Journal: PLoS ONE

Article Title: Notch3 signaling promotes tumor cell adhesion and progression in a murine epithelial ovarian cancer model

doi: 10.1371/journal.pone.0233962

Figure Lengend Snippet: (A) No Notch3 transcripts are detected in ID8 IP2 by semiquantitative RT-PCR (box). Notch receptors 1, 2, and 4 as well as ligands Jagged1 and Delta-like1 are detected in ID8 IP2 cells. (B) ID8 IP2 cells have undetectable levels of Notch3 protein when compared to cell lines previously characterized by detectable levels of Notch3 (OVCAR3, A2780, Caov3, and PA-1, Western blot). Uncropped images of all blots and gels are available in . (C) Representative Western blots show that expression of Notch3 intracellular domain is upregulated in Notch3IC lentivirally infected ID8 IP2 cell lines. Numbers indicate set number. (D) qRT-PCR indicates that Notch target genes are upregulated in Notch3IC Sets #1-#5 compared to matched Controls (error bars = S.E.M in all figures). This panel shows evidence of Notch activation across all 5 matched Sets, however, some variability was observed between sets . (E) Selected established Notch target genes are upregulated in RNA-Seq data, demonstrating upregulation of active Notch3 signaling. Starred genes are also examined by qRT-PCR in panel D and .

Article Snippet: OVSAHO, and OVCA429 were a kind gift of Dr. Joanna Burdette (University of Illinois Chicago), A2780 was a kind gift of Dr. Tian-Li Wang (Johns Hopkins), and SKOV3-IP1 was a kind gift of Dr. Olga Razorenova (University of California Irvine).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Infection, Quantitative RT-PCR, Activation Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Oncogene-like addiction to aneuploidy in human cancers

doi: 10.1101/2023.01.09.523344

Figure Lengend Snippet: (A) Chromosomal engineering strategies for the targeted deletion of chromosome arms: (1) ReDACT-NS: using CRISPR-Cas9 homology-directed repair, we integrated a positive-negative selection cassette encoding a fluorescent reporter, a positive selection marker, and a negative selection marker (HSV thymidine kinase) at a centromere-proximal region on chromosome 1q. We induced arm loss by generating a dsDNA break centromere-proximal to the cassette with Cas9, and isolated clonal populations of cells that were ganciclovir-resistant. (2) ReDACT-TR: We induced arm loss by generating a dsDNA break at a centromere-proximal location with Cas9 while providing cells with an ectopic telomere seed sequence for repair. (3) ReDACT-CO: We induced arm loss by generating a dsDNA break at a centromere-proximal location with Cas9, and isolated clonal populations of cells. For all three approaches, we screened clonal populations of cells for targeted chromosome loss through TaqMan CNV assays and validated their karyotypes through SMASH sequencing. (B) Representative SMASH karyotypes of the 1q-disomic clones generated from the 1q-trisomic cancer cell lines A2780, AGS, and A2058. Chromosome 1q is highlighted in blue. A complete list of aneuploidy-loss clones is included in . (C) 1q-disomic clones display decreased RNA expression and protein expression of genes encoded on chromosome 1q. RNA expression data was obtained through bulk RNA-seq and represents the average expression of genes by chromosome arm across multiple 1q-disomic clones for each cell line. Protein expression data was obtained through mass spectrometry, and representative data from one 1q-disomic clone is shown for each cell line. Data are log2 transformed, normalized to the parental cell line, and adjusted so that the mean expression across all chromosomes is 0. (D) 1q-disomic clones exhibit decreased anchorage-independent growth. The micrographs display representative images of colony formation for 1q-trisomic and 1q-disomic clones. (E) 1q-disomic clones exhibit impaired xenograft growth in vivo . 1q-trisomic and 1q-disomic cells were injected contralaterally and subcutaneously into immunocompromised mice. The graphs display the mean ± SEM for each trial. Representative mice are shown on the right. (F) SMASH karyotype of a 1q-disomic clone generated from the mammary epithelial cell line MCF10A. Chromosome 1q is highlighted in blue. (G) 1q-disomic MCF10A clones transduced with HRAS G12V exhibit decreased anchorage-independent growth relative to 1q-trisomic MCF10A cells. (H) 1q disomic MCF10A clones transduced with HRAS G12V clones exhibit impaired xenograft growth in vivo . 1q-trisomic and 1q-disomic cells were injected contralaterally and subcutaneously into immunocompromised mice. The graphs display the mean ± SEM for each trial. Representative mice are shown below. For anchorage-independent growth assays in D and G, the boxplots represent the 25th, 50th, and 75th percentiles of colonies per field, while the whiskers represent the 10th and 90th percentiles. Unpaired t-test, n = 15 fields of view, data from representative trial. Representative images are shown below. **p < 0.005, ***p < 0.0005

Article Snippet: A2780 samples were sent to Cell Line Genetics Inc. ( www.clgenetics.com ), and AGS and A2058 samples were sent to Karyologic Inc. ( www.karyologic.com ) for G-banding karyotyping.

Techniques: CRISPR, Selection, Marker, Isolation, Sequencing, Clone Assay, Generated, RNA Expression, Expressing, RNA Sequencing, Mass Spectrometry, Transformation Assay, In Vivo, Injection, Transduction

Journal: bioRxiv

Article Title: Oncogene-like addiction to aneuploidy in human cancers

doi: 10.1101/2023.01.09.523344

Figure Lengend Snippet: (A) GSEA analysis of A2780 RNA-seq data reveals upregulation of the p53 pathway in the 1q-disomic clones, relative to the parental trisomy. (B) A heatmap displaying the upregulation of 10 p53 target genes in A2780 1q-disomic clones. The TK+ clone indicates a clone that harbors the CRISPR-mediated integration of the HSV-TK transgene but that was not treated to induce chromosome 1q-loss. (C) Western blot analysis demonstrating activation of p53 signaling in 1q-disomic clones. GAPDH was analyzed as a loading control. The TK+ clone indicates a clone that harbors the CRISPR-mediated integration of the HSV-TK transgene but that was not treated to induce chromosome 1q-loss. (D) A waterfall plot highlighting the most-significant instances of mutual exclusivity between chromosome arm gains and mutations in cancer-associated genes. The complete dataset for mutual exclusivity and co-occurrence is included in . (E) Boxplots displaying the TP53-mutation phenocopy signature in cancers from the TCGA, split based on whether the cancers harbor a non-synonymous mutation in TP53. (F) A scatterplot comparing the association between chromosome arm gains and the TP53-mutation phenocopy signature in TP53-wildtype cancers from TCGA. Cancers with chromosome 1q gains are highlighted in blue. (G) Boxplots displaying the TP53-mutation phenocopy signature in cancers from the TCGA, split based on whether tumors harbor a gain of chromosome 1q. Only TP53-wildtype cancers are included in this analysis. (H) Boxplots displaying the expression of three p53 target genes – CDKN1A (p21), RRM2B, and GADD45A – in cancers from TCGA split based on the copy number of chromosome 1q. Only TP53-wildtype cancers are included in this analysis. (I) A CRISPRi competition assay demonstrates that gRNAs targeting MDM4 drop out over time in A2780 cells. In contrast, gRNAs targeting AAVS1 and PIP5K1A, another gene encoded on chromosome 1q, exhibit minimal depletion. (J) A schematic displaying the strategy for using paired CRISPR gRNAs to delete a single copy of MDM4 in a cell line with a trisomy of chromosome 1q. (K) SMASH karyotype demonstrating maintenance of the chromosome 1q trisomy in an MDM4 +/+/KO clone. Chromosome 1q is highlighted in blue. (L) 1q-disomic clones and MDM4 +/+/KO clones in A2780 exhibit comparable upregulation of p53 transcriptional targets, as determined through TaqMan gene expression assays. (M) MDM4 +/+/KO clones exhibit decreased anchorage-independent growth relative to the MDM4 +/+/+ parental cell line. (N) Induction of MDM4 cDNA in 1q-disomic clones in A2780 increases anchorage-independent growth. For the graphs in E, G, H, M, and N, the boxplots represent the 25th, 50th, and 75th percentiles of the indicated data, while the whiskers represent the 10th and 90th percentiles of the indicated data. For the soft agar experiments in M and N, the data are from n = 15 fields of view, and a representative trial is shown. ***p < 0.0005

Article Snippet: A2780 samples were sent to Cell Line Genetics Inc. ( www.clgenetics.com ), and AGS and A2058 samples were sent to Karyologic Inc. ( www.karyologic.com ) for G-banding karyotyping.

Techniques: RNA Sequencing, Clone Assay, CRISPR, Western Blot, Activation Assay, Control, Mutagenesis, Expressing, Competitive Binding Assay, Gene Expression

Journal: bioRxiv

Article Title: Oncogene-like addiction to aneuploidy in human cancers

doi: 10.1101/2023.01.09.523344

Figure Lengend Snippet: (A) A schematic of the metabolism of two pyrimidine analogs, RX-3117 and 3-deazauridine. UCK2, a kinase encoded on chromosome 1q, phosphorylates these compounds to produce cytotoxic derivatives that can poison DNA and RNA synthesis. (B) Boxplots displaying the expression of UCK2 in cancer cell lines (left) and human cancers (right), divided based on the copy number of chromosome 1q. The boxplots represent the 25th, 50th, and 75th percentiles of the indicated data, while the whiskers represent the 10th and 90th percentiles of the indicated data. (C) Expression of UCK2 protein in cancer cell lines with 1q trisomies or following aneuploidy-elimination. (D) Cellular sensitivity of A2780 and MCF10A treated with different concentrations of RX-3117 or 3-deazauridine. (E) A schematic displaying cellular competition between trisomic and disomic cells. Under normal conditions, certain trisomies enhance cellular fitness, allowing these cells to overtake the population and enhance malignant growth (top). However, treatment with an “anti-trisomy” compound could selectively impair the growth of the aneuploid cells, keeping the population in a low-malignant state (bottom). (F) A cellular competition between fluorescently-labeled A2780 1q-trisomic and unlabeled 1q-disomic cells. These cells were mixed at a ratio of 10% to 90% and then cultured in either DMSO or RX-3117. While the trisomic cells quickly dominate the population in drug-free media, treatment with RX-3117 prevents the outgrowth of the 1q-trisomy subpopulation. *p < 0.05, ** p < 0.005, *** p < 0.0005

Article Snippet: A2780 samples were sent to Cell Line Genetics Inc. ( www.clgenetics.com ), and AGS and A2058 samples were sent to Karyologic Inc. ( www.karyologic.com ) for G-banding karyotyping.

Techniques: Expressing, Labeling, Cell Culture

Journal: Acta Pharmaceutica Sinica. B

Article Title: Targeting cancer-associated fibroblast-activated HGF/c-MET pathway inhibits extrahepatic cholangiocarcinoma progression and restores gemcitabine therapeutic sensitivity

doi: 10.1016/j.apsb.2026.02.023

Figure Lengend Snippet: Effect of CAFs on eCCA progression. (A) Immunofluorescence staining of CAFs/NFs showed positive expression of α -SMA, Vimentin and FAP. Scale bar: 200 μm. (B) Effect of CAF-CM/NF-CM on proliferation of TFK-1 and CBC3T-1 cells. Negative control (NC) ( n = 3). (C, D) Effect of CAF-CM/NF-CM on migration and invasion of TFK-1 and CBC3T-1 cells ( n = 3). Scale bar: 200 μm. (E, F) Representative fluorescent images of PDOs monocultures or co-cultures with CAFs/NFs and quantitative analysis of total organoid area ( n = 3). PDOs (green), CAFs/NFs (red). Scale bar: 200 μm. (G) Representative tumor images of cell-derived xenografts: cancer cells, cancer cells + NFs and cancer cells + CAFs. (H, I) Mean tumor volume and tumor weight ( n = 8). (J) Representative images of H&E and IHC staining of α -SMA, CK7 and Ki67 ( n = 8). Data are shown as mean ± SD. ns, no significance; ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: The human eCCA TFK-1 cell line was procured from Creative Bioarray (NY, USA).

Techniques: Immunofluorescence, Staining, Expressing, Negative Control, Migration, Derivative Assay, Immunohistochemistry

Journal: Acta Pharmaceutica Sinica. B

Article Title: Targeting cancer-associated fibroblast-activated HGF/c-MET pathway inhibits extrahepatic cholangiocarcinoma progression and restores gemcitabine therapeutic sensitivity

doi: 10.1016/j.apsb.2026.02.023

Figure Lengend Snippet: Inhibition of HGF/c-MET signaling pathway suppresses CAFs-induced eCCA progression. (A) Representative IHC images of c-MET expression in eCCA ( n = 27) and para-tumor tissues ( n = 9). (B) IHC staining intensity of c-MET in eCCA tissues ( n = 27) and para-tumor tissues ( n = 9) of TMAs. (C) Representative images of multiplex immunofluorescence staining ( α -SMA, green; p-c-MET, yellow; CK19, red) in eCCA and para-tumor tissue. (D) Percentage of p-c-MET + CK19 + cells to CK19 + cells in eCCA tissues and para-tumor tissues. (E) Mean fluorescence intensity of α -SMA + in eCCA and para-tumor tissues. (F) Western blot analysis of p-c-MET, total c-MET, p-PI3K, total PI3K, p -AKT, and total AKT in TFK-1 and CBC3T-1 cells treated with recombinant HGF at different time points (15, 30, 60, 120 min). (G, H) Western blotting of the c-MET/PI3K/AKT signaling pathway in TFK-1 and CBC3T-1 cells under the following conditions: normal control (NC), CAF-CM, CAF-CM supplemented with an HGF-neutralizing antibody (CAF-CM + HGF Ab), or recombinant HGF ( n = 3). (I, J) Western blot analysis of c-MET/PI3K/AKT pathway activity in eCCA cells treated with CAF-CM, with or without the c-MET inhibitors JNJ-38877605 or crizotinib ( n = 3). Data are shown as mean ± SD. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001.

Article Snippet: The human eCCA TFK-1 cell line was procured from Creative Bioarray (NY, USA).

Techniques: Inhibition, Expressing, Immunohistochemistry, Multiplex Assay, Immunofluorescence, Staining, Fluorescence, Western Blot, Recombinant, Control, Activity Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Targeting cancer-associated fibroblast-activated HGF/c-MET pathway inhibits extrahepatic cholangiocarcinoma progression and restores gemcitabine therapeutic sensitivity

doi: 10.1016/j.apsb.2026.02.023

Figure Lengend Snippet: Targeting the HGF/c-MET signaling pathway enhances gemcitabine treatment sensitivity in vitro . (A) Cell viability of TFK-1 and CBC3T-1 cells treated with gemcitabine or gemcitabine combined with HGF ( n = 3). (B) Representative images of the sensitivity of gemcitabine to PDOs monoculture systems or direct co-culture systems with CAFs ( n = 3). PDOs (green), CAFs (red). The percentage of total area of the PDOs was quantified and the viability was determined. Scale bar: 500 μm. (C) Schematic diagram for the construction of TFK-1 gemcitabine-resistant cells (TFK-1R). (D) Western blot analysis of c-MET protein levels in parental TFK-1 cells and gemcitabine-resistant TFK-1 cells (TFK-1R). (E) Cell viability of 100 μmol/L gemcitabine-treated TFK-1 and TFK-1R cells ( n = 3). (F) Cell viability of TFK-1R cells treated with 100 μmol/L gemcitabine or in combination with c-MET inhibitor under conditions of added HGF ( n = 3). (G) Dose–response curves of gemcitabine alone or in combination with c-MET inhibitors for treatment of TFK-1 and CBC3T-1 cells under conditions of added HGF ( n = 3). (H) Western blot analysis of c-MET protein levels in PDOs established from 10 cases of eCCA. (I, J) Representative images and dose–response curves of gemcitabine alone or in combination with c-MET inhibitor in a direct co-culture system of PDOs and CAFs. Patient 8 (P8) ( n = 3). Scale bar: 500 μm. Data are shown as mean ± SD. ns, no significance; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: The human eCCA TFK-1 cell line was procured from Creative Bioarray (NY, USA).

Techniques: In Vitro, Co-Culture Assay, Western Blot