Journal: bioRxiv

Article Title: Netrin-1 regulates colorectal cancer stem cell self-renewal via a TFF3 dependent paracrine survival mechanism

doi: 10.1101/2025.04.28.650943

Figure Lengend Snippet: (A) Correlation between UNC5B and NTN1 mRNA expression in the TCGA colon adenocarcinoma dataset; (B) Correlation between UNC5B mRNA and NTN1 mRNA expression in stage IV CRC tumors; ( C ) Expression of netrin-1 receptors (DCC, UNC5A-D) mRNA in stage IV CRC tumors (n=58) (bars indicate means); (D-E) Comparison of mRNA Expression in Healthy adjacent liver and Stage IV colorectal tumor tissues for UNC5B (D) and NTN1 (E) ; (F) Summary of mRNA expression levels of various netrin-1 receptors in organoids derived from metastatic colorectal tumors (n=21); (G) Correlation between UNC5B mRNA and NANOG mRNA expression; (H-I) Quantification of UNC5B (H) and netrin-1 (I) protein expression in organoid cell lines using Western blotting; Top panels: representative Western Blot image; Bottom panel: quantification of protein expression across organoid cell lines; Normalized gene expression is expressed in logCPM; (TCGA cohort: n=521; stage IV tumors cohort: n=58, Adjacent healthy tissue cohort: n=34); p Values: * = <0.05, ** = <0.01, **** = <0.0001; Statistical test: Spearman/Pearson for (A); Spearman’s rank correlation coefficient for (B); One-Way ANOVA - Tukey’s multiple comparisons test for (C) and (F), unpaired t-tests for (D) and (E); (H) Results are expressed as a % of yP190LT mean expression, using β-actin as loading control; 3 replicates per organoid cell line. (I) Results are expressed as % compared to lane containing 20ng of Recombinant netrin-1, using GAPDH as loading control; 1 replicate per organoid cell line.

Article Snippet: Anti-UNC5B (Cell Signalling; 13851) and anti Netrin-1 (Abcam; ab126729) were used to detect respectively the UNC5B receptor and Netrin-1.

Techniques: Expressing, Comparison, Derivative Assay, Western Blot, Gene Expression, Control, Recombinant

Journal: bioRxiv

Article Title: Netrin-1 regulates colorectal cancer stem cell self-renewal via a TFF3 dependent paracrine survival mechanism

doi: 10.1101/2025.04.28.650943

Figure Lengend Snippet: - (A-C) Self-renewal capable fraction (as a % of total cell number) in yP190LT (A), P315LT (B) and P931LT (C) organoids in the presence or absence of recombinant netrin-1 (100 ng/mL), NP001 (10μg/mL) and/or NP137 (10μg/mL), quantified using ELDA; ( D ) (Left) Representative flow cytometry analysis (Gate was determined in comparison with an unstained control and left unchanged for all samples) and (Right) bar graph summarizing proportions of CD133-positive cells in yP190LT organoid after 72h of treatment with recombinant netrin-1 (100 ng/mL), NP001 (10μg/mL) and/or NP137 (10μg/mL); (E) Self-renewal capable fraction of in wildtype, scramble control (sgSCR) and CRISPR-edited (SgUNC5B#1 and SgUNC5B#2) P315LT organoids, either left untreated (Control) or treated with recombinant netrin-1 (100 ng/mL), observed by ELDA; (F) Self-renewal capable fraction of in wildtype, scramble control (sgSCR) and UNC5B-null (SgUNC5B#1 and SgUNC5B#2) P315LT organoid cells, untreated (Control) or treated with NP001 (10μg/mL) or NP137 (10μg/mL), quantified using ELDA; Statistical significance was determined using One-way Anova – Tukey’s multiple comparison test, ns = non-significant, * = p <0.05, ** = p <0.01, *** = p <0.001. N=3 for (B), (C), (E) and (F)); N=4 for (A) and (D)

Article Snippet: Anti-UNC5B (Cell Signalling; 13851) and anti Netrin-1 (Abcam; ab126729) were used to detect respectively the UNC5B receptor and Netrin-1.

Techniques: Recombinant, Flow Cytometry, Comparison, Control, CRISPR

Journal: bioRxiv

Article Title: Netrin-1 regulates colorectal cancer stem cell self-renewal via a TFF3 dependent paracrine survival mechanism

doi: 10.1101/2025.04.28.650943

Figure Lengend Snippet: (A) UMAP plot of LGR5 and UNC5B expression level in control yP190LT organoid cells (NP001-treated); (B) Violin plot of the expression of PROM1, SOX4 and SMOC2 genes in UNC5B neg and UNC5B pos cells treated with either NP001 or NP137 (Each dot represent an event (cell)), (C) Fraction of high-expressing TFF3 and low-expressing TFF3 in UNC5B-positive cells treated with either NP001 (10μg/mL) or NP137 (10μg/mL); (D) UMAP plot of UNC5B (Left) and TFF3 (Right) expression level yP190LT organoid cells (treated with NP001 control antibody (10μg/mL)); (E) UMAP plot of TFF3 expression level yP190LT organoid cells treated with either NP001 or NP137; (F) Correlation between UNC5B mRNA and TFF3 in stage IV CRC tumors (G) Correlation between PROM1 mRNA and TFF3 in stage IV CRC tumors; (H) Correlation between SOX4 mRNA and TFF3 in stage IV CRC tumors; (I) Correlation between PROM1 mRNA and TFF3 mRNA in mCRC organoids; (J) Correlation between SMOC2 mRNA and TFF3 mRNA in mCRC organoids; For (C) High TFF3 expression is defined as > log5 and low expression as < log4; Means and p.values displayed determined by stat_compare_means function; Bars indicate means, statistical test use: Wilcoxon test for (C) and (E), Spearman’s rank correlation coefficient for (G-I); One-way Anova – Tukey’s multiple comparison test for (k), *** = p<0.001 , **** = p<0.0001

Article Snippet: Anti-UNC5B (Cell Signalling; 13851) and anti Netrin-1 (Abcam; ab126729) were used to detect respectively the UNC5B receptor and Netrin-1.

Techniques: Expressing, Control, Comparison

Journal: bioRxiv

Article Title: Netrin-1 regulates colorectal cancer stem cell self-renewal via a TFF3 dependent paracrine survival mechanism

doi: 10.1101/2025.04.28.650943

Figure Lengend Snippet: (A) Diagram summarizing the longitudinal collection protocol of liver metastasis biopsies prior to (C1D1) or after (C3D1) systemic treatment with NP137 in a patient with mCRC; (B) TFF3, PROM1, LGR5, SMOC2, SOX4 and EPCAM mRNA in CRC tumor of a patient treated with NP137 levels determined by RNA sequencing; Data analyzed using DESseq2 package in R; (C) Quantification of relative UNC5B mRNA expression level in yP190LT PDO after 24h of treatment with either the vehicle or FOLFOX (1 µM) by real-time qPCR; (D) Real-time qPCR quantification of relative NTN1 mRNA expression level in yP190LT PDO after 24h of treatment with either vehicle or FOLFOX (1 µM); (E) Western blot quantification of UNC5B protein expression level in yP190LT PDO after 72h of treatment with either vehicle or FOLFOX (1 µM) followed by a 15-day recovery period; (F) Self-renewal capable fraction of yP190LT organoid cell line after 72h of FOLFOX treatment (72h; 1 µM) in presence or absence of recombinant Netrin-1 (100 ng/mL), NP001 (10μg/mL) and/or NP137 (10μg/mL) observed by ELDA; Statistical test used : One-way Anova – Tukey’s multiple comparison test (G); Schematic representation of In Vivo protocol to evaluate the therapeutical potential of NP137 in mCRC using PDO-xenografted mice; (H) Evolution of tumor size in BALBc nude mice subcutaneously engrafted with yP190LT PDO after treatment with either PBS or FOLFOX (solution combining 5-Fluoro Uracil (50mg/Kg IP), oxaliplatin (6mg/Kg IP) and disodium levofolinate (90mg/Kg IP) daily for two days) or FOLFOX and NP137 (20 mg/kg; three times a week); N = 8/group, Error bars indicate SEM; Statistical test used : Two-way Anova - Dunnett’s multiple comparisons test; (I) Real-time qPCR quantification of TFF3 expression of engrafted tumors in mice treated with either PBS, FOLFOX or FOLFOX and NP137 at week 10; N = 8/group, Error bars indicate SD, Statistical test used : One-way Anova - Dunnett’s multiple comparisons test

Article Snippet: Anti-UNC5B (Cell Signalling; 13851) and anti Netrin-1 (Abcam; ab126729) were used to detect respectively the UNC5B receptor and Netrin-1.

Techniques: RNA Sequencing, Expressing, Western Blot, Recombinant, Comparison, In Vivo