Fig 2: Cadherin heterogeneity within the same cell population in ETX embryos.(a) Pie charts show different mis-sorted ETX embryos under the indicated conditions. n = 4186 (control), n = 2940 (Cdh1-KD ES), n = 2471 (Cdh3-KD ES), n = 2407 (Cdh1-KD TS), n = 2151 (Cdh3-KD TS) structures were collected from 3 independent experiments for quantification. (b) Immuno-staining of ES (upper) and TS (lower) cells to reveal E-cadherin (green) and P-cadherin (red), respectively. Nuclei (purple) are stained by DAPI. Scale bars represent 100 µm. Zoomed images are of regions indicated by dashed lines. Experiments were repeated 5 times. (c) Flow cytometric analysis of E-cadherin in wild-type ES cells, E-cadherin knockdown ES cells and ES cells over-expressing E-cadherin (upper). Flow-cytometric analysis of P-cadherin in wild-type TS cells, P-cadherin knockdown TS cells and TS cells over-expressing P-cadherin (lower). CV (coefficient of variation) values shown against peak values in plots. (d) Top: FACS profiles for E-cadherin in E-cadherin KD, WT and E-cadherin OE ES cells, Bottom: FACS profiles for P-cadherin in P-cadherin KD, WT and P-cadherin OE TS cells. Source data

Fig 3: (A) Analysis of RNA-Seq data from TCGA Pan-cancer analysis with mean value for the FPKM for each cancer type and each cadherin family member. (B) RNA-seq from CTCs enriched from whole blood with the iChip microfluidic device and isolated as single cells. Expression values represent log10(RPM+1). Gray horizontal lines demarcate individual patients. (C) Three-dimensional modeling of the top two docking sites of the antigen binding region (ribbon model) of the 23C6 antibody on the extracellular domain of CDH11 (space filling model). Upper: Binding free energy difference (ΔE) = −46.4 and Lower model with ΔE = −32.6. (D) Protein sequence alignment using Clustal omega of the two docking sites of the 23C6 antibody on CDH11 compared to the protein sequences for CDH1. Colors represent amino acid properties: Red, identical amino acids; orange, conserved amino acids with combined strongly and weakly similar properties. (E) Western blot analysis of recombinant purified hCDH1, hCDH11 and mCDH1 proteins detected with 23C6 antibody or a CDH11 specific antibody 3H10. “h” indicates human and “m” indicated murine. Loading control with Ponceau staining. High molecular weight bands likely represent aggregation of the highly purified proteins. (F) H&E staining (Left) and 23C6 immunohistochemical staining (Right) of representative sections of two different tumors from a breast cancer tumor array. Upper right scored as 3+ for tumor cell membrane staining. Lower right scored as 1+ for tumor cell membrane staining. (G) Distribution (Upper) and breakdown in ER positive and negative tumors (Lower) of membranous staining intensity on a scale of 0 (no staining) to 3+ (intense staining). (H) Analysis of RNA-Seq data from TCGA Pan-cancer analysis of whole genomes with mean value for the FPKM for CDH1 and CDH11. Error bars represent SD. (I) RNA-seq from single cell CTCs enriched from whole blood from metastatic breast cancer patients. (Left) Proportion of patients with average expression of the combined sum of CDH1 and CDH11 in the indicated ranges. (Right) Proportion of CTCs with >5 RPM expression of CDH1 and CDH11 within individual patients.

Fig 4: Differential cadherin code in ETX-embryo and natural embryo.(a) UMAP dimensional reduction shows Cdh1, Cdh3 and Cdh6 expression profile in different clusters as indicated by dashed lines. Each dot represents a single cell that is color-coded by sample type. (b) Heatmap showing average expression of cadherin and protocadherin related genes revealed by scRNA-seq in natural embryos (NE, n = 50) collected at 4.5, 5.5 and 6.5 days after fertilization and well-sorted ETX embryos (n = 50) at 4, 5, 6 days of culture. (c) Colonies of cultured ES and TS cells stained to reveal E-cadherin (green) and P-cadherin (red). Quantifications showing the mean intensity (A.U.) of E-cadherin or P-cadherin at cell-cell junctions. 20 colonies from 3 different experiments were selected for quantification. Scale bars represent 100 µm. Data are presented as mean ± SD. Statistics calculated by unpaired two-tailed Student’s t test. (d) Natural embryos (E5.5) and ETX embryos (Day 4) stained to reveal E-cadherin (green) and P-cadherin (red). Magnified insets show E- or P-cadherin staining in ExE and EPI compartments in natural embryos, TS and ES compartments in ETX embryos. Quantifications showing the mean intensity (A.U.) of E-cadherin or P-cadherin at cell-cell junctions. n = 20 ETX embryos and n = 19 natural embryos were used for quantification. Data are presented as mean ± SD. Statistics calculated by unpaired two-tailed Student’s t test. Scale bars represent 100 µm (main Figure) and 20µm (inset). (e) Representative images of E4.5 chimeras (8-cell stage embryos aggregated with Cdh6 or (f) Cdh3 OE ES stained for RFP (magenta), Sox17 (green), and DAPI (grey). Experiments were repeated 3 times. Scale bars represent 50 µm. Zoomed images are of regions indicated by dashed lines (scale bars represent 10 µm). Source numerical data are available in source data. Source data

Fig 5: Differential adhesion force in ETX embryos.a, Schematic showing cell–cell adhesion force measurement by AFM. b, The resulting force–distance curve, following the procedure depicted in a, enables quantification of the maximum adhesion force (Fmax). c, Fmax for the indicated homotypic and heterotypic adhesions between three different cell types. The experiments were performed three times independently. Total measured cell pairs: n = 60 (ES–ES), n = 177 (TS–TS), n = 101 (XEN–XEN), n = 124 (ES–TS), n = 148 (XEN–TS) and n = 134 (XEN–ES). Statistical significance was determined by one-way ANOVA with a multiple comparison test. d, Schematics of weakly and strongly adherent cell pairs at force equilibrium. ? is the contact angle of the two adhering cells. e, Distribution of the measured contact angles at all cell–cell contacts. Total measured cell pairs: n = 31 (ES–ES), n = 38 (TS–TS), n = 30 (XEN–XEN), n = 32 (TS–ES), n = 36 (XEN–TS) and n = 29 (XEN–ES). N = 3 for all conditions. Statistical significance was determined by one-way ANOVA with a multiple comparison test. f, Adhesion forces between cells and different cadherins. Left, schematic showing cell–cadherin adhesion force measurement by AFM. Right, quantification of the results. n = 42 (ES–E-cadherin), n = 35 (ES–P-cadherin), n = 41 (TS–E-cadherin) and n = 37 (TS–P-cadherin). N = 3 for all of the conditions. Statistical significance was determined by unpaired two-tailed Student’s t-test. g, Fmax for homotypic adhesion between the three different cell types after downregulation of Cdh1 or Cdh3. n = 60 (WT ES–ES), n = 18 (Cdh1 KD ES–ES), n = 19 (Cdh3 KD ES–ES), n = 177 (wild-type TS–TS), n = 20 (Cdh1 KD TS–TS), n = 20 (Cdh3 KD TS–TS), n = 101 (wild-type XEN–XEN), n = 19 (Cdh1 KD XEN–XEN) and n = 19 (Cdh3 KD XEN–XEN). N = 3 for all conditions. Statistical significance was determined by one-way ANOVA with a multiple comparison test. h, Heatmap of the adhesion parameter matrix, generated by sampling measured AFM adhesion forces, which parameterizes the CPM. i, Bootstrapping procedure to infer the distributions of conformations under the CPM (N = 498). The schematic represents all of the possible sorted conformations, demonstrating that the ETX-like configuration is the most represented. Conformations observed at a frequency of <5% are grouped. MCS, Markov Chain Steps. In the box and whisker plots in c and e–g, the line inside the box indicates the median value and the error bars show the minimum and maximum values. Box edges indicate lower and upper quartile value. Numerical data are available as source data.Source data