Fig 2: Galectin-1 inhibition reduces ALL cell migration. US7 (a) or LAX56 (b) cells treated with DMSO or 10 µM PTX008 were allowed to migrate toward aMEM + 2%FBS (control), 200 ng/mL SDF1a, or a confluent layer of irradiated OP9 cells. Left panels, representative experiment. Error bars, standard deviation (**, p<0.01, ***, p<0.001, 95% CI, 2-way ANOVA). Right panels, summary data from 3 replicate experiments with 3 replicate samples each. Error bars, standard error of mean (**, p<0.01, ***, p<0.001, 95% CI, 2-way ANOVA)

Fig 3: Galectin-1 is expressed in different BP-ALL subtypes. a Meta-analysis of GSE28497 representing 270 ALL diagnosis samples and four CD19 + CD10+ control normal B-cell precursors (normal BM). ALLs are subdivided into categories based on karyotype abnormalities as indicated [18] The category MLL includes BP-ALLs with different MLL gene rearrangements (bars, mean ± SEM; ***p=0.001, 95% CI, 1-way ANOVA; other comparisons, ns.). Each symbol represents one sample. MFI, mean fluorescent intensity. b Western blot analysis of Ph-negative US7, Ph-positive TXL2, relapse LAX56 and diagnosis LAX57 BP-ALL patient-derived lines (Genetex Galectin-1 Abs). c Real-time RT/PCR for Galectin-1 mRNA on the indicated ALLs cultured for 24 h in complete medium but without OP9 stroma, then stimulated with the indicated recombinant proteins for an additional 24 h. Two samples represent ALL cells grown with irradiated OP9 stroma as indicated. Values shown were normalized to a reference gene, GAPDH, and are comparisons with non-treated US7 cells. One of two experiments, similar results. US7 GST-Gal3 compared to GST *p < 0.05. 95% CI, 1-way ANOVA. d FACS analysis for Galectin-1 and Galectin-3 with and without cross-linking with DTSSP to stabilize Galectin-1 and Galectin-3 protein complexes on the cell surface. Antibodies used are indicated above the panels. e FACS analysis (antibodies- R&D Systems Galectin-1) for cell surface and total Galectin-1 expression in the indicated ALLs co-cultured on OP9 stromal cells mitotically inactivated by mitomycin C treatment. Percentages indicated are for the lower right-hand quadrant. f Galectin-1 levels measured by ELISA consisting of GeneTex anti-Galectin-1 capture antibodies and Abcam anti-Galectin-1 primary detection antibodies with secondary HRP-conjugated goat anti-mouse antibodies. Values represent ng/ml in undiluted control and ALL human plasma samples (n = 5–6 for each) from PB and BM collected with anticoagulant after removal of the cellular component

Fig 4: Inhibition of Galectin-1 results in cell cycle arrest. ALL cells grown on (mitotically inactivated, irradiated) OP9 stromal cells were treated with DMSO or 10 µM PTX008, as indicated, for 72 h. a Representative flow cytometry images of BrdU and 7-AAD staining of LAX56 cells. Gates represent apoptotic/subG0 (Apop), G0/G1 phase (G0-G1), S phase (S), and G2/M phase (G2) of the cell cycle. b Overall cell cycle analysis of LAX56 and LAX57 cells. Graphs are combined data from 3 replicate experiments with 3 replicate samples. Error bars, standard error of mean (p<0.001, 95% CI, 2-way ANOVA). c, d. LAX56 treated for 72 h with 10 µM PTX008, 5 nM vincristine, or a combination of both and control, DMSO treated cells in the absence or presence of (mitotically inactivated, mitomycin-C-treated) OP9 stromal cells as indicated below the panels. Error bars, standard error of mean of triplicate samples. c Cell cycle analysis performed as in panel (a). (**, p<0.01, ***, p<0.001, ****, p < 0.0001 95% CI, 2-way ANOVA, control versus single or combination treatment). d Percentage of apoptotic cells measured by staining for Annexin V and 7-AAD (combination treatment compared to other samples **, p<0.01, ***, p<0.001, 95% CI, 1-way ANOVA)

Fig 5: PTX008 inhibits binding of Galectin-1 to the surface of ALL cells. LAX56 cells (2 × 104) were plated without OP9 stroma and treated for 1 h with DMSO or 10 µM PTX008. The ability of human recombinant Galectin-1 or human recombinant GST-Galectin-3 or control GST to cause agglutination of the LAX56 cells was then quantitated by light microscopy. a Representative images of cells. Left two panels show controls of cells incubated with GST only or DMSO only. b quantification of number of aggregates in each condition. (*p < 0.05)