epcam sirna  (Santa Cruz Biotechnology)

Journal: BMC Cancer

Article Title: Identification of genes regulating migration and invasion using a new model of metastatic prostate cancer

doi: 10.1186/1471-2407-14-387

Figure Lengend Snippet: The role of EpCAM, integrin β4 and uPA in migration and invasion of DU145-LN4 cells. Cell migration of DU145-LN4 cells in a transwell migration assay (A-C) and a Matrigel invasion assay (D-F) . siRNA knockdown of (A) EpCAM, (B) integrin β4, or (C) uPA significantly inhibited cell migration relative to control siRNA and untreated cells. siRNA knockdown of (D) EpCAM or (E) ITGB4 did not significantly affect cell invasion in a Matrigel invasion assay. (F) uPA siRNA significantly inhibited cell invasion relative to control siRNA and untreated cells. Western blot analysis of whole cell lysates from cells treated with siRNA in parallel with migration/invasion assays: (G) EpCAM, (H) integrin β4, and uPA western blots. Blots were probed with GAPDH as loading controls. (I) Western blot analysis of whole cell lysates from cells treated with control siRNA or si-uPA and serum starved (−) or serum stimulated (+). p-AKT and p-S6K were induced after serum addition in control cells but not in cells lacking uPA. Total AKT, S6K, and GAPDH served as controls. DU145-LN4 cell migration (J) , and cell invasion (K) were significantly inhibited by treatment with 10 μM amiloride or 10 μM UK122. Students t-test, *p ≤ 0.05, **p ≤ 0.01, n.s.-not significant. Top asterisked p value represents analysis between untreated cells and specific siRNA, lower value indicates analysis between control siRNA treated cells and specific siRNA.

Article Snippet: Cells were transfected with siRNA using SilentFect (Biorad) in Opti-MEM I Reduced Serum Medium (Invitrogen), incubated for 4 hours, media changed, and cells used for assays at 48-72 hr. siRNAs were obtained from Thermo Scientific: ON-TARGETplus non-targeting control siRNA pool (D-001818-10-05), ON-TARGETplus human EPCAM siRNA pool (L-004568-01-0005), ON-TARGETplus human PLAU siRNA (L-006000-00-0005), ON-TARGETplus human ITGB4 siRNA pool (L-008011-00-0005).

Techniques: Migration, Transwell Migration Assay, Invasion Assay, Western Blot

Journal: Molecules and Cells

Article Title: E-cadherin–driven adherens junction reinforcement promotes spheroid-mediated invasion and progression in ALK–rearranged lung cancer chemoresistance

doi: 10.1016/j.mocell.2026.100329

Figure Lengend Snippet: E-cadherin modulates crizotinib sensitivity in ALK–rearranged H2228 cells. (A) Cell survival of H2228, H2228-CR1, and H2228-CR2 cells following E-cadherin knockdown using siE-cad compared with control siRNA (siCon). (B) Half-maximal inhibitory concentration (IC 50 ) values for crizotinib in H2228 and H2228-CR2 cells after transfection with siCon or siE-cad. (C) Cell survival of parental H2228 cells following expression of FLAG-tagged E-cadherin (FLAG-E-cad) compared with empty vector (vec). (D) IC 50 values for crizotinib in parental H2228 cells expressing Vec or FLAG-E-cad.

Article Snippet: The following siRNA oligonucleotides were used: control siRNA-A (sc-37007), E-cadherin siRNA (sc-35242), and EpCAM siRNA (sc-43032), all purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Control, Concentration Assay, Transfection, Expressing, Plasmid Preparation

Journal: Molecules and Cells

Article Title: E-cadherin–driven adherens junction reinforcement promotes spheroid-mediated invasion and progression in ALK–rearranged lung cancer chemoresistance

doi: 10.1016/j.mocell.2026.100329

Figure Lengend Snippet: RNA sequencing reveals the upregulation of tissue morphogenic transcripts in crizotinib–resistant H2228 cells. (A) Volcano plot showing significantly differentially expressed genes (DEGs) between parental H2228 and crizotinib–resistant H2228-CR1 and H2228-CR2 cells. Red and blue dots indicate upregulated and downregulated genes, respectively; arrow indicates CDH1 . (B) Functional enrichment analysis of commonly upregulated DEGs in H2228-CR1 and H2228-CR2 cells using Metascape. (C) Heatmap showing the mean log2 fold change of selected DEGs in H2228-CR1 and H2228-CR2 cells. (D) Correlation analysis between CDH1 and selected epithelial junction–related genes ( EpCAM, CLDNs, and GJB3 ) in lung adenocarcinoma (LUAD) patients using GEPIA2 base. (E) Quantitative real–time PCR analysis of EpCAM mRNA expression in parental H2228, H2228-CR1, and H2228-CR2 cells. (F) Western blot analysis of EpCAM protein levels in parental H2228, H2228-CR1, and H2228-CR2 cells. β-actin was used as a loading control.

Article Snippet: The following siRNA oligonucleotides were used: control siRNA-A (sc-37007), E-cadherin siRNA (sc-35242), and EpCAM siRNA (sc-43032), all purchased from Santa Cruz Biotechnology.

Techniques: RNA Sequencing, Functional Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: Molecules and Cells

Article Title: E-cadherin–driven adherens junction reinforcement promotes spheroid-mediated invasion and progression in ALK–rearranged lung cancer chemoresistance

doi: 10.1016/j.mocell.2026.100329

Figure Lengend Snippet: EpCAM regulates migration and invasion in crizotinib-resistant spheroids. (A) Immunofluorescence analysis of EpCAM (green) and E-cadherin (red) localization in 3D Matrigel-cultured spheroids of parental H2228 and H2228-CR2 cells. (B) Fluorescence intensity mapping along the white dashed line shown in (A). Peri. region, peripheral protrusive region; AJ, adherens junction. (C) Bright-field images of collagen I-embedded spheroids derived from parental H2228 and H2228-CR2 cells following transfected with sicontrol (sicon) or siEpCAM. (D) Transwell invasion assays using spheroids from parental H2228 and H2228-CR2 cells transfected with sicon or siEpCAM.

Article Snippet: The following siRNA oligonucleotides were used: control siRNA-A (sc-37007), E-cadherin siRNA (sc-35242), and EpCAM siRNA (sc-43032), all purchased from Santa Cruz Biotechnology.

Techniques: Migration, Immunofluorescence, Cell Culture, Fluorescence, Derivative Assay, Transfection

Journal: Molecules and Cells

Article Title: E-cadherin–driven adherens junction reinforcement promotes spheroid-mediated invasion and progression in ALK–rearranged lung cancer chemoresistance

doi: 10.1016/j.mocell.2026.100329

Figure Lengend Snippet: Immunohistochemistry of E-cadherin and EpCAM on longitudinal biopsy samples of patients with ALK–rearranged pulmonary adenocarcinoma (N = 23). (A) Workflow of pathological study. (B) Representative images of E-cadherin expression (×200). (C) Significantly higher E-cadherin expression was observed in the second biopsy specimen than in the first biopsy specimen. (D) Representative images of EpCAM expression (×20). (E) EpCAM expression increased significantly in the second biopsy specimen compared to that in the first biopsy specimen.

Article Snippet: The following siRNA oligonucleotides were used: control siRNA-A (sc-37007), E-cadherin siRNA (sc-35242), and EpCAM siRNA (sc-43032), all purchased from Santa Cruz Biotechnology.

Techniques: Immunohistochemistry, Expressing

Journal: Molecules and Cells

Article Title: E-cadherin–driven adherens junction reinforcement promotes spheroid-mediated invasion and progression in ALK–rearranged lung cancer chemoresistance

doi: 10.1016/j.mocell.2026.100329

Figure Lengend Snippet: Schematic diagram illustrating the role of E-cadherin and EpCAM in promoting spheroid and invasion in crizotinib–resistant ALK–rearranged lung cancer. In the upper panel, crizotinib–resistant H2228 lung cancer cells with EML4-ALK fusion exhibit enhanced morphogenic features, forming compact spheroids with elevated E-cadherin and EpCAM expression. RNA-seq analysis reveals enrichment of morphogenesis–related gene signatures. Reinforced adherens junctions (via E-cadherin) and the accumulation of EpCAM–marked protrusive cells contribute to invasion. In the lower panel, longitudinal biopsy samples from ALK–rearranged lung cancer patients show increased E-cadherin and EpCAM expression at the time of cancer progression, supporting the clinical relevance of adhesion-driven survival and structural remodeling in tumor progression.

Article Snippet: The following siRNA oligonucleotides were used: control siRNA-A (sc-37007), E-cadherin siRNA (sc-35242), and EpCAM siRNA (sc-43032), all purchased from Santa Cruz Biotechnology.

Techniques: Expressing, RNA Sequencing

Journal: Extracellular Vesicles and Circulating Nucleic Acids

Article Title: A single-sEV analysis identifies plasma EPCAM + sEVs as a biomarker for early diagnosis and monitoring postoperative remission of thyroid cancer

doi: 10.20517/evcna.2025.93

Figure Lengend Snippet: Characterization of sEV surface protein biomarkers for early diagnosis of TC via PBA. The heatmaps displayed the top 20 differentially expressed sEV proteins between HC and TC in Cohort 1 (A) and 2 (B); The differential expression of CLDN11 (C), EPCAM (D), ITGAX (E), and LAG3 (F) between HC and TC in Cohort 1 (left panel) and 2 (right panel). The data of CLDN11, ITGAX, and LAG3 in Cohort 1 was analyzed using the Student’s t -test while their data in Cohort 2 was analyzed using the Mann‒Whitney U test. The data of EPCAM in both cohorts was analyzed using the Mann‒Whitney U test; ROC curves for CLDN11 (G), EPCAM (H), ITGAX (I), and LAG3 (J) in Cohort 1 (left panel) and 2 (right panel) were plotted; (K) The plasma EPCAM concentrations were analyzed using an ELISA kit in Cohort 3, which included 41 HC and 34 TC patients. The data of plasma EPCAM concentration was analyzed using the Mann‒Whitney U test. The ROC curve was plotted in the right panel; (L) EPCAM expression was assessed by IHC in tissue sections of TC (T) with peritumor tissues (PT) and representative images were exhibited for samples in Cohort 4 ( n = 21). sEV: Small extracellular vesicle; TC: thyroid carcinoma; PBA: proximity-dependent barcoding assay; HC: healthy controls; CLDN11: claudin 11; EPCAM: epithelial cell adhesion molecule; ITGAX: integrin alpha X; LAG3: lymphocyte-activating 3; ROC: receiver operating characteristic; ELISA: enzyme-linked immunosorbent assay; IHC: immunohistochemistry; AUC: area under the curve; CI: confidence interval.

Article Snippet: The 293T cells were infected with lentivirus of negative control short hairpin RNA (shNC) or lentivirus of EPCAM short hairpin RNA (shEPCAM) obtained from OriGene Technologies (USA), and then screened by puromycin.

Techniques: Biomarker Discovery, Quantitative Proteomics, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Immunohistochemistry

Journal: Extracellular Vesicles and Circulating Nucleic Acids

Article Title: A single-sEV analysis identifies plasma EPCAM + sEVs as a biomarker for early diagnosis and monitoring postoperative remission of thyroid cancer

doi: 10.20517/evcna.2025.93

Figure Lengend Snippet: Identification of plasma sEV subpopulation biomarkers for early diagnosis of TC. The differential plasma sEV subpopulations between HC and TC in Cohort 1 (A) and 2 (B) were displayed in the two heatmaps; The CPM values of EPCAM + (C), and LAG3 + (D), SIGLEC11 + (E) sEVs were compared between HC and TC in Cohort 1 (left panel) and 2 (right panel). The data of EPCAM + and LAG3 + sEVs in both cohorts was analyzed using the Mann‒Whitney U test. The data of SIGLEC11 + sEVs in cohort 1 and 2 was analyzed using the Student’s t -test and the Mann‒Whitney U test, respectively; ROC curves for EPCAM + (F), and LAG3 + (G), SIGLEC11 + (H) sEVs in Cohort 1 (left panel) and 2 (right panel) were plotted; (I) The conventional biomarker, serum Tg, was compared between HC and TC in Cohort 1 (left panel) and 2 (right panel). The data of serum Tg in both cohorts was analyzed using the Mann‒Whitney U test; (J) ROC analysis of Tg in Cohort 1 (left panel) and 2 (right panel). sEV: Small extracellular vesicle; TC: thyroid carcinoma; HC: healthy controls; CPM: counts per million; EPCAM: epithelial cell adhesion molecule; LAG3: lymphocyte-activating 3; SIGLEC11: sialic acid-binding Ig-like lectin 11; ROC: receiver operating characteristic; Tg: thyroglobulin; AUC: area under the curve; CI: confidence interval.

Article Snippet: The 293T cells were infected with lentivirus of negative control short hairpin RNA (shNC) or lentivirus of EPCAM short hairpin RNA (shEPCAM) obtained from OriGene Technologies (USA), and then screened by puromycin.

Techniques: Clinical Proteomics, Biomarker Discovery, Binding Assay

Journal: Extracellular Vesicles and Circulating Nucleic Acids

Article Title: A single-sEV analysis identifies plasma EPCAM + sEVs as a biomarker for early diagnosis and monitoring postoperative remission of thyroid cancer

doi: 10.20517/evcna.2025.93

Figure Lengend Snippet: The performance of three blood biomarkers in monitoring post-operative remission of TC. Blood samples of TC patients before and after the operation were collected. The performance of plasma sEV counts (A) and EPCAM + sEVs (B), and serum Tg concentrations (C) in monitoring post-operative remission of TC was analyzed; (D) The chi-square analysis was conducted to evaluate the difference among the three biomarkers in monitoring TC remission. TC: Thyroid carcinoma; sEV: small extracellular vesicle; EPCAM: epithelial cell adhesion molecule; Tg: thyroglobulin; CPM: counts per million.

Article Snippet: The 293T cells were infected with lentivirus of negative control short hairpin RNA (shNC) or lentivirus of EPCAM short hairpin RNA (shEPCAM) obtained from OriGene Technologies (USA), and then screened by puromycin.

Techniques: Clinical Proteomics

Journal: Extracellular Vesicles and Circulating Nucleic Acids

Article Title: A single-sEV analysis identifies plasma EPCAM + sEVs as a biomarker for early diagnosis and monitoring postoperative remission of thyroid cancer

doi: 10.20517/evcna.2025.93

Figure Lengend Snippet: The function of EPCAM + sEV. (A) The western blotting analysis of EPCAM in 293T cells stably transfected with shNC or shEPCAM. The sEVs were isolated from 293T cells stably transfected with shNC or shEPCAM, namely shNC-sEV or shEPCAM-sEV, respectively. TPC-1 and BCPAP cells were treated with shNC-sEV or shEPCAM-sEV; (B and C) TC cell proliferation determined by CCK-8 assays; (D and E) TC cell proliferation determined by colony formation assays; Representative images of migration assays (F and G) and invasion assays (H and I) of TC cells subjected to shNC-sEV or shEPCAM-sEV. The data was analyzed using the Student’s t -test. * P value < 0.05; ** P value < 0.01; *** P value < 0.001; ns: not significant. EPCAM: Epithelial cell adhesion molecule; sEVs: small extracellular vesicles; shNC: lentivirus of negative control short hairpin RNA; shEPCAM: lentivirus of EPCAM short hairpin RNA; TC: thyroid carcinoma; CCK-8: cell counting kit-8; OD450: optical density at 450 nm.

Article Snippet: The 293T cells were infected with lentivirus of negative control short hairpin RNA (shNC) or lentivirus of EPCAM short hairpin RNA (shEPCAM) obtained from OriGene Technologies (USA), and then screened by puromycin.

Techniques: Western Blot, Stable Transfection, Transfection, Isolation, CCK-8 Assay, Migration, Negative Control, shRNA, Cell Counting

Journal: Molecular Vision

Article Title: Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells

doi:

Figure Lengend Snippet: Determination of Size and charge of nanoconjugates. The size distribution of AuNPs mixed with siRNA and/or conjugated EpCAM antibody was determined at varying AuNP:siRNA ratios. A : The siRNA conjugated gold nanoparticles showed increased size of about 710 nm and 257 nm at the ratios of 0.5 and 1, respectively. The optimal size of about 28 nm was achieved at the AuNP:siRNA weight ratio of 3:1. The size of the gold nanoparticles conjugated with PEI and EpCAM antibody at a ratio of 0.5 and 1 was 111 and 124, respectively. B : The zeta potential measurements of the AuNPs mixed with siRNA and/or conjugated EpCAM antibody. The zeta potential was almost neutral at the weight ratios of 0.5 and 1, which suggests particle aggregation in the solution. The zeta potential was shifted to 5 and 11 at the weight ratios of 2 and 3, respectively. The zeta potential was 2.1 and 1.6 at EpCAMPEI ratio of 0.5 and 1, respectively. The optimal size of about 28 nm was achieved at the AuNP:siRNA weight ratio of 3:1. The size of the AuNP-PEI-EpAb at a ratio of 0.5 and 1 was 111 and 124, respectively. B : The zeta potential measurements of the AuNPs mixed with siRNA and/or conjugated EpCAM antibody. The zeta potential was almost neutral at the weight ratios of 0.5 and 1, which suggests particle aggregation in the solution. The zeta potential was shifted to 5 and 11 at the weight ratios of 2 and 3, respectively. The zeta potential was 2.1 and 1.6 at EpCAM-PEI 0.5 and 1, respectively.

Article Snippet: Fluorescently labeled 6-fluorescein amidite (FAM)-siRNA, negative control siRNA (NC siRNA), and EpCAM siRNA were obtained from Qiagen.

Techniques:

Journal: Molecular Vision

Article Title: Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells

doi:

Figure Lengend Snippet: Determination of the effect of nano-conjugates on the cell viability of Y79 retinoblastoma cells. A : Cytotoxic evaluation of transfection reagent and gold nanoparticles (AuNP) conjugates. The MTT assay shows no significant cellular cytotoxicity is associated when treated with (gold nanoparticles) AuNP or polyethyleneimine capped gold nanoparticles AuNP-PEI or EpCAM antibody conjugated polyethyleneimine capped gold nanoparticles AuNP-PEI-EpAb (EpCAM:PEI −0.5). B : Effect of siRNA loaded AuNP-PEI/AuNP-PEI-EpAb conjugates on Y79 cell viability. The MTT assay showed that the Y79 cells treated with AuNP PEI-EpAb loaded with 100 nmol of siRNA showed significantly decreased viability (47%) when compared with cells treated with AuNP-PEI-siRNA without antibody conjugation (68%; p<0.05). However, there was a significant decrease in the cell viability of the Y79 cells treated with AuNP-PEI loaded with 100 nmol of siRNA when compared with cells treated with100 nmol of naked siRNA (p<0.05).

Article Snippet: Fluorescently labeled 6-fluorescein amidite (FAM)-siRNA, negative control siRNA (NC siRNA), and EpCAM siRNA were obtained from Qiagen.

Techniques: Transfection, MTT Assay, Conjugation Assay

Journal: Molecular Vision

Article Title: Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells

doi:

Figure Lengend Snippet: Fluorescence microscope images showing the uptake of siRNA loaded AuNP-PEI/ AuNP-PEI-EpAb nanoparticles. ( A ) control cells; ( B ) Y79 cells treated with naked siRNA; ( C ) Y79 cells treated with AuNP-PEI-siRNA and ( D ) Y79 cells treated with AuNP-PEI- EpAb-siRNA. Free FAM-siRNA was not taken up by the Y79 cells, and therefore, we did not see any detectable fluorescence. In contrast, the Y79 cells demonstrated detectable fluorescence when treated with FAM-siRNA loaded AuNP-PEI ( C ) and AuNP-PEI-EpAb nanoparticles ( D ). Increased fluorescence (white arrows show fluorescence in the Y79 cells) was seen in the Y79 cells due to the EpCAM antibody conjugation to AuNP-PEI-siRNA (FAM) when compared to cells treated with AuNP-PEI-siRNA (FAM) without EpCAM antibody conjugation.

Article Snippet: Fluorescently labeled 6-fluorescein amidite (FAM)-siRNA, negative control siRNA (NC siRNA), and EpCAM siRNA were obtained from Qiagen.

Techniques: Fluorescence, Microscopy, Conjugation Assay

Journal: Molecular Vision

Article Title: Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells

doi:

Figure Lengend Snippet: Flow cytometry analysis showing the uptake of siRNA loaded AuNP-PEI/AuNP-PEI EpAb nanoparticles by Y79 cells. ( A ) Control untreated Y79 cells run as control (violet area-M1); ( B ) Y79 cells treated with naked siRNA shows only 0.2% naked siRNA uptake (green peak-M2) when compared to normal untreated Y79 cells (violet area); ( C ) Y79 cells treated with AuNP-PEI-siRNA shows 29.2% siRNA uptake (green peak-M2) when compared to normal untreated Y79 cells (violet area); ( D ) Y79 cells treated with AuNP-PEI-EpAb-siRNA shows 59% of siRNA uptake (green peak-M2) when compared to normal untreated Y79 cells (violet area); ( E ) Only 0.4% uptake of AuNP-PEI-EpAb nanoparticles was observed in EpCAM-siRNA treated Y79 cells. Flow cytometry analysis demonstrated higher uptake of EpCAM conjugated AuNP-PEI-siRNA nanoparticles (59%) by Y79 cells when compared to EpCAM unconjugated AuNP-PEIsiRNA (29.2%; p<0.05).

Article Snippet: Fluorescently labeled 6-fluorescein amidite (FAM)-siRNA, negative control siRNA (NC siRNA), and EpCAM siRNA were obtained from Qiagen.

Techniques: Flow Cytometry

Journal: Molecular Vision

Article Title: Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells

doi:

Figure Lengend Snippet: Effect of EpCAM antibody and siRNA conjugated nanoparticles on EpCAM expression in Y79 cells. A : Real-time reverse-transcriptase PCR analysis of EpCAM in Y79 cells. The graph shows significant down-regulation of EpCAM mRNA in Y79 cells treated with EpCAM siRNA loaded with polyethylenemine capped gold nanoparticles (AuNP-PEI-siRNA) and EpCAM antibody conjugated siRNA loaded PEI capped AuNP (AuNP-PEI-EpAb-siRNA) when compared to Y79 cells treated with scrambled siRNA or AuNP alone or AuNP-PEI. The asterisk mark represents statistically significant (p<0.05) expression increase in EpCAM compared to other groups. Enhanced downregulation of the EpCAM gene (−15 fold) was observed in the Y79 cells treated with EpCAM-conjugated AuNP-PEI-siRNA when compared with EpCAM-unconjugated AuNP- PEI-siRNA (p<0.05). B : Western blotting analysis shows the effect of siRNA loaded AuNP conjugates on the EpCAM protein levels in the Y79 cells. Enhanced knock-down of the EpCAM protein was observed in the Y79 cells when they were treated with EpCAM-conjugated AuNP-PEI-siRNA. β-actin was included as the loading control (panel 2 and 4). C : Bar graphs showing the densitometry analysis of western blotting of the EpCAM protein. The graph shows the percentage optical density of the EpCAM protein detected bands on the blotting membrane. Enhanced knockdown of the EpCAM protein was observed in the Y79 cells when they were treated with EpCAM-conjugated AuNP-PEI-siRNA.

Article Snippet: Fluorescently labeled 6-fluorescein amidite (FAM)-siRNA, negative control siRNA (NC siRNA), and EpCAM siRNA were obtained from Qiagen.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: An EpCAM/Trop2 mechanostat differentially regulates individual and collective migration of human carcinoma cells

doi: 10.1101/2022.10.03.510449

Figure Lengend Snippet: Spheroids of transfected MCF7 cells with control, EpCAM, Trop2 and EpCAM/Trop2 siRNA were plated on a layer of fibrillar collagen gel, let adhere for 30 minutes, then phase contrast images were taken every 30min for 24hrs. A-D . Images of whole spheroids at selected time points. Scale bar = 100μm. E . Quantification of relative area increase after 24hrs, expressed as ratio of the final and initial areas. Six to nine independent experiments, with a total of 45-63 spheroids. In all figures, the box plots show the interquartile range (box limits), median (center line), and min and max values without outliers (whiskers). Statistical analysis: One-way non-parametric ANOVA (Kruskal-Wallis Test) followed by Dunn post hoc test. For all experiments presented in this study, P values are indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001 and NS, not significant. F . Quantification of spheroids solidity, measured as the ratio [Area]/[Area of convex hull]. Quantification from the six experiments. G . Schematic representation of typical spheroid morphologies in top and orthogonal views. H, H’ . Examples of phalloidin-labelled spheroids after 24hrs spreading in top and orthogonal view. H” . Details of spheroid edges. Maximal projection of 3 z planes, 1μm apart. Protrusions are observed in all conditions (arrowheads). However, control and Trop2 KD spheroids have numerous cells protruding out of the main cell mass (asterisks), which are rare in EpCAM KD and dKD. These are rather characterized by frequent actin cable-like structures along the spheroid edge (arrows).

Article Snippet: EpCAM (sc-43032), Trop2 (sc-72392) and control non-targeting (sc-37007) siRNA were from Santa Cruz Biotechnology.

Techniques: Transfection, Control

Journal: bioRxiv

Article Title: An EpCAM/Trop2 mechanostat differentially regulates individual and collective migration of human carcinoma cells

doi: 10.1101/2022.10.03.510449

Figure Lengend Snippet: A . Scheme representing EpCAM negative regulation of the nPKC - myosin II pathway with the corresponding chemical inhibitors calphostin and blebbistatin. B . Myosin II and nPKC inhibition rescue the EpCAM KD spreading phenotype in a dose-dependent manner, and decrease spreading of wild type spheroids. Spreading of control and EpCAM KD spheroids was tested in the absence or presence of 0.25 and 0.5μM calphostin C (Calph), or 2 and 5μM blebbistatin (Bleb). Area increase was normalized to untreated controls. 7-32 spheroids from 2-8 independent experiments. Statistical analysis: one-way ANOVA followed by Tukey-HSD post hoc test. C-F . E-cadherin and pMLC levels are elevated upon both EpCAM and Trop2 KD. C , D . Representative confocal images of spheroids after 24hrs spreading, immunolabelled for p-MLC and E-cadherin. Levels are visualized using the “fire” pseudocolors of ImageJ. The selected z planes correspond to the widest area for each spheroid. Scale bar = 50μm. E-G . Quantification of p-MLC (E) and E-cadherin (F) mean intensities along cell outlines, and calculated E-cadherin/pMLC ratio (G). Both EpCAM and Trop2 KD cause an increase in both pMLC and E-cadherin. However, the E-cadherin to pMLC ratio is highest in EpCAM KD but lowest in Trop2 KD. Eight spheroids of each condition from three independent experiments. One-way non-parametric ANOVA (Kruskal-Wallis) followed by Dunn post hoc test.

Article Snippet: EpCAM (sc-43032), Trop2 (sc-72392) and control non-targeting (sc-37007) siRNA were from Santa Cruz Biotechnology.

Techniques: Inhibition, Control

Journal: bioRxiv

Article Title: An EpCAM/Trop2 mechanostat differentially regulates individual and collective migration of human carcinoma cells

doi: 10.1101/2022.10.03.510449

Figure Lengend Snippet: A-D . Traction Force Microscopy (TFM) of cell doublets on H patterns. A . Scheme of the experimental settings. Cells doublets were laid on H patterns coated with a thin layer of collagen (green) on a polyacrylamide gel with a stiffness of 5kPa, containing far red fluorescence nanobeads. Cells and underneath nanobeads were imaged, then cells were removed by trypsinization, and second images of the same positions were taken and used as reference, from which bead displacement was measured. A’ . Diagram of force vectors obtained by TFM on H-shape micropatterns. While traction forces (black arrows) are measured from the displacement of nanobeads, the cell-cell force (red arrow) is calculated indirectly based on it counterbalancing the sum of traction forces, as shown in the equation. B . Representative images of micropattern-confined cell doublets for the four experimental conditions. C . Corresponding average maps of traction forces. D . Quantification of traction forces. E . Quantification of cell-cell forces. F . Cell-cell force to traction force ratio. G . Quantification of junction lengths, measured from the phase contrast images. One-way ANOVA followed by Tukey-HSD post-hoc test. H-L. Determination of relative cortical tension and cell-cell adhesiveness of cell doublets laid on a non-adhering surface. H . Diagram of an asymmetric cell doublet, with the balance between cortical tensions at the free edges (γ m(A) and γ m(B) ) and contact tension (γ c(AB) ). The principles of such system are as follows: The directions of γ m(A) , γ m(A) and γ c(AB) are tangential to the membranes at the cell vertex, which allows to directly calculate the relative strengths of these tensions based on the geometry at vertices. The orange layer represents the actomyosin cortex, with its thickness symbolizing relative contractility. In the configuration represented in the diagram, the curved cell-cell interface reflects differences in cortical tension, as cell A tends to partly engulf B, due to its lower tension (γ m(A) < γ m(B) ). The strength of adhesion is related to the capacity of downregulating cortical tension along the cell-cell contact (2 * γ c(AB) < γ m(A) + γ m(B) ). θ is the angle formed by the two vectors γ m(A) and γ m(B) . It directly relates to adhesiveness, a dimensionless value that ranges from 0 to 1. I , J . Examples of homotypic and heterotypic doublets imaged by live confocal microscopy. The doublets were formed by mixing dissociated cells and let them re-associate on an adhesion-free support. Membranes were labeled with CellMask Alexa Fluor 647. siCtrl (A) cells were marked by Hoechst staining (showed in red) prior dissociation. Scale bar = 10μm. K . Quantification of relative cortical tensions expressed as the ratio γ m(A) /γ m(B) . As expected, under all conditions, the ratio is close to 1 for homotypic doublets. The ratio for heterotypic doublets is significantly lower, demonstrating that single and double depletions all cause an increase in cortical tension. L . Quantification of adhesiveness, calculated from γ c and γ m . Values for homotypic doublets show that doublets of EpCAM KD or dKD cells have increased adhesiveness compared to control cells. Trop2 KD only leads to a weak, non-significant increase. In the case of heterotypic doublets, however, adhesiveness appears decreased in all cases. Four independent experiments, total 500-1250 doublets. Experiment medians indicated by dots. One-way non-parametric ANOVA (Kruskal-Wallis Test) followed by Dunn post-hoc test.

Article Snippet: EpCAM (sc-43032), Trop2 (sc-72392) and control non-targeting (sc-37007) siRNA were from Santa Cruz Biotechnology.

Techniques: Microscopy, Fluorescence, Confocal Microscopy, Labeling, Staining, Control

Journal: bioRxiv

Article Title: An EpCAM/Trop2 mechanostat differentially regulates individual and collective migration of human carcinoma cells

doi: 10.1101/2022.10.03.510449

Figure Lengend Snippet: A . Representation of schematic cell doublets on a substrate, with the relative tensions represented at the cell-cell vertex and at the edge of the matrix substrate interface. Their relative strength is represented by the length of the vector. The general changes in tensions between control and EpCAM KD, and between EpCAM KD and Trop2 KD are summarized in the two boxes with blue borders. B . Balance of forces at the cell-cell vertex. While all three tensions are strongly increased both in EpCAM KD and Trop2 KD, the resulting adhesiveness, which can be directly deduced from the angle θ, is increased in EpCAM KD but not in Trop2 KD. C . Balance of forces at the matrix interface. γ s is the tension exerted on the substrate, corresponding to the traction measured by TFM. It counterbalances the cell-matrix tension γ x and the horizontal component of the cell-medium tension γ m . Both tensions are increased in EpCAM and Trop2 KD, but their relative balance determines different outcomes: EpCAM KD cells adopts a spread configuration (acute angle ϕ), while, on the contrary, Trop2 KD cells have a compact shape (obtuse angle ϕ). D-F . Simulation of spheroids spreading using a 3D cellular Potts model (CompuCell3D software) for control, EpCAM KD and Trop2 KD conditions. See annex for detailed information. Images correspond to examples of yx and yz planes after 300 iterations. Medium is in light blue, matrix substrate in dark blue. D’-D’ . Outlines of explants at 24hrs from , drawn for comparison. G . Energy parameters used to simulate the three conditions. H , H’ . Comparison of curves of relative area expansion from experimental data and from simulation. Error bars: SD.

Article Snippet: EpCAM (sc-43032), Trop2 (sc-72392) and control non-targeting (sc-37007) siRNA were from Santa Cruz Biotechnology.

Techniques: Plasmid Preparation, Control, Software, Comparison

Journal: bioRxiv

Article Title: An EpCAM/Trop2 mechanostat differentially regulates individual and collective migration of human carcinoma cells

doi: 10.1101/2022.10.03.510449

Figure Lengend Snippet: Confocal microscopy image of groups of wild-type MCF cells on collagen gel, labelled under non-permeabilized conditions for EpCAM (green), Trop2 (red) and F-actin (phalloidin-Alexa647, magenta). EpCAM and Trop2 levels where analyzed from whole z-stacks, as well from resliced stacks to obtain z projections (see details in suppl. Fig.S5). Both EpCAM and Trop2 were found distributed all along the membrane. A . Merged image. A’ , A” , A’’’ . Individual channels. A”“ . Maximal projection of the whole stack. B . Example of z projection. The position of the slice is indicated by the white dashed line in A. EpCAM and Trop2 channels are displayed as “fire” pseudocolours. The patterns of the two regulators appear virtually identical, except for slightly higher Trop2 levels at the ventral side (yellow arrowheads). C-F. Quantification of relative levels of EpCAM and Trop2. C . Levels in three regions of control embryos, indicated in the diagram C’, and reciprocal impact of EpCAM and Trop2 depletions. A larger set of 12 categories was quantified, see suppl. Fig.S5 for full analysis. All values were normalized to the average intensity for EpCAM or for Trop2 at cell contacts of control cells (contact ave, see suppl. Fig.S5A). In control groups, cell contacts and dorsal side had similar relative levels of EpCAM and Trop2. The ventral side showed the lowest levels, but a relative enrichment in Trop2 versus EpCAM was reproducibly observed. Trop2 KD had no significant impact on EpCAM levels and distribution. EpCAM KD led to a decrease of Trop2 at contacts and dorsal side (and most locations, suppl. Fig.S5C), but a significant enrichment on the ventral side. D . Direct pairwise comparison of dorsal to ventral ratios for EpCAM and Trop2 in wild type cell groups. The ratio is significantly lower for Trop2. E , F . Effect of Trop2 and EpCAM depletions on the dorsal-ventral ratio (E) and on the ratio dorsal side versus average cell contact (F). EpCAM is not significantly affected by Trop2 KD, but Trop2 ratios are strongly decreased in EpCAM KD. All quantifications were on 13-15 groups of cells (4-8 cells per group) from three independent experiments. Statistical analysis: C: Non-parametric ANOVA (Kruskal-Wallis Test) followed by post-hoc Pairwise Mann-Whitney tests. D-F: Student’s t-test. G. Summary diagram of EpCAM and Trop2 cell surface distribution in wild type cells and changes occurring upon EpCAM or Trop2 KD . Relative levels are symbolized by red and green colour scales. Depletion is symbolized by grey cells. Blue arrows of different sizes point to Trop2 levels and changes observed upon EpCAM KD. The result is a relative enrichment at the ventral and cell contact sites relative to dorsal. This change is compatible with a decrease in corresponding tension balances, stimulating spreading on the substrate and cell-cell adhesion. Purple arrows point to the absence of significant changes for EpCAM levels in Trop2 KD, indicating that the Trop2 KD phenotype is mainly due to the decrease of the Trop2-dependent regulation. In particular, lower spreading can be explained by the loss of the ventral Trop2 pool, leading to relatively higher contractility compared to the dorsal side.

Article Snippet: EpCAM (sc-43032), Trop2 (sc-72392) and control non-targeting (sc-37007) siRNA were from Santa Cruz Biotechnology.

Techniques: Confocal Microscopy, Membrane, Control, Comparison, MANN-WHITNEY