Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: TNF-α modulates the WISP1 expression in stroma cells of the human bladder. (A) The RT-qPCR determined the expression of WISP1 in the human bladder cells. (B) Two WISP1 isoforms (WISP1v1 and WISP1v2) were found in the human bladder fibroblast (HBdSF) and bladder smooth muscle (HBdSMC) cells by RT-PCR with the pair of primers as shown. The expressions of WISP1, IL-6, CXCL5, CXCL12, and GDF15 of the HBdSF (C) and HBdSMC (D) cells after 10 ng/ml TNF-α treatment assessed by RT-qPCR. WISP1 (E) and IL-6 (F) secretions after TNF-α (0 – 20 ng/mL) treatments in HBdSF cells. Secretion of WISP1 (G) and IL-6 (F) in HBdSMC_shCOL and HBdSMC_shWISP1 cells treated with/without 10 ng/ml of TNF-α. Data are presented as the mean percentage (± SE; n = 4) in relation to the vehicle-treated HBdSMC_shCOL cells. (I) WISP1 secretion after 20 μM CAPE and/or TNF-α in HBdSF cells determined by ELISA assays. Data are presented as the mean percentage (±SE; n = 4) in relation to the vehicle-treated HBdSF cells. (J) The luciferase activity of the WISP1 reporter vector when HBdSMC cells were treated with TNF-α (10 ng/mL) and/or 20 μM CAPE for 24 h. * p < 0.05; ** p < 0.01; ND: no detectable.

Article Snippet: Cells were transduced with WISP1 shRNA lentiviral transduction particles (sc-39,335-V; Santa Cruz Biotechnology, CA, USA) or control shRNA transduction particles (sc-10,808-v, Santa Cruz Biotechnology), and selected with 50 ng/mL puromycin dihydrochloride (Cyrusbioscience, Taipei, Taiwan) as described previously [ ].

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Plasmid Preparation

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: Modulating effect of the conditioned media from the ectopic WISP1 overexpressed-293T cells on the cell proliferation in the bladder stroma cells. Expressions of WISP1v1 and WISP1v2 after the ectopic-overexpressed WISP1v1 and WISP1v2 in the 293T cells were assessed by the RT-qPCR (A) and ELISA (B) assays. The ability of the proliferation of HBdSMC (C, D) and HBdSF (E, F) cells after being treated with the supernatant from 293T-DNA, 293T-WISP1v1, and 293T-WISP1v2 cells, respectively, was measured by the EdU staining or EdU flow cytometry assays. The quantitative analysis is presented as the mean percentage (±SE; n = 3) of EdU-positive cells. * p < 0.05; ** p < 0.01.

Article Snippet: Cells were transduced with WISP1 shRNA lentiviral transduction particles (sc-39,335-V; Santa Cruz Biotechnology, CA, USA) or control shRNA transduction particles (sc-10,808-v, Santa Cruz Biotechnology), and selected with 50 ng/mL puromycin dihydrochloride (Cyrusbioscience, Taipei, Taiwan) as described previously [ ].

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: WISP1 modulates expressions of α-SMA, IL-6, GDF15, and CXCL5 in bladder stroma cells. The mRNA levels of two WISP1 isoforms (WISP1v1 and WISP1v2) after WISP1 knock-downed in the HBdSF (A) and HBdSMC (B) cells were determined by RT-PCR. The protein levels with quantification analysis of WISP1, GDF15, and α-SMA of HBdSMC (C) and HBdSF (D) cells after mock-knockdown or WISP1-knockdown were assessed by immunoblot assays. The mRNA levels of the WISP1, α-SMA, IL-6, GDF15, and CXCL5 after knockdown of WISP1 in the HBdSMC (E) and HBdSF (F) cells, as indicated, were determined by RT-qPCR. Data are presented as target genes/β-actin of mock-knockdown cells relative to WISP1-knockdown cells. ** p < 0.01.

Article Snippet: Cells were transduced with WISP1 shRNA lentiviral transduction particles (sc-39,335-V; Santa Cruz Biotechnology, CA, USA) or control shRNA transduction particles (sc-10,808-v, Santa Cruz Biotechnology), and selected with 50 ng/mL puromycin dihydrochloride (Cyrusbioscience, Taipei, Taiwan) as described previously [ ].

Techniques: Reverse Transcription Polymerase Chain Reaction, Knockdown, Western Blot, Quantitative RT-PCR

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: Modulating effect of WISP1-knockdown on the cell proliferation, migration, and contraction in bladder stroma and cancer cells. (A) The ability of cell proliferation of mock-knockdown HBdSMC (HBdSMC_shCOL) and WISPI-knockdown (HBdSMC_shWISP1) cells was assessed by EdU proliferation assays. The quantitative analysis is presented as the mean percentage (±SE; n = 3) of EdU-positive cells. (B) The ability of cell contraction of the mock-knockdown HBdSF (HBdSF_shCOL) and the WISP1-knockdown HBdSF (HBdSF_shWISP1) cells was measured by the collage contraction assays. Data are presented as gel area of the times as indicated in relation to the 0 h. (C) The ability of the migration of T24 cells after being treated with the supernatant from HBdSMC_shCOL and HBdSMC_shWISP1 cells, respectively, was determined by wound healing assays. The white line indicated the average of the leading cellular edges, and the wound area size was calculated by Image J. ** p < 0.01.

Article Snippet: Cells were transduced with WISP1 shRNA lentiviral transduction particles (sc-39,335-V; Santa Cruz Biotechnology, CA, USA) or control shRNA transduction particles (sc-10,808-v, Santa Cruz Biotechnology), and selected with 50 ng/mL puromycin dihydrochloride (Cyrusbioscience, Taipei, Taiwan) as described previously [ ].

Techniques: Knockdown, Migration

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: Modulating effect of WISP1 on the cell proliferation and invasion in bladder cancer cells. (A) Cell proliferation of HT1376 and T24 cells after treatments of recombinant human WISP1 was assessed by CyQUANT cell proliferation assay. Data are presented as the mean percentage of the rhWISP1-treated cells relative to vehicle-treated cells. Expressions of total WISP1, WISP1v1, or WISP1v2 after the ectopic-overexpressed WISP1v1 and WISP1v2 in the HT1376 cells were assessed by RT-qPCR (B), RT-PCR (C), and ELISA (D) assays. The abilities of the proliferation of HT-DNA, HT-WISP1v1 (E), and HT-WISP1v2 (F) cells were measured by Ki67 flow cytometry assays. Data are presented as the mean percentage of the Ki67-positive cells relative to HT-DNA cells. The invasion ability of HT1376 cells after ectopic overexpression of WISP1v1 (G) and WISP1v2 (H) was determined by in vitro Matrigel invasion assays (± SE; n = 4). Data are presented as the mean percentage of the invasion cells relative to HT-DNA cells. * p < 0.05; ** p < 0.01.

Article Snippet: Cells were transduced with WISP1 shRNA lentiviral transduction particles (sc-39,335-V; Santa Cruz Biotechnology, CA, USA) or control shRNA transduction particles (sc-10,808-v, Santa Cruz Biotechnology), and selected with 50 ng/mL puromycin dihydrochloride (Cyrusbioscience, Taipei, Taiwan) as described previously [ ].

Techniques: Recombinant, CyQUANT Assay, Proliferation Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Over Expression, In Vitro

Journal: Translational Oncology

Article Title: WISP1 is the stromal-secreting oncoprotein via paracrine downregulation of NDRG1, KAI1, and Maspin in human bladder cancer cells

doi: 10.1016/j.tranon.2026.102680

Figure Lengend Snippet: Determination of the molecular mechanism of WISP1v1 on the cell invasion in the bladder cancer cells. (A) The protein levels of NDRG1, KAI1, and Maspin of HT-DNA and HT-WISP1v1 cells were determined by immunoblot assays. (B) The luciferase activity of the NDRG1 reporter vector was shown after transiently overexpressing various amounts of WISP1v1 expression vectors in HT1376 and T24 cells, as indicated. (C) The luciferase activity of KAI1 and Maspin reporter vectors was shown as indicated after transiently overexpressing various dosages of WISP1v1 expression vectors in HT1376 cells. (D) The luciferase activity of NDRG1, KAI1, and Maspin reporter vectors, respectively, after transiently overexpressed pcDNA, WISP1v1, and WISP1v2 expression vectors as indicated in HT1376 cells. (E) The mRNA levels of the E-cadherin, N-cadherin, Slug, Snail, and Vimentin were determined by RT-qPCR. Data are presented as target genes/β-actin of HT-WISP1v1 cells relative to HT-DNA cells. The mRNA levels of the WISP1 (F), NDRG1, KAI1, Maspin, and IL-6 (G) after transient overexpression of WISP1v1 in T24 cells were determined by RT-qPCR. Data are presented as target genes/β-actin of T24-WISP1v1 cells relative to T24-DNA cells. (H) The invasion ability of T24 cells after the ectopic overexpression of WISP1v1 was determined by in vitro Matrigel invasion assays (± SE; n = 4). The ability of the migration (I) and quantification analysis (I) of T24 cells after being treated with the supernatant from 293T-DNA and 293T-WISP1v1 cells was determined by wound healing assays. The white line indicated the average of the leading cellular edges, and the wound area size was calculated by Image J. * p < 0.05, ** p < 0.01.

Article Snippet: Cells were transduced with WISP1 shRNA lentiviral transduction particles (sc-39,335-V; Santa Cruz Biotechnology, CA, USA) or control shRNA transduction particles (sc-10,808-v, Santa Cruz Biotechnology), and selected with 50 ng/mL puromycin dihydrochloride (Cyrusbioscience, Taipei, Taiwan) as described previously [ ].

Techniques: Western Blot, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Quantitative RT-PCR, Over Expression, In Vitro, Migration

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer

doi: 10.64898/2026.01.07.698253

Figure Lengend Snippet: CCN4 concentration as quantified by ELISA in media conditioned for 48 hours by WT Py230 and Py8119 cells and two independent CCN4 KO variants for each parental cell line. Cell-free media was used as a negative control and media conditioned by B16F0 cells was used as a positive control. Individual data points represent technical replicates and values reported below quantification threshold are indicated as n.d.

Article Snippet: To measure CCN4 secretion from each WT cell line and KO variant, cells were grown for 48 hours to reach about 80% confluence and the media was filtered for ELISA (enzyme-linked immunosorbent assay) analysis using a Mouse CCN4 Quantikine ELISA Development Kit (R&D Systems, Minneapolis, MN).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer

doi: 10.64898/2026.01.07.698253

Figure Lengend Snippet: Kaplan-Meier time-to-event survival curves for immunocompetent C57BL/6 mice (B and D) and severely immunocompromised NSG mice (A and C) challenged subcutaneously with wild-type (black curves) and two CCN4 KO variants (KO1 in blue and KO2 in dotted red curves) derived from Py8119 (A and B) and Py230 (C and D) cell lines. The tumor size exceeding 100 mm 3 was considered the triggering event. The number of animals that experienced a triggering event among the total number of animals challenged with tumor cells is indicated in the legend. Statistical difference among the three curves was assessed using a Peto & Peto Modification the Gehan-Wilcoxon test with the resulting Chi-squared, degrees of freedom and resulting P-values are indicated. As this was an exploratory study, the p-values indicate the extent to which these three Kaplan-Meier survival curves are different.

Article Snippet: To measure CCN4 secretion from each WT cell line and KO variant, cells were grown for 48 hours to reach about 80% confluence and the media was filtered for ELISA (enzyme-linked immunosorbent assay) analysis using a Mouse CCN4 Quantikine ELISA Development Kit (R&D Systems, Minneapolis, MN).

Techniques: Derivative Assay, Modification

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer

doi: 10.64898/2026.01.07.698253

Figure Lengend Snippet: (A) Box-and-whisker plots for spleen weights obtained from C57BL/6 mice challenged with WT Py230 cells and CCN4 KO variants In a time-matched experimental design with the end point at 69 days (n = 5 for each group). (B) Spleen weights and corresponding excised tumor weights obtained from C57BL/6 mice challenged with WT Py8119 cells and CCN4 KO variants in a size-matched experimental design (n = 5 for each group). As there was no statistical difference between CCN4 KO variants, results were combined for analysis. A homoscedastic two-sided Student’s t-test was used to assess statistical significance.

Article Snippet: To measure CCN4 secretion from each WT cell line and KO variant, cells were grown for 48 hours to reach about 80% confluence and the media was filtered for ELISA (enzyme-linked immunosorbent assay) analysis using a Mouse CCN4 Quantikine ELISA Development Kit (R&D Systems, Minneapolis, MN).

Techniques: Whisker Assay

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer

doi: 10.64898/2026.01.07.698253

Figure Lengend Snippet: Following enzymatic dissociation of tumor tissues, the immune cell compartment was quantified by full spectrum flow cytometry. A self-organizing map shows the distribution of negative Live/Dead violet and positive CD45 cells in size-matched WT (left) and CCN4 KO1 (center) tumors (n = 5 for both WT and CCN4 KO1 Py230). Right panel shows the percentage of events associated with each cluster that relate to cells identified by manual gating.

Article Snippet: To measure CCN4 secretion from each WT cell line and KO variant, cells were grown for 48 hours to reach about 80% confluence and the media was filtered for ELISA (enzyme-linked immunosorbent assay) analysis using a Mouse CCN4 Quantikine ELISA Development Kit (R&D Systems, Minneapolis, MN).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer

doi: 10.64898/2026.01.07.698253

Figure Lengend Snippet: In a size-matched experimental design, the tumor-infiltrating lymphocytes were quantified by flow cytometry using antibody panels focused on the T cell (A, B, and C) and the myeloid (D, E, and F) compartments. Mice received either WT Py8119 cells (black circles) or one of two independently generated CCN4 KO variants (red + and blue x, n = 5 for all groups). Results highlight the CD45+ fraction among total Live events measured using the T cell (A) and myeloid (D) panels. The proportion of infiltrating CD4+ T cells within the live CD45+ compartment (B) was not different but the CD25+ fraction of live CD4+ T cells (C) was increased in CCN4 KO tumors. The MDSC compartment was shifted from PMN-MDSC (E, Live CD45+ CD11b+ Ly6G+ Ly6C int events) to Mo-MDSC (F, Live CD45+ CD11b+ Ly6G-Ly6C+ events) upon CCN4 KO. p-values calculated between WT and pooled CCN4 KOs using two-sided Student’s t test.

Article Snippet: To measure CCN4 secretion from each WT cell line and KO variant, cells were grown for 48 hours to reach about 80% confluence and the media was filtered for ELISA (enzyme-linked immunosorbent assay) analysis using a Mouse CCN4 Quantikine ELISA Development Kit (R&D Systems, Minneapolis, MN).

Techniques: Flow Cytometry, Generated

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer

doi: 10.64898/2026.01.07.698253

Figure Lengend Snippet: Results from R&D Systems’ Mouse XL Cytokine Array Kit of cytokine, chemokine, and growth factor expression by WT versus CCN4 KO cells derived from Py8119 (A) and Py230 (B) lines in vitro . (C) Compares the WT profiles between the Py8119 and Py230 cell lines. In presenting the combined results for assaying each cell line’s secretome using a single biological replicate, open circles represent results for specific cytokine probes, which are spotted in duplicate on the array, and filled circles represent positive and negative controls. Dotted red lines indicate Z-scores of 3 and −3. Particular secreted factors that are differentially expressed are annotated with their respective names.

Article Snippet: To measure CCN4 secretion from each WT cell line and KO variant, cells were grown for 48 hours to reach about 80% confluence and the media was filtered for ELISA (enzyme-linked immunosorbent assay) analysis using a Mouse CCN4 Quantikine ELISA Development Kit (R&D Systems, Minneapolis, MN).

Techniques: Expressing, Derivative Assay, In Vitro

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer

doi: 10.64898/2026.01.07.698253

Figure Lengend Snippet: (A) Projections of WT versus CCN4 KO variants along PC1 and PC2 axes for B16F0, YUMM1.7, Py230, and Py8119 cell lines. The location of duplicates of each cell line label correspond to technical replicates on the array. The percentage of variance explained by each principal component is indicated in the axis label. (B) Projection of the measured proteins based on the loading scores associated with PC1 (y-axis) listed from left to right in alphabetical order. The curves on the right represent the density distribution of the PC1 loading scores (black) and a normal distribution centered at −0.091 with a standard deviation of 0.00074 (red).

Article Snippet: To measure CCN4 secretion from each WT cell line and KO variant, cells were grown for 48 hours to reach about 80% confluence and the media was filtered for ELISA (enzyme-linked immunosorbent assay) analysis using a Mouse CCN4 Quantikine ELISA Development Kit (R&D Systems, Minneapolis, MN).

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in breast cancer

doi: 10.64898/2026.01.07.698253

Figure Lengend Snippet: (A) Schematic diagram of the network topology associated with the adherens pathway activity of beta-catenin. Adherens junctions are formed by homotypic interactions between the extracellular cadherin domains of a single pass transmembrane protein E-cadherin (E-cad: blue double oval). The cytoplasmic tail of E-cadherin provides a scaffold for a multi-protein complex that includes beta-catenin ( β -cat: red oval). (B) Upon the proteolytic cleavage of adherens junctions, a cytoplasmic fragment of E-cadherin and associated catenins (tBE) are transported to the cytoplasm. In the cytoplasm, the cytoplasmic fragment of E-cadherin and associated catenins can either enter the nucleus or undergo proteasomal degradation. In the nucleus, this multi-protein complex promotes the transcription and translation of CCN4, beta-catenin, and E-cadherin, among other factors. mRNA is represented by light green parallelogram. Once synthesized, CCN4 (light blue circle) is secreted. Newly synthesized beta-catenin and E-cadherin reform the multi-protein complex and are transported to the cell membrane to re-establish adherens junctions. If no E-cadherin binding sites are present, the multi-protein complex is internalized and degraded. (C) Graphical summary of mutations that include both changes in single nucleotides and in copy numbers for breast invasive carcinoma (TCGA, Cell 2015) samples. Of the 816 samples contained in the dataset obtained through cBioPortal (retrieved 11/10/2025), the queried genes are altered in 336 (41%) of the samples.

Article Snippet: To measure CCN4 secretion from each WT cell line and KO variant, cells were grown for 48 hours to reach about 80% confluence and the media was filtered for ELISA (enzyme-linked immunosorbent assay) analysis using a Mouse CCN4 Quantikine ELISA Development Kit (R&D Systems, Minneapolis, MN).

Techniques: Activity Assay, Synthesized, Membrane, Binding Assay