Original article Characterization of the inflammatory response during Ehrlich ascitic tumor development Patrícia Dias Fernandes a, ⁎, Fabiana S. Guerra a , Natália M. Sales a , Thais B. Sardella a , Sonia Jancar b , Josiane S. Neves c a Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Farmacologia da Dor e da Inflamação, Brazil b Universidade de São Paulo, Instituto de Ciências Biomédicas, Departamento de Imunologia, Universidade de São Paulo, Brazil c Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório Compartilhado, Brazil abstract article info Article history: Received 5 June 2014 Accepted 1 September 2014 Available online 6 September 2014 Keywords: Nitric oxide Prostaglandin E2 Inducible nitric oxide sythase Cyclooxygenase Introduction: Ehrlich tumor is a mammary adenocarcinoma with aggressive behavior. Inoculated in mice peritoneal cavity, the Ehrlich tumor grows in ascitic form (EAT). Since inflammation modulates tumor progres- sion we further investigated the inflammatory response during EAT growth. Methods: Balb/C mice were intra- peritoneal inoculated with 5 × 10 5 Ehrlich cells and after every 2 days, blood samples were collected for hemoglobin, hematocrit, platelets and leukocytes counts. The ascitic fluid was collected for protein concentration and cell count. Phenotype analysis of the peritoneal cells was made by FACS, prostaglandin E2 (PGE2) and cyto- kines by ELISA, nitric oxide (NO) by nitrate conversion protocol, and cyclooxygenase-1 (COX1), COX2 and induc- ible nitric oxide synthase (iNOS) by immunoblotting. Results: Following EAT inoculation into the peritoneal cavity there was a rapid increase in ascitis volume and protein concentration. The cell number in ascitis remained stable until day 8 (lag phase) followed by a sharp increase. As tumor progressed, blood leukocytes increased and erythrocyte decreased. Phenotypic analysis showed that during the lag phase the percentage of F4/80 + cells remained similar to control levels and around 7% of this population was also positive for the GR1 marker. These double-positive cells (probably inflammatory monocytes) markedly increased at day 6. The percentage of F4/80-GR1 + cells (probably neutrophils) was low and did not significantly vary during tumor progression. CD4 + and CD8 + cells were not detected in the time points analyzed. iNOS and COX1 expression increased after day 2 reaching peak levels on day 10. COX2 enzyme expression did not change significantly over time. Sustained increase in PGE2 and NO levels was observed. IL-10 and MCP-1 peaked at day 14 and IL-1β increased progressively till day 10. IFN-γ levels were low till day 10, increasing progressively after that. Discussion: These data extended the characterization of the inflammatory response during Ehrlich ascitis tumor growth, further validating it as a useful model for antitumor drugs screening. © 2014 Elsevier Inc. All rights reserved. 1. Introduction Evidence has emerged in the last two decades that, at the molecular level, most chronic diseases, including cancer, are caused by a deregulated inflammatory response (Prasad, Ravindran, & Aggarwal, 2009). Components of tumor microenvironment, including tumor cells themselves, can produce inflammation (Sgambato & Cittadini, 2010). Inflammatory mediators and growth factors are involved in all stages of tumor development through multiple mechanisms which include enhanced proliferation and resistance to apoptosis, induction of DNA mutations, promotion of angiogenesis, invasion and metastasis. Several studies demonstrated that the use of both non-selective non-steroidal anti-inflammatory drugs (NSAIDs), and selective cyclooxygenase-2 (COX2) inhibitors is associated with an important decrease in the incidence of colorectal, breast, bladder, prostate as well as lung cancer (De Groot, de Vries, Groen, & de Jong, 2009). Ehrlich tumor is a spontaneous murine mammary adenocarcinoma with rapidly growing and aggressive behavior (Chen & Watikins, 1970; Segura, Barbero, & Marquez, 2000). Following intraperitoneal inoculation of Ehrlich tumor cells, a local inflammatory reaction develops and the ascitic volume and number of tumor cells increase progressively (Vincent & Nicholls, 1967). Ascitis is probably formed as a consequence of tumor-induced inflammation, due to the increase in peritoneal vascular permeability (Fastaia & Dumont, 1976). The EAT growth curve in mice has an initial phase of few days, depending on the amount of EAT cells inoculated, where the number of cells in Journal of Pharmacological and Toxicological Methods 71 (2015) 83–89 ⁎ Corresponding author at: Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Farmacologia da Dor e da Inflamação, Av. Carlos Chagas Filho, 373, prédio do CCS, Bloco J, sala 10. 21941-902, Rio de Janeiro, Brazil. Tel.: +55 21 39386442. E-mail address: patricia.dias@icb.ufrj.br (P.D. Fernandes). http://dx.doi.org/10.1016/j.vascn.2014.09.001 1056-8719/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox