Journal: Science translational medicine

Article Title: Radiotherapy and immunotherapy converge on elimination of tumor-promoting erythroid progenitor cells through adaptive immunity

doi: 10.1126/scitranslmed.abb0130

Figure Lengend Snippet: (A) Colony formation assay of MC38 cells treated with 0, 1, or 3 Gy irradiation. MC38 cells were either co-cultured with 2x106 Ter-cells sorted from spleens of tumor-bearing mice or treated with 100 ng/ml artemin. (B) 1x105MC38-OTI-zsGreen cells were co-cultured with 2x105 CD8+ T cells purified from OTI mice in a 96-well U bottomed plate. Tumor cells were either co-cultured with 2x105 Ter-cells sorted from spleens of tumor-bearing mice or treated with 100 ng/ml artemin for 6 hours as indicated. Apoptosis of MC38 cells was analyzed by flow cytometry. (C) C57BL/6 mice were inoculated with LLC cells. On day 10 post-inoculation, tumor-bearing mice were treated with IR or anti-PD-L1. Mice were transferred i.v. with 1x107 purified Ter-cells every other day for a total of three times. Tumor growth was monitored. (D) C57BL/6 mice were inoculated with LLC cells. On day 10 post-inoculation, tumor-bearing mice were treated with IR or anti-PD-L1. Mice were treated via intratumoral (i.t.) injection with 0.5 artemin μg/mouse every other day. Tumor growth was monitored. (E-F) C57BL/6 mice were inoculated with LLC cells. On day 10 post-inoculation, tumor-bearing mice were treated with IR (E), or anti-PD-L1 (F). Mice were treated i.v. with 20 U EPO/mouse every other day. The number of Ter-cells in spleen was analyzed by flow cytometry. (G) C57BL/6 mice were inoculated with LLC cells. On day 10 post-inoculation, tumor-bearing mice were treated with IR or anti-PD-L1. Mice were treated i.v. with 20 U EPO/mouse every other day. The artemin concentrations in serum were determined by ELISA on day 10 post-treatment. (H-I) Tumor growth of LLC tumor-bearing mice treated with EPO and either IR (H) or anti-PD-L1 (I). (J) C57BL/6 mice were inoculated with LLC cells and treated with IR, EPO, and/or anti-Ter119. Tumor growth was monitored. (K) C57BL/6 mice were inoculated with LLC cells and treated with anti-PD-L1, EPO, and/or anti-Ter119. Tumor growth was monitored. Representative data are shown from three experiments. Data are represented as mean ± SD. All data in this figure were calculated with one-way ANOVA with Bonferroni’s multiple comparison tests. *p < 0.05, **p < 0.01, and ***p < 0.001. NS, not significant.

Article Snippet: Recombinant mouse artemin (1085-AR), EPO (959-ME), and mouse artemin antibody (AF1085) were purchased from R&D Systems.

Techniques: Colony Assay, Irradiation, Cell Culture, Purification, Flow Cytometry, Injection, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Science translational medicine

Article Title: Radiotherapy and immunotherapy converge on elimination of tumor-promoting erythroid progenitor cells through adaptive immunity

doi: 10.1126/scitranslmed.abb0130

Figure Lengend Snippet: (A-B) Tumor growth of LLC tumor-bearing mice treated with Ter119-depleting antibody and either IR (A), or anti-PD-L1 (B). (C) Tumor growth of LLC tumor-bearing mice treated with artemin-neutralizing antibody i.t. and either IR or anti-PD-L1. (D) WT or GFRα3 KD MC38 cells were inoculated in C57BL/6 mice, and tumor growth was monitored following treatment with either IR or anti-PD-L1. (E) WT or RET KO MC38 cells were inoculated in C57BL/6 mice, and tumor growth was monitored following treatment with either IR or anti-PD-L1. (F-G) Tumor growth of LLC tumor-bearing mice treated with the RET inhibitor LOXO-292 and either IR (F) or anti-PD-L1 (G). (H) C57BL/6 mice were inoculated with LLC cells and treated with either IR or EPO alone or in combination with LOXO-292. Tumor growth was monitored. (I) C57BL/6 mice were inoculated with LLC cells and treated with either anti-PD-L1 or EPO, and/or LOXO-292. Tumor growth was monitored. Representative data are shown from two or three experiments. Data are represented as mean ± SD. All data in this figure were calculated with one-way ANOVA with Bonferroni’s multiple comparison tests. **p < 0.01 and ***p < 0.001.

Article Snippet: Recombinant mouse artemin (1085-AR), EPO (959-ME), and mouse artemin antibody (AF1085) were purchased from R&D Systems.

Techniques: Comparison

Journal: Genes & Cancer

Article Title: Erythropoietin Receptor Signaling through STAT3 Is Required for Glioma Stem Cell Maintenance

doi: 10.1177/1947601909356352

Figure Lengend Snippet: Targeting erythropoietin receptor (EPOR) expression in glioma stem cells (GSCs) reduces cell growth associated with decreased survival and neurosphere formation capacity. (A) Western blotting demonstrated successful knockdown of EPOR expression using lentivirus expressing two different EPOR-targeted short-hairpin RNA (shRNA) constructs (EPOR shRNA1 and EPOR shRNA2) in comparison to a nontargeting shRNA (NT) in T3691 and T3359 GSCs. (B) EPOR knockdown with two distinct shRNAs decreased the growth of T3691 GSCs in comparison to nontargeting control. *P < 0.001, one-way analysis of variance (ANOVA), n = 3. (C) Targeting EPOR significantly inhibited GSC proliferation. [3H]thymidine incorporation was decreased in T3691 GSCs infected with either one of two different EPOR shRNAs in comparison to a nontargeting control shRNA. *P < 0.001, one-way ANOVA, n = 5. (D) Loss of EPOR expression significantly increased apoptosis. Targeting EPOR with two different shRNAs increased relative caspase 3/7 activity (Promega, Madison, WI) in comparison to nontargeting control shRNA in T3691 GSCs. *P < 0.001, one-way ANOVA, n = 3. (E) Representative images demonstrating reduced neurosphere formation in T3359 and T3691 GSCs with two different EPOR targeting shRNA constructs (EPOR shRNA1 and EPOR shRNA2) in comparison to nontargeting (NT) control. (F) Targeting EPOR inhibits neurosphere formation capacity in primary passages. Increasing numbers of T3359 GSCs infected with the indicated shRNAs were plated per well and the percentage of wells demonstrating neurospheres determined after 4 days (n = 12). (G) Targeting EPOR inhibits neurosphere formation in sequential passages. Ten T3359 GSCs were plated in the presence of 1 µg/mL recombinant human soluble EPO receptor or bovine serum albumin as the control. For the primary passage, the percentage of wells with neurospheres and the number of neurospheres per well were quantified after 7 days. For the secondary passage, neurospheres formed in the primary passage were harvested and dissociated and the experiment repeated. Representative images of neurospheres formed in primary and secondary passages are shown. *P < 0.05, Student’s t test, n = 5.

Article Snippet: For the serial neurosphere formation assay with soluble EPOR treatment, 10 T3359 or D456MG glioma stem cells were plated into each well of 96-well plates with 200 μL completed neurobasal medium with either 1 μg/mL recombinant human soluble EPO receptor (sEPOR, R&D Systems) or equal amount of bovine serum albumin (BSA; GIBCO, Carlsbad, CA) as the control.

Techniques: Expressing, Western Blot, Knockdown, shRNA, Construct, Comparison, Control, Infection, Activity Assay, Recombinant

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: In vitro erythroid differentiation of canine PB MNCs with recombinant cEPO and recombinant hEPO. (a) Total cumulative cell numbers on differentiation days with cEPO at concentrations of 10 ng/mL ( n = 5), 20 ng/mL ( n = 5), 40 ng/mL ( n = 8), 80 ng/mL ( n = 5), and 160 ng/mL ( n = 2). *Indicates statistical significance with p ≤ 0.05. (b) Photographs of cell pellets on day 10 and day 14 of culture. (c) Total cumulative cell numbers with cEPO (40 and 80 ng/mL) compared to hEPO (40 and 80 ng/mL) ( n = 1). (d) Total cumulative cell numbers with cEPO (30, 40, and 50 ng/mL) compared to those with hEPO (30, 40, and 50 ng/mL) ( n = 1).

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: In Vitro, Recombinant

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Characterization of cell types arising from in vitro erythroid differentiation of canine PB MNCs using cEPO at 40 ng/mL. (a) Representative Wright-Giemsa-stain images of cells during in vitro erythroid differentiation over 17 days. Orange arrowheads; Orthochromatic erythroblast, red arrowheads; Reticulocyte. (b) Identification of the major erythroid lineage cell types emerging from canine PB MNCs during in vitro differentiation, with a comparison to corresponding human cell types. (c) Quantification of cumulative erythroid lineage cell types during in vitro erythroid differentiation over 17 days. Erythroid cell types were quantified by counting over 200 cells per specific day of differentiation from Wright-Giemsa-stain images, and the stacked percentages of cell types were plotted. Scale bars: 10 μM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: In Vitro, Giemsa Stain, Comparison

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Quantification of cumulative erythroid lineage cell types during in vitro erythroid differentiation of canine PB MNCs with different concentrations of cEPO and hEPO. (a and b) Erythroid cell types on day 17 of the in vitro differentiation experiments presented in a were quantified by counting over 200 cells for each specific concentration of cEPO from Wright-Giemsa-stain images. Quantification of cumulative erythroid lineage cell types with 10, 20, 40, 80 and 160 ng/mL of cEPO (a), and the stacked percentages of cell types (b). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (c) Stacked percentages of erythroid cell types with cEPO (40 and 80 ng/mL) compared to those with hEPO (40 and 80 ng/mL). Erythroid cell types on day 21 of the in vitro differentiation experiments presented in (b) were quantified by counting over 100 cells for each specific concentration of cEPO and hEPO from Wright-Giemsa-stain images. RBC; red blood cells, Ortho-E; Ortho-erythroblasts, and Pro-/Baso-/Poly-E; pro-erythroblasts/baso-erythroblasts/poly-erythroblasts.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: In Vitro, Concentration Assay, Giemsa Stain

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Expression profiles of erythroid differentiation marker genes during in vitro erythroid differentiation of canine PB MNCs with cEPO (40 ng/mL) over 3 weeks. (a) mRNA expression profiles of canine erythroid markers: canine alpha hemoglobin stabilizing protein ( cAHSP ; n = 5), canine Krüppel-like factor 1 ( cKLF1 ; n = 8), canine erythropoietin receptor ( cEPOR ; n = 6), and canine transferrin receptor ( cTFRC ; n = 6). qRT-PCR was performed on total RNA extracted from specific days of differentiation, and the values (2^-ΔΔCt) were normalized to the expression levels at day 0. Blue lines represent the mean values, and the gray shades indicate the standard deviation (± SD). (b) A schematic representation of mRNA expression profiles of corresponding marker genes during human erythropoiesis. The mRNA expression data were obtained from previous studies (dos Santos et al. ; Marsee et al. ; Dzierzak and Philipsen ; Lee et al. ). It is noteworthy that hAHSP expression begins to increase in the baso-erythroblast stage, reaching its highest level in the reticulocyte. In contrast, hEPOR is expressed at the earliest stage of erythroid cell development, the proerythroblast, increases until the poly-erythroblast stage, but then starts to decrease and becomes undetectable in the reticulocyte. Although both hKLF1 and hTFRC start to be expressed in the baso-erythroblast stage, hKLF1 reaches its highest level in the ortho-erythroblast, while hTFRC peaks in the early reticulocyte stage.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: Expressing, Marker, In Vitro, Quantitative RT-PCR, Standard Deviation

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Functional analysis of day 17 cells from in vitro erythroid differentiation of canine PB MNCs with cEPO (40 ng/mL). (a) Western blot analysis of hemoglobin α (HBA) expression in differentiated cells from three different donors on day 21 of differentiation: lanes #1, #2, and #3. The lane labeled ‘RBC’ indicates peripheral blood RBCs collected from a reference dog. The arrow indicates the monomeric hemoglobin A (HBA), whereas the asterisk (*) denotes the HBA dimer. (b) Oxygen-binding capacity of the cells on day 18 of differentiation, demonstrating comparability to peripheral blood RBCs obtained from a different donor than the one used for in vitro differentiation.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: Functional Assay, In Vitro, Western Blot, Expressing, Labeling, Binding Assay

Journal: Animal Cells and Systems

Article Title: Establishment of an in vitro erythroid differentiation system from canine peripheral blood mononuclear cells

doi: 10.1080/19768354.2025.2492148

Figure Lengend Snippet: Structural differences between human and canine EPO. (a) Amino acid sequence alignment of mature human EPO (hEPO, P01588) and canine EPO (cEPO, P33707). Multiple sequence alignment was performed using Clustal Omega (PDB id: clustalo-I20241209-014629-0930-11133740-p1 m). The dotted square regions indicate the predicted amphipathic α-helical structures. (b) Aligned 3D structures of hEPO (magenta) and cEPO (cyan) in the D3 and D4 amphipathic α-helical regions, visualized using ChimeraX. The amino acid sequences of D3 (residues 89–107) are as follows: hEPO: EPLQLHVDKAVSGLRSLTT; cEPO: ETPQLHVDKAVSSLRSLTS. The amino acid sequences of D4 (residues 131–152) are as follows: hEPO: RTITADTFRKLFRVYSNFLRGK; cEPO: RTFTVDTLCKLFRIYSNFLRGK. Residues in a significant difference in three-dimensional structure are highlighted with white circles in the enlarged images.

Article Snippet: The cultures were also supplemented with cytokines, including recombinant canine erythropoietin (cEPO) (10–160 ng/mL, R&D Systems, cat#3816-CE), recombinant human erythropoietin (hEPO) (40–80 ng/mL, Millipore, cat#329871), recombinant canine stem cell factor (cSCF) (100 ng/mL, R&D Systems, cat#2278-SC), and recombinant human interleukin-3 (hIL-3) (10 ng/mL, R&D Systems, cat#203-IL).

Techniques: Sequencing