Modelo de xenoenxerto derivado de paciente com células CAL27 em língua de camundongos NOD/SCID

Abstract

Oral cancer has a high incidence, with squamous cell carcinoma (SCC) being the most frequent subtype, and the tongue as the preferred site, often resulting in mutilating and unsatisfactory treatments, as well as a high recurrence rate. Therefore, the continuous search for new therapeutic targets and preclinical pharmacological tests is necessary. Patient-derived xenografts (PDX), using tongue SCC cell lines, have been employed in several studies. However, the methodologies differ in terms of animal lineage, cell amount and inoculation site, necessitating the standardization of a reproducible and rapid model that resembles SCC seen in humans. The aim of this study is to establish a model and characterize the tumor formed from PDX of CAL27 cells in the tongue of NOD/SCID mice, creating a reproducible, effective, and rapid protocol for future studies. To this end, CAL27 cells, derived from human SCC, were cultured and inoculated into the left lateral border of the tongue of male NOD/SCID mice, divided into three groups: G1 – eight mice that received 2x10⁶ CAL27 cells; G2 – eight mice that received 5x10⁶ CAL27 cells; G3 (negative control) – eight mice that received 50 µL of phosphate-saline solution. Tumor development was assessed daily by measuring tumor volume, as well as the animals' health by measuring their weight. After 31 days, the animals were euthanized and their tongues and organs (esophagus, lungs, and liver) were removed. Tumor implantation was confirmed by histological analysis of the tongues with hematoxylin and eosin (HE) and by Tissue Micro Array (TMA) with histochemistry (PAS with diastase) and immunohistochemistry (p63, Ki-67, and CK7). The other organs were analyzed using HE staining to verify the occurrence of metastasis. Data were expressed as mean ± standard error and analyzed using the Kolmogorov-Smirnov test, 2-way ANOVA, Bonferroni test, or Student's t-test (SPSS, p<0.05). The tumor on the tongue was visible from the third day. At the end of the experiment, the average tumor size per group was G1=11.91±1.44mm3 and G2=16.94±4.91mm3. G3 animals did not develop a tumor. Histopathological analysis revealed the development of SCC, with adenosquamous variation, presence of ductal formation, and signet ring cells. PAS-d was positive for the material contained within the ductal structures. In G1 and G2, there was strong staining for p63 (>90%) and Ki-67 (±70%). CK7 expression was observed in the ductal lining epithelium and in some signet ring cells. There was no tumor growth in other organs. The protocol used in G1 showed advantages due to the use of fewer cells and lower animal loss. This study successfully demonstrated the establishment of an PDX model, reproducing the histopathological characteristics of the original tumor, consolidating itself as a tool in the study of tumor biology. A rapid model was established, which presents clinical, macroscopic, and microscopic similarities with human tongue tumors, constituting an excellent resource for in vivo studies.