Expression of TIMPs and MMPs in Ovarian Tumors, Ascites, Ascites-Derived Cells, and Cancer Cell Lines: Characteristic Modulatory Response Before and After Chemotherapy Treatment

Figures

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  Figure 1. mRNA expression of TIMP-1, -2 and -3 in 1191 epithelial ovarian serous cystadenocarcinoma patients from two different studies published in the TCGA database: (A) the TCGA PanCan 2018 study (585 patient samples); and (B) the TCGA Provisional study (606 patient samples). Light blue filled dots, indicate genes not mutated, open light grey dots specify samples not profiled for mutation, open dark red dots show a gene amplification, open pink dots signify a mutation gain, open grey dots a diploid gene, light open blue dots a shallow deletion, a dark open circle indicates a deep deletion, and a dark black open circle specifies samples not profiled for copy number alteration (CNA).

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  Figure 2. (A) Expression and co-localization of TIMP-2 and CA-125 in high-grade serous ovarian tumors compared to benign serous tumors. All images are representative of the same high-grade, Stage III and Grade 3 serous ovarian tumor. Column 1 is representative of that tumor stained with H&E. Columns 2 and 3 represent corresponding CA-125 and TIMP-2 staining on the same area of that tumor. Each row indicates different magnification, first row indicates magnification 0.2X, scale green bar = 9mm; second row a magnification 2X, scale purple bar = 1mm; third row magnification 40X, scale black bar = 50µm. (B) Matching sections showing H & E stain, CA-125, TIMP-1, -2 and -3 staining of benign and high-grade tumor. Magnification 40X, scale black bar = 50µm.

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  Figure 3. Expression of TIMP-2 in primary ovarian tumors according to different tumor classifications. Representative images of TIMP-2 staining in primary ovarian tumors (n=26) and benign ovarian tumors (n=8). Images of samples are classified into Stages and WHO classification. Magnification 40X, scale-bar=50µm. Graphs represent DAB positivity over area divided by the negative control for each patient tissue block. Red points indicate BRCA positive patients and black/white points indicated patients with ascites present at the time collection. Significance was determined by One-way ANOVA (Tukey’s multiple comparison test) and indicated by *p <0.05.

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  Figure 4. Expression of TIMP-3 in primary ovarian tumors according to different tumor classifications. Representative images of TIMP-3 staining in primary ovarian tumors (n=26) and benign ovarian samples (n=8). Images of samples were sorted into Stages and WHO classification. Magnification 40X, scale bar = 50µm. Graphs represent DAB positivity over area divided by the negative control for each patient tissue block. Red points indicate BRCA positive patients and black/white points indicated patients with ascites present at the time collection. Significance between normal/benign tissues and carcinomas was determined by One-way ANOVA (Tukey’s multiple comparison test) and is indicated by *p <0.05.

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  Figure 5. mRNA expression of MMP genes in primary ovarian tumors. Graphs represents MMP-2, -9, -11 and -14 mRNA expression across (A) Stages I-IV, and (B) WHO Types 1 and 2 classifications. The total mRNA expression (fg) was normalized to 18S in 21 ovarian serous epithelial and 9 benign ovarian tumors (N/B). Red points indicate BRCA positive patients and black/white points indicated patients with ascites present at the time collection. Values are mean ± SEM; significance was determined by One-way ANOVA (Tukey’s multiple comparison test) and is indicated by *p<0.05, **p<0.01, ***p<0.001.

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  Figure 6. The concentration of TIMPs in the ascites of ovarian cancer patients. Graphs represents (A) the observed concentrations (pg/mL) of TIMP proteins in ascites from CN (n=7) patients; (B), concentration of TIMP-1, -2 and -3 in CR (n=7) and CN (n=7) ascites of patients with serous ovarian cancer. Values are mean ± SEM; significance is determined by One-way ANOVA (Tukey’s multiple comparison test) and indicated by *p<0.05, ***p<0.001; (C) Expression of TIMPs mRNAs in ascites-derived epithelial (Ep) and mesenchymal (Mes) cells from CN and CR patients; (D) mRNA Expression of TIMPs in different ovarian tissues. Graphs represents total mRNA amounts (fg) of TIMP-1, -2 and -3 normalized to total amount of 18S, from benign/normal ovarian tissue (N/B), high-grade primary tumors (HG) and epithelial cells collected from CN ascites (EpCN). Values are mean ± SEM; significance was determined by One-way ANOVA (Tukey’s multiple comparison test) and indicated by *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

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  Figure 7. mRNA expression of MMP genes in ascites-derived epithelial (Ep) and mesenchymal (Mes) cells from CN and CR patients. Graphs represents the MMP-2, -9, -11 and -14 mRNA expression across these samples. The total mRNA expression (fg) was normalized to 18S in these samples. Values are mean ± SEM; significance was determined by One-way ANOVA (Tukey’s multiple comparison test) and is indicated by **p < 0.001.

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  Figure 8. Differential mRNA expression of (A) TIMPs and (B) MMPs in ovarian cancer and FT282 cell lines deduced by qRT-PCR as described in Methods. The bar graphs are mean ± SEM (fg) (n=3) for each cell line collected at different split passages. (C) The bar graphs represent total TIMP-1, TIMP-2 and TIMP-3 protein expression (pg/µg of cell lysate) in the cell lysates of FT282 and ovarian cancer cell lines. Values are mean ± SEM (n=3) for each cell line collected at different split passages. Significance was obtained using One-way ANOVA (Dunnett’s multiple comparison test), **p<0.01, ****p<0.0001 when compared to the control cell line FT282. Black bar indicates the normal epithelial cell line FT282, yellow bars indicate cell lines derived from ascites and green bars cell lines derived from primary tumors.

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  Figure 9. IC50 values of ovarian cancer cell lines in response to paclitaxel (PTX) and cisplatin (CIS) treatment. (A) IC50 values of tumor-derived CAOV3, JHOS2 and HEY and ascites-derived OVCAR4, OVCAR5 and SKOV3 cell lines treated with PTX with corresponding graph showing differences in the tumor and ascites-derived cell lines. (B) IC50 values of tumor-derived CAOV3, JHOS2 and HEY and ascites-derived OVCAR4, OVCAR5 and SKOV3 treated with CIS with corresponding graph showing differences in the tumor and ascites-derived cell lines. Cell lines were treated for 48 hours with varying concentrations (0 to 320 µg/ml) of either CIS or PTX and their IC50 values (the concentration that kills 50% of the cells) was determined by MTT assay. Data are representative of three independent experiments using cells at different passages treated in triplicate. Significance in bar graphs was determined by student’s t test (Mann-Whitney test).

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  Figure 10. (A) TIMP protein levels in response to PTX and CIS treatments in ovarian cancer cell lines. OVCAR4, OVCAR5, HEY and SKOV3 cells lines were treated for 48 hours with their respective IC50 concentrations of CIS (blue bars) and PTX (pink bars). Black bars indicate the control untreated cell lines. The total TIMP-1, -2 and -3 protein expression (pg/µg of cell lysate) was deduced by ELISA as described in Methods. Values are mean ± SEM (n=3) for each cell line collected at different cell passages. (B) The mRNA expression of MMPs in CIS and PTX treated ovarian cancer cell lines. OVCAR4, OVCAR5, HEY and SKOV3 cells lines were treated for 48 hours with their respective IC50 concentrations of CIS (blue bars) and PTX (pink bars). The mRNA levels of MMP-2, -9 and -14 were obtained by relative qRT-PCR. Each experiment was repeated three times in triplicate. (C) mRNA expression of CSC marker genes in ovarian cancer cell lines treated with CIS and PTX. OVCAR4, OVCAR5, HEY and SKOV3 cells lines were treated for 48 hours with their respective IC50 concentrations of CIS (blue bars) and PTX (pink bars). Black bar indicates the control untreated cell lines. The mRNA expression levels of…

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  Figure 11. (A) Mutational Oncoprint results for MMP and TIMP genes from data in the cBioPortal for Cancer Genomics (TCGA data set). Data is from 3 original studies totaling 1680 samples (606 in the Firehose Legacy study, 489 in the Nature 211 study, and 585 in the PanCancer Atlas study). The colored bands in the horizontal line at the top represent the study of origin. Each vertical line in that band indicates a single sample and the horizontal bands below correspond to the TIMP and MMP genes analyzed in those vertical samples. Percentages indicate the mutational percentage of that gene in the total samples studied. Red, blue, green and grey indicate the type of genetic alteration in each sample. (B,C) Platinum Status of patients in relation to MMP and TIMP genes; (B) patients with unaltered genetic mutation; and (C) patients with genes altered by mutation. The data is based on 1680 samples as described in (A). Percentage of patients are indicated in each of the histograms. Above each bar, the number of patients is indicated (#). (D, E) Overall and disease-free survival curves of patients with MMP-14 gene expression. Data as described above querying 1668 patients/1680 samples for the MMP-14…