Journal: bioRxiv

Article Title: Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

doi: 10.64898/2026.01.08.698324

Figure Lengend Snippet: A) Schematic of the breeding strategy used to generate EPN3-KI:MMTV-NeuN mice. FVB.K5-Cre and FVB.EPN3-KI lines were first crossed to combine the Cre driver with the knock-in allele. Progeny carrying K5-Cre and EPN3-KI were then crossed with the MMTV-NeuN mice. B) Representative IHC images showing ERBB2 (NeuN) and EPN3 expression in mammary epithelial ducts of 18-week-old mice. Bar, 100 μm. C) Left, representative images of whole-mount sections of mammary glands from 18-week-old CTRL, EPN3-KI, MMTV-NeuN and EPN3-KI:MMTV-NeuN mice; Bar, 500 μm. Dotted lines indicate the limit of fat-pad penetration. Right, quantification of mammary fat-pad penetration (as distance from the lymph nodes) shown as percentage of the full-length of the fat pad (CTRL, N=7; EPN3-KI, N=8; MMTV-NeuN, N=12; EPN3-KI:MMTV-NeuN, N=10). D) Kaplan-Meier analysis of tumor-free survival in a cohort of EPN3-KI:MMTV-NeuN mice (N=20) and MMTV-NeuN mice (N=30), over a 1-year period. Time zero, when all mice were tumor-free, was set at 15 weeks. An event was recorded when tumors first became palpable. Survival curves were compared using the log-rank (Mantel-Cox) test, p=0.02. E) RT-qPCR analysis of endogenous Mm-Erbb2, transgenic Rn-Erbb2 (NeuN) and Mm-Lalba mRNA levels in lung tissues collected at 28–30 weeks of age from EPN3-KI:MMTV-NeuN (N=38) and MMTV-NeuN (N=44) mice. Results are expressed as relative to housekeeping (HK) genes ( Gapdh, Tbp, Gusb ). Samples with Rn-Erbb2 levels >3-fold higher than HK genes are shown in red. F) Representative anti-ERBB2 IHC staining of lung tissue samples used in (E). Upper panels: MMTV-NeuN lung samples with scattered metastatic cells (red arrowheads) and Rn-Erbb2 mRNA levels < 3. Middle panels: EPN3-KI:MMTV-NeuN lung samples with scattered metastatic cells (red arrowheads) and Rn-Erbb2 mRNA levels < 3. Lower panels: EPN3-KI:MMTV-NeuN lung samples with macro-metastatic lesions (brown areas) and Rn-Erbb2 mRNA levels >3; bar = 100 μm. G) Simple linear regression of the correlation between primary tumor size (in cm 3 ) and relative Rn-Erbb2 levels over HK in lung tissue samples from MMTV-NeuN and EPN3-KI:MMTV-NeuN used in (E). Dotted line indicates the absence of palpable tumors (smallest tumor from EPN3-KI:MMTV-NeuN was 3x4 mm). H) Top, ECAD internalization was monitored by IF (37°C for 30 min) in primary MECs purified from NeuN (N=4) and EPN3-KI:MMTV-NeuN mice (N=4) and pre-treated with (+)-Lactose (100 mM, 1 h), I3 (20 µM, 10 min) or vehicle (DMSO). Quantification of ECAD fluorescence intensity per condition normalized to NeuN DMSO control. N (cells)>50 (n=2). Bottom, Transwell Matrigel invasion assay in the presence of Lactose (100 mM), I3 (20 µM) or vehicle (DMSO). Quantification of invading cells is expressed as the number of invading cells per field of view. N (mice): (NeuN, DMSO, N=8; EPN3-KI:MMTV-NeuN, DMSO, N=7; NeuN, I3/Lactose, N=6; EPN3-KI:MMTV-NeuN, I3/Lactose, N=6; N (fields of view): NeuN, DMSO=30 (n=3), I3=20 (n=2), Lactose=20 (n=2); EPN3-KI:MMTV-NeuN, DMSO=30 (n=3), I3=20 (n=2), Lactose=20 (n=2). p-values in all panels were calculated with Unpaired Student’s t-test, two-tailed: * p < 0.05; **, <0.01; ****, <0.000; ns, not significant.

Article Snippet: The RT-qPCR Taqman primers used are listed below: Gapdh: mm_9999915 Tbp: mm01277042_g1 Gusb: mm01197698_m1 Erbb2: Rn01461176_g1 Erbb2: mm01306784_g1 Lalba: Mm00495258_m1 Data analysis involved calculating the cycle threshold (Ct) values.

Techniques: Knock-In, Expressing, Quantitative RT-PCR, Transgenic Assay, Immunohistochemistry, Purification, Fluorescence, Control, Invasion Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

doi: 10.64898/2026.01.08.698324

Figure Lengend Snippet: A) Representative anti-ERBB2 IHC staining of lung tissue samples from CTRL and EPN3-KI:MMTV-NeuN mice (#1 to #3) with varying metastatic loads (from the same sample cohort used in ) . Bar, 2 mm. Arrowheads point to metastatic lesions magnified in insets, bar, 200 µm. B) RT-qPCR analysis of endogenous Mm-Erbb2 and transgenic Rn-Erbb2 (NeuN) in lung tissues collected in (A) and shown as relative levels over HK genes. Standard deviation is calculated from technical qPCR triplicate. Samples were used to validate the RT-qPCR assay for metastasis detection shown in . C) ECAD internalization was monitored by IF at 37°C for 30 min in primary MECs derived from CTRL (N=3), NeuN (N=4), EPN3-KI (N=4) and EPN3-KI:MMTV-NeuN mice (N=4). Left, representative IF images are shown: internalized ECAD (green), DAPI (blue). Bar, 20 µm. Right, quantification of ECAD fluorescence intensity normalized to control, n= 3. p-values (Unpaired Student’s t-test, two-tailed): **, <0.01; ns, not significant. D) Transwell Matrigel invasion assay. Left, representative fields of view of invading cell nuclei stained with Hoechst; bar, 200 µm. Right, quantification of invading cells expressed as the number of invading cells per field of view; (the plot contains the samples used for generating panels and ). N (mice): (CTRL, N=9; EPN3-KI, N=8; NeuN, N=8; EPN3-KI:MMTV-NeuN, N=7). N (fields of view): CTRL=40, NeuN=30, EPN3-KI=40, and EPN3-KI:MMTV-NeuN=30. p-values (Unpaired Student’s t-test, two-tailed): ****, <0.0001; ns, not significant.

Article Snippet: The RT-qPCR Taqman primers used are listed below: Gapdh: mm_9999915 Tbp: mm01277042_g1 Gusb: mm01197698_m1 Erbb2: Rn01461176_g1 Erbb2: mm01306784_g1 Lalba: Mm00495258_m1 Data analysis involved calculating the cycle threshold (Ct) values.

Techniques: Immunohistochemistry, Quantitative RT-PCR, Transgenic Assay, Standard Deviation, Derivative Assay, Fluorescence, Control, Two Tailed Test, Invasion Assay, Staining

Journal: bioRxiv

Article Title: Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

doi: 10.64898/2026.01.08.698324

Figure Lengend Snippet: A) Schematic of the breeding strategy used to generate EPN3-KI:MMTV-NeuN mice. FVB.K5-Cre and FVB.EPN3-KI lines were first crossed to combine the Cre driver with the knock-in allele. Progeny carrying K5-Cre and EPN3-KI were then crossed with the MMTV-NeuN mice. B) Representative IHC images showing ERBB2 (NeuN) and EPN3 expression in mammary epithelial ducts of 18-week-old mice. Bar, 100 μm. C) Left, representative images of whole-mount sections of mammary glands from 18-week-old CTRL, EPN3-KI, MMTV-NeuN and EPN3-KI:MMTV-NeuN mice; Bar, 500 μm. Dotted lines indicate the limit of fat-pad penetration. Right, quantification of mammary fat-pad penetration (as distance from the lymph nodes) shown as percentage of the full-length of the fat pad (CTRL, N=7; EPN3-KI, N=8; MMTV-NeuN, N=12; EPN3-KI:MMTV-NeuN, N=10). D) Kaplan-Meier analysis of tumor-free survival in a cohort of EPN3-KI:MMTV-NeuN mice (N=20) and MMTV-NeuN mice (N=30), over a 1-year period. Time zero, when all mice were tumor-free, was set at 15 weeks. An event was recorded when tumors first became palpable. Survival curves were compared using the log-rank (Mantel-Cox) test, p=0.02. E) RT-qPCR analysis of endogenous Mm-Erbb2, transgenic Rn-Erbb2 (NeuN) and Mm-Lalba mRNA levels in lung tissues collected at 28–30 weeks of age from EPN3-KI:MMTV-NeuN (N=38) and MMTV-NeuN (N=44) mice. Results are expressed as relative to housekeeping (HK) genes ( Gapdh, Tbp, Gusb ). Samples with Rn-Erbb2 levels >3-fold higher than HK genes are shown in red. F) Representative anti-ERBB2 IHC staining of lung tissue samples used in (E). Upper panels: MMTV-NeuN lung samples with scattered metastatic cells (red arrowheads) and Rn-Erbb2 mRNA levels < 3. Middle panels: EPN3-KI:MMTV-NeuN lung samples with scattered metastatic cells (red arrowheads) and Rn-Erbb2 mRNA levels < 3. Lower panels: EPN3-KI:MMTV-NeuN lung samples with macro-metastatic lesions (brown areas) and Rn-Erbb2 mRNA levels >3; bar = 100 μm. G) Simple linear regression of the correlation between primary tumor size (in cm 3 ) and relative Rn-Erbb2 levels over HK in lung tissue samples from MMTV-NeuN and EPN3-KI:MMTV-NeuN used in (E). Dotted line indicates the absence of palpable tumors (smallest tumor from EPN3-KI:MMTV-NeuN was 3x4 mm). H) Top, ECAD internalization was monitored by IF (37°C for 30 min) in primary MECs purified from NeuN (N=4) and EPN3-KI:MMTV-NeuN mice (N=4) and pre-treated with (+)-Lactose (100 mM, 1 h), I3 (20 µM, 10 min) or vehicle (DMSO). Quantification of ECAD fluorescence intensity per condition normalized to NeuN DMSO control. N (cells)>50 (n=2). Bottom, Transwell Matrigel invasion assay in the presence of Lactose (100 mM), I3 (20 µM) or vehicle (DMSO). Quantification of invading cells is expressed as the number of invading cells per field of view. N (mice): (NeuN, DMSO, N=8; EPN3-KI:MMTV-NeuN, DMSO, N=7; NeuN, I3/Lactose, N=6; EPN3-KI:MMTV-NeuN, I3/Lactose, N=6; N (fields of view): NeuN, DMSO=30 (n=3), I3=20 (n=2), Lactose=20 (n=2); EPN3-KI:MMTV-NeuN, DMSO=30 (n=3), I3=20 (n=2), Lactose=20 (n=2). p-values in all panels were calculated with Unpaired Student’s t-test, two-tailed: * p < 0.05; **, <0.01; ****, <0.000; ns, not significant.

Article Snippet: The RT-qPCR Taqman primers used are listed below: Gapdh: mm_9999915 Tbp: mm01277042_g1 Gusb: mm01197698_m1 Erbb2: Rn01461176_g1 Erbb2: mm01306784_g1 Lalba: Mm00495258_m1 Data analysis involved calculating the cycle threshold (Ct) values.

Techniques: Knock-In, Expressing, Quantitative RT-PCR, Transgenic Assay, Immunohistochemistry, Purification, Fluorescence, Control, Invasion Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Targeting GL-Lect driven endocytosis to suppress cell plasticity in breast cancer

doi: 10.64898/2026.01.08.698324

Figure Lengend Snippet: A) Representative anti-ERBB2 IHC staining of lung tissue samples from CTRL and EPN3-KI:MMTV-NeuN mice (#1 to #3) with varying metastatic loads (from the same sample cohort used in ) . Bar, 2 mm. Arrowheads point to metastatic lesions magnified in insets, bar, 200 µm. B) RT-qPCR analysis of endogenous Mm-Erbb2 and transgenic Rn-Erbb2 (NeuN) in lung tissues collected in (A) and shown as relative levels over HK genes. Standard deviation is calculated from technical qPCR triplicate. Samples were used to validate the RT-qPCR assay for metastasis detection shown in . C) ECAD internalization was monitored by IF at 37°C for 30 min in primary MECs derived from CTRL (N=3), NeuN (N=4), EPN3-KI (N=4) and EPN3-KI:MMTV-NeuN mice (N=4). Left, representative IF images are shown: internalized ECAD (green), DAPI (blue). Bar, 20 µm. Right, quantification of ECAD fluorescence intensity normalized to control, n= 3. p-values (Unpaired Student’s t-test, two-tailed): **, <0.01; ns, not significant. D) Transwell Matrigel invasion assay. Left, representative fields of view of invading cell nuclei stained with Hoechst; bar, 200 µm. Right, quantification of invading cells expressed as the number of invading cells per field of view; (the plot contains the samples used for generating panels and ). N (mice): (CTRL, N=9; EPN3-KI, N=8; NeuN, N=8; EPN3-KI:MMTV-NeuN, N=7). N (fields of view): CTRL=40, NeuN=30, EPN3-KI=40, and EPN3-KI:MMTV-NeuN=30. p-values (Unpaired Student’s t-test, two-tailed): ****, <0.0001; ns, not significant.

Article Snippet: The RT-qPCR Taqman primers used are listed below: Gapdh: mm_9999915 Tbp: mm01277042_g1 Gusb: mm01197698_m1 Erbb2: Rn01461176_g1 Erbb2: mm01306784_g1 Lalba: Mm00495258_m1 Data analysis involved calculating the cycle threshold (Ct) values.

Techniques: Immunohistochemistry, Quantitative RT-PCR, Transgenic Assay, Standard Deviation, Derivative Assay, Fluorescence, Control, Two Tailed Test, Invasion Assay, Staining