Preconditioning methods influence tumor property in an orthotopic bladder urothelial carcinoma rat model

Abstract

Urothelial carcinoma (UC) is an extremely common type of cancer that occurs in the bladder. It has a particularly high rate of recurrence. Therefore, preclinical studies using animal models are essential to determine effective forms of treatment. In the present study, in order to establish an orthotopic bladder UC animal model with clinical relevance, the effects of preconditioning methods on properties of the developed tumor were evaluated. The bladder cavity was pretreated with phosphate-buffered saline (PBS), acid-base, trypsin (TRY) or poly (L-lysine) (PLL) and then rat UC cells (AY-27) (4×10⁶ cells) were inoculated. The results demonstrated that, two weeks later, the tumorigenic rate (88%) and tumor count (2.3 per rat) were not significantly different among the preconditioning methods, whereas tumor volume and invasion depth into bladder tissue were significantly different. Average tumor volumes were >50 mm³ in the PBS and acid-base-treated groups and <10 mm³ in the TRY- and PLL-treated groups. The percentage of invasive tumors (T2 or more advanced stage) was ∼75% of total tumors in the PBS- and acid-base-treated groups, whereas the percentages were reduced in the TRY- and PLL-treated groups (58 and 32%, respectively). Non-invasive tumors (Ta or T1) accounted for 54% of tumors in the PLL-treated group, which was 2-5-fold higher than the percentages in the remaining groups. Properties of the developed tumor in the rat orthotopic UC model were different depending on preconditioning methods. Therefore, different animal models suitable for a discrete preclinical examination may be established by using the appropriate preconditioning condition.

Keywords: acid-base; phosphate-buffered saline; polylysine; trypsin; ultrasonography.

Figure 1.

(A) Axial views of ultrasound imaging of the rat urinary bladder and (B) macroscopic images of the bladder tumor. Two weeks following tumor cell inoculation, the ultrasound imaging was conducted and bladders were extracted. The bladders were longitudinally incised at the anterior wall and extended: the upper and lower panels of each image correspond to the apex and base of the bladder, respectively. (A) In ultrasound images, upper panel, ventral direction; lower panel, dorsal direction; left side, leftward; right side, rightward. White arrow shows tumor lesion. Red arrow shows microtumors. Bar, 5 mm.

Figure 2.

(A) Tumor volume and (B) number of tumors in each preconditioning group. A mass of >1 mm in the long axis that protruded on the mucosa was identified as a tumor. The tumor volume and number of tumors per bladder were determined macroscopically. A mass of <1 mm in the long axis was not evaluated. Individual data and the means ± SD are shown in each graph. n=13, 13, 10 and 8 for phosphate-buffered saline (PBS), acid-base, trypsin (TRY) and poly (L-lysine) (PLL), respectively.

Figure 3.

Histopathological images of bladder tumors (H&E staining) at each tumor stage (left-upper corner of each image). Right-sided images are magnified views of the section inside a square in each left image. Bar, 1 mm. In Ta, papillae are developed and usually short. (A) The nuclear-to-cytoplasmic (N/C) ratio of cells is moderately increased, nuclei have irregularly distributed chromatin, and nucleoli are prominent. In Tis, bladder CIS replaces normal urothelium and partially or entirely covers the bladder lumen surface. (B) A moderate increase in the N/C ratio in cells is evident. In T1, tumor cells invade the submucosa. (C) Low to moderate dysplastic cells (grade I–II) and concentrated inflammatory cells are evident. In the advanced stages, tumor cells invade the bladder muscle (T2) and penetrate the bladder wall (T3). (D-F) Tumor cells show an increase in the N/C ratio with markedly increased variation in nuclear size and shape, and mitotic figures (grade III).