Fig 2: Identification of P4HA3 by integrated metabolomic and transcriptomic analysis.a Metabolic reactions of ARG2, ANPEP, GLS, and P4HA3. b Correlations between EMT markers and P4HA3 mRNA expression in non-small cell lung cancer (NSCLC) cell lines in the Cancer Cell Line Encyclopedia (CCLE) dataset. EMT scores were calculated based on the expression of the reported 76 genes as an EMT marker in NSCLC. c Expression of P4HA3 during TGF-ß-stimulation and withdrawal. A549 cells were stimulated with TGFß (2 ng/mL) for the indicated number of days (marked TGF-ß), and mRNA level of P4HA3 was measured by real-time PCR. Values are presented as the mean ± SD from triplicate samples. d Protein levels of EMT markers (CDH1 and CDH2) and P4HA3 in A549 cells. A549 cells were stimulated with TGF-ß (5 ng/mL) for 3 days (marked TGF-ß) and then cultured in normal growth medium without TGF-ß for 3 days (marked Withdraw). Actin was used as a loading control for western blot analysis.

Fig 3: Spatiotemporal expression of CXC-chemokine receptor 4 (CXCR4) and its ligand, CXCL12, and developmental genes in embryonic organs. (A) Schematic diagram of embryonic submandibular gland (eSMG) isolation and ex vivo culture. (B) Ex vivo branching morphogenesis of eSMGs from embryonic day (E) 13 to 17, showing epithelial growth and retraction of mesenchyme. Scale bars: 500 µm. (C) Temporal mRNA expression patterns of keratin 7 (Krt7), aquaporin 5 (Aqp5), e-cadherin (Cdh1), Krt15, Cxcr4, and Cxcl12 were measured from E13 to E17 by qPCR (n = 3). (D) Epithelial (Epi) and mesenchymal (Mes) expression of Cxcr4, Cxcl12, odd-skipped related transcription factor 1 (Osr1), and Cdh1 were quantified by qPCR at E13. The comparative Ct values are expressed as fold increase relative to the epithelium (n = 3). (E) Representative images showing expression of CXCR4 and CXCL12 in eSMG (upper) and their colocalization (lower) (n = 3, scale bar: 500 µm). (F) Representative immunofluorescence images of CXCR4 and CXCL12 expression in E12 embryonic lung and pancreas (n = 4); whole view (left two panels; scale bar: 500 µm) and magnified lumen structures (right two panels; scale bar: 50 µm). Data are presented as the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS: not significant; two-way ANOVA (C); t-test (D).

Fig 4: Antitumor effect of 5‐FU in an ESCC xenograft mouse model and immunohistochemical staining of ESCC samples from patients treated with NAC. (A) Summary of the xenograft model using TE11* and TE11‐ICN3 cell lines. Female nu/nu mice (8 weeks of age) were subcutaneously injected with 3 × 106 cells. When the average tumor volume reached approximately 100 mm3, mice were provided with water containing DOX. PBS or 5 mg/kg 5‐FU was administered to these mice by intraperitoneal injection every 3 days. (B) Tumor volume in TE11‐ICN3 mice treated with 5‐FU was significantly lower than in corresponding mice treated with PBS. (C) Analysis of apoptosis by TUNEL staining (blue: DAPI staining for nuclei, cyan: TUNEL staining) in tumors of xenograft mice. (D) Immunohistochemical analysis of NOTCH3, VIM, CDH2, and CDH1 in tumors of xenograft mice. Scale bar, 100 μm. (E) Western blot analysis of lysates prepared from tumor tissue of mice treated with vehicle control or 5‐FU for 21 days. ACTB served as a loading control. (F) NOTCH3‐negative, CDH2‐positive, and VIM‐positive IHC staining. Scale bars, 100 μm. (G) NOTCH3‐positive, CDH2‐negative, and VIM‐negative IHC staining. Scale bars, 100 μm. (H and I) Survival curves based on NOTCH3 expression in ESCC patients treated with NAC. (H) Overall survival and (I) recurrence‐free survival. Blue and red lines correspond to NOTCH3‐positive and NOTCH3‐negative groups, respectively. TE11*: TE11 Tet‐on control cell line, TE11‐ICN3: TE11 Tet‐on ICN3 cell line, DOX: Doxycycline, ICN3: intracellular NOTCH3, NAC: neoadjuvant chemotherapy

Fig 5: OVOL-induced MET reduces the metastatic potential of mesenchymal PC3-EMT14 in vivo.(A) Tumor burden: Luciferase expression is depicted as regions of interest (ROI-photons/s) in mice with orthotopic injections.(B) Metastasis: (Left) the percentage of orthotopically inoculated mice with multiple luciferase signals at 21 and 28 days (Right). depicts the total number of metastases per group divided by the number of mice (n) per group at 28 days.(C) Imaging: Representative images of luciferase expression in mice 28 days after receiving orthotopic injections. Metastases for each group are circled in red in either the ventral or dorsal images and excluding those suspected to correspond to the same tumor.(D) Tumor Weight: The average weights of orthotopic (prostate) tumors resected at 42 days (n = 4).(E) IHC: E-cad, and ZEB1 staining of metastatic tumors in mice that received orthotopic injections from each group inoculated with OVOL expressing cells or control PC3-EMT14. Note the higher E-cad and lower ZEB1 expression in OVOL-expressing cancer cells. Scale bar represents 100 µm. The IHC shows a representative staining of one out of three sections with similar results.Graphs show mean +/- sem; p-values were calculated and represented as ** p < 0.01. See also Figure S4.