Penile Cancer-Derived Cells Molecularly Characterized as Models to Guide Targeted Therapies

Abstract

Penile cancer (PeCa) is a common disease in poor and developing countries, showing high morbidity rates. Despite the recent progress in understanding the molecular events involved in PeCa, the lack of well-characterized in vitro models precludes new advances in anticancer drug development. Here we describe the establishment of five human primary penile cancer-derived cell cultures, including two epithelial and three cancer-associated fibroblast (CAF) cells. Using high-throughput genomic approaches, we found that the epithelial PeCa derived- cells recapitulate the molecular alterations of their primary tumors and present the same deregulated signaling pathways. The differentially expressed genes and proteins identified are components of key oncogenic pathways, including EGFR and PI3K/AKT/mTOR. We showed that epithelial PeCa derived cells presented a good response to cisplatin, a common therapeutic approach used in PeCa patients. The growth of a PeCa-derived cell overexpressing EGFR was inhibited by EGFR inhibitors (cetuximab, gefitinib, and erlotinib). We also identified CAF signature markers in three PeCa-derived cells with fibroblast-like morphology, indicating that those cells are suitable models for PeCa microenvironment studies. We thus demonstrate the utility of PeCa cell models to dissect mechanisms that promote penile carcinogenesis, which are useful models to evaluate therapeutic approaches for the disease.

Interplay Between Immune and Cancer-Associated Fibroblasts: A Path to Target Metalloproteinases in Penile Cancer

“…Considering the global alteration of MMPs in PeCa and their potential to promote an immunosuppressive TME by remodeling it ( 42 ), we investigated the expression of CAF markers in PeCa-derived cells previously published by our group ( 40 ). The expression profile of Cell1 (healthy individual) was distinct from cells with fibroblast-like morphology (Cell4, Cell5, and Cell6, Figure 5B ), which presented a high expression of CAF markers ( Figure 5A ).…”

“…We performed chemosensitivity assays using GM6001 (Merck Life Science, Hellerup, Denmark), a broad-spectrum MMP inhibitor (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-14, and MMP-26). Briefly, PeCa cells were seeded in a 96-well plate at a density of 1 × 10 5 cells/ml and incubated at 37°C in a complete medium composed of 3:1 keratinocyte serum-free medium–DMEM/F12 (Dulbecco’s modified Eagle medium/nutrient mixture F-12) (GIBCO, Carlsbad, CA, USA) supplemented following the previously described protocol ( 40 ). Treatment with GM6001 was administered after 24 h in concentrations of 0, 1, 3, 10, and 20 μM, and six replicates were used for each concentration.…”

“…In a previous study, we established three cells derived from PeCa (Cell4, Cell5, and Cell6) that were molecularly and morphologically characterized as CAFs (40). The morphology of CAF in PeCa was evaluated by immunofluorescence using Texas Red: actin/phalloidin (Thermo Fisher Scientific, Waltham, MA, USA), FITC (fluorescein isothiocyanate): tubulin (Thermo Fisher Scientific, Waltham, MA, USA), and DAPI (4′,6diamidino-2-phenylindole): nucleus (Vector Laboratories, Burlingame, CA, USA) as described by Kuasne et al (40).…”

DataSheet_1.docx

“…Considering the global alteration of MMPs in PeCa and their potential to promote an immunosuppressive TME by remodeling it (42), we investigated the expression of CAF markers in PeCaderived cells previously published by our group (40). The expression profile of Cell1 (healthy individual) was distinct from cells with fibroblast-like morphology (Cell4, Cell5, and Cell6, Figure 5B), which presented a high expression of CAF markers (Figure 5A).…”

“…We performed chemosensitivity assays using GM6001 (Merck Life Science, Hellerup, Denmark), a broad-spectrum MMP inhibitor (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-14, and MMP-26). Briefly, PeCa cells were seeded in a 96-well plate at a density of 1 × 10 5 cells/ml and incubated at 37°C in a complete medium composed of 3:1 keratinocyte serum-free medium-DMEM/F12 (Dulbecco's modified Eagle medium/nutrient mixture F-12) (GIBCO, Carlsbad, CA, USA) supplemented following the previously described protocol (40). Treatment with GM6001 was administered after 24 h in concentrations of 0, 1, 3, 10, and 20 mM, and six replicates were used for each concentration.…”