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An improved orthotopic rat bladder tumor model using DiI-loaded fluorescent AY-27 cells

An improved orthotopic rat bladder tumor model using DiI-loaded fluorescent AY-27 cells Resea RCh pape R Cancer Biology & Therapy 9:12, 986-993; June 15, 2010; © 2010 Landes Bioscience An improved orthotopic rat bladder tumor model using DiI-loaded fluorescent AY-27 cells 1 1 2 2 3 4 Joachim Vandepitte, Jan Maes, Ben Van Cleynenbreugel, h ein Van poppel, evelyne Lerut, patrizia a gostinis 1, and peter a .M. de Witte * 1 2 3 Laboratorium voor Farmaceutische Biologie; Faculteit Farmaceutische Wetenschappen; Dienst Urologie; Departement Morfologie en Moleculaire pathologie; and Departement voor Moleculaire Celbiologie; Faculteit Geneeskunde; Katholieke Universiteit Leuven; Leuven, Belgium Key words: bladder cancer, orthotopic animal model, AY-27, DiI, mitomycin-c Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl) nitrosamine; CIS, carcinoma in situ; DiI, 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine; FANFT, N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide; H&E, haematoxilin and eosin; MM-C, mitomycin-C; MNU, N-methyl-N-nitrosourea; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; TUR, transurethral resection; UCC, urothelial cell carcinoma h ere we evaluate an improved orthotopic rat bladder tumor model, to be used for the evaluation of the therapeutic potential of novel cancer therapeutics. Before instilling aY-27 tumor cells into chemically denudated rat bladders, aY-27 cells were labeled with the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). We found that the presence of DiI did not alter the in vitro aY-27 cell proliferation and that the DiI label was strongly associated with the cells. We further provide evidence that the use of fluorescently labeled aY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inoculation process. Using this technique it was possible to track down the tumor cells after inoculation into the bladder, which makes it straightforward to distinguish tumor cells from remaining or regenerated normal urothelium over a period of 5 d. The results also demonstrated that malignant a Y-27 tissue exists as an intact non-muscle invasive bladder tumor only for 1–3 d after cell implantation. a ccordingly the a Y-27 bladder tumor model was used to evaluate the antitumoral ee ff ct of a single intravesical MM-C instillation. a ll rats instilled with 1 mM MM-C survived the final endpoint of 30 d after intravesical MM-C. Moreover, 10 and 30 d after treatment the urothelium of the MM-C-treated animals was completely restored. Remarkably, after MM-C treatment distinct patchy fluorescent dots were found into the submucosa and the regenerated urothelium, suggesting that dye retention is secondary to the digestion of DiI-loaded a Y-27 cells and cellular debris by macrophages and related immune cells. regard to the recurrence rate and invasion. As a result different Introduction experimental therapies such as photodynamic therapy are cur- 5,6 rently being investigated. Bladder cancer is the fourth most common malignant disease accounting for approximately 5% of all cancer cases. At initial Different pre-clinical animal bladder tumor models have been diagnosis, most of the patients show non-muscle invasive blad- developed that are essential in testing new treatment modalities der cancer. These lesions extend visually into the lumen of the against bladder cancer. For instance, rodent bladder tumors can bladder so that they can be removed by transurethral resection be induced by feeding the animals with chemical carcinogens (TUR) with relative ease. Although a high incidence of tumor such as N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), or 2 8 recurrence is observed afterwards, these tumors seldom prog- N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT). Using ress into invasive disease. A small percentage of these patients this methodology, both UCC and squamous cell carcinoma also show carcinoma in situ (CIS or Tis), flat lesions that have can be generated, but it takes at least 8–11 mo before tumors 8,9 a very malignant potential and a high risk of progressing into develop. Another possibility is to repeatedly instill N-methyl- muscle invasive disease. Early recognition of CIS therefore N-nitrosourea (MNU) into the rat bladder. Doing so, UCC is essential to offer the patients the most appropriate therapy without the presence of squamous cell carcinoma are induced and cure rate. CIS is generally treated with intravesical Bacillus within 4 mo. Furthermore, also xenograft tumor models have Calmette-Guerin (BCG) instillations or with radical cystec- been developed by transplanting heterotopically or orthotopically 4 11,12 tomy for refractory cases. The medical options to treat urothe- human urothelial cancer cells into immunodeficient rats or 13,14 lial cell carcinoma (UCC) are still insufficient, especially with mice. *Correspondence to: Peter A.M. de Witte; Email: [email protected] Submitted: 01/25/10; Revised: 02/24/10; Accepted: 02/26/10 Previously published online: www.landesbioscience.com/journals/cbt/article/11638 DOI: 10.4161/cbt.9.12.11638 986 Cancer Biology & Therapy Volume 9 Issue 12 Resea RCh pape R Resea RCh pape R Using the AY-27 syngeneic orthotopic rat bladder tumor model, we found it often very difficult to discrim - inate between inoculated AY-27 cells and tissue consist- ing out of locally regenerated normal urothelial cells. Unlike their human counterparts, normal urothelial rat cells proliferate very rapidly, reconstituting a urothelial lining within 2 or 3 d after chemical destruction. These normal urothelial cells can disrupt and infiltrate the tumor cell layers, creating mixed tissues of both normal urothelial cells and tumoral cells. This situation makes it problematic to assess correctly any antitumoral effect induced by a therapeutic agent under investigation. In this study we loaded AY-27 tumor cells with the u fl orescent marker DiI before inoculating the cells into chemically denudated bladders. Lipophilic carbocya- nines, such as DiI, have been widely used as non-toxic 18,19 u fl orescent markers for cells of the immune system. They are also frequently used to visualize neurons in in vitro systems and in other applications such as the assessment of cell invasion, or for the tracing of labeled 22,23 tumor cells. Once applied to the cells, DiI is incor- porated into cellular membranes and the dye diffuses laterally within the membrane, resulting in staining of the entire cell. In this study we show that the use of fluorescently labeled AY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inocula- tion process, and makes it possible to track down the proliferation and location of tumor cells over a period of 5 d. The results further demonstrate that malignant AY-27 tissue exists as an intact non-muscle invasive blad- der tumor only for up to 1–3 d after cell implantation. Subsequently, the formation of mixed tissues consist- ing of malignant and normal regenerating urothelium cells are observed and finally invasive AY-27 tumors. We also provide in vivo evidence that a single intravesical 1 mM MM-C instillation in rat bladders bearing AY-27 tumors of 1 d old completely destroys the malignant Figure 1. (a ) Flow cytometry analysis of DiI-loaded a Y-27 cells 1 = day 1; 2 = day 2; urothelium. 3 = day 3; 4 = day 4; 5 = day 5) and control a Y-27 cells (control). (B) Mean fluorescence values ( ±s .D.) of DiI-loaded and control a Y-27 cells. Results The most widely used model, originally described by Xiao Flow cytometry analysis of DiI-loaded AY-27 cells revealed that et al. consists out of a syngeneic orthotopic rat bladder tumor the intensity of the fluorescence decreased exponentially as a induced by a subsequent treatment of the bladder with solutions function of time, and that labeled cells could be clearly distin- of hydrochloric acid (HCl) and potassium hydroxide (KOH), guished from the non-labeled ones up to 5 d after DiI-loading followed by washing and intravesical instillation of viable AY-27 (Fig. 1A and B). By multiplying the average fluorescence value rat tumor cells. Tumors obtained by this method are purely with the total amount of cells per d and comparison of the prod- UCC carcinomas, are multifocal and have an unpredictable ucts as a function of time (Fig. 2), we found that the total u fl o - localization. Alternative methods where the bladder is locally rescence present in the overall cell population did not change conditioned with HCl/NaOH before tumor inoculation, or over time (results not shown). Hence the decrease of fluorescence where the bladder wall is first mechanically abraded, followed is inversely proportional to the exponential increase of the total by the intravesical instillation of AY-27 tumor cells, have been AY-27 cell count, demonstrating that the DiI label is very strongly described as well. In this last model, all tumors appear as invasive associated with the cells and that no DiI is lost into the surround- carcinomas already from the start and superficial bladder tumors ing medium. Fluorescence microscopy of DiI-loaded AY-27 cells such as CIS are absent. taken from day 1–5 confirmed the gradual decline of fl uorescence www.landesbioscience.com Cancer Biology & Therapy 987 present in the individual cells (Fig. 2B–E). Furthermore it was found that the presence of DiI did not result in an altered in vitro AY-27 cell proliferation (Fig. 2A). We then performed an anti-proliferative assay in which control AY-27 cells and DiI-loaded AY-27 cells were exposed to increasing concentrations of the cytotoxic agent MM-C. Our results show that loading AY-27 cells with DiI did not modify their sensitivity to the chemotherapeutic used (Fig. 3). All animals (n = 12) survived the chemical bladder denudation procedure with or without the subsequent inoculation of AY-27 tumor cells. However, we stress the fact that a thorough PBS washing procedure is extremely important, both for the success- ful inoculation of tumor cells, and the survival of the animals. In control conditions (no tumor cells instilled) the acid/base treatment caused a generalized urothelial denudation with some hemorrhage and dilated vessels, but in some areas there were still patches of intact urothelium, or remaining basal cells left (Fig. 4A). Twenty-four h later, there was a very pronounced inflammation reaction noticeable with a massive infiltrate of polymorphonuclear cells and fibrinopurulent material ( Fig. 4B). Early regenerated urothelium was already visible at days 2 and 3 after treatment. Generally this urothelium had a normal appear- ance with some irregularities such as irregular nuclei and the presence of mitosis figures ( Fig. 4C). The further maturation of the regenerated epithelium took place during the next days and at Figure 2. (a ) proliferation of DiI-loaded and control a Y-27 cells over day 10 the superficial umbrella cell layer was already completely a 5 d period (n = 3; mean ± s .D.). The proliferation rate of DiI-loaded restored (Fig. 4D). Thirty days after conditioning of the bladder, cells was statistically not different from control cells. Fluorescence the regenerated and intact urothelium still showed a thickened (B and D) and corresponding phase contrast (C and e) microphoto- appearance as compared to untreated normal bladder urothelium graphs 1 d (B and C) and 4 d (D and e) after DiI loading. Original magnification = x200. (Fig. 4E and F). Instillation of DiI-loaded AY-27 cells into the chemically denudated bladders resulted in tumor growth. Starting from day 1 after inoculation, fluorescence microscopy showed that the tumors are present as superficial lesions ( Fig. 5). Subsequent H&E staining of the tissues, showed cells with an increased nuclear to cytoplasmatic index together with some mitosis figures in the upper urothelial layers and a more irreg - ular growth pattern. In general, there was a fast tumor growth and already at day 5, tumors were starting to invade into the submucosa (Fig. 5E and F). However, in between well-defined fluorescent sections, we could occasionally observe patches and sometimes whole areas of non-u fl orescent cells where apparently normal or regener - ated urothelial cells were present. In these areas the amount of non-u fl orescent cells exceeded and overgrew the fluores - cent tumor cells (Fig. 6A and B). Histological examination Figure 3. s ensitivity of DiI-loaded and control a Y-27 cells to MM-C (Mean of these H&E stained sections alone, without the fluorescence of 3 replicates ± s .D.). Differences in sensitivity against MM-C were not images, revealed that it was very difficult to clearly distinguish statistically significant. the tumor cells from the normal urothelial cells. Ten days after tumor inoculation, the fluorescence observed was highly weakened, but tumors could now clearly be discerned multiple papillary tumors of different stages were macroscopi- from normal urothelial cells: typical tumor nests were visible in cally visible. Fluorescence was no longer observable. H&E pho- the submucosa and tumors were already invasive (Fig. 6C and tomicrographs showed tumor cells massively present in all layers D). Only two out of five animals which were inoculated with of the bladder (Fig. 6E and F). tumor cells survived a period of 30 d post tumor implantation. Accordingly the AY-27 bladder tumor model was used to At that time, bladders were enlarged, with areas of necrosis and evaluate the antitumoral effect of a single intravesical MM-C 988 Cancer Biology & Therapy Volume 9 Issue 12 Figure 4. Bladder sections showing the restoration of the bladder after global h Cl/NaOh conditioning of the bladder. (a ) Immediately after condition- ing, a complete denudation of the urothelium can be seen, with some patches of normal urothelium (arrow). (B) a strong inflammation reaction is observed 24 h later, with a massive infiltrate of polymorphonuclear cells (arrows). (C) early hyperplastic urothelium, lining the entire bladder, 2 d after conditioning. (D) a t day 10 post, the urothelium is further maturated and a superficial layer of umbrella cells is present. ( e) Thirty days after condition- ing the urothelium is completely restored. (F) Untreated normal bladder urothelium. Representative images are shown. Original magnification x200. instillation. After a short time period (1–3 d), both concentra- DiI before inoculating the cells into chemically denudated blad- tions (1 and 10 mM) instilled for 2 h in the bladder caused more ders. Fluorescent carbocyanine dyes have already been used to or less similar effects: large amounts of fibrinopurulent mate - identify living neurons or for the labeling of cancer cells in order 20,23 rial together with polymorphonuclear cells were present and to quantify their attachment to endothelial cell monolayers. the tumor cells appeared to be largely destroyed (Fig. 7A–C). To the best of our knowledge, it is however the first time that this Although no histological evidence of damage to the deeper lay- carbocyanine dye is used for the identification of cancer cells in ers of the bladder was found (data not shown), none of the rats an in vivo bladder tumor model. (n = 5) instilled with 10 mM MM-C survived a post-treatment Firstly we assessed whether the presence of DiI altered some period of more than 7 d, most likely due to a systemic toxic effect cellular characteristics. Both the proliferative as well as anti-pro- of the drug. liferative assay showed that DiI-loaded AY-27 cells do not behave Conversely, all rats instilled with 1 mM MM-C survived differently in vitro as compared to the non-loaded cells. Our the final endpoint of 30 d after intravesical MM-C instillation. results therefore are in line with the idea that DiI-loading does Moreover, 10 and 30 d after treatment the urothelium of the not appreciably affect cell viability, development or basic physi- MM-C-treated animals was completely restored (Fig. 7F and I). ological properties. Besides, evidence was found that the label is Interestingly, numerous fluorescent spots were present after 10 very strongly associated with the cells, which is compatible with and 30 d which were mostly located in the submucosa, but also the finding that DiI is not transferred from labeled cells towards apparent in between the cells of the (regenerated) urothelium. unlabeled cells. Fluorescence was never observed in the muscle layer of the bladder. Global conditioning of the bladder with HCl and NaOH without tumor cell inoculation caused a general denudation of Discussion the bladder urothelium. The present study shows that when no AY-27 cells are instilled, an intact benign urothelium is regen- The orthotopic rat bladder tumor model, originally created by erated strikingly fast within day 1 and 2 after chemical treat- Xiao et al. is the most widely used animal model in bladder can- ment of the bladder. The regeneration probably originates from cer. A limitation of this model is that it is very difficult to make a cell proliferation in patches of surrounding healthy urothelium clear distinction between grafted AY-27 cells and tissue consist- that are unaffected by the acid/base treatment (Fig. 4A) or from ing out of locally regenerated normal urothelial cells, especially remaining basal cells, which are located mostly into mucosal the first days after the denudation procedure. To understand folds. On the other hand, it is also possible that the regeneration better and validate further the orthotopic rat bladder tumor is initiated starting from the healthy trigone, ureters and urethra, model, we loaded AY-27 cells with the fluorescent cell tracker as described for canine bladder by Wishnow et al. www.landesbioscience.com Cancer Biology & Therapy 989 Figure 5. Fluorescence (a , C and e) and correspond- ing h &e staining photomicrographs (B, D and F) of 5 µ m bladder sections showing urothelial superficial tumors. Representative images are shown. Bladder sections were taken 2 days (a –D) or 5 days (e and F) after inoculation of DiI-loaded a Y-27 cells. U = uro- thelium, s M = submucosa, M = muscle, L = liver tissue (the thin bladder sections are mounted on pork liver tissue before sectioning for support); the selected area in (a ) is shown in (C); (e) a rrow: a Y-27 tumor starts to invade into the submucosa. Original magnifi- cation x25 (a and B); x100 (C and D); x200 (e and F). shown by Bindels et al. This situation might hamper a proper investigation of any antitumoral effect induced by a therapeutic agent under inves- tigation, and preferably exposure of the tumoral cells to the chemotherapeutic agent should take place within the limited time window of 1–3 d after cell inoculation. The outcome therefore chal- lenges somewhat the results by Hendricksen et al. who stated that the recommended time to start intravesical therapy is 5 d after tumor cell inocu- lation. However the denudation procedure and the amount of cells used in their tumor model is slightly different from ours, and obviously the ideal time to start intravesical therapy depends on the specific conditions used to set-up the tumor model. We believe that the use of the DiI- methodology as described in this paper can greatly aid researchers typically working with their own AY-27 tumor bladder model to decide on the exact timing of the therapeutic intervention. As a consequence, when the technically demanding denuda- Already at day 5 after tumor inoculation, the aggressive tion procedure is not properly performed, or when for some rea- behavior of the AY-27 tumor cells makes them to invade into sons the AY-27 cells inadvertently do not inoculate efficiently, the submucosa. As proliferation goes fast, we could accordingly the urothelium will promptly be repaired and reconstituted with observe a rapid decrease of the fluorescence signal over time. For benign cells. As shown in Figure 4C and D, without a proper instance, 10 d after tumor inoculation the overall fluorescence histological examination this regenerated tissue can easily be mis- was already strongly weakened (Fig. 6C and D). taken for superficial malignant tissue, especially by an inexpe - To evaluate further the orthotopic AY-27 bladder tumor rienced investigator. When using the orthotopic AY-27 bladder model, we investigated the antitumoral activity of MM-C, an tumor model, we therefore recommend to routinely DiI-label the alkylating agent that produces DNA interstrand cross-links and AY-27 cells before instillation and to confirm the presence of these is capable of arresting cell proliferation and induction of apop- 28-30 cells by u fl orescence microscopy in control bladders. This simple tosis. The compound is often used in the urological clinic procedure dramatically facilitates the validation of the different against superficial tumor bladder at a concentration usually 4,31 steps required to induce a malignant superficial urothelium. around 1–2 mM. Intrabladder treatment with 1 mM MM-C The present study also shows that even when the AY-27 cell one day after tumor inoculation caused complete tumor destruc- inoculation is adequately performed, malignant AY-27 tissue tion (Fig. 7A–C), and no histological evidence of remaining exists as an intact non-muscle invasive bladder tumor only for tumor cells or tumor growth was found the subsequent weeks up to 1–3 d after cell inoculation. Afterwards the rapid regenera- (Fig. 7G–I). tion of normal urothelial cells produces randomly atypical nests Remarkably, 3 d after MM-C treatment distinct patchy fluo - of tumor cells mixed up with regenerated urothelial cells that rescent dots lined up between the destroyed urothelium and have completely overgrown the tumor (compare Figs. 5E and submucosa, after which they migrated over the following days F with 6A and B). This mixed growth is microscopically dif- and weeks into the submucosa and the regenerated urothelium. ficult to assess (see Fig. 6B ). A similar situation where urothelial Obviously large amounts of fluorescent DiI introduced by the cells overgrow the tumor cells or where tumor cells show pag- inoculation of DiI-loaded AY-27 cells were not cleared from the etoid growth underneath the regenerating urothelium has been bladder wall over a long period of time. Although the underlying 990 Cancer Biology & Therapy Volume 9 Issue 12 Figure 6. Fluorescence (a , C and e) and corresponding h &e staining (B, D and F) photomicrographs of 5 µ m bladder sections at day 4 (a and B), day 10 (C and D) and day 30 (e and F) after tumor inoculation. Representative images are shown. Note that the non-fluorescent part of the urothelium in ( a ) is due to normal urothelial cells that have overgrown the tumor cells, whereas in (C and e) the lack of fluorescence is the result of the dilution of the fluorescent dye by a Y-27 cellular proliferation. Original magnification x200 (a –D); x100 (e and F). principle was not investigated into detail, we believe that the dye retention is secondary to the digestion of DiI-loaded AY-27 cells and cellular debris by macrophages and related immune cells. Of interest, among other anticancer drugs, MM-C is capable to induce macrophage-mediated tumour cell lysis in vivo and it is anticipated that the fluo - rescent spots represent immune-related cells that became located in the urothelium and the sub- mucosa. Obviously, these first findings should be further be explored and future studies are needed to characterize fully the interaction between AY-27 cells treated with different cytotoxic compounds and the immune system. In conclusion, the present observations state that the DiI-loading of AY-27 cells used in the orthotopic bladder tumor model permits a simple inspection of the presence of a uniform malig- nant urothelium in control animals before the model is used to therapeutically assess the antitumoral efficiency of anticancer agents. This model makes it possible to easily distinguish tumor cells from the normal regenerated urothelial cells during early tumor development, especially when mixed forms are present. The methodol- ogy described here will also be a major advantage in the further unraveling of the mechanism underlying fundamental aspects of immune responses after antitu- moral intrabladder therapy. Figure 7. Fluorescent (a , D and G) and cor- responding h &e staining photomicrographs (C, F and I) of 5 µ m bladder sections 3 (a and C), 10 (D and F) and 30 (G and I) d after MM-C treatment (1 mM). Overlay pictures of the fluorescent and the corresponding phase contrast photomicrographs are depicted in (B, e and h ), respectively. Representative images are shown. a rrows in (B, e and h ) indicate the lining of the urothelium. Note the intact uro- thelium after 10 and 30 d (arrows in F and I). Original magnification x100 ( a –C); x200 (D–I). www.landesbioscience.com Cancer Biology & Therapy 991 Materials and Methods in 200 µ l of DMSO per well. The plates were read on a microtiter plate reader (SLT, Salzburg, Austria) at 550 nm. Tumor cell line and chemicals. AY-27 cells, originally developed Orthotopic AY-27 bladder tumor model. Female Fisher rats by Dr. S. Selman and Dr. J. Hampton (Ohio Medical College, (CDF [F-344]), were purchased from Charles R iver Laboratories USA), were kindly provided by Dr. D. Notter (Université henri (Lyon, France). They were provided with purine chow and water Poincare, Nancy, France). Cells were cultured in 175 cm tis- ad libitum. All animal procedures were performed in compliance sue culture flasks at 37°C in a humidified atmosphere containing with national and European regulations and were approved by the 5% CO and 95% air in Minimum Essential Medium (MEM) Animal Care and Use Committee of the Katholieke Universiteit with Earle’s salts containing 2 mM L-glutamine, 1% antibiotic/ Leuven. Rats weighing more than 160 g were used for the experi- antimycotic solution, 1% non-essential amino acids, tylosine ments. Animals were anesthetized with intraperitoneal injection (60 µ g/ml) (Eli Lilly, Brussels, Belgium) and 10% foetal calf of sodium pentobarbital (Nembutal; 45 mg/kg) and placed on serum (FCS). All culture medium compounds were purchased supine position on an animal board kept at 25°C. Bladders were from Invitrogen (Merelbeke, Belgium). DiI was purchased from catheterized via the urethra using a plastic 18 gauge intravenous Molecular Probes Inc., (Eugene, OR, USA) and stored as a 2 catheter. Afterwards the bladder mucosa was mildly disrupted mM stock solution in DMSO at -20°C. Just before use, the stock using a 20 s wash with 0.3 ml of 0.1 N HCl, followed by a treat- solution was diluted in MEM to obtain the appropriate con- ment with 0.3 ml of a 0.1 N NaOH solution for 20 s. The blad- centration. Mitomycin-C (Kyowa) (MM-C) was stored as a 2 der was then several times washed thoroughly with PBS (0.3 mg powder in a dry, cool place and prior to use solutions of the ml each time) until a neutral pH was obtained. A suspension of appropriate concentrations were made in distilled water. DiI-loaded AY-27 cell (4 x 10 cells in 0.3 ml of medium) was DiI-loading and fluorescence of AY-27 cells. For DiI-loading, instilled into the bladder via the catheter and maintained in the conu fl ent AY-27 cells were incubated for 30 min with DiI (2 µ M) bladder for at least 1 h. Every 15 min rats were turned 45° to allow in the presence of culture medium. Afterwards, cells were washed an equal exposure time for the tumor cells to the entire bladder twice with PBS (pH 7.4), trypsinized and collected after centrifu- wall. Afterwards, the cannula was removed and the rats were gation (2,000 rpm, 5 min). Control AY-27 cells and DiI-loaded allowed to void spontaneously. Rats were sacrificed at various AY-27 cells were then plated on 25 cm culture plates (n = 15) time points after tumor inoculation. In the control group, blad- (Sarstedt, Inc., Newton, USA) at 5 x 10 cells per plate. Every day ders were treated with solutions of HCl and NaOH as described, for five consecutive days, the cells of three individual plates were rinsed thoroughly with PBS and instilled with culture medium collected by trypsinization, resuspended in medium (3 ml) and without tumor cell suspension for 1 h. counted using a Coulter Z1 particle counter (Coulter Electronic, To investigate the antitumoral activity of MM-C, rats were Luton, UK). treated intravesically with the compound (1 or 10 mM, 0.3 ml) Fluorescence quantitation was evaluated using a FACScan for 2 h one day after AY-27 tumor implantation. Control animals flow cytometer (Becton Dickinson, San Diego, CA, USA), were instilled with an equal volume of 0.9% NaCl solution. equipped with a 488 nm argon-ion laser and a standard filter For histological investigations, the bladder was firstly sectioned combination provided by the manufacturer. 10,000 events were in two halves; each half was embedded in Tissue tek medium collected in each sample and analysis of the acquired data was (Miles Inc., Elkhart, IN, USA) opposite to each other and snap fro- performed with CELLquest software (Becton Dickinson) and zen in liquid nitrogen. A series of 5 µ m sections were prepared with Prism 5 (GraphPad software). a cryostat microtome and examined by fluorescence microscopy Antiproliferative assay. Control AY-27 cells and DiI-loaded (Axioskop 2 plus u fl orescence microscope, Carl Zeiss, Göttingen, AY-27 cells were seeded onto 96-well microtiter culture plates Germany). For the visualization of DiI a 510–560 nm band-pass (Costa, Cambridge, MA, USA) at 1 x 10 cells per well and excitation filter and a 590 nm long-pass emission filter was used. incubated at 37°C in a humidified atmosphere containing 5% Fluorescence images were acquired using a light-sensitive charge- CO and 95% oxygen. After 24 h, cells were washed with MEM coupled device digital camera (AxioCam HR, Carl Zeiss). After without FCS and treated for 2 h with increasing concentrations examination of the fluorescence, bladder sections were stained with of MM-C. Afterwards the cells were washed twice with MEM standard H&E staining for histological examination. (without FCS) and cultured for 48 h in the presence of culture Statistical analysis. Statistical analysis was performed using medium. Cell proliferation was determined by the use of the the unpaired Student’s t test. p values <0.05 were considered as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide significant. Acknowledgements (MTT; Sigma, St. Louis, MO, USA) dye reduction assay. MTT was dissolved in medium and added to the cells (1 mg/ml) and This work was supported by grants awarded by “Fonds voor the plates were incubated at 37°C for approximately 4 h. MTT Wetenschappelijk Onderzoek-Vlaanderen (F.W.O-Vlaanderen) was removed and the resulting formazan crystals were dissolved and by the K.U.Leuven (onderzoekstoelage). 992 Cancer Biology & Therapy Volume 9 Issue 12 12. Oshinsky GS, Chen Y, Jarrett T, Anderson AE, Weiss 23. Hiscox S, Jiang WG. Quantification of tumour References GH. A model of bladder tumor xenografts in the nude cell-endothelial cell attachment by 1,1'-dioctadecyl- 1. Kamat AM, Lamm DL. Chemoprevention of urologi- rat. J Urol 1995; 154:1925-9. 3,3,3',3'-tetramethylindocarbocyanine (DiI). Cancer cal cancer. J Urol 1999; 161:1748-60. Lett 1997; 112:209-17. 13. Russell PJ, Raghavan D, Gregory P, Philips J, Wills EJ, 2. 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Glioma cell invasion visualized BJU Int 2006; 97:509-12. nitrosourea-induced autochthonous rat bladder cancer by scanning confocal laser microscopy in an in vitro co- 32. Shindo H, Ogura T, Masuno T, Hayashi S, Kishimoto model. Cancer Res 1990; 50:6668-74. culture system. Invasion Metastasis 1995; 15:179-88. S. Induction of activated macrophages by intraperi- 11. Oldroyd RI, Poole RR, Reed RR, Lawson RK, Hodges toneal injection of mitomycin C in mice. Cancer 22. Timmers M, Vermijlen D, Vekemans K, De Zanger R, CV. An animal model for the growth of human tumor Wisse E, Braet F. Tracing DiO-labelled tumour cells in Immunol Immunother 1985; 20:145-50. cell lines. Invest Urol 1977; 14:434-9. liver sections by confocal laser scanning microscopy. J Microsc 2002; 208:65-74. www.landesbioscience.com Cancer Biology & Therapy 993 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

An improved orthotopic rat bladder tumor model using DiI-loaded fluorescent AY-27 cells

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Copyright © 2010 Landes Bioscience
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10.4161/cbt.9.12.11638
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Abstract

Resea RCh pape R Cancer Biology & Therapy 9:12, 986-993; June 15, 2010; © 2010 Landes Bioscience An improved orthotopic rat bladder tumor model using DiI-loaded fluorescent AY-27 cells 1 1 2 2 3 4 Joachim Vandepitte, Jan Maes, Ben Van Cleynenbreugel, h ein Van poppel, evelyne Lerut, patrizia a gostinis 1, and peter a .M. de Witte * 1 2 3 Laboratorium voor Farmaceutische Biologie; Faculteit Farmaceutische Wetenschappen; Dienst Urologie; Departement Morfologie en Moleculaire pathologie; and Departement voor Moleculaire Celbiologie; Faculteit Geneeskunde; Katholieke Universiteit Leuven; Leuven, Belgium Key words: bladder cancer, orthotopic animal model, AY-27, DiI, mitomycin-c Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl) nitrosamine; CIS, carcinoma in situ; DiI, 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine; FANFT, N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide; H&E, haematoxilin and eosin; MM-C, mitomycin-C; MNU, N-methyl-N-nitrosourea; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; TUR, transurethral resection; UCC, urothelial cell carcinoma h ere we evaluate an improved orthotopic rat bladder tumor model, to be used for the evaluation of the therapeutic potential of novel cancer therapeutics. Before instilling aY-27 tumor cells into chemically denudated rat bladders, aY-27 cells were labeled with the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI). We found that the presence of DiI did not alter the in vitro aY-27 cell proliferation and that the DiI label was strongly associated with the cells. We further provide evidence that the use of fluorescently labeled aY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inoculation process. Using this technique it was possible to track down the tumor cells after inoculation into the bladder, which makes it straightforward to distinguish tumor cells from remaining or regenerated normal urothelium over a period of 5 d. The results also demonstrated that malignant a Y-27 tissue exists as an intact non-muscle invasive bladder tumor only for 1–3 d after cell implantation. a ccordingly the a Y-27 bladder tumor model was used to evaluate the antitumoral ee ff ct of a single intravesical MM-C instillation. a ll rats instilled with 1 mM MM-C survived the final endpoint of 30 d after intravesical MM-C. Moreover, 10 and 30 d after treatment the urothelium of the MM-C-treated animals was completely restored. Remarkably, after MM-C treatment distinct patchy fluorescent dots were found into the submucosa and the regenerated urothelium, suggesting that dye retention is secondary to the digestion of DiI-loaded a Y-27 cells and cellular debris by macrophages and related immune cells. regard to the recurrence rate and invasion. As a result different Introduction experimental therapies such as photodynamic therapy are cur- 5,6 rently being investigated. Bladder cancer is the fourth most common malignant disease accounting for approximately 5% of all cancer cases. At initial Different pre-clinical animal bladder tumor models have been diagnosis, most of the patients show non-muscle invasive blad- developed that are essential in testing new treatment modalities der cancer. These lesions extend visually into the lumen of the against bladder cancer. For instance, rodent bladder tumors can bladder so that they can be removed by transurethral resection be induced by feeding the animals with chemical carcinogens (TUR) with relative ease. Although a high incidence of tumor such as N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), or 2 8 recurrence is observed afterwards, these tumors seldom prog- N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT). Using ress into invasive disease. A small percentage of these patients this methodology, both UCC and squamous cell carcinoma also show carcinoma in situ (CIS or Tis), flat lesions that have can be generated, but it takes at least 8–11 mo before tumors 8,9 a very malignant potential and a high risk of progressing into develop. Another possibility is to repeatedly instill N-methyl- muscle invasive disease. Early recognition of CIS therefore N-nitrosourea (MNU) into the rat bladder. Doing so, UCC is essential to offer the patients the most appropriate therapy without the presence of squamous cell carcinoma are induced and cure rate. CIS is generally treated with intravesical Bacillus within 4 mo. Furthermore, also xenograft tumor models have Calmette-Guerin (BCG) instillations or with radical cystec- been developed by transplanting heterotopically or orthotopically 4 11,12 tomy for refractory cases. The medical options to treat urothe- human urothelial cancer cells into immunodeficient rats or 13,14 lial cell carcinoma (UCC) are still insufficient, especially with mice. *Correspondence to: Peter A.M. de Witte; Email: [email protected] Submitted: 01/25/10; Revised: 02/24/10; Accepted: 02/26/10 Previously published online: www.landesbioscience.com/journals/cbt/article/11638 DOI: 10.4161/cbt.9.12.11638 986 Cancer Biology & Therapy Volume 9 Issue 12 Resea RCh pape R Resea RCh pape R Using the AY-27 syngeneic orthotopic rat bladder tumor model, we found it often very difficult to discrim - inate between inoculated AY-27 cells and tissue consist- ing out of locally regenerated normal urothelial cells. Unlike their human counterparts, normal urothelial rat cells proliferate very rapidly, reconstituting a urothelial lining within 2 or 3 d after chemical destruction. These normal urothelial cells can disrupt and infiltrate the tumor cell layers, creating mixed tissues of both normal urothelial cells and tumoral cells. This situation makes it problematic to assess correctly any antitumoral effect induced by a therapeutic agent under investigation. In this study we loaded AY-27 tumor cells with the u fl orescent marker DiI before inoculating the cells into chemically denudated bladders. Lipophilic carbocya- nines, such as DiI, have been widely used as non-toxic 18,19 u fl orescent markers for cells of the immune system. They are also frequently used to visualize neurons in in vitro systems and in other applications such as the assessment of cell invasion, or for the tracing of labeled 22,23 tumor cells. Once applied to the cells, DiI is incor- porated into cellular membranes and the dye diffuses laterally within the membrane, resulting in staining of the entire cell. In this study we show that the use of fluorescently labeled AY-27 tumor cells allows the visualization and hence the validation of the orthotopic tumor inocula- tion process, and makes it possible to track down the proliferation and location of tumor cells over a period of 5 d. The results further demonstrate that malignant AY-27 tissue exists as an intact non-muscle invasive blad- der tumor only for up to 1–3 d after cell implantation. Subsequently, the formation of mixed tissues consist- ing of malignant and normal regenerating urothelium cells are observed and finally invasive AY-27 tumors. We also provide in vivo evidence that a single intravesical 1 mM MM-C instillation in rat bladders bearing AY-27 tumors of 1 d old completely destroys the malignant Figure 1. (a ) Flow cytometry analysis of DiI-loaded a Y-27 cells 1 = day 1; 2 = day 2; urothelium. 3 = day 3; 4 = day 4; 5 = day 5) and control a Y-27 cells (control). (B) Mean fluorescence values ( ±s .D.) of DiI-loaded and control a Y-27 cells. Results The most widely used model, originally described by Xiao Flow cytometry analysis of DiI-loaded AY-27 cells revealed that et al. consists out of a syngeneic orthotopic rat bladder tumor the intensity of the fluorescence decreased exponentially as a induced by a subsequent treatment of the bladder with solutions function of time, and that labeled cells could be clearly distin- of hydrochloric acid (HCl) and potassium hydroxide (KOH), guished from the non-labeled ones up to 5 d after DiI-loading followed by washing and intravesical instillation of viable AY-27 (Fig. 1A and B). By multiplying the average fluorescence value rat tumor cells. Tumors obtained by this method are purely with the total amount of cells per d and comparison of the prod- UCC carcinomas, are multifocal and have an unpredictable ucts as a function of time (Fig. 2), we found that the total u fl o - localization. Alternative methods where the bladder is locally rescence present in the overall cell population did not change conditioned with HCl/NaOH before tumor inoculation, or over time (results not shown). Hence the decrease of fluorescence where the bladder wall is first mechanically abraded, followed is inversely proportional to the exponential increase of the total by the intravesical instillation of AY-27 tumor cells, have been AY-27 cell count, demonstrating that the DiI label is very strongly described as well. In this last model, all tumors appear as invasive associated with the cells and that no DiI is lost into the surround- carcinomas already from the start and superficial bladder tumors ing medium. Fluorescence microscopy of DiI-loaded AY-27 cells such as CIS are absent. taken from day 1–5 confirmed the gradual decline of fl uorescence www.landesbioscience.com Cancer Biology & Therapy 987 present in the individual cells (Fig. 2B–E). Furthermore it was found that the presence of DiI did not result in an altered in vitro AY-27 cell proliferation (Fig. 2A). We then performed an anti-proliferative assay in which control AY-27 cells and DiI-loaded AY-27 cells were exposed to increasing concentrations of the cytotoxic agent MM-C. Our results show that loading AY-27 cells with DiI did not modify their sensitivity to the chemotherapeutic used (Fig. 3). All animals (n = 12) survived the chemical bladder denudation procedure with or without the subsequent inoculation of AY-27 tumor cells. However, we stress the fact that a thorough PBS washing procedure is extremely important, both for the success- ful inoculation of tumor cells, and the survival of the animals. In control conditions (no tumor cells instilled) the acid/base treatment caused a generalized urothelial denudation with some hemorrhage and dilated vessels, but in some areas there were still patches of intact urothelium, or remaining basal cells left (Fig. 4A). Twenty-four h later, there was a very pronounced inflammation reaction noticeable with a massive infiltrate of polymorphonuclear cells and fibrinopurulent material ( Fig. 4B). Early regenerated urothelium was already visible at days 2 and 3 after treatment. Generally this urothelium had a normal appear- ance with some irregularities such as irregular nuclei and the presence of mitosis figures ( Fig. 4C). The further maturation of the regenerated epithelium took place during the next days and at Figure 2. (a ) proliferation of DiI-loaded and control a Y-27 cells over day 10 the superficial umbrella cell layer was already completely a 5 d period (n = 3; mean ± s .D.). The proliferation rate of DiI-loaded restored (Fig. 4D). Thirty days after conditioning of the bladder, cells was statistically not different from control cells. Fluorescence the regenerated and intact urothelium still showed a thickened (B and D) and corresponding phase contrast (C and e) microphoto- appearance as compared to untreated normal bladder urothelium graphs 1 d (B and C) and 4 d (D and e) after DiI loading. Original magnification = x200. (Fig. 4E and F). Instillation of DiI-loaded AY-27 cells into the chemically denudated bladders resulted in tumor growth. Starting from day 1 after inoculation, fluorescence microscopy showed that the tumors are present as superficial lesions ( Fig. 5). Subsequent H&E staining of the tissues, showed cells with an increased nuclear to cytoplasmatic index together with some mitosis figures in the upper urothelial layers and a more irreg - ular growth pattern. In general, there was a fast tumor growth and already at day 5, tumors were starting to invade into the submucosa (Fig. 5E and F). However, in between well-defined fluorescent sections, we could occasionally observe patches and sometimes whole areas of non-u fl orescent cells where apparently normal or regener - ated urothelial cells were present. In these areas the amount of non-u fl orescent cells exceeded and overgrew the fluores - cent tumor cells (Fig. 6A and B). Histological examination Figure 3. s ensitivity of DiI-loaded and control a Y-27 cells to MM-C (Mean of these H&E stained sections alone, without the fluorescence of 3 replicates ± s .D.). Differences in sensitivity against MM-C were not images, revealed that it was very difficult to clearly distinguish statistically significant. the tumor cells from the normal urothelial cells. Ten days after tumor inoculation, the fluorescence observed was highly weakened, but tumors could now clearly be discerned multiple papillary tumors of different stages were macroscopi- from normal urothelial cells: typical tumor nests were visible in cally visible. Fluorescence was no longer observable. H&E pho- the submucosa and tumors were already invasive (Fig. 6C and tomicrographs showed tumor cells massively present in all layers D). Only two out of five animals which were inoculated with of the bladder (Fig. 6E and F). tumor cells survived a period of 30 d post tumor implantation. Accordingly the AY-27 bladder tumor model was used to At that time, bladders were enlarged, with areas of necrosis and evaluate the antitumoral effect of a single intravesical MM-C 988 Cancer Biology & Therapy Volume 9 Issue 12 Figure 4. Bladder sections showing the restoration of the bladder after global h Cl/NaOh conditioning of the bladder. (a ) Immediately after condition- ing, a complete denudation of the urothelium can be seen, with some patches of normal urothelium (arrow). (B) a strong inflammation reaction is observed 24 h later, with a massive infiltrate of polymorphonuclear cells (arrows). (C) early hyperplastic urothelium, lining the entire bladder, 2 d after conditioning. (D) a t day 10 post, the urothelium is further maturated and a superficial layer of umbrella cells is present. ( e) Thirty days after condition- ing the urothelium is completely restored. (F) Untreated normal bladder urothelium. Representative images are shown. Original magnification x200. instillation. After a short time period (1–3 d), both concentra- DiI before inoculating the cells into chemically denudated blad- tions (1 and 10 mM) instilled for 2 h in the bladder caused more ders. Fluorescent carbocyanine dyes have already been used to or less similar effects: large amounts of fibrinopurulent mate - identify living neurons or for the labeling of cancer cells in order 20,23 rial together with polymorphonuclear cells were present and to quantify their attachment to endothelial cell monolayers. the tumor cells appeared to be largely destroyed (Fig. 7A–C). To the best of our knowledge, it is however the first time that this Although no histological evidence of damage to the deeper lay- carbocyanine dye is used for the identification of cancer cells in ers of the bladder was found (data not shown), none of the rats an in vivo bladder tumor model. (n = 5) instilled with 10 mM MM-C survived a post-treatment Firstly we assessed whether the presence of DiI altered some period of more than 7 d, most likely due to a systemic toxic effect cellular characteristics. Both the proliferative as well as anti-pro- of the drug. liferative assay showed that DiI-loaded AY-27 cells do not behave Conversely, all rats instilled with 1 mM MM-C survived differently in vitro as compared to the non-loaded cells. Our the final endpoint of 30 d after intravesical MM-C instillation. results therefore are in line with the idea that DiI-loading does Moreover, 10 and 30 d after treatment the urothelium of the not appreciably affect cell viability, development or basic physi- MM-C-treated animals was completely restored (Fig. 7F and I). ological properties. Besides, evidence was found that the label is Interestingly, numerous fluorescent spots were present after 10 very strongly associated with the cells, which is compatible with and 30 d which were mostly located in the submucosa, but also the finding that DiI is not transferred from labeled cells towards apparent in between the cells of the (regenerated) urothelium. unlabeled cells. Fluorescence was never observed in the muscle layer of the bladder. Global conditioning of the bladder with HCl and NaOH without tumor cell inoculation caused a general denudation of Discussion the bladder urothelium. The present study shows that when no AY-27 cells are instilled, an intact benign urothelium is regen- The orthotopic rat bladder tumor model, originally created by erated strikingly fast within day 1 and 2 after chemical treat- Xiao et al. is the most widely used animal model in bladder can- ment of the bladder. The regeneration probably originates from cer. A limitation of this model is that it is very difficult to make a cell proliferation in patches of surrounding healthy urothelium clear distinction between grafted AY-27 cells and tissue consist- that are unaffected by the acid/base treatment (Fig. 4A) or from ing out of locally regenerated normal urothelial cells, especially remaining basal cells, which are located mostly into mucosal the first days after the denudation procedure. To understand folds. On the other hand, it is also possible that the regeneration better and validate further the orthotopic rat bladder tumor is initiated starting from the healthy trigone, ureters and urethra, model, we loaded AY-27 cells with the fluorescent cell tracker as described for canine bladder by Wishnow et al. www.landesbioscience.com Cancer Biology & Therapy 989 Figure 5. Fluorescence (a , C and e) and correspond- ing h &e staining photomicrographs (B, D and F) of 5 µ m bladder sections showing urothelial superficial tumors. Representative images are shown. Bladder sections were taken 2 days (a –D) or 5 days (e and F) after inoculation of DiI-loaded a Y-27 cells. U = uro- thelium, s M = submucosa, M = muscle, L = liver tissue (the thin bladder sections are mounted on pork liver tissue before sectioning for support); the selected area in (a ) is shown in (C); (e) a rrow: a Y-27 tumor starts to invade into the submucosa. Original magnifi- cation x25 (a and B); x100 (C and D); x200 (e and F). shown by Bindels et al. This situation might hamper a proper investigation of any antitumoral effect induced by a therapeutic agent under inves- tigation, and preferably exposure of the tumoral cells to the chemotherapeutic agent should take place within the limited time window of 1–3 d after cell inoculation. The outcome therefore chal- lenges somewhat the results by Hendricksen et al. who stated that the recommended time to start intravesical therapy is 5 d after tumor cell inocu- lation. However the denudation procedure and the amount of cells used in their tumor model is slightly different from ours, and obviously the ideal time to start intravesical therapy depends on the specific conditions used to set-up the tumor model. We believe that the use of the DiI- methodology as described in this paper can greatly aid researchers typically working with their own AY-27 tumor bladder model to decide on the exact timing of the therapeutic intervention. As a consequence, when the technically demanding denuda- Already at day 5 after tumor inoculation, the aggressive tion procedure is not properly performed, or when for some rea- behavior of the AY-27 tumor cells makes them to invade into sons the AY-27 cells inadvertently do not inoculate efficiently, the submucosa. As proliferation goes fast, we could accordingly the urothelium will promptly be repaired and reconstituted with observe a rapid decrease of the fluorescence signal over time. For benign cells. As shown in Figure 4C and D, without a proper instance, 10 d after tumor inoculation the overall fluorescence histological examination this regenerated tissue can easily be mis- was already strongly weakened (Fig. 6C and D). taken for superficial malignant tissue, especially by an inexpe - To evaluate further the orthotopic AY-27 bladder tumor rienced investigator. When using the orthotopic AY-27 bladder model, we investigated the antitumoral activity of MM-C, an tumor model, we therefore recommend to routinely DiI-label the alkylating agent that produces DNA interstrand cross-links and AY-27 cells before instillation and to confirm the presence of these is capable of arresting cell proliferation and induction of apop- 28-30 cells by u fl orescence microscopy in control bladders. This simple tosis. The compound is often used in the urological clinic procedure dramatically facilitates the validation of the different against superficial tumor bladder at a concentration usually 4,31 steps required to induce a malignant superficial urothelium. around 1–2 mM. Intrabladder treatment with 1 mM MM-C The present study also shows that even when the AY-27 cell one day after tumor inoculation caused complete tumor destruc- inoculation is adequately performed, malignant AY-27 tissue tion (Fig. 7A–C), and no histological evidence of remaining exists as an intact non-muscle invasive bladder tumor only for tumor cells or tumor growth was found the subsequent weeks up to 1–3 d after cell inoculation. Afterwards the rapid regenera- (Fig. 7G–I). tion of normal urothelial cells produces randomly atypical nests Remarkably, 3 d after MM-C treatment distinct patchy fluo - of tumor cells mixed up with regenerated urothelial cells that rescent dots lined up between the destroyed urothelium and have completely overgrown the tumor (compare Figs. 5E and submucosa, after which they migrated over the following days F with 6A and B). This mixed growth is microscopically dif- and weeks into the submucosa and the regenerated urothelium. ficult to assess (see Fig. 6B ). A similar situation where urothelial Obviously large amounts of fluorescent DiI introduced by the cells overgrow the tumor cells or where tumor cells show pag- inoculation of DiI-loaded AY-27 cells were not cleared from the etoid growth underneath the regenerating urothelium has been bladder wall over a long period of time. Although the underlying 990 Cancer Biology & Therapy Volume 9 Issue 12 Figure 6. Fluorescence (a , C and e) and corresponding h &e staining (B, D and F) photomicrographs of 5 µ m bladder sections at day 4 (a and B), day 10 (C and D) and day 30 (e and F) after tumor inoculation. Representative images are shown. Note that the non-fluorescent part of the urothelium in ( a ) is due to normal urothelial cells that have overgrown the tumor cells, whereas in (C and e) the lack of fluorescence is the result of the dilution of the fluorescent dye by a Y-27 cellular proliferation. Original magnification x200 (a –D); x100 (e and F). principle was not investigated into detail, we believe that the dye retention is secondary to the digestion of DiI-loaded AY-27 cells and cellular debris by macrophages and related immune cells. Of interest, among other anticancer drugs, MM-C is capable to induce macrophage-mediated tumour cell lysis in vivo and it is anticipated that the fluo - rescent spots represent immune-related cells that became located in the urothelium and the sub- mucosa. Obviously, these first findings should be further be explored and future studies are needed to characterize fully the interaction between AY-27 cells treated with different cytotoxic compounds and the immune system. In conclusion, the present observations state that the DiI-loading of AY-27 cells used in the orthotopic bladder tumor model permits a simple inspection of the presence of a uniform malig- nant urothelium in control animals before the model is used to therapeutically assess the antitumoral efficiency of anticancer agents. This model makes it possible to easily distinguish tumor cells from the normal regenerated urothelial cells during early tumor development, especially when mixed forms are present. The methodol- ogy described here will also be a major advantage in the further unraveling of the mechanism underlying fundamental aspects of immune responses after antitu- moral intrabladder therapy. Figure 7. Fluorescent (a , D and G) and cor- responding h &e staining photomicrographs (C, F and I) of 5 µ m bladder sections 3 (a and C), 10 (D and F) and 30 (G and I) d after MM-C treatment (1 mM). Overlay pictures of the fluorescent and the corresponding phase contrast photomicrographs are depicted in (B, e and h ), respectively. Representative images are shown. a rrows in (B, e and h ) indicate the lining of the urothelium. Note the intact uro- thelium after 10 and 30 d (arrows in F and I). Original magnification x100 ( a –C); x200 (D–I). www.landesbioscience.com Cancer Biology & Therapy 991 Materials and Methods in 200 µ l of DMSO per well. The plates were read on a microtiter plate reader (SLT, Salzburg, Austria) at 550 nm. Tumor cell line and chemicals. AY-27 cells, originally developed Orthotopic AY-27 bladder tumor model. Female Fisher rats by Dr. S. Selman and Dr. J. Hampton (Ohio Medical College, (CDF [F-344]), were purchased from Charles R iver Laboratories USA), were kindly provided by Dr. D. Notter (Université henri (Lyon, France). They were provided with purine chow and water Poincare, Nancy, France). Cells were cultured in 175 cm tis- ad libitum. All animal procedures were performed in compliance sue culture flasks at 37°C in a humidified atmosphere containing with national and European regulations and were approved by the 5% CO and 95% air in Minimum Essential Medium (MEM) Animal Care and Use Committee of the Katholieke Universiteit with Earle’s salts containing 2 mM L-glutamine, 1% antibiotic/ Leuven. Rats weighing more than 160 g were used for the experi- antimycotic solution, 1% non-essential amino acids, tylosine ments. Animals were anesthetized with intraperitoneal injection (60 µ g/ml) (Eli Lilly, Brussels, Belgium) and 10% foetal calf of sodium pentobarbital (Nembutal; 45 mg/kg) and placed on serum (FCS). All culture medium compounds were purchased supine position on an animal board kept at 25°C. Bladders were from Invitrogen (Merelbeke, Belgium). DiI was purchased from catheterized via the urethra using a plastic 18 gauge intravenous Molecular Probes Inc., (Eugene, OR, USA) and stored as a 2 catheter. Afterwards the bladder mucosa was mildly disrupted mM stock solution in DMSO at -20°C. Just before use, the stock using a 20 s wash with 0.3 ml of 0.1 N HCl, followed by a treat- solution was diluted in MEM to obtain the appropriate con- ment with 0.3 ml of a 0.1 N NaOH solution for 20 s. The blad- centration. Mitomycin-C (Kyowa) (MM-C) was stored as a 2 der was then several times washed thoroughly with PBS (0.3 mg powder in a dry, cool place and prior to use solutions of the ml each time) until a neutral pH was obtained. A suspension of appropriate concentrations were made in distilled water. DiI-loaded AY-27 cell (4 x 10 cells in 0.3 ml of medium) was DiI-loading and fluorescence of AY-27 cells. For DiI-loading, instilled into the bladder via the catheter and maintained in the conu fl ent AY-27 cells were incubated for 30 min with DiI (2 µ M) bladder for at least 1 h. Every 15 min rats were turned 45° to allow in the presence of culture medium. Afterwards, cells were washed an equal exposure time for the tumor cells to the entire bladder twice with PBS (pH 7.4), trypsinized and collected after centrifu- wall. Afterwards, the cannula was removed and the rats were gation (2,000 rpm, 5 min). Control AY-27 cells and DiI-loaded allowed to void spontaneously. Rats were sacrificed at various AY-27 cells were then plated on 25 cm culture plates (n = 15) time points after tumor inoculation. In the control group, blad- (Sarstedt, Inc., Newton, USA) at 5 x 10 cells per plate. Every day ders were treated with solutions of HCl and NaOH as described, for five consecutive days, the cells of three individual plates were rinsed thoroughly with PBS and instilled with culture medium collected by trypsinization, resuspended in medium (3 ml) and without tumor cell suspension for 1 h. counted using a Coulter Z1 particle counter (Coulter Electronic, To investigate the antitumoral activity of MM-C, rats were Luton, UK). treated intravesically with the compound (1 or 10 mM, 0.3 ml) Fluorescence quantitation was evaluated using a FACScan for 2 h one day after AY-27 tumor implantation. Control animals flow cytometer (Becton Dickinson, San Diego, CA, USA), were instilled with an equal volume of 0.9% NaCl solution. equipped with a 488 nm argon-ion laser and a standard filter For histological investigations, the bladder was firstly sectioned combination provided by the manufacturer. 10,000 events were in two halves; each half was embedded in Tissue tek medium collected in each sample and analysis of the acquired data was (Miles Inc., Elkhart, IN, USA) opposite to each other and snap fro- performed with CELLquest software (Becton Dickinson) and zen in liquid nitrogen. A series of 5 µ m sections were prepared with Prism 5 (GraphPad software). a cryostat microtome and examined by fluorescence microscopy Antiproliferative assay. Control AY-27 cells and DiI-loaded (Axioskop 2 plus u fl orescence microscope, Carl Zeiss, Göttingen, AY-27 cells were seeded onto 96-well microtiter culture plates Germany). For the visualization of DiI a 510–560 nm band-pass (Costa, Cambridge, MA, USA) at 1 x 10 cells per well and excitation filter and a 590 nm long-pass emission filter was used. incubated at 37°C in a humidified atmosphere containing 5% Fluorescence images were acquired using a light-sensitive charge- CO and 95% oxygen. After 24 h, cells were washed with MEM coupled device digital camera (AxioCam HR, Carl Zeiss). After without FCS and treated for 2 h with increasing concentrations examination of the fluorescence, bladder sections were stained with of MM-C. Afterwards the cells were washed twice with MEM standard H&E staining for histological examination. (without FCS) and cultured for 48 h in the presence of culture Statistical analysis. Statistical analysis was performed using medium. Cell proliferation was determined by the use of the the unpaired Student’s t test. p values <0.05 were considered as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide significant. Acknowledgements (MTT; Sigma, St. Louis, MO, USA) dye reduction assay. MTT was dissolved in medium and added to the cells (1 mg/ml) and This work was supported by grants awarded by “Fonds voor the plates were incubated at 37°C for approximately 4 h. MTT Wetenschappelijk Onderzoek-Vlaanderen (F.W.O-Vlaanderen) was removed and the resulting formazan crystals were dissolved and by the K.U.Leuven (onderzoekstoelage). 992 Cancer Biology & Therapy Volume 9 Issue 12 12. Oshinsky GS, Chen Y, Jarrett T, Anderson AE, Weiss 23. Hiscox S, Jiang WG. Quantification of tumour References GH. A model of bladder tumor xenografts in the nude cell-endothelial cell attachment by 1,1'-dioctadecyl- 1. Kamat AM, Lamm DL. Chemoprevention of urologi- rat. J Urol 1995; 154:1925-9. 3,3,3',3'-tetramethylindocarbocyanine (DiI). Cancer cal cancer. J Urol 1999; 161:1748-60. Lett 1997; 112:209-17. 13. Russell PJ, Raghavan D, Gregory P, Philips J, Wills EJ, 2. 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