2.3. Ehrlich Ascites Carcinoma (EAC) Mouse Model

For the culture and treatment of Ehrlich Ascites Carcinoma (EAC) cells, male Swiss albino mice were used (25 g body weight). They were housed under standard laboratory conditions in clean plastic cages with an ad libitum supply of food and water. The experimental procedures were carried out under the guidelines of the ethics committee at the State University of Londrina/PR in Brazil (approval number: #1633.2019.88). To minimize animal suffering, the 3R principles (reduce, refine, replace) were applied. For the induction of EAC, 5 × 10⁷ cells were injected in 50 µL phosphate-buffered saline (PBS) into the mice’s peritoneal cavities. To investigate the ex vivo effect of POS on EAC cells, ascites fluid was extracted ten days post tumor inoculation. The evaluation of the in vivo efficacy of POS was done by repeated intraperitoneal injections of 1 mL of freshly prepared POS at days 2, 4, 6, 8, and 10 before they were euthanized on the following day. Several control groups were included. In addition to a group receiving untreated saline, one group received a concentration-matched (i.e., at the concentration found in POS) control of H₂O₂ experimentally spiked into the saline. In another control group, oxaliplatin (OXA; 0.5 mg/kg of body weight; Sigma-Aldrich, Taufkirchen, Germany) was added to saline and injected at days 5, 7, and 9. Finally, a fifth group received a combination of both OXA (at days 5, 7, and 9) and POS (at days 2, 4, 6, 8, and 10). At sacrifice, the animals were weighted, and the peritoneal ascitic fluid was collected, centrifuged at 1300× g for 10 min, supernatants were separated, and cells were washed three times with PBS. All samples were stored at −20 °C for further downstream processing.