Dear Editor,
The recurrence rate of bladder cancer reaches up to 40–50%, and progression from non-muscle invasive bladder cancer (NMIBC) to muscle invasive bladder cancer (MIBC) occurs in up to 9.8–13.8% of cases [1]. When bladder cancer progresses to MIBC, radical cystectomy with orthotopic neobladder substitution or urinary diversion is required to improve survival, although this decreases patient quality of life [2].
Intravesical instillation of a chemotherapeutic agent is usually performed in cases of NMIBC to prevent recurrence after transurethral resection of the bladder tumor (TURBT) [3]. In moderate-risk or high-risk NMIBC patients, successive instillation of an intravesical chemotherapeutic agent once a week for six weeks is usually performed [4,5,6]. Given that proper selection of chemotherapeutic agents is essential for positive outcomes, chemosensitivity tests are required to provide a rationale for drug selection. To date, chemosensitivity tests have been based on cell survival assessments after a single treatment at a specific time with various methods. However, these in vitro tests are not accepted as routine tests before chemotherapy [7].
We developed in vitro chemosensitivity tests mimicking the situation of clinical chemotherapy by supporting the survival of a cancer cell line in vitro under treatment with an actual intravesical chemotherapy schedule.
Mitomycin C, epirubicin, gemcitabine, and docetaxel were used at six concentrations for the chemosensitivity tests in six bladder cancer cell lines: J82, T24, SW780, UM-UC-3, TCCSUP, and HT-1376.
For conventional analysis, cancer cells were seeded in welled plates (5×103 cells/well) on day one. The chemotherapeutic agent was administered from a dose of 0 (no drug) to 200% on the second day, and chemosensitivity tests were performed after five days. A negative control (no cells) was included on each evaluation plate. The effects of the drugs on cell viability were tested by using CellTiter 96 aqueous nonradioactive cell proliferation assay kits (Promega Co., Madison, WI, USA). Tests were repeated three times, and the mean values were analyzed. Inhibition as a percentage of cancer cells was measured for each plate by using the formula (1-T/C)×100, where T/C=absorbance of cultured cancer cells treated with each test drug/absorbance of cultured cells not treated with the test drug.
Simulation of intravesical chemotherapy was performed according to an in vivo schedule. Chemotherapy was performed once a week in cells in plate wells for two hrs for six weeks (six cycles). The medium containing the chemotherapeutic agent was removed, and fresh medium was added to the wells; two-thirds of the medium was replaced every two days until the next cycle of chemotherapy. Five days after the last chemotherapy treatment at the sixth week, cell survival was measured to evaluate chemosensitivity.
To compare the effectiveness of the conventional and simulation methods, the ratio of the half-maximal inhibitory concentration (IC50) for a test drug concentration (TDC) from the simulation method to that from the conventional method was calculated.
The IC50 value of each chemotherapeutic agent was compared by the %TDC to calculate the drug with the lowest IC50 concentration by the conventional and simulation methods to determine the most sensible chemotherapeutic agent among the tested drugs.
All bladder cancer cell lines were more resistant to epirubicin than to other drugs (Table 1). The IC50 ratio of the conventional method and simulation protocol showed significant differences for mitomycin C and gemcitabine (P=0.003 and P=0.020, respectively; Table 1). All tested cancer cell lines were most sensitive to mitomycin C by the conventional method (Table 2). In five of six cancer cell lines, sensitivity was highest to gemcitabine by the simulation method.
Table 1. IC50 values by cancer cell line and IC50 ratios for the comparison of conventional and simulation methods.
| Cancer cell line | Chemotherapeutic agent | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Mitomycin C | Epirubicin | Gemcitabine | Docetaxel | ||||||
| Con | Sim | Con | Sim | Con | Sim | Con | Sim | ||
| IC50 (%TDC) | SW780 | 6.86 | 18.12 | 959.37 | 471.07 | 10.60 | 1.64 | 8.45 | 1.73 |
| T24 | 0.90 | 4.15 | 7.34 | 94.02 | 1.43 | 0.06 | 3.15 | 0.64 | |
| UM-UC-3 | 0.77 | 2.52 | 20.30 | 349.68 | 3.29 | 3.23 | 1.18 | 2.20 | |
| TCCSUP | 3.57 | 11.66 | 48.51 | 165.66 | 5.64 | 0.86 | 5.60 | 17.62 | |
| J82 | 36.91 | 67.21 | 276.89 | 475.40 | 57.43 | 35.14 | 45.99 | 36.83 | |
| HT-1376 | 2.87 | 7.66 | 7.73 | 27.07 | 3.37 | 2.72 | 6.02 | 27.30 | |
| Mean | 8.65 | 18.56 | 220.02 | 263.82 | 13.63 | 7.28 | 11.73 | 14.39 | |
| SD | 14.02 | 24.49 | 376.69 | 194.74 | 21.69 | 13.70 | 16.97 | 15.35 | |
| IC50 ratio | SW780 | 1 | 2.64 | 1 | 0.49 | 1 | 0.155 | 1 | 0.20 |
| T24 | 1 | 4.61 | 1 | 12.80 | 1 | 0.043 | 1 | 0.20 | |
| UM-UC-3 | 1 | 3.29 | 1 | 17.22 | 1 | 0.98 | 1 | 1.86 | |
| TCCSUP | 1 | 3.27 | 1 | 3.41 | 1 | 0.15 | 1 | 3.15 | |
| J82 | 1 | 1.82 | 1 | 1.72 | 1 | 0.61 | 1 | 0.80 | |
| HT-1376 | 1 | 2.67 | 1 | 3.50 | 1 | 0.81 | 1 | 4.53 | |
| Mean | 1 | 3.05 | 1 | 6.53 | 1 | 0.46 | 1 | 1.79 | |
| SD | 0 | 0.93 | 0 | 6.81 | 0 | 0.39 | 0 | 1.75 | |
| P value | 0.003 | 0.104 | 0.020 | 0.319 | |||||
We calculated the IC50 value for each chemotherapeutic agent for each cancer cell line. All concentrations are presented as the percent test drug concentration (%TDC) and ranged from 0.06% to 959.37%. Paired t-tests were used to analyze the differences in IC50 ratios between the conventional and simulation methods. Differences were significant for mitomycin C and gemcitabine. Statistical significance was established at P<0.05.
Abbreviations: Con, conventional method; Sim, simulation method.
Table 2. Sensitivity ranking for the conventional and simulation methods.
| Cancer cell line | Sensitivity ranking | |
|---|---|---|
| Conventional | Simulation | |
| SW780 | M>D>G>E | G>D>M>E |
| T24 | M>G>D>E | G>D>M>E |
| UM-UC-3 | M>D>G>E | D>M>G>E |
| TCCSUP | M>D>G>E | G>M>D>E |
| J82 | M>D>G>E | G>D>M>E |
| HT-1376 | M>G>D>E | G>M>D>E |
Chemotherapeutic agents were ranked by cell sensitivity for each cell line. Left, agents to which cells showed the highest sensitivity. All tested cancer cell lines were the most sensitive to mitomycin C when assessed by using the conventional method; however, mitomycin C was ranked second for UM-UC-3, TCCSUP, and HT-1376, and third for SW780, T24, and J82 by using the simulation method.
Abbreviations: M, mitomycin C; G, gemcitabine; D, docetaxel; E, epirubicin.
For patients who receive chemotherapy six times per week, one-time chemotherapy in a chemosensitivity test is a completely different regimen than that for in vitro cancer cells. Although it is impossible to create an environment identical to that of cancer cells in the body, it is important to establish in vitro environments that are as similar as possible to conditions in the body for accurately predicting chemosensitivity. We attempted this with our simulation protocols by providing fresh media two hr after chemotherapy and replacing the media every other day. This was assumed to affect the survival of cancer cell lines in the simulation protocol.
Although other factors such as pharmacokinetics and pharmacogenomics were not considered, different treatment schedules and media replacement in the simulation protocol were believed to cause the observed differences in chemosensitivities.
This study used established bladder cancer cell lines. Additional studies employing in vivo models or cancer cells and clinical data from bladder cancer patients are needed to confirm our findings regarding these chemosensitivity methods and the clinical relevance of the newly developed chemosensitivity test.
Footnotes
Authors' Disclosures of Potential Conflicts of Interest: No potential conflicts of interest relevant to this article were reported.
References
- 1.Sylvester RJ, Oosterlinck W, van der Meijden AP. A single immediate postoperative instillation of chemotherapy decreases the risk of recurrence in patients with stage Ta T1 bladder cancer: a meta-analysis of published results of randomized clinical trials. J Urol. 2004;171:2186–2190. doi: 10.1097/01.ju.0000125486.92260.b2. quiz 2435. [DOI] [PubMed] [Google Scholar]
- 2.Rouprêt M, Babjuk M, Compérat E, Zigeuner R, Sylvester R, Burger M, et al. European guidelines on upper tract urothelial carcinomas: 2013 update. Eur Urol. 2013;63:1059–1071. doi: 10.1016/j.eururo.2013.03.032. [DOI] [PubMed] [Google Scholar]
- 3.O'Brien T, Ray E, Chatterton K, Khan MS, Chandra A, Thomas K. Prospective randomised trial of hexylaminolevulinate photodynamic-assisted transurethral resection of bladder tumor (TURBT) plus single-shot intravesical mitomycin C vs conventional white-light TURBT plus mitomycin C in newly presenting non-muscle-invasive bladder cancer. BJU Int. 2013:1096–1104. doi: 10.1111/bju.12355. [DOI] [PubMed] [Google Scholar]
- 4.van Lingen AV, Witjes JA. Current intravesical therapy for non-muscle invasive bladder cancer. Expert Opin Biol Ther. 2013;13:1371–1385. doi: 10.1517/14712598.2013.824421. [DOI] [PubMed] [Google Scholar]
- 5.Koie T, Ohyama C, Hosogoe S, Yamamoto H, Imai A, Hatakeyama S, et al. Oncological outcomes of a single but extensive transurethral resection followed by appropriate intra-vesical instillation therapy for newly diagnosed non-muscle-invasive bladder cancer. Int Urol Nephrol. 2015;47:1509–1514. doi: 10.1007/s11255-015-1048-3. [DOI] [PubMed] [Google Scholar]
- 6.Hayne D, Stockler M, McCombie SP, Chalasani V, Long A, Martin A, et al. BCG+MMC trial: adding mitomycin C to BCG as adjuvant intravesical therapy for high-risk, non-muscle-invasive bladder cancer: a randomised phase III trial (ANZUP 1301) BMC Cancer. 2015;15:432. doi: 10.1186/s12885-015-1431-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Burstein HJ, Mangu PB, Somerfield MR, Schrag D, Samson D, Holt L, et al. American Society of Clinical Oncology clinical practice guideline update on the use of chemotherapy sensitivity and resistance assays. J Clin Oncol. 2011;29:3328–3330. doi: 10.1200/JCO.2011.36.0354. [DOI] [PubMed] [Google Scholar]