imr5  (AcceGen Biotechnology)

Journal: Nature

Article Title: Reprogramming neuroblastoma by diet-enhanced polyamine depletion

doi: 10.1038/s41586-025-09564-0

Figure Lengend Snippet: a) eIF5A hypusination defects detected by isoelectric focusing followed by immunoblotting. Tumors of ProArg-free and CD DFMO arms, showing defective hypusination in only two CD DFMO tumors and five ProArg-free DFMO tumors. Neuroblastoma cell line IMR5 as negative control (PC1), not treated with DFMO, and positive control (PC2), treated for five days with 500 uM of DFMO. Stars denote tumors with a non-hypusinated eIF5A (eIF5A-K47acetyl) band compared to hypusinated band (eIF5A-hyp). b) Neuroblastoma cell line IMR5 (used as positive control in other panels) treated with increasing DFMO concentrations for five days and assessed by immunoblotting with an anti-hypusine antibody. c) eIF5A hypusination defects detected by immunoblotting. Tumors of ProArg-free DFMO show partially reduced hypusination. IMR5 (C1) and CHLA20 (C2) were added as negative controls for comparison to incomplete hypusination. d) Quantification of hypusination by isoelectric blots reported in a) and immunoblots reported in b). Mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. n = 7-8 per group. e) Validation of isoelectric blots bands. IMR5 protein lysates treated with 500 uM DFMO for 5 days. In a single IEF gel, these 3 lysates were run in triplicate, the membrane cut, and independently probed without stripping, using anti-eIF5A (detecting all forms), anti-hypusine (detecting only hypusinated eIF5A), and anti-eIF5A-K47 acetyl (detecting only K47-acetylated forms). f) Schematic of eIF5A hypusination via DHPS using spermidine as a substrate. g) Relative ribosome density in relation to proline codons upon Eif5a and Dhps knock down (KD) as compared to short-hairpin control (sh-contrl). The left panels are centered by poly proline tracts and the right by proline codons outside thereof. In Dhps KD, increased occupancy shows at all proline codons independent of the nucleotide three position (e.g. adenosine-ending). Reprocessed and reanalyzed data from Nakanishi et al. n = 2 per group. h) In vitro translation setup of IMR5 neuroblastoma cell lysates that were harvested in log-phase growth. Lysates were co-incubated with in vitro transcribed mRNA fragments encoding a repeat of either 7 instances of CCA or CCG just 5’ upstream of luciferase. Changes in fluorescent intensity thereby reflect the effect of polyamines on the translation of the codon repeats. i) Polyamine supplementation with 1.5 mM spermidine preferentially facilitates translation of CCA codon repeats, as compared to CCG stretches. Relative fluorescence to baseline translation of two independent experiments are shown. j) Translation defects are codon specific. Relative ribosome density on all amino acid codons in combined drug-diet treatment (ProArg-free DFMO vs. CD). First letter of name denotes amino acid followed by the encoding codon. k) The diet effect (ProArg-free vs. CD) in the ribosomal P site is not driven by pausing at arginine and proline amino acid codons. l) DFMO treatment effect (CD DFMO vs. CD) is characterized by ribosome pausing depending on the nucleotide at the codon-three position in ribosome P site. m) Adding a ProArg-free diet to DFMO treatment is characterized by enhanced ribosome pausing depending on the nucleotide identity at the codon-three position in ribosomal P site (ProArg-free DFMO vs. CD DFMO). Boxplot where the center line represents the median, the box spans the interquartile range (IQR; 25th to 75th percentiles), and whiskers extend to 1.5I QR. Data points beyond this range are shown as outliers (solid black). One-way ANOVA. A-ending codons n = 14, T-ending codons n = 16, G-ending codons n = 15, C-ending codons n = 16. n) Increased pausing at A-ending codons when comparing ProArg-free DFMO vs. CD DFMO at the ribosomal P site. j-n shows mean of n = 5. Abbreviations: CD, control diet; ProArg-free, proline and arginine-free diet; DFMO, difluoromethylornithine. Panels f and h created in BioRender. Morscher, R. (2025) https://BioRender.com/sf37unt (f); https://BioRender.com/7ngicj2 (h).

Article Snippet: For xenografts used in therapeutic trials, tumours were established on 4- to 6-week-old female NCr-nu mice (Charles River) by injection of 100 μl 50/50 RPMI/Matrigel solution containing 3 × 10 6 IMR5 cells ( MYCN amplified, ALK amplified).

Techniques: Western Blot, Negative Control, Positive Control, Comparison, Two Tailed Test, Biomarker Discovery, Membrane, Stripping Membranes, Knockdown, Control, In Vitro, Incubation, Luciferase, Fluorescence

Journal: Nature

Article Title: Reprogramming neuroblastoma by diet-enhanced polyamine depletion

doi: 10.1038/s41586-025-09564-0

Figure Lengend Snippet: a) Heatmap of gene expression from cell cycle genes of interest according to MYCN status highlights their overexpression in MYCN amplified primary tumors ( n = 93), as compared to their non-amplified counterpart ( n = 551). b) The three cell cycle genes are overexpressed in relation to MYCN status (non-amplified n = 551, amplified n = 93). c) CeNPR, KIF2C and CEP57 show a stage dependent expression profile with an increased expression in high-risk neuroblastoma. Stage 4 special (st4s) was removed for simplicity. Statistical comparison to stage 1. Number of patients per stage: st1 = 152, st2 = 113, st3 = 90, st4 = 211. d) Cell cycle proteins are concomitantly reduced upon ODC1 knock down (KD) using two short hairpin (sh) compared to scramble (SCR) in IMR5 neuroblastoma cell line. e) Relative expression of CENPR upon KD with two independent sh-CENPR to SCR in IMR5 and the confirmation of reduced protein levels on western blot. Mean ± s.e.m. n = 3 per group. f) Reduction of growth upon CENPR KD with two sh-CENPR constructs as compared to SCR. n = 16 measurements per day per condition. g) Relative expression of KIF2C upon KD with two independent sh-KIF2C as compared to SCR. And the confirmation of reduced protein levels on western blot. Mean ± s.e.m. n = 3 per group. h) Reduction of cell growth upon KIF2C KD compared SCR in the IMR5 neuroblastoma cell line. n = 10 measurements per day per condition. i) Western blot confirming reduced KIF2C and CENPR protein levels upon combined KD with sh-CENPR and sh-KIF2C. j) Cell growth upon combination of CENPR and KIF2C KD. n = 10 measurements per day per condition. k) Schematic of the involvement of the respective cell cycle proteins of interest S to in G2 phase transition as components of the CENPA-CAD complex. After being recruited to centromeres, they are involved in assembly of kinetochore proteins, mitotic progression and chromosome segregation. l) Cell cycle phase distribution as evaluated using flow cytometry of scrambled and combined sh-CENPR and sh-KIF2C KD. m) Concomitantly to a G0-G1 increase upon combined knock down of CENPR and KIF2C S-phase is decreased. Statistical comparison to SCR. Mean ± s.e.m. n = 3 per group. n) Decoupling hypusination dependent and independent polyamine effects. Inhibition of polyamine dependent hypusination via genetic knock down of Dhps combined with intracellular polyamine depletion by 20%ProArg media with 500uM DFMO. In neuroblastoma putrescine supplementation (100uM) rescues growth rates and protein levels as demonstrated by immunoblotting without rescuing hypusination of eIF5a. Mean ± s.e.m. n = 19-20 per group. Abbreviations: sh, short hairpin; ODC1, ornithine decarboxylase 1; KD, knock down; SCR, scramble; Pro, proline; Arg, arginine; DFMO, difluoromethylornithine. For b and c: Boxplot where the center line represents the median, the box spans the interquartile range (IQR; 25th to 75th percentiles), and whiskers extend to 1.5 IQR. Data points beyond this range are shown as outliers (solid black). For b, c, m and n: two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Panel k created in BioRender. Morscher, R. (2025) https://BioRender.com/u25yr73 .

Article Snippet: For xenografts used in therapeutic trials, tumours were established on 4- to 6-week-old female NCr-nu mice (Charles River) by injection of 100 μl 50/50 RPMI/Matrigel solution containing 3 × 10 6 IMR5 cells ( MYCN amplified, ALK amplified).

Techniques: Gene Expression, Over Expression, Amplification, Expressing, Comparison, Knockdown, Western Blot, Construct, Sublimation, Flow Cytometry, Inhibition, Two Tailed Test

Journal: Nature

Article Title: Reprogramming neuroblastoma by diet-enhanced polyamine depletion

doi: 10.1038/s41586-025-09564-0

Figure Lengend Snippet: a) Immunohistochemistry showing Ki67 (proliferation marker) of representative T h - MYCN tumors. Arrows denote cells in areas with local cytodifferentiation. b) Hallmark gene set enrichment across omics layers. Displayed is the full gene set tested across each treatment group CD DFMO, ProArg-free and combined ProArg-free DFMO compared to CD. The top 5 significantly changed gene sets on protein level are highlighted in bold. Point size denotes the significance level and the color scale the normalized enrichment score (NES), with red showing enrichment in the intervention group (CD DFMO, ProArg-free or ProArg-free DFMO) and blue in CD. c) Western blot analysis of MYCN in tumors from CD DFMO and ProArg-free treatment arms. Control 1(C1) is CHLA20, NB cell line with MYC amplification (no MYCN expressed) C2 is control IMR5, NB cell line with MYCN amplification and high expression. d) Combined diet-drug treatment disrupts the MYCN -driven super enhancer core regulatory circuitry on the transcript expression (square), and the protein level (ellipsoid). Similarly, other elements of the core regulatory circuitry are affected. e) Combined diet-drug treatment disrupts the MYCN- driven super enhancer adrenergic and retino-sympathetic regulatory circuitry on the transcript expression (square), and the protein level (ellipsoid). f) The polyamine biosynthetic pathway with targets reported to be regulated by MYCN on the transcriptional level highlighted by red arrows. g) Regulation of core polyamine biosynthesis pathway members given by fold change across the omics layers. Despite MYCN loss on the protein level, core targets including AMD1 (RNA level) and ODC1 (protein level) are upregulated. Others are broadly unchanged. h) Levels of enzymes in polyamine biosynthesis across treatment groups. Adjusted p-value is from the comparison between CD and ProArg-free DFMO. i) Western blot analysis of neuroblastoma cell line IMR5 treated with 5 uM MYCi975 for four days show downregulation of MYCN, no change of ODC1 and eIF5A hypusination. j) The mouse orthologue for TP53, TRP53 and its core regulator MDM2 are unchanged across all four treatment groups in the T h - MYCN neuroblastoma tumors (CD, CD DFMO, ProArg-free or ProArg-free DFMO). k) Ribosome density along the TRP53 coding sequence normalized to CDS mean. Insert highlights the proline rich region with no signs of ribosome stalling at the respective proline codons in the ProArg-free DFMO combination treatment. For c, d and e RNA-Seq: CD DFMO, ProArg-free and ProArg-free DFMO n = 5; CD n = 4, Ribo-Seq: n = 5 per group, Proteomics: CD DFMO, ProArg-free and ProArg-free DFMO n = 6; CD n = 5. For h and j: Boxplot where the center line represents the median, the box spans the interquartile range (IQR; 25th to 75th percentiles), and whiskers extend to 1.5 IQR. Data points beyond this range are shown as outliers (solid black). CD DFMO, ProArg-free and ProArg-free DFMO n = 6; CD n = 5. Abbreviations: CD, control diet; ProArg-free, proline arginine free diet; DFMO, difluoromethylornithine; c-d: Reprocessed and reanalyzed data from Elkon et al. n = 2 per group. e and f: Reprocessed and reanalyzed data from Volegova et al. n = 2 per group. b,i and j: Data generated as part of this study. n = 3 per group.

Article Snippet: For xenografts used in therapeutic trials, tumours were established on 4- to 6-week-old female NCr-nu mice (Charles River) by injection of 100 μl 50/50 RPMI/Matrigel solution containing 3 × 10 6 IMR5 cells ( MYCN amplified, ALK amplified).

Techniques: Immunohistochemistry, Marker, Western Blot, Control, Amplification, Expressing, Comparison, Sequencing, RNA Sequencing, Generated

Journal: Nature

Article Title: Reprogramming neuroblastoma by diet-enhanced polyamine depletion

doi: 10.1038/s41586-025-09564-0

Figure Lengend Snippet: a , GSEA across omics layers in all three treatment groups using the Hallmark gene set. Only the ProArg-free plus DFMO treatment group showed a significant effect compared with CD. The effect was mainly on the translation and protein level. Shown are the five top enriched sets (complete sets in Extended Data Fig. ). Size indicates P value (Benjamini–Hochberg correction) and colour represents NES, with red indicating enrichment in the intervention group (CD + DFMO, ProArg-free or ProArg-free + DFMO) and blue indicating enrichment in the CD group. b , Western blot analysis of MYCN in tumours from CD and ProArg-free plus DFMO treatment arms. Negative control (C1), CHLA20 neuroblastoma cell line ( MYCN non-amplified, MYC expressing); positive control (C2), IMR5 neuroblastoma cell line ( MYCN- amplified). GAPDH is used as a loading control. Blots are representative of two independent experiments yielding similar results. c , Representative haematoxylin and eosin (H&E)-stained sections. CD and ProArg-free diet treatments show undifferentiated primitive neuroblasts, absent neuropil and prominent mitotic figures. CD plus DFMO shows poorly differentiated primitive neuroblasts with scant neuropil (arrowhead) and foci of cytodifferentiation (<5% differentiating, arrow). ProArg-free diet plus DFMO tumours show high fractions of differentiating neuroblasts (>5% differentiating) with increased cytoplasmic to nuclear ratio (arrow) and abundant neuropil (arrowhead). Sections are representative of many images with the same observations. Scale bars, 50 μm. d , Summary of treatment effects. Cell cycle and MYCN programmes are downregulated at the protein level owing to translation inhibition and immature cancer cells are driven into neuronal differentiation. In a , c , RNA-seq: ProArg-free DFMO: n = 5; CD: n = 4. Ribo-seq: n = 5. Proteomics: ProArg-free DFMO: n = 6; CD: n = 5. Panel d created in BioRender. Morscher, R. (2025) https://BioRender.com/kk9n051 .

Article Snippet: For xenografts used in therapeutic trials, tumours were established on 4- to 6-week-old female NCr-nu mice (Charles River) by injection of 100 μl 50/50 RPMI/Matrigel solution containing 3 × 10 6 IMR5 cells ( MYCN amplified, ALK amplified).

Techniques: Western Blot, Negative Control, Amplification, Expressing, Positive Control, Control, Staining, Inhibition, RNA Sequencing

Journal: Nature

Article Title: Reprogramming neuroblastoma by diet-enhanced polyamine depletion

doi: 10.1038/s41586-025-09564-0

Figure Lengend Snippet: a) Blinded histological assessment by a pathologist of differentiation status and abundance of neuropil status in tumor sections (linked to Fig. ) in T h - MYCN mice. n = 6 per group. b) Schematic of xenografts induced in nude mice by the MYCN-amplified human neuroblastoma cell line IMR5. Upon reaching 200 mm 3 mice were randomized to receive either a CD or ProArg-free diet with or without DFMO drug application via the drinking water (1 %). c) Tumor volume of individual xenografts in mice across treatment groups. d) Average neuroblastoma xenograft tumor volume according to the four treatment groups CD, CD DFMO, ProArg-free and ProArg-free DFMO. Upon reaching the maximal tumor volume in an individual tumor (2 cm 3 ) this measurement was counted towards the mean until death of the last mouse in this group. Mean ± s.e.m. e) Example of neuroblastoma tumor regression in a ProArg-free DFMO treated mouse (day 77 compared to day 100). f) Kaplan-Meier survival curve related to b) with death encoded for human endpoints or tumor volume above 2 cm 3 . g) Mouse weight corrected for tumor weight across time after engraftment of IMR5 xenografts. h) qPCR quantification of MYCN and ODC1 gene expression in IMR5 xenograft tumors across treatment groups. n = 7-8 per group. Mean ± s.e.m. i) Western blot analysis of MYCN and the DFMO target ODC1 and in IMR5 xenograft tumors after treatment. j) Proliferation rate as assessed by immune histochemistry staining (Ki67 staining) on tumor sections of the four treatment groups. Data acquired by a pathologist blinded to treatment groups. k) Differentiation status according to clinical-diagnostic standards and scoring for neuropil abundance in H&E-stained histology sections derived from the IMR5 intervention trial. Data acquired by a pathologist blinded to treatment groups. Abbreviations: CD, control diet; ProArg-free, proline arginine free diet; DFMO, difluoromethylornithine. For b-h IMR5 xenograft model: CD n = 11, CD DFMO n = 13, ProArg-free n = 12, ProArg-free DFMO n = 12. For f: Log-rank test p-value compared to CD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For h: two-tailed t-test compared to CD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Panel b created in BioRender. Morscher, R. (2025) https://BioRender.com/sd1cd2k .

Article Snippet: For xenografts used in therapeutic trials, tumours were established on 4- to 6-week-old female NCr-nu mice (Charles River) by injection of 100 μl 50/50 RPMI/Matrigel solution containing 3 × 10 6 IMR5 cells ( MYCN amplified, ALK amplified).

Techniques: Amplification, Gene Expression, Western Blot, Staining, Diagnostic Assay, Derivative Assay, Control, Two Tailed Test

Journal: Molecular therapy. Oncology

Article Title: A dual-luciferase bioluminescence system for the assessment of cellular therapies.

doi: 10.1016/j.omton.2024.200763

Figure Lengend Snippet: Figure 1. Using CBG99 and Akaluc bioluminescence to track cell behavior in vitro (A) Illustrations of the CBG99 (left) and the Akaluc (right) viral vectors used to engineer cells. (B) Flow cytometry data illustrating the expression of CBG99 indicated by GFP (top row) and Akaluc indicated by mOrange (bottom row) positivity in different tumor cell lines measured after flow cytometric sorting to exclude nontransduced cells. (C) Measurement of CBG99 bioluminescence at different cell densities for IMR5-CBG99 cells. (D) Measurement of Akaluc bioluminescence at various cell densities for IMR5- Akaluc cells. (E) IMR5-CBG99 and IMR5-Akaluc bioluminescence at different seeding densities when exposed to D-Luciferin, captured using the CLARIOstar plate reader with open filter. (F) IMR5-CBG99 and IMR5-Akaluc bioluminescence at different seeding densities when exposed to AkaLumine-HCl, captured using CLARIOstar plate reader with open filter. (C–F) Data shown as mean ± SEM, n = 4.

Article Snippet: Bioluminescence for IMR5 cells was captured using CLARIOstar microplate reader (BMG LABTECH, Cary, NC), Capan-1 cells were obtained using IVIS Lumina III (PerkinElmer), and A549 spheroids measured by IVIS Lumina III or TECAN Spark microplate reader (Tecan Group, Männedorf, Switzerland), depending on the experiment.

Techniques: In Vitro, Flow Cytometry, Expressing

Journal: Molecular therapy. Oncology

Article Title: A dual-luciferase bioluminescence system for the assessment of cellular therapies.

doi: 10.1016/j.omton.2024.200763

Figure Lengend Snippet: Figure 2. Using CBG99 and Akaluc to monitor effector and target populations in vitro (A) Diagram illustrating the experimental procedure for the 2D co-culture assay using CBG99+ T cells and Akaluc+ IMR5 tumor cells. (B) Tumor (Akaluc) bioluminescence in absence of T cells (orange triangles), in presence of NT T cells at the indicated E:T ratios (orange circles), and NT cell (CBG99) bioluminescence at the indicated E:T ratios (green circles). (C) Tumor (Akaluc) bioluminescence in absence of T cells (orange triangles), in presence of GD2 CAR T cells at the indicated E:T ratios (orange squares), and GD2 CAR T cell (CBG99) bioluminescence at the indicated E:T ratios (green squares). (B and C) Tumor and T cell bioluminescence signals captured using CLARIOstar plate reader. (D) Flow cytometry plots showing tumor (x axis) and T cell (y axis) populations for a representative donor for each condition in (B) and (C). Numbers indicate percentage of total gated cells. (E and F) Quantitative summary of data shown in (D). (B, C, E, and F) Data shown as mean ± SEM, n = 4.

Article Snippet: Bioluminescence for IMR5 cells was captured using CLARIOstar microplate reader (BMG LABTECH, Cary, NC), Capan-1 cells were obtained using IVIS Lumina III (PerkinElmer), and A549 spheroids measured by IVIS Lumina III or TECAN Spark microplate reader (Tecan Group, Männedorf, Switzerland), depending on the experiment.

Techniques: In Vitro, Co-culture Assay, Flow Cytometry

Journal: Cell reports

Article Title: Engineering CD276/B7-H3-targeted antibody-drug conjugates with enhanced cancer-eradicating capability

doi: 10.1016/j.celrep.2023.113503

Figure Lengend Snippet: (A and B) Cell viability assays measured m276-SL-PBD activity against glioblastoma/neuroblastoma (A) and pancreatic/breast cancer (B) cell lines. IC 50 values are in the key. Error bars were omitted for clarity; SD was always <10%. (C) Table of IC 50 values from cell viability assays showing the relative sensitivity of cancer cell lines to m276-SL-PBD. (D) CD276 mRNA expression in CD276-low B cell lymphocytic leukemia (BLL) and CD276-high glioblastoma (GBM), neuroblastoma (NB), pancreatic cancer (PC), and breast cancer (BC) cell lines using the database: DepMap mRNA expression. The number of cancer cell lines in each group is indicated in parentheses. Sensitive (blue dots) and resistant (back dot) cell lines used in the cell viability assays (A and B) are highlighted. (E and F) Tables showing glioblastoma/neuroblastoma (E) or pancreatic/breast cancer (F) cell line sensitivity (IC 50 values from cell viability assays) after SGD-1882-free drug treatment and CD276 surface expression levels (mean fluorescence intensity; MFI) as measured by flow cytometry. (G) Subcutaneous (IMR5) and orthotopic (SUM159 and MDA-MB-231) tumor volumes following ADC treatment. Treatments with 0.5 mg/kg m276-SL-PBD (blue arrows) were initiated when tumors reached ~1,000 mm 3 ; n = 8–10/group. Error bars denote SEM. (H) Bioluminescence imaging monitored systemic MDA-MB-231-luc tumor burden following intravenous injection into NRG mice, followed by randomization and treatment with vehicle or m276-SL-PBD. Representative mice shown (n = 15/group; DPI, days post inoculation). (I) Quantification of tumor burden from the MDA-MB-231 metastasis study in (H). Error bars denote SD. (J) Kaplan-Meier survival curves for the MDA-MB-231-luc metastases study in (H).

Article Snippet: B2-17, KP4 and YH13 were obtained from the JCRB Cell Bank, ES1 and ES4 were from the St. Jude Children’s Research Hospital, PC9 were obtained from Sigma, JIMT (JIMT-1) were obtained from AddexBio, DKMG were from the DSMZ repository, IMR5 were from Accegen Biotechnology and Cas1 were from the Interlab Cell Line Collection Catalogue (ICLC).

Techniques: Activity Assay, Expressing, Fluorescence, Flow Cytometry, Imaging, Injection