Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression

Abstract

There has been no reliable orthotopic model available to visualize the growth of human superficial bladder cancer over time and to evaluate the efficacy of intravesical therapies. We have developed a novel approach to accomplish this task by generating human superficial bladder tumor cells to stably express high levels of green fluorescent protein ( GFP ) in vivo. Superficial bladder tumors were produced in athymic mice by intravesical instillation. In our initial studies tumors were quantitated by image analysis at a single time point, and the results compared to the estimation of the percentage of GFP cells present using flow cytometry after obtaining single cell suspensions of normal and tumor cells in the same bladder. A high correlation between the two methods was seen. Therefore, in subsequent studies, approximately 1 week after the intravesical instillation of the GFP expressing cancer cells a small incision was made to expose the bladder. The anterior, posterior, and lateral images of each bladder were captured to visualize GFPexpressing tumors. The ratio of green fluorescence pixel area, which represented the tumor burden, to the total area of the bladder was then calculated. A similar procedure was performed at 2, 3, and 4 weeks after instillation of the tumor cells. Using this procedure tumor progression over time could be measured in each mouse. By using this approach, it will now be possible to monitor the initial tumor sizes in the bladder of each mouse and then to evaluate the efficacy of various intravesical therapy protocols including intravesical gene therapy alone or in combination with other treatment modalities. Cancer Gene Therapy ( 2002 ) 9, 681 -686 doi:10.1038/sj.cgt.7700489Keywords: bladder cancer; green fluorescent protein; image analysis B ladder cancer is the fifth most frequent cancer in the United States. More than 50,000 individuals are diagnosed with bladder cancer each year. Between 70% and 80% of these cases involve superficial tumor. Such lesions are usually managed with transurethral resection often followed by intravesical immunotherapy with Bacillus Calmette -Guerin ( BCG ). Although BCG can prolong the length of progression -free survival after the tumor is resected, approximately 50% of superficial lesions will continue to recur and as many as 30% will progress to a higher grade and /or stage. 1 Therefore, new therapeutic approaches, including gene therapy alone or in combination with other modalities of therapy, need to be considered for the treatment of refractory superficial bladder disease.We recently have developed an intravesical model to produce superficially growing human bladder tumors in athymic mice with high frequency. 2 In addition, our laboratory in collaboration with others has shown that this model can be used to document efficient intravesical adenoviral -mediated gene transfer when it is combined with the polyamide compound, Syn 3. 3 Our next challenge was to use this tumor model in vivo to examine the efficacy of various treatments and schedules. Howeve...

A Novel Human AlkB Homologue, ALKBH8, Contributes to Human Bladder Cancer Progression

“…KU7 was derived from human papillary bladder cancer (14). We generated stable green fluorescent protein (GFP) clones as previously described (15).…”

Efficacy of a single intravesical treatment with Ad-IFN/Syn 3 is dependent on dose and urine IFN concentration obtained: implications for clinical investigation

“…The methods for growing superficial KU7/GFP human bladder tumors in athymic mice and their imaging have previously been described. 7,8 At 2 weeks after the tumor cells were intravesically instilled, the bladders were imaged for the presence of GFP-containing tumors. Anterior, posterior, as well as left lateral and right lateral views were taken as previously described.…”

“…Anterior, posterior, as well as left lateral and right lateral views were taken as previously described. 8 Mice then received a 100 ml dose of Ad-IFNa2a1/Syn3 intravesically instilled for 1 h. Three concentrations of Ad-IFNa2a1 were used in our experiments: 1 Â 10 11 P/ml, 1 Â 10 10 P/ml, and 1 Â 10 9 P/ml. In one additional group, 1 Â 10 11 P/ml of Ad-IFNa2a1 plus Syn3 was instilled for 1 h on two consecutive days.…”

“…To facilitate our efficacy studies, a recently developed superficial bladder tumor model was utilized to monitor the progression of human bladder tumors growing in athymic mice by GFP fluoresence. 7,8 In our initial study, we found that two daily 1 h intravesical administrations of Ad-IFNa at a concentration of 1 Â 10 11 particles/ml (P/ml), together with Syn3, produced a marked tumor regression in superficial human bladder tumors. 9 All other treatments including Syn3 alone, Ad-bgal/Syn3, Ad-IFN without Syn3, or the IFN protein alone at high concentration showed no effect on tumor growth.…”

Monitoring the Response of Orthotopic Bladder Tumors to Granulocyte Macrophage Colony-Stimulating Factor Therapy Using the Prostate-Specific Antigen Gene as a Reporter

“…In addition, accurate quantification of real tumor burden is extremely difficult, because the tumor mass may contain a necrotic center or infiltrating immune cells, which would cause overestimation of tumor volume. Both luciferase and green fluorescent protein (GFP)-transfected cancer cells (5)(6)(7)(8) have been produced. Although the modified tumor cells can be easily visualized, these techniques do not readily enable the quantitative measurement of tumors.…”