Journal: Leukemia research

Article Title: ROR1 targeted immunoliposomal delivery of OSU-2S shows selective cytotoxicity in t(1;19)(q23; p13) translocated B-cell acute lymphoblastic leukemia

doi: 10.1016/j.leukres.2022.106872

Figure Lengend Snippet: A) ROR1 Mean Fluorescence Intensity (MFI) normalized to isotype (ΔMFI) of non t(1;19) and t(1;19) translocated B-ALL samples. Translocated patient derived cells show specific ROR1 expression (n = 3, p = 0.0002, mean difference in MFI 403.3 ± 48.04). Histograms show representative surface ROR1 expression in t(1:19) translocated and non t(1:19) B-ALL patient derived CD19 cells. B) ROR1 Mean Fluorescence Intensity (MFI) normalized to isotype (ΔMFI) of non t(1;19) (NALM-6, RS4;11) and t(1;19) translocated (697) B-ALL cell lines. t(1;19) translocated cells show specific ROR1 expression (n = 3, p = <0.0001, mean increase in MFI 697 vs NALM-6 = 2528, 697 vs RS4;11 = 2591). C) Representative histograms showing ROR1 expression on 697 cells but not non t(1;19) NALM-6 and RS4;11 cells as detected by flow cytometry. D) Scheme depicting preparation and expected action of ROR1 targeting immunoliposomes encapsulating OSU-2S (2A2-OSU-2S-ILP). E) Representative analysis of size and concentration of 2A2-OSU-2S-ILPs using Nanoparticle Tracking Analysis by NanoSight. Mean size was 186.9 +/− 0.8nm, mean concentration was 1.2×1013 +/− 1.05 × 1011 particles/ml. F) 2A2-OSU-2S-ILPs mediate selective cytotoxicity in ROR1 + 697 cells as compared to IgG-OSU-2S-ILP (n = 8, p < 0.0001, mean decrease in viability 61.62%). G) 2A2-OSU-2S-ILPs mediate selective cytotoxicity in ROR1 + t(1:19) primary ALL cells as compared to IgG-OSU-2S-ILP and Empty ILPs (n = 3, p = 0.0174, mean decrease in viability 35.14%). H) No significant relative cytotoxicity was observed with 2A2-OSU-2S-ILPs in ROR1- non t(1;19) cell line (NALM-6) and B-ALL primary cells. I) 2A2-OSU-2S-ILP significantly reduced tumor burden in the bone marrow of treated mice as compared to IgG control as detected by human CD45+/CD19+cells [p = 0.022, n = 5 (IgG-OSU-2S-ILP), n = 6 (2A2-OSU-2S-ILP), mean decrease = 1.751 ± 0.6372 × 106 cells. J) 2A2-OSU-2S-ILP (n = 9) treatment significantly improves survival in 697 CDX model as compared to (n = 6) IgG-OSU-2S-ILP (p = 0.013) and (n = 5) 2A2-Empty-ILP (p = 0.0044) treated mice.

Article Snippet: Antibodies used include c-Myc, p21 and β-Actin (Cell Signaling, Danvers, MA), CD45-FITC, CD3-PE-Cy7, CD19-BV785 (BioLegend, San Diego, CA), murine CD45-PECF594 (BD Biosciences) and ROR1- PE (R&D Systems).

Techniques: Fluorescence, Derivative Assay, Expressing, Flow Cytometry, Concentration Assay

Journal: Leukemia

Article Title: New anti-IL-7Rα monoclonal antibodies show efficacy against T cell acute lymphoblastic leukemia in pre-clinical models

doi: 10.1038/s41375-019-0531-8

Figure Lengend Snippet: IL-7Rα is expressed on T-ALL and anti-IL-7Rα mediates ADCC. a IL-7Rα expression and ADCC of murine D1-hIL-7Rα P1 and D1-pMIG. (Left) D1-hIL-7Rα P1 and D1-pMIG gated on GFP+ cells. Histogram of IL-7Rα using a commercial MAb (blue) with an unstained (red) control. (Right) LDH release assay using D1-hIL-7Rα P1 or D1-pMIG as target cells and freshly isolated human NK cells as effectors. Cells were incubated for 4 h in medium alone, or medium with 10 μg/mL of anti-IL-7Rα 4A10. b IL-7Rα expression and ADCC of 4 human PDX T-ALL. (Left) Patient derived leukemia cells gated on human CD45. Histogram of IL-7Rα (blue) with an unstained (red) control. (Right) LDH release assay using patient derived leukemia cells as targets and freshly isolated human NK cells as effectors. Cells were incubated for 4 h in medium with isotype control, medium with 10 μg/mL anti-IL-7Rα 4A10, 10 μg/mL anti-IL-7Rα 2B8, or the combination of both MAbs

Article Snippet: An anti-murine CD45 FITC (30-F11, cat #553079) was purchased from BD Biosciences.

Techniques: Expressing, Lactate Dehydrogenase Assay, Isolation, Incubation, Derivative Assay

Journal: Leukemia

Article Title: New anti-IL-7Rα monoclonal antibodies show efficacy against T cell acute lymphoblastic leukemia in pre-clinical models

doi: 10.1038/s41375-019-0531-8

Figure Lengend Snippet: Anti-IL-7Rα controls the growth of patient- derived T-ALL xenografts in NOD. SCID mice. a Experimental protocol. Patient derived T-ALL xenografts were established by intravenous injection in NOD. SCID mice on day 0. Mice were given weekly injections of anti-IL-7Rα or isotype control beginning on day 1 for a total of 11 injections. b Reduction of leukemia burden in the peripheral blood of a subset of treated mice (n = 2–3) compared to isotype treated controls (n = 2–3). c Kaplan Meier curve depicting survival prolongation in xenografts by anti-IL-7Rα 4A10 (treated; n = 7) compared to isotype control treated mice (control; n = 6). One mouse that did not develop leukemia was excluded from the control group. Survival analysis by Log-rank (Mantel-Cox) test. d Leukemia burden in the bone marrow by anti-hIL7Rα 4A10 (treated) at D33, compared to isotype control treated mice. Leukemia burden was determined by flow cytometry using antibodies specific for human CD45. Bars represent mean values and n = 3 for both groups

Article Snippet: An anti-murine CD45 FITC (30-F11, cat #553079) was purchased from BD Biosciences.

Techniques: Derivative Assay, Injection, Flow Cytometry

Journal: Leukemia

Article Title: New anti-IL-7Rα monoclonal antibodies show efficacy against T cell acute lymphoblastic leukemia in pre-clinical models

doi: 10.1038/s41375-019-0531-8

Figure Lengend Snippet: Chemotherapy resistant patient derived leukemia cells express high levels of IL-7Rα. a Percentage of human CD45 positive leukemia cells in the T-ALL PDX cell bank (left). Expression of IL-7Rα on freshly thawed T-ALL PDX cells (right). b Patient derived T-ALL xenografts were injected into NOD.SCID mice on day 0. When leukemia burden reached 1% in peripheral blood, mice were treated with a combination of dexamethasone (5 mg/kg, daily) and vincristine (0.15 mg/kg, weekly) for 4 weeks. One week after the last chemotherapy treatment, relapsing/refractory hCD45+ leukemia cells (left) were analyzed for hIL-7Rα expression by flow cytometry (right) at day 68 post leukemia transfer. Relapsing leukemia cells had increased expression and MFI of IL-7Rα (right side; n = 3). c Patient derived T-ALL xenografts were injected into NOD.SCID mice on day 0. When leukemia burden reached 1% in peripheral blood, mice were randomized into dexamethasone (5 mg/kg, daily), or a combination of dexamethasone and vincristine (0.15 mg/kg, weekly) treatment groups and treated for 6 weeks. Relapsing leukemia cells in surviving mice were analyzed at day 95 (2.5 weeks after last chemotherapy treatment) for hIL-7Rα expression by flow cytometry. Relapsing leukemia cells (left) in mice receiving dexamethasone alone had increased MFI, and mice treated with a combination of dexamethasone and vincristine had increased levels and MFI of IL-7Rα (right; n = 1)

Article Snippet: An anti-murine CD45 FITC (30-F11, cat #553079) was purchased from BD Biosciences.

Techniques: Derivative Assay, Expressing, Injection, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: Hematopoietic Stem Cell–Derived Cancer–Associated Fibroblasts Are Novel Contributors to the Pro-Tumorigenic Microenvironment 1 2

doi: 10.1016/j.neo.2015.04.004

Figure Lengend Snippet: qRT-PCR: RT and PCR step controls. mRNA expression of α-SMA, Col I, vimentin, FAP, CD45, MMP-2, MMP-3, MMP-9, and MMP-14 in primary skin fibroblasts is shown. Relative expression is normalized to RPL13A as described in the Materials and methods section.

Article Snippet: Fluorochrome-conjugated, biotinylated or purified versions of the following antibodies were used: anti–Sca-1 (anti–Ly-6A/E[D7]), anti–c-kit (anti-CD117[2B8]), anti–Gr-1 (anti–Ly-6G[RB6-8C5]), anti-CD45R/B220 (RA3-6B2), anti–Thy-1.2 (30-H12), anti–TER-119 (TER-119), anti-CD3e (145-2C11), anti-CD45 (leukocyte common antigen, Ly-5;30-F11), anti-CD8a (53-6.7), anti-CD4 (GK1.5), and anti-CD45.1 (A20) from BD Biosciences (San Jose, CA); anti-F4/80 (BM8) and anti-CD34 (RAM34) from eBioscience (San Diego, CA); anti–β-actin–HRP (5125 s) from Cell Signaling Technology (Danvers, MA); anti–Col I from Rockland (Limerick, PA); anti–α-SMA (ab5694), anti-vimentin, anti–wide spectrum cytokeratin (WS CyK), anti–Col I (ab21286), anti-CD45 (ab10558), anti-CD31 (ab13970), and anti-GFP (anti-green fluorescent protein, ab28364) from Abcam (Cambridge, MA); VEGF-A, TGF-β1 neutralizing antibodies from R&D Systems (Minneapolis, MN); isotype control antibodies from BD Biosciences; secondary antibodies from Jackson ImmunoResearch (West Grove, PA) or BD Pharmingen (San Diego, CA).

Techniques: Quantitative RT-PCR, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Hematopoietic Stem Cell–Derived Cancer–Associated Fibroblasts Are Novel Contributors to the Pro-Tumorigenic Microenvironment 1 2

doi: 10.1016/j.neo.2015.04.004

Figure Lengend Snippet: Isolation of HSC-CAFs from clonally engrafted mice. (A) Representative image of EGFP + (HSC-derived) and EGFP − cells (asterisks) isolated from an LLC tumor grown in a clonally engrafted mouse. (B) qRT-PCR analysis of CD45 expression in HSC-CAFs (black bar) and LLC tumor cells (hatched bar; * P ≤ .0001). (C) DIC and immunofluorescence staining of representative isolated cells show morphology (DIC), nuclei (Hoechst dye, HO), expression of EGFP, and CD45 (Ab stain). Merged images (right panels) represent EGFP, HO, and antibody stain (Ab stain). Secondary only (no primary antibody) controls for immunofluorescence images are depicted in . Bars, 100 μm (B) and 25 μm (D). Isolation of HSC-CAFs from clonally engrafted mice. (A) Representative image of EGFP + (HSC-derived) and EGFP − cells (asterisks) isolated from an LLC tumor grown in a clonally engrafted mouse. (B) qRT-PCR analysis of CD45 expression in HSC-CAFs (black bar) and LLC tumor cells (hatched bar; * P ≤ .0001). (C) DIC and immunofluorescence staining of representative isolated cells show morphology (DIC), nuclei (Hoechst dye, HO), expression of EGFP, and CD45 (Ab stain). Merged images (right panels) represent EGFP, HO, and antibody stain (Ab stain). Secondary only (no primary antibody) controls for immunofluorescence images are depicted in Supplementary Figure S2. Bars, 100 μm (B) and 25 μm (D).

Article Snippet: Fluorochrome-conjugated, biotinylated or purified versions of the following antibodies were used: anti–Sca-1 (anti–Ly-6A/E[D7]), anti–c-kit (anti-CD117[2B8]), anti–Gr-1 (anti–Ly-6G[RB6-8C5]), anti-CD45R/B220 (RA3-6B2), anti–Thy-1.2 (30-H12), anti–TER-119 (TER-119), anti-CD3e (145-2C11), anti-CD45 (leukocyte common antigen, Ly-5;30-F11), anti-CD8a (53-6.7), anti-CD4 (GK1.5), and anti-CD45.1 (A20) from BD Biosciences (San Jose, CA); anti-F4/80 (BM8) and anti-CD34 (RAM34) from eBioscience (San Diego, CA); anti–β-actin–HRP (5125 s) from Cell Signaling Technology (Danvers, MA); anti–Col I from Rockland (Limerick, PA); anti–α-SMA (ab5694), anti-vimentin, anti–wide spectrum cytokeratin (WS CyK), anti–Col I (ab21286), anti-CD45 (ab10558), anti-CD31 (ab13970), and anti-GFP (anti-green fluorescent protein, ab28364) from Abcam (Cambridge, MA); VEGF-A, TGF-β1 neutralizing antibodies from R&D Systems (Minneapolis, MN); isotype control antibodies from BD Biosciences; secondary antibodies from Jackson ImmunoResearch (West Grove, PA) or BD Pharmingen (San Diego, CA).

Techniques: Isolation, Derivative Assay, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Hematopoietic Stem Cell–Derived Cancer–Associated Fibroblasts Are Novel Contributors to the Pro-Tumorigenic Microenvironment 1 2

doi: 10.1016/j.neo.2015.04.004

Figure Lengend Snippet: Expression profiles of HSC-CAFs. (A) qRT-PCR analysis of CD45 in HSC-CAFs isolated from non-transplanted mice injected with LLC tumor cells (black bar) and LLC tumor cells (hatched bar; * P ≤ .0001). (B) DIC and representative immunofluorescence staining of EGFP, nuclear (Hoescht dye, HO), and CD45 (Ab stain) expression in HSC-CAFs are depicted. (C) qRT-PCR analysis of activated fibroblast markers Col I (* P = .0001), α-SMA (* P ≤ .0001), FAP (* P = .0176), and vimentin (* P ≤ .0001) in HSC-CAFs (black bars) and LLC tumor cells (hatched bars). (D) qRT-PCR analysis of MMP-2 (* P = .5931), MMP-3 (* P ≤ .0001), MMP-9 (* P = .0012), and MMP-14 (* P = .0007) in HSC-CAFs (black bars) and LLC tumor cells (hatched bars). (E) DIC and representative immunofluorescence staining of nuclei (Hoescht dye, HO), EGFP, and Col I, α-SMA, and vimentin (Ab stain) expression in HSC-CAFs are depicted. Bars, 25 μm (B, E).

Article Snippet: Fluorochrome-conjugated, biotinylated or purified versions of the following antibodies were used: anti–Sca-1 (anti–Ly-6A/E[D7]), anti–c-kit (anti-CD117[2B8]), anti–Gr-1 (anti–Ly-6G[RB6-8C5]), anti-CD45R/B220 (RA3-6B2), anti–Thy-1.2 (30-H12), anti–TER-119 (TER-119), anti-CD3e (145-2C11), anti-CD45 (leukocyte common antigen, Ly-5;30-F11), anti-CD8a (53-6.7), anti-CD4 (GK1.5), and anti-CD45.1 (A20) from BD Biosciences (San Jose, CA); anti-F4/80 (BM8) and anti-CD34 (RAM34) from eBioscience (San Diego, CA); anti–β-actin–HRP (5125 s) from Cell Signaling Technology (Danvers, MA); anti–Col I from Rockland (Limerick, PA); anti–α-SMA (ab5694), anti-vimentin, anti–wide spectrum cytokeratin (WS CyK), anti–Col I (ab21286), anti-CD45 (ab10558), anti-CD31 (ab13970), and anti-GFP (anti-green fluorescent protein, ab28364) from Abcam (Cambridge, MA); VEGF-A, TGF-β1 neutralizing antibodies from R&D Systems (Minneapolis, MN); isotype control antibodies from BD Biosciences; secondary antibodies from Jackson ImmunoResearch (West Grove, PA) or BD Pharmingen (San Diego, CA).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Injection, Immunofluorescence, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Hematopoietic Stem Cell–Derived Cancer–Associated Fibroblasts Are Novel Contributors to the Pro-Tumorigenic Microenvironment 1 2

doi: 10.1016/j.neo.2015.04.004

Figure Lengend Snippet: HSC-CAFs contribute to the pro-tumorigenic microenvironment. (A) Representative flow cytometric analysis shows percent of EGFP-expressing cells that co-express CD45 and Col I (68%, left panel) and those that co-express CD45 and α-SMA (90%, right panel). Gates were set on the basis of fluorescence minus one (FMO) controls. (B) Western blot analysis shows 150 and 42 kDa bands for Col I and α-SMA, respectively, from HSC-CAFs isolated from three biological replicates (M1, M2, and M3); β-actin loading control is shown below. (C) Functional MMP activity assay in HSC-CAF-CM (black squares) and αMEM (empty circles) shows that HSC-CAF-CM contains active MMPs (* P ≤ .05).

Article Snippet: Fluorochrome-conjugated, biotinylated or purified versions of the following antibodies were used: anti–Sca-1 (anti–Ly-6A/E[D7]), anti–c-kit (anti-CD117[2B8]), anti–Gr-1 (anti–Ly-6G[RB6-8C5]), anti-CD45R/B220 (RA3-6B2), anti–Thy-1.2 (30-H12), anti–TER-119 (TER-119), anti-CD3e (145-2C11), anti-CD45 (leukocyte common antigen, Ly-5;30-F11), anti-CD8a (53-6.7), anti-CD4 (GK1.5), and anti-CD45.1 (A20) from BD Biosciences (San Jose, CA); anti-F4/80 (BM8) and anti-CD34 (RAM34) from eBioscience (San Diego, CA); anti–β-actin–HRP (5125 s) from Cell Signaling Technology (Danvers, MA); anti–Col I from Rockland (Limerick, PA); anti–α-SMA (ab5694), anti-vimentin, anti–wide spectrum cytokeratin (WS CyK), anti–Col I (ab21286), anti-CD45 (ab10558), anti-CD31 (ab13970), and anti-GFP (anti-green fluorescent protein, ab28364) from Abcam (Cambridge, MA); VEGF-A, TGF-β1 neutralizing antibodies from R&D Systems (Minneapolis, MN); isotype control antibodies from BD Biosciences; secondary antibodies from Jackson ImmunoResearch (West Grove, PA) or BD Pharmingen (San Diego, CA).

Techniques: Expressing, Fluorescence, Western Blot, Isolation, Functional Assay, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Hematopoietic Stem Cell–Derived Cancer–Associated Fibroblasts Are Novel Contributors to the Pro-Tumorigenic Microenvironment 1 2

doi: 10.1016/j.neo.2015.04.004

Figure Lengend Snippet: HSC-CAFs contribute to murine breast cancer. (A) qRT-PCR analysis of CD45 expression in HSC-CAFs from E0771 tumors (black bar) and E0771 tumor cells (hatched bar; * P ≤ .0001). (B) DIC and immunofluorescence staining of representative E0771 HSC-CAFs show morphology (DIC), nuclei (Hoechst dye, HO), expression of EGFP, and CD45 (Ab stain). (C, D) qRT-PCR analysis (C) for fibroblast markers Col I (* P ≤ .0001), α-SMA (* P = .0104), FAP (* P = .0016), and vimentin ( P = .0082) and (D) MMP-2 (* P ≤ .0001), MMP-3 (* P = .0013), MMP-9 (* P ≤ .0001), and MMP-14 (* P ≤ .0001) in E0771 HSC-CAFs (black bars) and E0771 tumor cells (hatched bars). (E) DIC and immunofluorescence staining of representative E0771 HSC-CAFs show morphology (DIC), nuclei (HO), EGFP expression, antibody (Col I, α-SMA, vimentin, F4/80, WS CyK) stain (Ab stain), and merged images from representative cells. (F) HUVEC tube formation assay of cells treated with αMEM (negative control), HUVEC medium (positive control), or E0771 HSC-CAF-CM. Tubes and vascular pattern were visualized with DIC microscopy (left panels) and Calcein AM dye (right panels). (G) Number of nodes (left panel), segments (middle panel), and Ma:Mv (right panel) from F were quantified. In G, * P < .05 compared to αMEM and ψ P = .0280 compared to HUVEC medium. Bars, 25 μm (A, C) and 250 μm (F).

Article Snippet: Fluorochrome-conjugated, biotinylated or purified versions of the following antibodies were used: anti–Sca-1 (anti–Ly-6A/E[D7]), anti–c-kit (anti-CD117[2B8]), anti–Gr-1 (anti–Ly-6G[RB6-8C5]), anti-CD45R/B220 (RA3-6B2), anti–Thy-1.2 (30-H12), anti–TER-119 (TER-119), anti-CD3e (145-2C11), anti-CD45 (leukocyte common antigen, Ly-5;30-F11), anti-CD8a (53-6.7), anti-CD4 (GK1.5), and anti-CD45.1 (A20) from BD Biosciences (San Jose, CA); anti-F4/80 (BM8) and anti-CD34 (RAM34) from eBioscience (San Diego, CA); anti–β-actin–HRP (5125 s) from Cell Signaling Technology (Danvers, MA); anti–Col I from Rockland (Limerick, PA); anti–α-SMA (ab5694), anti-vimentin, anti–wide spectrum cytokeratin (WS CyK), anti–Col I (ab21286), anti-CD45 (ab10558), anti-CD31 (ab13970), and anti-GFP (anti-green fluorescent protein, ab28364) from Abcam (Cambridge, MA); VEGF-A, TGF-β1 neutralizing antibodies from R&D Systems (Minneapolis, MN); isotype control antibodies from BD Biosciences; secondary antibodies from Jackson ImmunoResearch (West Grove, PA) or BD Pharmingen (San Diego, CA).

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, HUVEC Tube Formation Assay, Negative Control, Positive Control, Microscopy

Journal: REPRODUCTION

Article Title: Investigations into the mechanisms controlling parturition in cattle

doi: 10.1530/rep-11-0471

Figure Lengend Snippet: Figure 5 Identification of leucocytes in placentomes by immunostaining for the pan-leucocyte marker CD45. (A and B) Results from control experiments using bovine lymphatic tissue. In (B), the specific primary antibody was replaced by an irrelevant isotypic murine mAb. (C) Placentome of a parturi- ent cow. Positive staining in leucocytes situated in a blood vessel of a major caruncular septum (surrounded by dotted line). Positive reactions in the adjacent tissue formed by interdigitating tertiary chorionic villi and maternal crypts only occur sporadically (arrows). (D) Leucocytes in caruncular capillaries.

Article Snippet: Primary antibodies used were the following: a rabbit mAb against a synthetic peptide from the C-terminus of rat COX2 (Thermo Fisher Scientific, Fremont, CA, USA), a polyclonal rabbit antiserum against recombinant bovine CYP17 (gift from Prof. A Conley, UC Davis; reference: Peterson et al. 2001), a monoclonal murine antibody against the highly conserved C-terminus of PR (clone 10A9; Immunotech, Hamburg, Germany), a polyclonal rabbit IgG antibody against amino acids 346–367 of human glucocorticoid receptor (PA1-511A; Affinity BioReagents, Golden, CO, USA), and a murine mAb against bovine CD45 (MCA832S; AbD Serotec, Düsseldorf, Germany).

Techniques: Immunostaining, Marker, Control, Staining