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Volume 3, Issue 4, Pages 377-386 (April 2003) Remodeling of the extracellular matrix through overexpression of collagen VI contributes to cisplatin resistance in ovarian cancer cells Cheryl A. Sherman-Baust, Ashani T. Weeraratna, Leticia B.A. Rangel, Ellen S. Pizer, Kathleen R. Cho, Donald R. Schwartz, Teresa Shock, Patrice J. Morin Cancer Cell Volume 3, Issue 4, Pages 377-386 (April 2003) DOI: 10.1016/S1535-6108(03)00058-8

Figure 1 Characterization of cisplatin-resistant clones ACRP and ACR6 by clonogenic assays A: Surviving colonies of a typical experiment are shown for the cell lines A2780, ACR6, and ACRP following treatment with 10 or 15 μM cisplatin. B: Colonies from (A) were counted and plotted for the various lines at 10 uM and 15 uM cisplatin. Cancer Cell 2003 3, 377-386DOI: (10.1016/S1535-6108(03)00058-8)

Figure 2 Many genes are differentially expressed between A2780 and the cisplatin-resistant cell line ACRP A: Tag frequency scatter plot of ACRP versus A2780. The frequency of each tag (per 30,000) is plotted on a logarithmic scale. Tags that are significantly differentially expressed in ACRP compared to A2780 (p < 0.05) are indicated in black. Some diamonds may represent multiple tags that are expressed at identical levels. B: Genes differentially expressed between A2780 and ACRP are grouped into main functional categories. The number of genes in each category is indicated. Cancer Cell 2003 3, 377-386DOI: (10.1016/S1535-6108(03)00058-8)

Figure 3 Semi-quantitative real-time RT-PCR validation of differentially expressed genes A: Validation of selected upregulated genes in ACRP. The y axis represents the fold upregulation of the indicated genes in the cisplatin-resistant cell lines compared to parental A2780 cells. B: Validation of selected downregulated genes in ACRP. The y axis represents the level of expression of the indicated genes in the cisplatin-resistant cell lines ACR6 and ACRP compared to parental A2780 cells. Cancer Cell 2003 3, 377-386DOI: (10.1016/S1535-6108(03)00058-8)

Figure 4 Collagen VI protein specifically increases cisplatin resistance of parental ovarian cancer cell line A2780 A: A2780 and ACRP cells were plated in 12-well dishes previously coated with collagen I, collagen VI, or left uncoated. Typical wells of A2780 clonogenic assays after treatment with the indicated concentrations of cisplatin for 24 hr are shown. B: The results of a clonogenic assay of A2780 cells plated on various collagen substrates after cisplatin treatment (0, 0.5, 0.75, 1.0 μM) are shown. Number of surviving colonies are indicated. C: MTS proliferation assays were performed on cells plated on collagen I (I), collagen VI (VI), or left uncoated (U). Cancer Cell 2003 3, 377-386DOI: (10.1016/S1535-6108(03)00058-8)

Figure 5 COL6A3 expression in microdissected ovarian tumors A: Fold overexpression of COL6A3 compared to an ovarian surface epithelial culture is shown for ovarian tumors of various subtypes: serous, mucinous (Muc), endometrioid (Endo), and clear cell (CC). ACRP (Ac), the cisplatin-resistant cell line generated in this study, is included for comparison, along with A2780 (A), the parental line. B: Expression levels of COL6A3 in serous ovarian tumors as a function of tumor grade. The dotted line represents an arbitrary value emphasizing the difference in expression levels between poorly differentiated (PD) and moderately to well differentiated (MD + WD) ovarian serous tumors. The p value for PD versus MD + WD is 0.018. Cancer Cell 2003 3, 377-386DOI: (10.1016/S1535-6108(03)00058-8)

Figure 6 Collagen VI staining of human ovarian cancers Cy-5-conjugated antibody against collagen VI was used to analyze ovarian tumor sections. H&E staining for three different representative tumors (D–F) is shown as well as the corresponding collagen VI staining (A–C). Red Cy-5 staining is apparent in panels (A) and (B). Tumor photographed in panel (C) did not exhibit collagen VI staining. Collagen VI is frequently overexpressed in the vicinity of the cancer cells but absent in the stromal areas. The approximate demarcations between stroma (S) and cancer (C) are shown by broken lines. Cancer Cell 2003 3, 377-386DOI: (10.1016/S1535-6108(03)00058-8)