Figure 7. Tumorigenicity and oncogenicity of ciPTEC-OAT1.

(A) Schematic representation of tumorigenicity and oncogenicity study in vivo. A total of 10⁷ cells (ciPTEC-OAT1 or HeLa) resuspended in 100 μl of HBSS were injected subcutaneously in the flank of the rats. In an additional group cell lysate derived from 10⁷ ciPTEC-OAT1 cells and resuspended in 100 μl of HBSS was injected per animal. In the negative control group rats received 100 μl of the vehicle (HBSS). Following the observational period of 5 months, animals were sacrificed (†) and histopathological and molecular (PCR) analyses were performed to confirm tumor formation and origin, respectively. (B) Summary of ciPTEC-OAT1 tumorigenicity and oncogenicity study results. (C–F) Representative pictures of histopathological analysis of the injection sites, performed by eosin and hematoxylin staining. (C) Negative control group injection site showing normal skin with subcutaneous lymph node (‡) and mammary tissue (arrows). (D) Positive control group injected with HeLa cells, showing the presence of anaplastic carcinoma with central area of necrosis (^*) expanding in subcutaneous tissue at the site of injection. (E) Experimental tumorigenicity group injected with ciPTEC-OAT1 cells, presenting normal skin with subcutaneous lymph node (‡) and mammary tissue (arrows). (F) Oncogenicity group injected with ciPTEC-OAT1 cell lysate, showing normal skin with subcutaneous lymph node (‡) and mammary tissue (arrows). Pictures taken at magnification 2×. (G) PCR analysis of human-specific Alu elements confirming human origin of identified tumors and tumor volume (mm³) at necropsy. Ct values of all tumor samples were similar to that obtained for positive control human genomic DNA. Both the negative control (rat genomic DNA) and blank sample produced Ct values higher than 35. Limit of the blank [74], defined as the highest signal expected to be found when a blank sample containing no human DNA is tested, had a Ct value of 37.2 ± 0.71. N.M. (not measurable).