Journal: bioRxiv

Article Title: A Developmental Lectin-Glycan Program Enables Early Breast Cancer Dissemination and Metastatic Onset

doi: 10.64898/2026.02.23.707584

Figure Lengend Snippet: A) Representative whole mount images of mammary glands (MGs) from C57BL/6 Lgals1 +/+ or −/− mice treated or not with MPA. B) Quantification of MG branching as the number of terminal end-buds (TEBs) per field. C) Branching index. D-F) Flow cytometric analysis of epithelial lineage markers in Lgals1 +/+ and Lgals1 −/− MGs, showing the percentage of D) mammary stem cells (MaSC, Epcam low CD49f + ), E) basal cells (Epcam med CD49f + ), and F) luminal cells (Epcam high CD49f low ), with and without MPA stimulation. G) Analysis of the MG luminal compartment (ER +/− luminal progenitors and differentiated ER + cells) based on Sca1 and CD49b, H) Heatmap displaying mRNA expression z-scores of MG morphogenesis relevant genes measured by RNAseq. I-J) Representative images of PR staining by immunohistochemistry (IHC) (I) and quantification of PR+ cells per duct (J). K) Schematic representation of GAL1 effect on MG branching morphogenesis. Statistical analysis : Data are presented as mean ± SD. For panels B, C, E, F, G differences between groups were analyzed by one-way ANOVA followed by Tukey’s post-test. (H) n = 3 mice per group. (J) For panel J, an unpaired Welch’s t-test was used to compare Lgals1 +/+ and Lgals1 −/− MGs. P-values < 0.05 were considered statistically significant. All experiments were performed with n ≥ 5 mice per group.

Article Snippet: MMTV-HER2+ mice (EL - 14–18 weeks old females) were treated twice a week I.P. with anti-GAL1 mAb or IgG2b isotype control (BioXCell Cat# BE0086) for 4 weeks (dose per mice of 30 mg/kg).

Techniques: Expressing, RNA sequencing, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: A Developmental Lectin-Glycan Program Enables Early Breast Cancer Dissemination and Metastatic Onset

doi: 10.64898/2026.02.23.707584

Figure Lengend Snippet: A-B) A 2D UMAP (Uniform Manifold Approximation and Projection) of the ATLAS data is organized into clusters, classified according to murine mammary cell lineages. The expression of Lgals1 and Acta2 (alpha-SMA) is visualized on the UMAP projection. C-D) Lgals1 expression across the different cell lineages (D) or developmental stages (D) identified in the MG. E) Representative immunofluorescence (IF) images showing the expression of αSMA and GAL1 in MGs of C57BL/6 mice at different developmental stages. DAPI was used for nuclei staining. F) Flow cytometry analysis of GAL1 and SNA binding in C57BL/6 Lgals1 +/+ or Lgals1 −/− mice MGs. G) UMAP projection (Uniform Manifold Approximation and Projection) color-coded by clusters corresponding to human mammary cell lineages. The UMAP was generated using 15 dimensions and a cluster resolution of 0.01 with Seurat V4. H-I) Expression of LGALS1 across the previously defined cell lineages.

Article Snippet: MMTV-HER2+ mice (EL - 14–18 weeks old females) were treated twice a week I.P. with anti-GAL1 mAb or IgG2b isotype control (BioXCell Cat# BE0086) for 4 weeks (dose per mice of 30 mg/kg).

Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry, Binding Assay, Generated

Journal: bioRxiv

Article Title: A Developmental Lectin-Glycan Program Enables Early Breast Cancer Dissemination and Metastatic Onset

doi: 10.64898/2026.02.23.707584

Figure Lengend Snippet: A) Representative histological images of mammary gland (MG) from PyMT Lgals1 +/+ and Lgals1 −/− mice stained with hematoxylin and eosin (H&E) between weeks 10 to 16. B) Representative IHC images er of PR-positive stains in the nuclei of mammary gland. C) Quantification of PR + cells per duct. Quantification was performed by counting the number of PR + nuclei relative to the total number of cells per duct. D) Detection and localization of GAL1 in mammary ducts of PyMT mice by IHC. Arrows indicate positive immunoreactivity in basal cells. E) Flow cytometry analysis of MaSC in both PyMT Lgals1 +/+ and Lgals1 −/− mice. F) Kaplan Meier curve showing tumor-free survival time in both PyMT Lgals1 +/+ and Lgals1 −/− mice. G-I) Flow cytometry analysis of G) Mammary CSCs in tumors of both PyMT Lgals1 +/+ and Lgals1 −/− mice, H) CSCs in lungs of both PyMT Lgals1 +/+ and Lgals1 −/− mice, I) Epcam + PyMT + DTCs in lungs of both PyMT Lgals1 +/+ and Lgals1 −/− mice at different age. J) Quantification of macrometastatic foci in lungs of both PyMT Lgals1 +/+ and Lgals1 −/− mice at euthanasia. Statistical analysis: (C-E-G-H-I-J) Unpaired Welch’s t-test. p-values < 0.05 were considered statistically significant. All experiments were performed with n ≥ 4 mice per group (C) A total of 12 randomly selected ducts were analyzed per mice sample. (F) Mantel-Cox and Gehan-Breslow-Wilcoxon test. n= 75 (PyMT Lgals1 +/+ ), n= 25 (PyMT Lgals1 −/− ).

Article Snippet: MMTV-HER2+ mice (EL - 14–18 weeks old females) were treated twice a week I.P. with anti-GAL1 mAb or IgG2b isotype control (BioXCell Cat# BE0086) for 4 weeks (dose per mice of 30 mg/kg).

Techniques: Staining, Flow Cytometry

Journal: bioRxiv

Article Title: A Developmental Lectin-Glycan Program Enables Early Breast Cancer Dissemination and Metastatic Onset

doi: 10.64898/2026.02.23.707584

Figure Lengend Snippet: A) Graphical abstract representing the different stages of the MMTV-Her2Neu transgenic mouse model. B) Heatmap of galectins and glycosyltansferases (GTs) expression derived from mammary gland (MG) 3D cultures measured by RNAseq (EL/PT ratio). C) RT-qPCR confirmation of galectins and GTs expression in MG 3D cultures (EL/PT ratio). D-E) Immunofluorescence images showing GAL1 staining in normal FVB MG tissue (D) and in MMTV-Neu EL (16 weeks) (E). F) Quantification of GAL1 + cells per duct in both normal and pre-neoplastic mammary gland. Each dot represents the average of GAL1 + cells per duct per MG per mice. G) Microscopy images of EL 3D cultures treated for 7 days with different concentrations of recombinant GAL1 (rGAL1) or with vehicle (scale bar: 50 µm) H) Quantification of invasive and non-invasive organoids after rGAL1 treatment in EL derived 3D cultures. I) Microscopy images of PT 3D cultures treated with different concentrations of rGAL1 for 7 days or with vehicle (scale bar: 50 µm). J) Quantification of invasive and non-invasive organoids of PT. K) Heatmap of genes related to EMT and the Wnt pathway measured by RT-qPCR (GAL1 treated/untreated ratio) in EL. L) IF images of the epithelial marker E-cadherin in 3D cultures treated with vehicle or rGAL1 N) The quantification of E-cadherin negative cells. Each dot represents the average of E-cadherin negative cells per organoid per replicate. M) IF images of the mesenchymal marker Twist1 in 3D cultures treated with vehicle or rGAL1. O) Quantification of Twist1 + cells is shown in panel. Each dot represents the average of Twist1 + cells per organoid per replicate. P-Q) Time-lapse imaging (24hours) of EL organoids treated with vehicle (P) or rGAL1 (Q). Scale bar: 10 µm Statistical analysis : (F-H-J-M-O) Unpaired Welch’s t-test (J). p-values < 0.05 are considered significant. All experiments were performed with n = 4 mice per group.

Article Snippet: MMTV-HER2+ mice (EL - 14–18 weeks old females) were treated twice a week I.P. with anti-GAL1 mAb or IgG2b isotype control (BioXCell Cat# BE0086) for 4 weeks (dose per mice of 30 mg/kg).

Techniques: Transgenic Assay, Expressing, Derivative Assay, RNA sequencing, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Recombinant, Marker, Imaging

Journal: bioRxiv

Article Title: A Developmental Lectin-Glycan Program Enables Early Breast Cancer Dissemination and Metastatic Onset

doi: 10.64898/2026.02.23.707584

Figure Lengend Snippet: A-B) LGALS1 mRNA (A) and protein (B) expression in JIMT-1 and BT-474 cell lines. C) GAL1 protein expression in JIMT-1 and BT-474 cell lines conditioned media (by ELISA). D) GAL1 binding on JIMT-1 and BT-474 cell lines (by flow cytometry). F) ST6GAL1 mRNA expression in JIMT-1 and BT-474 cell lines (qPCR). F) SNA binding on JIMT-1 and BT-474 cell lines (flow cytometry). G) Graphical abstract of analyzed cell lines summarizing their glycophenotype. H) Bright-field images of JIMT-1 cells treated with vehicle or rGAL1 (5μM, 72 h). I) Proliferation measured by tritiated thymidine incorporation after addition of rGAL1 (1, 3 or 5 µM) in JIMT-1 cells. J) Cell cycle analysis (PI+BrdU, flow cytometry). K) Quantification of the G1/ S /G2 phases. L) Heatmap of EMT and stemness markers (RT-qPCR, normalized). M) Dot plot of JIMT-1 cells treated with rGAL1 (5 µM) and stained for CD24 and CD44 (flow cytometry) (left panel), percentage of CD24 low /CD44 high cells across different experimental conditions (right panel). N) CD24 modulation normalized to mode, O) Mammosphere formation: cells pre-treated with rGal-1 (5 µM, 72 h), then plated in sphere media; spheres counted per 500 seeded cells. P) Wound-healing assay (Incucyte® 96-Well Woundmaker) Q) Wound confluency over 48hs. R) Graphical abstract of the tail vein injection experimental design. S) Lung images and quantification of macrometastasis at 6 weeks in KD or Scramble JIMT-1 injected-mice. Statistical analysis: (A-B-C-D-E-F-O-S) Unpaired Welch’s t-test. P-values < 0.05 were considered significant. (I-M) One-way ANOVA with Dunnet’s post-test (vs Vehicle). All experiments were performed with n ≥ 3 per group.

Article Snippet: MMTV-HER2+ mice (EL - 14–18 weeks old females) were treated twice a week I.P. with anti-GAL1 mAb or IgG2b isotype control (BioXCell Cat# BE0086) for 4 weeks (dose per mice of 30 mg/kg).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Flow Cytometry, Cell Cycle Assay, Quantitative RT-PCR, Staining, Wound Healing Assay, Injection

Journal: bioRxiv

Article Title: A Developmental Lectin-Glycan Program Enables Early Breast Cancer Dissemination and Metastatic Onset

doi: 10.64898/2026.02.23.707584

Figure Lengend Snippet: A) Graphical abstract representing the therapeutic strategy in the context of the disease development in the MMTV-HER2 mouse model. Mice were treated with IgG control or anti-GAL1 mAb (30mg/kg) twice a week (for 4 weeks). B) GAL1 levels in blood measured by ELISA. C) Representative mammary whole mounts of treated mice. D) Quantification of the number of ducts per area of the mammary gland after anti-GAL1 treatment. E) Representative microscopy images after PR staining in MMTV-HER2 MGs. G) Quantification of the number of PR + cells per MG. G) Quantification of the number of PR + cells per duct. H) Quantification of the number of CTCs in blood before euthanasia. I) Representative microscopy images of lungs sections stained with H&E. Arrows highlights macrometastasis foci. J) Incidence of PT tumor per group. K) Frequency of macrometastasis presence per lung section. L) Quantification of macrometastasis per lung. Statistical analysis: (B-D-F-G-H) Unpaired Welch’s t-test (J). Unpaired Mann-Whitney’s t-test. p-values < 0.05 are considered significant. All experiments were performed with n > 4 mice per group.

Article Snippet: MMTV-HER2+ mice (EL - 14–18 weeks old females) were treated twice a week I.P. with anti-GAL1 mAb or IgG2b isotype control (BioXCell Cat# BE0086) for 4 weeks (dose per mice of 30 mg/kg).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Microscopy, Staining