Journal: Frontiers in Microbiology

Article Title: Gut microbiota dysbiosis impairs TGF-β/Smad4 signaling to drive postoperative metastasis in colorectal cancer

doi: 10.3389/fmicb.2025.1654227

Figure Lengend Snippet: Comparison of liver EMT-related protein expression via immunohistochemistry in four groups. (A) Representative immunohistochemical staining of N-cadherin, E-cadherin, MMP-9, and VEGF in livers (Scale bar = 100 μm); (B) the histochemistry score (H-score) of N-cadherin; (C) the H-score of E-cadherin; (D) the H-score of MMP-9; (E) the H-score of VEGF. Values are expressed as mean ± SEM ( n = 3 per group); The Kruskal–Wallis test followed by Dunn’s post-hoc test was used for non-parametric data.

Article Snippet: The slides were repaired in sodium citrate buffer before being blocked with a 3% BSA solution for 30 min. After that, primary antibodies such as E-cadherin (1:400, Boster, China), N-cadherin (1:400, Boster, China), MMP9 (1:400, Boster, China), and VEGF (1:400, Boster, China) were added to the slides and incubated at 4 °C for the entire night.

Techniques: Comparison, Expressing, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: CDH3 as a Novel Therapeutic Target in Basal-like Double-Negative Prostate Cancer

doi: 10.1101/2025.11.18.689150

Figure Lengend Snippet: ( A ) Survival curves of wild-type (WT) mice compound transgenic mice carrying prostate-specific Cre driver ( PB Cre ), floxed fluorescent Cre-reporter ( ROSA mT/mG ) and the floxed alleles of key tumor suppressor genes ( Pten alone or in combination with p53 , Smad4 or Apc ). ( B ) H&E staining of prostate tissue from wild type, Pten and Pten Apc mice at 4 months old. Scale bar: 100 µm. ( C ) Heatmap of RPPA analysis depicting the top 20 most variably expressed proteins from lysates of wild type prostate and four different genotypes ( Pten , Pten Apc , Pten p53 , Pten Smad4 ), sampled from the anterior prostate (AP) or dorsolateral prostate (DLP) lobes. The intensity scale ranges from downregulated (blue) to upregulated (red). ( D ) Scatter plot showing the log2 fold change of protein expression ( Pten Apc vs others) versus expression variability across 281 proteins in all samples. CDH3, the protein with the highest differential expression, is highlighted in red. ( E ) Quantification of CDH3 expression levels (normalized values) across the same five genotypes. ( F ) Immunohistochemical staining showing CDH3 expression in Pten Apc , Pten p53 and Pten Smad4 tumors. Scale bar: 100 µm. ( G ) KEGG pathway analysis of GFP+ tumor cell transcriptomes ( Pten Apc vs. Pten Smad4 ) using genes significantly upregulated (log2FC > 2.5, adj. p-value < 0.05). ( H ) KEGG pathway analysis of bulk tumor transcriptomes ( Pten Apc vs. Pten Smad4 ) using genes significantly upregulated (log2FC > 2, adj. p-value < 0.05). ( I ) Heatmap of the top differentially expressed Wnt pathway–related genes in both the whole-tumor and GFP+ cohort transcriptomes, separated by canonical (yellow labels), non-canonical (purple labels) Wnt signaling and Wnt negative regulators (gray labels). Genes were selected based on differential expression ( Pten Apc vs. Pten Smad4 , |log₂FC| > 2, adj. p-value < 0.05) in the GFP⁺ cohort. ( J ) Expression levels of Axin2 (canonical Wnt marker) and Wnt5a (non-canonical Wnt marker) in the GFP+ cell cohort from wild type prostate, Pten Apc , and Pten Smad4 tumors, multiple probe IDs are shown for each gene. Error bars represent SEM. Kruskal-Wallis test (non-parametric ANOVA, red asterisks) for ( D ) and Mann-Whiney test (black asterisks) for ( E ) and ( J ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: We used a humanized IgG1 antibody directed against the extracellular domains of CDH3 (Perseus Proteomics), and chose to focus on DU145 to test the therapeutic agents because of its highest surface CDH3 among the various prostate cancer cell lines ( ).

Techniques: Transgenic Assay, Staining, Expressing, Quantitative Proteomics, Immunohistochemical staining, Marker

Journal: bioRxiv

Article Title: CDH3 as a Novel Therapeutic Target in Basal-like Double-Negative Prostate Cancer

doi: 10.1101/2025.11.18.689150

Figure Lengend Snippet: ( A ) t-SNE projection of transcriptomes of CRPC patient-derived xenograft (PDX), cell line, and organoid samples from Tang et al. (2022), illustrating four major subtypes: AR-dependent (ARPC), stem cell-like (SCL), neuroendocrine (NEPC), and WNT-driven (WNT). ( B ) Heatmap of selected pathway-associated genes across the same sample set. Red denotes upregulation, blue denotes downregulation. ( C ) CDH3 expression level, canonical WNT score, non-canonical WNT score, and basal score, compared among the different groups. ( D ) CDH3 expression in different subtype groups across multiple prostate cancer cohorts (Mayo, Cleveland, Thomas Jefferson, Johns Hopkins). Samples were classified into basal-like, luminal A, or luminal B subtypes using a PAM50-based approach. ( E ) CDH3 expression in TCGA primary prostate cancer (PRAD), classified by PAM50 subtypes, ( F ) Metastatic sample from Fred Hutch Cancer Center (FHCRC) prostate cancer datasets, classified by PAM50 subtypes. Error bars represent SEM. Kruskal-Wallis test (non-parametric ANOVA) for ( C ) and Mann-Whiney test for ( D ), ( E ) and ( F ). Box plots represent the 5th to 95th percentiles, with the horizontal line indicating the median for ( D ), ( E ) and ( F ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

Article Snippet: We used a humanized IgG1 antibody directed against the extracellular domains of CDH3 (Perseus Proteomics), and chose to focus on DU145 to test the therapeutic agents because of its highest surface CDH3 among the various prostate cancer cell lines ( ).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: CDH3 as a Novel Therapeutic Target in Basal-like Double-Negative Prostate Cancer

doi: 10.1101/2025.11.18.689150

Figure Lengend Snippet: ( A ) Single-cell UMAP from the Song et al. (2022) dataset showing prostate cancer cells grouped into basal epithelial, luminal epithelial, immune, and stromal populations. ( B ) CDH3 expression in the epithelial sub-compartment in feature plot (heatmap scale) and dot plot (dot size indicates percentage of expressing cells; color represents average expression). ( C ) Feature plots for selected non-canonical WNT genes within the epithelial sub-compartment. ( D ) Comparison of average expression of canonical vs. non-canonical WNT pathway components in basal vs. luminal cells. ( E ) Single-cell UMAP from the Wong et al. (2022) dataset, illustrating a similar array of cell populations. ( F ) CDH3 expression in the epithelial sub-compartment in feature plot (heatmap scale) and dot plot (dot size indicates percentage of expressing cells; color represents average expression). ( G ) Feature plots for selected non-canonical WNT genes within the epithelial sub-compartment. (H) Comparison of average expression of canonical vs. non-canonical WNT pathway components in basal vs. luminal cells. Wilcoxon rank-sum test for ( D ) and ( H ). *Adjusted P < 0.01, ns, not significant.

Article Snippet: We used a humanized IgG1 antibody directed against the extracellular domains of CDH3 (Perseus Proteomics), and chose to focus on DU145 to test the therapeutic agents because of its highest surface CDH3 among the various prostate cancer cell lines ( ).

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: CDH3 as a Novel Therapeutic Target in Basal-like Double-Negative Prostate Cancer

doi: 10.1101/2025.11.18.689150

Figure Lengend Snippet: ( A ) Immunofluorescence of prostate cancer cell lines (LNCaP, PC3, LHMK, and DU145 cells), YAP1 (green) and nuclei (DAPI, red) are shown to illustrate subcellular localization. An extra group of DU145 cells were treated with the YAP inhibitor verteporfin (Ver, 0.5 µM) for 72 hours. Scale bar, 20µm. ( B ) Western blot of CDH3 protein levels in LHMK, 22RV1, and DU145 cells comparing vehicle controls to verteporfin-treated conditions (0.3, 0.5 µM) for 72 hours. β-actin serves as a loading control. ( C ) Flow cytometry histograms of CDH3 expression in DU145 and 22RV1 cells, comparing vehicle controls to verteporfin-treated conditions (0.3, 0.5 µM) for 72 hours. ( D ) Flow cytometry analysis of CDH3 median fluorescence intensity (MFI) in DU145 and 22RV1 cells following CRISPR-mediated ROR2 knockout (sgRNA1–3), compared to a non-targeting control (scr-sgRNA). ( E ) Flow cytometry analysis of CDH3 MFI in DU145 cells, comparing vehicle controls to ETC159 (5, 25, 100 μM, an inhibitor of WNT ligand secretion) conditions for 72 hours. ( F ) Flow cytometry histograms of CDH3 expression in DU145 cells treated with ETC159 in the presence or absence of Wnt5a-conditioned medium (CM, 20% v/v) for 72h. ( G ) Visualization of CHIP-seq data from Tang et al. (2022) illustrating control (Ctrl), YAP, and FOSL1 binding profiles at the CDH3 locus in DU145 (orange) cell line and MSK-PCa3 (purple) organoid. ( H ) Expression levels of CDH3, ROR2, and WNT5A in DU145 cells upon YAP knockdown (KD) versus scramble control. ( I ) Expression levels of CDH3 upon overexpression (OE) of YAP or FOSL1 in LNCaP cells. Error bars represent SEM.

Article Snippet: We used a humanized IgG1 antibody directed against the extracellular domains of CDH3 (Perseus Proteomics), and chose to focus on DU145 to test the therapeutic agents because of its highest surface CDH3 among the various prostate cancer cell lines ( ).

Techniques: Immunofluorescence, Western Blot, Control, Flow Cytometry, Expressing, Fluorescence, CRISPR, Knock-Out, ChIP-sequencing, Binding Assay, Knockdown, Over Expression

Journal: bioRxiv

Article Title: CDH3 as a Novel Therapeutic Target in Basal-like Double-Negative Prostate Cancer

doi: 10.1101/2025.11.18.689150

Figure Lengend Snippet: ( A ) Flow cytometry analysis of CDH3 MFI in different prostate cancer cell lines (DU145, 22RV1, LHMK, LNCaP, PC3). ( B ) Immunofluorescence of DU145-TR-CDH3 WT (top) and DU145-TR-CDH3 KO (bottom) cells treated with different concentrations of CDH3 antibody (10, 4, 2, and 0 µg/mL) for 48 hours. Green fluorescence (TR-labeled cells) represents the cells, and red fluorescence (pHrodo-labeled antibody) indicates antibody internalization. Scale bar, 50µm. ( C ) Schematic representation of the CDH3Ab–DM1 antibody–drug conjugate, illustrating the anti-CDH3 antibody, linker, and cytotoxic DM1 payload. ( D ) Dose-dependent viability of five prostate cancer cell lines (DU145, 22RV1, LHMK, PC3, LNCaP) treated with the CDH3Ab–DM1 conjugate with their respective IC50 values. ( E ) Correlation between CDH3 surface expression (MFI) and CDH3Ab-DM1 IC50 across multiple cell lines, with correlation coefficient (r) and significance (p) shown. ( F ) Schematic illustrating the co-culture setup of DU145-TR (CDH3 wild type, GFP-labeled) and DU145-CDH3 knockout (non-labeled) cells, followed by cyclical ADC treatments at varying concentrations and different initial mixing ratios. Red and blue histograms indicate the expected result before and after treatment, respectively. ( G ) The proportion of DU145-CDH3 KO cells over three rounds of ADC treatment, comparing 3 µg/mL and 10 µg/mL doses across different initial co-culture ratios (DU145-TR-CDH3 WT : DU145-CDH3 KO at 1:3, 1:1 and 3:1). ( H ) Schematic of intracardiac injection and intravenous ADC administration schedule for nude mice bearing DU145-TR or DU145-TR-CDH3 KO cells, with treatment days indicated by red arrows. ( I ) Normalized bioluminescent intensity (BLI, scaled to Day 0 as 10) signals measured in legs over 30 days in mice injected with DU145-TR-CDH3 WT or DU145-TR-CDH3 KO cells. N=10. ( J ) Bioluminescent imaging of DU145-TR-CDH3 WT or DU145-TR-CDH3 KO tumor burden at Day 0 and Day 25 under different ADC doses (2.5 mg/kg or 5 mg/kg) versus untreated controls. Error bars represent SEM. Mann-Whiney test for ( I ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, #, not significant.

Article Snippet: We used a humanized IgG1 antibody directed against the extracellular domains of CDH3 (Perseus Proteomics), and chose to focus on DU145 to test the therapeutic agents because of its highest surface CDH3 among the various prostate cancer cell lines ( ).

Techniques: Flow Cytometry, Immunofluorescence, Fluorescence, Labeling, Expressing, Co-Culture Assay, Knock-Out, Injection, Imaging

Journal: bioRxiv

Article Title: CDH3 as a Novel Therapeutic Target in Basal-like Double-Negative Prostate Cancer

doi: 10.1101/2025.11.18.689150

Figure Lengend Snippet: ( A ) Schematic diagram of the αCDH3-CAR construct containing scFv, CD8α hinge, transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain. ( B ) Flow cytometry histograms indicating αCD19-CAR and αCDH3-CAR constructs (both carry a GFP gene) expression in Jurkat cells. ( C ) Flow cytometry–based quantification of Jurkat cell activation (CD69 and CD25 expression) following coculture with DU145-CDH3 WT or DU145-CDH3 KO cells, comparing the αCDH3-CAR with untransduced (UTD) and αCD19-CAR control. ( D ) Schematic of the T-cell engineering protocol, showing isolation of PBMCs, bead-based activation, lentiviral transduction of CAR, and subsequent expansion. ( E ) Flow cytometry histogram of granzyme B expression in T cells transduced with either αCD19-CAR or αCDH3-CAR, following coculture with DU145-CDH3 WT or DU145-CDH3 KO cells. ( F ) Cytotoxicity assays of DU145-CDH3 WT or DU145-CDH3 KO tumor targets at increasing effector-to-target (E:T) ratios, comparing αCD19-CAR T cells and αCDH3-CAR T cells. Error bars represent SEM. Mann-Whiney test for ( F ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

Article Snippet: We used a humanized IgG1 antibody directed against the extracellular domains of CDH3 (Perseus Proteomics), and chose to focus on DU145 to test the therapeutic agents because of its highest surface CDH3 among the various prostate cancer cell lines ( ).

Techniques: Construct, Flow Cytometry, Gene Expression, Activation Assay, Expressing, Control, Isolation, Transduction

Journal: bioRxiv

Article Title: CDH3 as a Novel Therapeutic Target in Basal-like Double-Negative Prostate Cancer

doi: 10.1101/2025.11.18.689150

Figure Lengend Snippet: ( A ) Schematic of the in vivo experimental design for metastatic prostate cancer models. DU145-TR are delivered via intracardiac injection, randomized into three groups, and treated with untransduced T (UTD T), αCD19-CAR T and) and αCDH3-CAR T cells. ( B ) Flow cytometric gating of infused CAR T cells, illustrating the proportions of key T-cell subsets (naïve, T effector, T effector memory, and T central memory). ( C ) Survival curves comparing UTD T cells, αCD19-CAR T, and αCDH3-CAR T treatment groups in metastatic prostate cancer model. ( D ) Schematic of the in vivo experimental design for combinational therapy. DU145-TR are delivered via intracardiac injection, randomized into four groups, and treated with αCD19-CAR T alone, αCD19-CAR T + nivolumab, αCDH3-CAR T alone, and αCDH3-CAR T + nivolumab. ( E ) Bioluminescence signaling intensity comparing four treatment group. ( F ) Bioluminescence imaging of the DU145-TR metastatic prostate cancer model, comparing the four treatment groups over several weeks. ( G ) Survival curves of αCDH3-CAR T alone, αCDH3-CAR T + nivolumab and αCD19-CAR T + nivolumab treatment groups. Log-rank (Mantel-Cox) test for ( C ) and ( G ). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

Article Snippet: We used a humanized IgG1 antibody directed against the extracellular domains of CDH3 (Perseus Proteomics), and chose to focus on DU145 to test the therapeutic agents because of its highest surface CDH3 among the various prostate cancer cell lines ( ).

Techniques: In Vivo, Injection, Imaging