Abstract 3248: Establishment and characterization of primary penile carcinoma cell culture versus xenograft by using transcriptome analysis
Authors: Muñoz Juan José Augusto Moyano , Drigo Sandra A. , Goss Tiago , Kuasne Hellen , Marchi Fabio , Guimaraes Gustavo , Lopes Ademar , Neto Cristovam S. , Rogatto Silvia R. 
Published: August 2015

Abstract
Background: The establishment of tumor cell lines in penile carcinoma (PeCa), a rare and high morbidity disease, is essential to conduct functional genomic and epigenomic studies. However, penile cancer cell line is not commercially available. In addition, xenograft in immunodeficient mouse is a valuable model between in vitro testing and pre-clinical trials.
Aim: This study aimed to establish and characterize PeCa primary cultures and xenografts by using large-scale gene expression analysis.
Methods: Primary tumor cultures were established from one untreated verrucous PeCa sample (HPV- negative). Tumor tissue was minced and digested with 0.2% trypsin for 30 minutes at 37°C. After extensively washing with 3 KSFM (GIBCO):1 DF12 (GIBCO). After passage 5 (36 days), two cell cultures with distinct morphologies were isolated and implanted subcutaneously on the right (epithelial cells) and left (fibroblast-like cells) flanks of BALB/C nude mouse. Analyses of AE1AE3(ABCAM), Vimentin (ABCAM), Desmin (DAKO), Myogenine (SIGMA), HHF35 (DAKO), 1A3 (SIGMA), 34βE12 (ABCAM) and CD44 (DAKO) by immunohistochemistry and ALDH (ABCAM) by flow cytometry were performed in xenograft tissues. Total RNA was extracted from cell cultures and xenograft tissues in duplicate and evaluated for gene expression by using Human Transciptome Array (HTA) 2.0 (Affymetrix). Expression Console (Affymetrix), SAM, hierarchical clustering analysis, Ingenuity Pathway Analysis (IPA).
Results: After 5 passages of verrucous PeCa cells, distinct epithelial and fibroblast-like cell morphologies were observed. After four weeks of inoculation of both cell types into right and left flanks of nude mouse, tumor growth was observed in both flanks. Both tumor tissues obtained from the patient and xenograft showed positive immunostaining for epithelial cell markers (AE1AE3, 5-10% of cells and high molecular weight cytokeratin 34βE12, 15% of cells), for mesenchymal cells (vimentin, 95%; myogenin, 95%; desmin, 80-90%; HHF35, 10%; 1A4, 95%) and also for stem cells (CD44, 5% of cells). ALDH activity was evaluated by flow cytometry and high ALDH activity was detected in vitro and in vivo cells. Preliminary results from transcriptome analysis showed 485 differentially expressed genes between primary tumor and Pre Xenograft passage 1; 502 genes in primary tumor and Pre Xenograft passage 5; 468 genes in verrucous PeCa sample vs xenografts passage 1 and 2; 402 genes in verrucous PeCa sample vs xenograft passage 3. It was verified upregulation of STAT3, IFNg, KRT14 and IFNA2 genes as well as genes involved in ROCK2 signaling networks.
Conclusions: We successfully established a primary tumor cell culture and xenograft derived from a verrucous penile carcinoma. Our data also support that despite cell cultures showed different morphologies compared to primary tumor, they present genotypic and phenotypic features similar to the tissue of origin.
Citation Format: Juan José Augusto Moyano Muñoz, Sandra A. Drigo, Tiago Goss, Hellen Kuasne, Fabio Marchi, Gustavo Guimaraes, Ademar Lopes, Cristovam S. Neto, Silvia R. Rogatto. Establishment and characterization of primary penile carcinoma cell culture versus xenograft by using transcriptome analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3248. doi:10.1158/1538-7445.AM2015-3248