL blasts.
Figure 5.
Absence of cell surface ROR1 in normal adult and pediatric tissues.
(A) Immunohistochemical analysis of FFPE slides of a selection of normal adult tissues (top, heart; middle, cerebrum; bottom, adipocytes) probed with goat anti-human ROR1 pAbs revealed cytoplasmic staining in cardiomyocytes and neurons and plasma membrane staining in adipocytes. (Magnification 20×). (B) A protein lysate (5 µg/lane) from Burkitt lymphoma cell line CA-46 was compared to protein lysates (20 µg/lane) from 14 normal pediatric tissues by Western blotting using polyclonal goat anti-human ROR1 pAbs followed by HRP-conjugated donkey anti-goat IgG pAbs. None of the normal pediatric tissues revealed a firm ∼120 kDa band indicative of cell surface ROR1 expression although a faint ∼120 kDa band was detected in heart (Hrt), kidney (Kid), and uterus (Ute). Bands of higher and lower molecular weight are discussed in the text. The previously described [25] recombinant fusion protein Fc-ROR1, which runs as ∼75-kDa band under reducing conditions, was used as additional control. The membrane was stripped and probed with rabbit anti-human GAPDH (∼36 kDa) followed by HRP-conjugated goat anti-rabbit IgG pAbs to confirm uniform loading of protein lysates. Abbreviations: Liv, liver; Lun, lung; Pan, pancreas; Ova, ovary; B, CD19+ B cells; Cbr, cerebrum; Cbe, cerebellum; Hrt, heart; Spl, spleen; Bla, bladder; Int, small intestine; Sto, stomach; Kid, kidney; Ute, uterus; Mus, skeletal muscle.
Table 2.
ROR1 expression in normal adult tissues as analyzed by immunohistochemistry.
Figure 6.
ROR1-mediated antibody internalization.
Confocal immunofluorescence microscopy demonstrated internalization of mouse anti-human ROR1 mAb 2A2 by pre-B ALL cell line 697. The cells were incubated with mAb 2A2 for 1 h on ice followed by incubation at 37°C for 0–2 h in the absence or presence of PAO. Subsequently, the cells were fixed, permeabilized, stained with DyLight 649-conjugated goat anti-mouse IgG pAbs, co-stained with DAPI, and analyzed with a Zeiss LSM 510 UV confocal microscope (objective 63×). In the absence of PAO, mAb 2A2 (pink) was found uniformly on the cell surface at 0 h (top panel) and predominantly in cytoplasmic clusters at 2 h (middle panel). No internalization at 2 h was seen in the presence of PAO (bottom panel). DAPI staining the nucleus is seen in blue. (Original magnification×2).