Journal: Advanced Science

Article Title: Optimized Monothiol Thioredoxin Derivative (ORP100S) Protects In Vitro and In Vivo from Radiation and Chemotoxicity Without Promoting Tumor Proliferation

doi: 10.1002/advs.202504426

Figure Lengend Snippet: ORP100S protects hematopoietic stem cells from chemotherapy induced injury and is additive/synergistic with GM‐CSF. A–D) C57Bl/6 mice were implanted with luciferase‐expressing EG7 cells (1 × 10 6 cells per animal), and once tumors were established, mice were treated with 5‐FU (IP, 50 mg kg −1 , one dose) followed by PBS vehicle or ORP100S (128 µg, SC every other day for five doses). (A) Tumor volume was monitored weekly by bioluminescence. Bioluminescence intensity of individual animals from week one to week three in four groups of mice, ie., PBS (CTL), 5‐FU, ORP100S, or the combination (5‐FU and ORP100S), was measured (10 mice per group). B) Bioluminescence activity was quantified by determining the total flux (photons/sec) in each mouse from week one to week three. C) Peripheral blood was collected weekly for the measurement of white blood cell count (WBC), hemoglobin (HB) and platelet (PLT). D) Experiments were terminated at two weeks post 5‐FU injection and mice were sacrificed. Total numbers of bone marrow long‐term (LT)‐HSPC (Lin‐Scal+C‐Kit+CD150+CD48‐), short‐term (ST)‐HSPC (Lin‐Scal+C‐Kit+CD150‐CD48‐), and multi‐potential progenitor cells (MPP) (Lin‐Scal+C‐Kit+CD150‐CD48+) were measured. E–H) C57Bl/6 mice were implanted with luciferase‐expressing EG7 cells and treated subsequently with PBS or 5‐FU IP (50 mg kg −1 , one dose). The mice were then given PBS control buffer, ORP100S (128 µg, SC every other day for five doses total), GM‐CSF (2 µg, SC, daily for five d), or a combination of ORP100S and GM‐CSF. Tumor volume was quantified weekly by measuring bioluminescence intensity and peripheral blood was drawn for hematological analysis. Mice were sacrificed at two weeks post 5‐FU injection. E) Bioluminescence imaging of tumor burden at week three for C57BL/6 mice implanted with EG7 tumor cells and treated as described (eight groups). F) Change in tumor volume over time for the indicated treatments. Bioluminescence was quantified by determining the total flux (photons/sec) in each mouse from week one to week three. G) Change in WBC, HB, and PLT in peripheral blood for the indicated groups and treatments. H) Total numbers of bone marrow LT‐HSC, ST‐HSC, and MPP cells at termination for each of the indicated treatments. Data represent mean ± SD for n = five to six mice per group. *: p < 0.05, **: p ,0.01; ***: p < 0.001.

Article Snippet: EML (CRL‐11691), TRAMP (CRL‐2731), HT29 (HTB‐38), B16‐F10 (CRL‐6475), MM1.R (CRL‐2975), MV4‐11 (CRL‐9591), SW48 (CCL‐231), LS1034 (CRL‐2158), and EG7 (CRL‐2113) cell lines were acquired from American Type Culture Collection (ATCC, Manassas VA).

Techniques: Luciferase, Expressing, Activity Assay, Cell Counting, Injection, Control, Imaging

Journal: Advanced Science

Article Title: Optimized Monothiol Thioredoxin Derivative (ORP100S) Protects In Vitro and In Vivo from Radiation and Chemotoxicity Without Promoting Tumor Proliferation

doi: 10.1002/advs.202504426

Figure Lengend Snippet: ORP100S attenuates ferroptosis induced by radiation and chemotherapy in stem cells but not in cancer cells. A) EML cells, HT29 cells, TRAMP cells, and B16‐F10 cancer cells were treated with PBS, 40 µg mL −1 ORP100S or 40 µg mL −1 rhTRX in PBS for 48 h. Cells were harvested and protein lysates were subjected to Western blotting using SLC7A11 antibody, GPX4 antibody or GAPDH antibody. B) EML cells and B16‐F10 cancer cells were treated with PBS, 40 µg mL −1 ORP100S or 40 µg mL −1 rhTRX in PBS for 48 h. The cells were transferred to pre‐coated slides and stained with immunofluorescence labeled SLC7A11 antibody, immunofluorescence labeled GPX4 antibody or 4',6‐diamidino‐2‐phenylindole (DAPI). The levels of SLC7A11 and GPX4 were detected under fluorescent microscope. Representative images were shown. C) EML cells and B16‐F10 cancer cells were treated with PBS, 40 µg mL −1 ORP100S or 40 µg mL −1 rhTRX in PBS with or without 10 µ m Erastin (ferroptosis inducer) for 48 h. Cells were harvested and protein lysate was subjected to Western blot analysis with indicated antibodies. D) EML cells were irradiated with 1 Gy, 3 Gy or 5 Gy and treated with PBS, 40 µg mL −1 ORP100S or 40 µg mL −1 rhTRX in PBS for 48 h. The cells were incubated with 5 µM boron‐dipyrromethene (BODIPY) 581/911 C11 reagent in PBS at 37 °C for 30 min. Labeled cells were washed and analyzed by flow cytometry. For lipid peroxidation analysis, the peroxidation state of each group was calculated by mean fluorescence intensity (MFI) ratio of the FL1 channel (590 nm) to that of FL3 channel(510 nm). E) EML cells were irradiated with 1 Gy, 3 Gy or 5 Gy and treated with PBS, 40 µg mL −1 ORP100S or 40 µg mL −1 rhTRX in PBS for 48 h. The cells were labeled with Bio Tracker Far‐red Labile Fe2+ Dye at 5 µ m for 90 min in PBS at 37 °C. Intracellular ferrous iron was measured by quantifying mean fluorescence intensity using Image J. F) Human CD34+ HSPCs were irradiated with 1 Gy, 3 Gy or 5 Gy and treated with PBS, 40 µg mL −1 ORP100S or 40 µg mL −1 rhTRX in PBS for 48 h. The cells were labeled with Bio Tracker Far‐red Labile Fe2+ Dye at 5 µ m for 90 min in PBS at 37 °C. Intracellular ferrous iron was measured by quantifying mean fluorescence intensity using Image J. G) MM1.R and MV4‐11 cells were treated with 5 Gy or 5‐FU (25 µ m ) or cisplatin (1 µM) with or without ORP100S (40 µg mL −1 ) or rhTRX (40 µg mL −1 ) for 48 hr. Lipid peroxidation (top panels) and intracellular ferrous iron level (lower panels) were measured. H) C57Bl/6 mice were implanted with EG7 and subsequently treated with 5‐FU ± ORP100S as described in Figure . At 3 weeks tumors and bone marrow were collected for SLC7A11 and GPX4 western blot analysis. I) C57Bl/6 mice were implanted with B16‐F10. Following tumor development, mice were treated with cisplatin ± ORP100S as described Figure (Supporting Information). Protein lysates were subjected to Western blotting with indicated antibodies. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: EML (CRL‐11691), TRAMP (CRL‐2731), HT29 (HTB‐38), B16‐F10 (CRL‐6475), MM1.R (CRL‐2975), MV4‐11 (CRL‐9591), SW48 (CCL‐231), LS1034 (CRL‐2158), and EG7 (CRL‐2113) cell lines were acquired from American Type Culture Collection (ATCC, Manassas VA).

Techniques: Western Blot, Staining, Immunofluorescence, Labeling, Microscopy, Irradiation, Incubation, Flow Cytometry, Fluorescence

Journal: Advanced Science

Article Title: Optimized Monothiol Thioredoxin Derivative (ORP100S) Protects In Vitro and In Vivo from Radiation and Chemotoxicity Without Promoting Tumor Proliferation

doi: 10.1002/advs.202504426

Figure Lengend Snippet: ORP100S mediates ferroptosis inhibition via the KLF4/p53 axis. (A) EML cells (left panel), B16‐F10 cancer cells (middle panel), and EG7 cancer cells (right panel) were transduced with control or p53‐specific CRISPR/Cas9 then treated with ORP100S (40 µg mL −1 ) or rhTRX (40 µg mL −1 ) and after 48 h cells were harvested, lysed and protein lysates were subjected to Western blotting with indicated antibodies. (B) SW48 cells were treated with 5‐FU (10 µM) or cisplatin (3 µM) with or without ORP100S (40 µg mL −1 ) or rhTRX (40 µg mL −1 ) for 48 hr. Cellular lipid peroxidation (left panel) and intracellular labile ferrous iron Fe 2+ levels (lower panel) were measured by BODIPY 581/591 C11 and BioTracker Fe 2+ fluorescence staining, respectively. C) LS1034 cells were treated with 5‐FU (5 µM) or cisplatin (10 µM) with or without ORP100S (40 µg mL −1 ) or rhTRX (40 µg mL −1 ) for 48 h and lipid peroxidation (left panel) and intracellular ferrous iron levels (right panel) were measured as described above. D) p53 promoter regions were cloned into the PGL3 firefly/renilla reporter system and the resultant PGL3‐p53‐reporter plasmids were transduced into EML cells, B16‐F10 and EG7 cell lines. Cells were treated with ORP100S (40 µg mL −1 ) or rhTRX (40 µg mL −1 ) for 48 hr, and luciferase bio‐luminescence activity was measured. E) RNA sequence heat map of enriched HSPCs from TRX knockout (KO) and WT mice (KLF4 is highlighted). F) KLF4 expression in TRX KO versus WT mice. G) Co‐immunoprecipitation (Co‐IP) of TRX with KLF4. Total cell lysates were immunoprecipitated using an anti‐TRX antibody or IgG control and the pull‐down was probed for KLF4 by immunoblotting. H) Representative images of ChIP‐PCR products amplified by primers (100 bp) on 2% agarose gel in EML cells. I) EML cells were treated with PBS or ORP100S (40 µg mL −1 ) for 48 hr. Cells were harvested and KLF4 mRNA levels were measured by qPCR (top panel). Additionally, KLF4 protein levels were measured by Western blot (lower panel). J,K) EML and EG7 cells were treated with 5‐FU (25 µM), ORP100S (40 µg mL −1 ) or TRX (40 µg mL −1 ) for 48 hr. Protein lysates were subjected to western blotting with indicated antibodies. L,M) EML cells (L) and EG7 cells (M) were transduced with a control vector or KLF4‐overexpression plasmid and then treated with PBS or 40 µg mL −1 ORP100S. Chromatin immunoprecipitation (ChIP)‐PCR was performed to measure the binding (occupancy) of KLF to the p53 promoter (left panel). EML and EG7 cells transduced initially with a control vector or KLF4 overexpression plasmid were subsequently transfected with a PGL3‐p53 firefly luciferase reporter vector construct and treated with ORP100S (40 µg mL −1 ), following which p53 transcription‐driven luciferase bioluminescence was measured (right panel). N,O): KLF4 in EML cells (N) and EG7 cells (O) was knocked out by CRISPR/Cas9 and cells were then treated with 40 µg mL −1 ORP100S. ChIP‐PCR was performed to measure the binding (occupancy) of KLF to the p53 promoter (left panel). EML and EG7 cells transduced initially with control vector or KLF4 KO plasmids were subsequently transfected with a PGL3‐p53 firefly luciferase reporter vector construct and treated with ORP100S (40 µg mL −1 ), following which p53 transcription‐driven luciferase bioluminescence was measured (right panel). Data represent means ± SD of three independent experiments *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: EML (CRL‐11691), TRAMP (CRL‐2731), HT29 (HTB‐38), B16‐F10 (CRL‐6475), MM1.R (CRL‐2975), MV4‐11 (CRL‐9591), SW48 (CCL‐231), LS1034 (CRL‐2158), and EG7 (CRL‐2113) cell lines were acquired from American Type Culture Collection (ATCC, Manassas VA).

Techniques: Inhibition, Transduction, Control, CRISPR, Western Blot, Fluorescence, Staining, Clone Assay, Luciferase, Activity Assay, Sequencing, Knock-Out, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Amplification, Agarose Gel Electrophoresis, Plasmid Preparation, Over Expression, Chromatin Immunoprecipitation, Binding Assay, Transfection, Construct