We produced a model of orthotopic transplantation in C3H/He mice by transplanting MBT-2 cells. A single-cell suspension of 1.0 × 105 MBT-2 cells/0.02 ml was successfully injected into the submucosal layer of the bladder, with an incidence of 100% after four experimental weeks. Inoculated tumor grew expansively into the bladder cavity from the bladder submucosa and invaded the serosa and the surrounding tissue. This model more closely resembled the characteristics of human bladder tumor when compared to other bladder cancer models. The results of the histological observation, electron microscopic examination and DNA content analysis by flow cytometry showed that the transplanted carcinoma maintained the biologic characteristics of the primary tumo

Jiang, F.

Zhou, X.

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Urol Res (1997) 25:179-182 © Springer-Verlag 1997 F. J i ang  • X . -M.  Zhou A model of orthotopic murine bladder (MBT-2) tumor implants Received: 3 November 1995/Accepted: 13 November !996 Abstract We produced a model of  orthotopic trans- plantation in C3H/He mice by transplanting MBT-2 cells. A single-cell suspension of  1.0 × 105 MBT-2 cells/ 0.02 ml was successfully injected into the submucosal layer of  the bladder, with an incidence of 100% after four experimental weeks. Inoculated tumor grew ex- pansively into the bladder cavity from the bladder sub- mucosa and invaded the serosa and the surrounding tissue. This model more closely resembled the charac- teristics of  human bladder tumor when compared to other bladder cancer models. The results of  the histo- logical observation, electron microscopic examination and DNA content analysis by flow cytometry showed that the transplanted carcinoma maintained the biologic characteristics of  the pr imary tumor. Key words Bladder tumor - Mouse .  Orthotopic transplantation Introduction The orthotopic murine bladder tumor is an invaluable experimental tool for assessing potential therapeutic agents against bladder carcinoma. It has been used for evaluating intravesical therapy [bacillus calmette gu6rin (BCG) and interleukin-2 (IL-2) [1, 2, 7]. Soloway [9] has successfully established a localized transitional cell carcinoma model in mice using intravesical instillation of  tumor cell suspension (MBT-2) into denuded [by N-methyl-N-nitrosourea (MNU)]  focally fulgurated bladder [9]. However, this method can only be applied to female mice and often produces urinary stones. The success rate of  the tumor implantation method varies between 50% and 70% [9, 10]. A study was undertaken to investigate the possibility of  developing a orthotopic tumor model in mice by injecting tumor cell suspension into the submucosal layer of  the bladders of  syngenic animals. Materials and methods Tumor cells and tumor implantation N-(4-(5-Nitro-2-furgl)-2-thiazolyl) formamide (FANFT)-induced MBT-2 transitional cell carcinoma of the bladder (originally de- rived by Dr. Soloway) was maintained in vivo as a solid subcuta- neous growing tumor by serial transplantation in syngenic C3H/He mice (Charles River), and in vitro in RPMI1640 medium plus 10% fetal calf serum. Single tumor suspension was prepared by mincing the tumor under sterile conditions and adding to the minced tissue 5 ml of an enzyme cocktail containing a mixture of 1% collagenase (Sigma, type II), 0.01% proteinase K (Sigma) and 0.01% DNAase (Sigma) in Dulbecco's phosphate-buffered saline (PBS). The cells were than washed twice with RPMI1640 medium, resuspended to the required concentration. The viability was always more than 90% by the trypan blue exclusion method. The final concentration of the suspension was adjusted to 5.0 x 107, 1.0 x 105 and 1.0 x 103 cells in 0.02 ml by dilution with the 1640 medium. The operation was performed with the mice under general anesthesia from a single dose of intraperitoneal sodium phenobarbital (130 mg/kg body weight). The urinary bladder was exposed through a low midline incision, the bladder was explored for tumor, 0.02 ml tumor sus- pension was injected into the submucosal layer of the bladders o as to make a "semitransparent bubble" for the submucosal layer tu- mor model, then omentum was drawn over the operation area. The procedure took place under clean but not sterile conditions and was performed without a magnifying lass. Animals were placed on antibiotics (tetracycline, 250 mg/500 ml drinking water) for 5 days after the operation. F. Jiang ([~) Institute of Pathology, University of Basel, Sch6nbeinstrasse 40, CH-4003 Basel, Switzerland X.-M. Zhou Department of Urology, Nanjing Railway Medical College, Nanjing 210009, People's Republic of China Experimental groups An intermediate dosis of tumor cells (1.0 x 105 viable cells in 0.02 ml) was injected into 48 mice. These mice were randomized into six groups (n - 8 each). The animals were inspected for: (a) weight loss, (b) the time of the appearance of the tumor and (c) palpation of the suprapubic mass. Mice of groups 1-5 were 180 sacrificed at 1, 2, 3, 4 and 5 weeks post-tumor implant, respectively. The sixth group was used as a group for observation ofthe survival time of the tumor-bearing animals. The large dose of tumor cells (5.0 x 107 cells in 0.02 ml) and the small dose of tumor cells (1.0 × 10 3 cells in 0.02 ml) was injected into 20 mice each in order to demonstrate the relationship between the time of the appearance and the number of the injected cells. An additional ten normal mice injected with 0.02 ml RPMI1640 medium were used as normal control group. For pathological and histological examination, cystectomy was performed via a midline incision after the bladder was distended with 0.1 ml formalin and the urethra ligated at the bladder neck. Tumor incidence and tumor weight were determined. Tumor weight was expected to be similar to the weight of a neo- plastic bladder. Histological examination was performed with he- matoxylin and eosin (H&E) staining on fixed whole bladder sections from the cystectomy specimens. The bladders were care- fully inspected for: (a) tumor incidence and (b) histological grade. Other organs were examined for gross abnormalities, and the kidneys, lungs, liver, uterus, spleen and pancreas were routinely processed for histology. A fragment of the grade II tumors from group 5 was fixed in 3% glutaraldehyde and then postfixed in OsO4, dehydrated in graded alcohol and embedded inEpon 812 (Carl Roth, Karlsruhe). Ultrathin sections were prepared with diamond knives, stained with uranyl acetate and lead citrate, and viewed with an H-600 electron microscope. A 4 x 4-mm piece of the grade II bladder tumor from group 5 was minced and stored frozen at -80°C in a solution of 5% dimethylsulfoxide, 250 mm sucrose and 40 mm trisodium citrate until analysis. The tissue was minced into pieces approximately l mm in diameter and subsequently enzymatically dissociated using collagenase type (200 U/ml) and DNAse (500 K units U/ml) at 37°C. DNA histogram display and analysis was performed on a Terak 8600 minicomputer (Terak, Scottsdale, AZ). Full bright fluorescent microspheres (Lot 5325, Coulter Electronics) were used to aid instrument alignment, and as an external f uorescence stan- dard. Murine peripheral blood mononuclear cells were used as an internal reference standard for DNA ploidy analysis. The cells were aliquoted, frozen as above and stained in parallel with the urinary bladder specimens in each flow cytometry run. An aneuploid population was considered present only if unequivocal Go/GI populations were evident. Results General The mice that received the middle cell doses all had palpable pubic mass-like soybean at 3 weeks, and half of them had gross hematur ia and were fatigued at 3-4 weeks. At 5 weeks, all of them appeared mor ibund and mean weights of the mice were 13.8 ± 1.8 g, whereas mice of the control group had a mean weight of Table 1 Reduction of tumor growth with implantation f 1.0 x 10 s tumor cells Groups No. of No. of Tumor Mean Mean mice dead incidence bladder mouse mice (%) weight (g) weight (g) 1 8 0 0.0 19.5 :t- 4.3 17.9 ± 1.6 2 8 0 75.0 22.4 ± 20.5 17.5 ± 2.0 3 8 0 88.0 350.0 ± 20.7 15.7 ± 2.4 4 8 0 100.0 483.5 ± 44.5 15.4 ± 3.0 5 8 1 100.0 510.4 ± 38.5 13.8 ± 1.8 6 8 8 100.0 540.7 ± 40.4 12.4 ± 1.5 Control 10 0 0.0 20.0 ± 4.7 20.2 ± 1.5 Table 2 Effect of different numbers of the cells on the growth of the tumor Time to Tumor Mean survival appearance incidence time (days) of tumor (weeks) 1.0 x 103 4 60.0% 58 1.0 x 105 3 88.0% 52 1.0 x 10 v 2 100.0% 26 20.2 ± 1.5 g; mean bladder weight of the mice was 510.4 ~: 38.5 mg, whereas that of the control group was 20.0 ± 4.7 mg. At 6 weeks post-tumor implant, all mice died (Table 1). There was a good correlation between the tumor incidence and the number  of cells at 3 weeks. The mice injected with a small dose of MBT-2 cells had hematur ia t 4 weeks and the incidence of the tumor was 60%; they survived 8 weeks after the operation. The mice injected with a large dose of cells had gross hematuria s early as 2 weeks after the procedure and all died at 4 weeks (Table 2). Pathological changes One week after the transplantat ion,  many vital tumor cells were found in the submucosa of the bladder. At 2 weeks, the tumors were grossly visible, their surface appearing papil lary or nodular.  The color of the cut sections of these tumors was grayish (Fig. 1). After the 3rd week, all tumors found in the bladders were large, occupying more than 50% of the bladder cross-sectional area. The tumors had hemorrhage or necrosis, and ex- tended into the perivesical fat or surrounding regional areas such as lymph nodes, mesenterium and repro- ductive organs. At autopsy examinat ion bilateral hydro- Fig. 1 The bladder partially filled with tumor at 2 weeks post-tumor implant; the color of the cross-section f tumor was grayish white Fig. 2 Bilateral hydronephrosis was observed with dead mouse at 5 weeks post-tumor implant 181 Fig. 3 There was no specialization f the luminal membrane, and no zonula occludes or macula dhaemns. TEM, ×6000 nephrosis was frequently observed (Fig. 2), uremia being the main cause of death. No hematogenous dissemina- tion, particularly to the liver or lungs, was found. His- tological examination of the tumor specimens revealed tumor cells characteristic of a poorly differentiated transitional cell carcinoma of the bladder, which demonstrated greater variability in both nuclear size and shape. The transplanted tumors were histologically identical to the original MBT-2 tumors. Electron microscopy The transplanted tumors showed a comparatively clear cytoplasm. Mitochondria were abundant and spherical, and showed vestigial cristae. Both rough- and smooth- surfaced endoplasmic reticulum was seen in quite long stretches. The Golgi apparatus was well developed, with large, open lamellae. The nuclei were round, containing prominent nucleoli. The nuclear membrane was not re- entrant, and there was no margination of chromatin. Throughout the tumor, boundaries between cells did not show the complex folding seen in the well-differentiated tumors and the normal epithelium. The tight junctions were poorly formed. The lamina densa of the basement membrane was markedly thickened and multilayered (Fig. 3). Flow cytometry The DNA index (DI) of MBT-2 cells and the trans- planted tumor cells was 1.98 and 1.96, respectively, which meant hat tumors were tetraploid, and that DNA histograms from these primary tumors and transplanted tumors were identical. Discussion The techniques of inducing urothelial tumor by oral carcinogens such as FANFT or by intravesical instilla- tion of carcinogens such as MNU in rats have been well established. However, these useful models have some shortcomings. They usually require a long period for the tumor to develop: 10-45 weeks for the FANFT model and 12 20 weeks for the MNU model [3, 4]. Further- more, the entire urothelium of the induced bladder is either hyperplastic or neoplastic, and therefore studies comparing the induced tumor with the surrounding normal mucosa as well as the mechanisms of tumor growth and spreading cannot be performed in these models. Nude mice have been used for many years for heterotransplantation of human tumors. However, studies concerning experimental metastases of human tumors using these experimental nimals have been limited because metastases seldom develop in nude mice when they are grafted with human tumor cells. The ex- perimental conditions required for nude mice are very strict. It has been reported that only 29% of tumor cell lines tested could be successfully used following subcu- taneous inoculation in such mice and that metastases oftransplanted tumors are not usually observed by gross and microscopic observation; the average survival time of the tumor-bearing animals is usually less than 2 weeks [8]. Previously we described another model for focal bladder carcinoma in rats that had a similar success rate to the present study [5]. However, a disadvantage of our previous model was a high incidence of calculi due to the use of sutures, which may have provided a nucleus for the formation of urinary stones. The operation was very complicated, and our experiments of grafting bladder tumor tissue to bladder of syngenic animals were con- sistent with the results of previous tudies [6]; our results were reported in a previously published study [5]. Moreover, this method has other complications includ- ing graft necrosis, with extravasation f urine, graft at- rophy and perivesical abscess formation. It is very easy to observe the appearance of the tumor in the murine flank, but the model does not reflect the characteristics of the bladder tumor as an orthotopic tumor model. Furthermore treatment of orthotopic tumors would seem to be a better method since it takes into consider- ation the concentration f antitumor agent in the urine. Orthotopic models may be used for evaluation of in- travesical therapy as well as for systemic therapeutic experiments. It is very important, for intravesical ther- apy is the best way to decrease a high recurrence rate of bladder cancer. Obviously mice bearing the same tumor in the flank are not used for this kind of test. Our established model of orthotopic transplantation in mice using MBT-2 has the following characteristics: (a) the incidence of injection and subsequent growth of the transitional tumor cell is 100% in all groups; (b) the tumors were found in all bladders as early as 2 weeks following tumor implantation into the bladders. The time for the tumor development and survival time of the animals might be controlled by adjusting the dose of the tumor cells implanted. There was a good number-related response of the urinary bladder to the MBT-2 cells in- jected into the submucosa. It is easy and economical to 182 do the operation. (d) The present study utilized a murine transitional cell tumor which closely resembled its hu- man counterpart histologically. Early stage orthotopic murine bladder tumors produced by an identical number of cells were always localized, allowing a comparison of the induced tumor with surrounding normal mucosa as well as the study of the mechanisms of tumor growth and spreading. In addition it is possible to produce and to study multifocal tumors using the described tech- nique, e) The incidence and depth of the tumor corre- lated very well with the time and dose of tumor cells implanted. Seven days following the procedure, histo- logic examination of the bladder showed that almost all the cells were in the submucosa but not in the muscular layer. Subsequently, the results of our repetitive xperi- ments demonstrated this phenomenon, which is due to the anatomical characteristics of the murine bladder wall. The submucosal layer is much looser than the muscular layer. It is very easy to inject the cells into the submucosal layer when the bladder is almost empty. The earliest change was simple or orderly hyperplasia fol- lowed by nodular and/or papillary and finally invasive carcinomas associated with the area of hemorrhage or necrosis. The sex of mice does not affect the transplan- tation of tumors since cells were not placed transure- therally into the bladder. (g) Some complications such as retrograde infection and bladder stone might be elimi- nated effectively because the technique did not involve the bladder cavity. There are several technical points which are critical to the success of the transplantation. First, it is important that the viability is always more than 90% using the trypan blue exclusion method. Second, the bladder wall should not be perforated in order to avoid injecting the tumor cells into the cavity. Third, postoperative pro- phylactic antibiotics are essential in preventing infection and maintaining the tumor cells. We believe that the potential applications of this model are numerous. In addition to being of use for evaluation of chemotherapeutic agents, regimens, and other modalities of treatment of TCC, the model can also be used for the study of basic mechanisms of tumor growth and the spreading and extrinsic and intrinsic factors affecting these processes. References 1. Chin J, Kadhim S, Garcia B, Kim Y, Karlik S (1991) Magnetic resonance imaging for detecting and treatment monitoring of orthotopic murine bladder tumor implants. J Urol 145:1297 2. Connor J, Bannerji R, Satio S, Heston W, Fair W, Gilboa E (1993) Regression of bladder tumors in mice treated with IL-2 gene-modified tumor cells. J Exp Med 177:1127 3. Erturk E, Cohen S (1969) Pathogenesis, histology and trans- plantability of urinary bladder carcinomas induced in albino rats by oral administration of FANFT. Cancer Res 29:2219 4. Hicks RM, Wakefield J St (1993) Rapid induction of bladder cancer in rats with MNU. Chen Biol Interact 5:139:I72 5. Jiang F, Zhou X-M (i993) Murine bladder cancer isograft model for focal carcinoma. Chinese J Exp Surg 10:151 6. Okajima E, Ozono S (1991) Experimental bladder tumor. Nippon-Hingokika-Gakkai-Zasshi 82:705 7. Recker F, Otto T (1991) Animal experiment aspects of bladder cancer. Urologe A 30:172 8. Shibayama T, Tachibana M, Deguchi N, Jitsukaws S, Tazaki H (1991) SCIO mice: a suitable model for experimental studies of urologic malignancies. J Urol 146:1136 Soloway M (1977) Intravesical and systemic hemotherapy ofmurine bladder cancer. Cancer Res 37:2918:1977 Soloway M, Masters S (1979) Implantation of transitional tumor cells on the cauterized murine urothelial surface. Proc Amer Ass Cancer Res 20:256 9. 10. 

A model of orthotopic murine bladder (MBT-2) tumor implants

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A model of orthotopic murine bladder (MBT-2) tumor implants

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