Bladder cancer has a recurrence rate of up to 70% necessitating patients with a history of bladder cancer to undergo years if not lifetime surveillance via periodic cystoscopy, which significantly burdens the healthcare system [13
]. There is also no reliable screening test for early detection of bladder cancer. Urinary biomarkers are an active area of investigation and current molecular cytologic tests include ImmunoCyt, which detects the glycosylated form of CEA and mucin glycoproteins (median sensitivity of 81%; median specificity of 75%), DD23 (Median sensitivity of 81%; median specificity of 60%), Lewis X antigen (median sensitivity of 84%; median specificity of 80%), and UroVysion test used to detect aneuploidy for chromosomes 3, 7, 17 and loss of 9p21 locus (median sensitivity of 73%; median specificity of 90%) [14
]. Recently, fibroblast growth factor receptor 3 (FGFR3) mutations have been used to detect concomitant recurrences of low-grade non-muscle-invasive bladder cancer with a sensitivity of 58% [15
]. While it is apparent that different molecular bladder tumor markers and clinical tests exist to detect bladder cancer [16
], these tests do not preclude the recommended invasive and frequent surveillance urethrocystoscopies. Here, we have examined the feasibility of screening short-term explant cultures from voided urine with an adenoviral reporter construct.
A total of 31 samples were evaluated for the ability to grow short-term explant cultures from voided urine. In agreement with a recent study we found that cell attachment and propagation occurred within 10 days after culture initiation [17
]. About half of all samples (16/31) showed evidence of in vitro
growth. This included 4/6 samples from patients with a previous history of bladder cancer (all white males with a median age of 69.5, 72.7 +/- 10.4), 4/5 samples from patients presenting with hematuria (1 WF, 2BF, 1WM, 1BM, median age 64, 60.8 +/- 14.7) and 8/20 healthy volunteers (8WM, 2BM, 10WF, 1AF, median age 52.5, 52.9+/- 9). In a previous study, primary culture outgrowth from urine, defined as the presence of islet-like cells, was observed in 54.7% of healthy volunteers and in 86.6% of bladder cancer patients [18
]. Since none of our samples were from patients currently having bladder cancer, our rate of 52% outgrowth is similar to the previously observed 54.7%. Upon closer examination however, the outgrowth rate from healthy volunteers approached 90% (7/8) in samples that were processed within 30 minutes of urine collection. These results suggest that a short time to processing is a critical factor to successfully establish short-term cultures from voided urine.
Our results also demonstrate that exfoliated cells obtained from spontaneous micturition can be transduced with adenoviral reporter vectors, which provides a novel opportunity to detect malignant cells via a non-invasive screening modality. Survivin, a member of the inhibitor of apoptosis family of proteins involved in regulating cell division and apoptosis, is overexpressed in tumor cells relative to normal cells. Therefore, the survivin-promoter has been used to transcriptionally target tumors for gene therapy [9
], including delivery of adenovirus-based vectors and conditionally replicative adenoviruses in treating malignant gliomas [19
]. Survivin mRNA was detected in urine with 94% sensitivity and 95% specificity [21
], suggesting the survivin promoter is preferentially activated in bladder cancer cells. In this study we used the full-length survivin promoter to drive GFP reporter gene expression in normal and malignant bladder cancer cells. Non-malignant UROtsa and malignant UM-UC-3 bladder cells were used to test the survivin-driven reporter gene expression. As expected, CMV-driven GFP expression was similar between the cell lines whereas survivin-driven GFP expression was preferentially observed in the malignant UM-UC-3 cells (Figure ).
However, when short-term explant cultures obtained from urine of healthy volunteers were infected with Ad.Surv.GFP, the percentage of GFP positive cells was comparable to the UM-UC-3 positive control rather than the non-malignant UROtsa cells, which served as a negative control (Figure ). There are several possible explanations for this observation. Survivin has dual roles in regulation of G1/S transition and apoptosis protection [22
]. Thus one possibility is that the level of survivin promoter activity in the explant cultures is dictated by proliferation and progression through the cell cycle. However, we did not observe the growth of explant cultures to be more rapid than UROtsa cells, which would argue against proliferation as a cause for higher GFP expression following Ad.Surv.GFP expression. While it has been widely accepted that survivin is expressed at low levels, if at all, in normal differentiated tissues, there are also reports of survivin expression in normal breast tissue and fibroadenomas [23
]. Weikert et al. have shown that survivin is expressed in human testicular germ cell tumors as well as in human normal cells of the testes [24
]. These studies suggest that baselines of survivin expression and/or promoter activity have to be established for each cell line or primary culture. Thus our data may indicate that primary cultures established from voided urine simply exhibit a higher basal level of survivin promoter activity than established bladder cell lines.