Pathological significance and prognostic significance of FES expression in bladder cancer vary according to tumor grade

Three human urothelial carcinoma cell lines, T24 (corresponding to grade 3), 5637 (grade 2), and RT4 (grade 1), were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 50 µg/ml gentamicin (Gibco/Life Technologies) at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Each line was seeded at 5 × 10⁵ cells per 100-mm dish. After 24 h, cells were treated with jetPRIME transfection reagent (Polyplus-transfection Inc., New York, NY, USA) and 50-nM FES siRNA, according to the manufacturer’s protocol. FES expression was then assessed by western blotting. The specificity and reliability of these commercial agents were confirmed in our previous report (Mitsunari et al. 2016). In addition, non-specific effects were ruled out using non-specific control siRNA (Negative Control siRNA, Qiagen, Venlo, The Netherlands) according to our previous report (Mitsunari et al. 2016).

Relative viable cell number was determined using the methylthiazolyltetrazolium (MTT) assay (Cell Proliferation Kit I (MTT); Roche, Basel, Switzerland). T24, 5637, and RT4 cells were placed in each well of a 96-well plate and allowed to adhere and spread for 24 h. The MTT labeling reagent was added to each well, and the cultures were incubated for 4 h at 37 °C. Solubilization solution was then added, and the cells were incubated in a humidified atmosphere overnight. Cell densities were determined by measuring absorbance at 550 nm.

Cells (0.3 × 10⁶) were incubated for 48 h in polycarbonate membrane inserts for use with a CytoSelect Cell Invasion Assay fluorometric kit (Cell Biolabs Inc., San Diego, CA, USA). Invasive cells having passed through the membrane were then lysed, and this lysate was transferred to a 96-well plate for the measurement of fluorescence (expressed in relative fluorescence units) in a plate reader at 480 nm.

Cells (2.1 × 10⁴) were seeded in a 2-well culture insert (ibidi GmbH, Martinsried, Germany) placed on the bottom of a 35-mm culture dish, and incubated for 24 h. A cell-free gap 500-μm wide was created between the two cell populations after removing the insert, and the medium was replaced with DMEM containing 10% FBS. T24 cells were incubated for 6 and 12 h, 5637 cells for 6 and 8 h, and RT4 cells for 24 and 48 h. Cell migration was subsequently evaluated by microscopically measuring the gap.

Western blotting was performed as described previously (Mitsunari et al. 2016). Briefly, cultured cells were harvested by scraping and were lysed in ice-cold hypotonic cell lysis buffer containing protease inhibitors. Aliquots of total cellular lysate (40–50 μg/lane) were electrophoresed on sodium dodecyl sulfate–polyacrylamide gels and transferred to nitrocellulose membranes. After membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at 22 °C, each membrane was incubated with the primary antibody overnight at 4 °C. After three washes with TBST, membranes were incubated with the appropriate secondary antibody for 1 h at room temperature. Specific protein bands were visualized using ECL Prime (GE Healthcare, Little Chalfont, UK).

Bladder tissue sections (5-µm thick) were deparaffinized in xylene and rehydrated using a graded ethanol series. Antigen retrieval was performed at 95 °C for 40 min in 0.01-M sodium citrate buffer (pH 6.0). Sections were then immersed in 3% hydrogen peroxide for 30 min to quench endogenous peroxidase activity, and incubated overnight at 4 °C with the primary antibody (anti-FES; C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the sections were washed in 0.05% Tween 20 in phosphate-buffered saline, and incubated with peroxidase using an LSAB™ + kit (Dako Corp., Carpinteria, CA, USA) according to the manufacturer’s instructions. The peroxidase reaction was visualized with a liquid 3,3′-diaminobenzidine tetrahydrochloride substrate kit (Zymed Laboratories Inc., South San Francisco, CA, USA). Sections were counterstained with hematoxylin, dehydrated through graded alcohol solutions, and cleared using xylene, before being mounted with Poly-Mount (Polysciences, Inc., Warrington, PA, USA). The specificity and sensitivity of the FES antibody used was verified in our previous report (Miyata et al. 2012). In short, normal kidney specimens confirmed in preliminary studies to be immunoreactive with this antibody were used as positive controls. Consecutive sections from each sample processed as above but without the addition of the primary antibody were used as negative controls. Positive and negative controls were included in each batch of samples. In addition, to confirm the specificity of the primary antibody, we also conducted a competition study using a blocking peptide (sc-7670 P, Santa Cruz Biotechnology) according to a previous report (Miyata et al. 2012). Furthermore, the specificity of this primary antibody for human c-Fes protein was again confirmed by comparing its expression by immunoblotting with that by immunohistochemistry, as described previously by our group (Ohba et al. 2011). The proliferation index (PI) was defined as the percentage of Ki-67-positive cancer cells detected using an anti-Ki-67 antibody (Dako Corp., Glostrup, Denmark). Detailed methods, including a description of the positive control, are given in our previous report (Miyata et al. 2014).

FES expression was measured semi-quantitatively using the method described by Zoubeidi et al. (2009). Staining intensity was graded as negative, weak, moderate, or strong. For the human tissues examined here, expression level was quantified using the immunoreactivity score (IRS), where IRS = staining intensity × percentage of positive cells. Staining intensity was defined as follows: 0, negative; 1, weak; 2, moderate; and 3, strong; while the percentage of positive cells was scored in the following way: 0, 0–10%; 1, 11–20%; 2, 21–40%; and 3, 41–100%. Three investigators (Y.M., S.K., and S.W.) independently performed these semi-quantitative analyses. When the conclusions of the three investigators differed, a final decision was reached by majority rule. For statistical analysis, patients were divided into two groups based on IRSs—“negative” and “positive”—the positive group comprising those with an IRS above the median value. Semi-quantitative analysis was performed on at least 500 cancer cells in 3–6 visual fields per section. Each field was also examined at 200× magnification using an E-400 microscope (Nikon, Tokyo, Japan), and representative images were taken with a digital camera (DU100; Nikon) and subjected to image analysis (Win ROOF version 5.0; MITANI Corp., Fukui, Japan). FES expression in cancerous stromal tissues was judged positive when the intensity and area of staining were similar to or higher than those observed in normal stromal tissues.

Normality was evaluated by normal distribution and histograms for each variable, and results are expressed as medians; interquartile range (IQR) and/or mean ± SDs. The Mann–Whitney U test and Student’s t test were used for comparisons involving non-parametric and parametric continuous variables, respectively. The Scheffé test was employed for multiple comparisons. The Chi-squared test was used to compare categorical data. Differences in survival were assessed using Kaplan–Meier curves, the log-rank test, and Cox regression analysis, and are expressed using hazard ratios (HRs), 95% confidential intervals (95% CIs), and p values, respectively. All statistical analyses were two-sided and performed in StatView for Windows (version 5.0; Abacus Concepts, Inc., Berkeley, CA, USA); p < 0.05 was considered to represent statistical significance.

FES expression in non-transfected and FES-KD cells of each line is shown in Fig. 1a. FES expression was clearly decreased by KD in all cancer cell lines. The proliferation of T24 cells was significantly inhibited by KD of FES (p < 0.001, Fig. 1b). In contrast, the proliferation of 5637 cells or RT4 cells was not significantly influenced by FES-KD (p = 0.105 and 0.119, respectively; Fig. 1c, d).

As shown in Fig. 1e, invasion was significantly suppressed by FES-KD in T24 cells (p = 0.018). Invasiveness of FES-KD 5637 and RT4 cells tended to be lower than that of the non-transfected controls; however, these differences were not statistically significant (p = 0.067 and 0.069, respectively).

With regard to the migration of cancer cell lines, representative examples for each cell line are shown in Fig. 1f. Finally, as shown in Fig. 1f, the area of the cell-free gap covered by FES-KD T24 cells was significantly reduced compared to that occupied by untreated controls (p < 0.001). In contrast, no such difference was noted in relation to FES-KD in 5637 cells (p = 0.865). The area covered by FES-KD RT4 cells was smaller than that observed in the corresponding control group but not to a significant extent (p = 0.054).

Figure 2a shows a representative normal human tissue section containing FES-expressing urothelial cells. The expression of FES was principally detected in the cytoplasm, and was strong in almost all normal cells. In contrast, although FES immunostaining was often observed in bladder cancer cells, strong expression was only occasionally evident (Fig. 2b). In addition, positive FES immunostaining was noted in fibroblast-like, endothelial, hematopoietic, and stromal-infiltrating cells (Fig. 2c).

FES IRSs of cancerous specimens (median = 2.0, IQR = 1.5–3.0, mean ± SD = 2.51 ± 1.58) were significantly lower (p < 0.001) than those of normal urothelial tissues (4.5, 3–5, and 4.25 ± 1.55). Relationships between cancer cell FES expression and clinicopathological features of malignancies are shown in Table 1. FES IRS in muscle-invasive bladder cancer (MIBC) (2.5, 1.5–3.0, and 2.83 ± 1.89) tended to be higher than in non-MIBC (2.0, 1.5–3.0, and 2.44 ± 1.5), albeit not significantly so (p = 0.109). In contrast, FES expression negatively correlated with grade (low grade; 2.5, 1.5–4.0, and 2.83 ± 1.67 versus high grade; 1.5, 1.5–2.5, 2.18 ± 1.40; p = 0.005). No significant association was established for FES expression with age at diagnosis or sex.

Figure 3 depicts FES IRS in normal urothelial tissues and according to pT stage and muscle invasion status. As shown in Fig. 3a, FES expression was higher in normal urothelium than in pT1 specimens, compared to which pT3 tissues exhibited higher levels. Moreover, multiple comparison tests revealed that FES IRSs in NMIBC and MIBC were significantly lower (p < 0.001 and 0.006, respectively) than those of normal epithelial samples. No significant difference was identified between NMIBC and MIBC in this respect (p = 0.414; Fig. 3b). Based on our in vitro results, we analyzed the relationship between FES IRS and invasiveness in low- and high-grade cancers separately. In the low-grade group, although decreases in FES IRS were noted at each subsequent pT stage up to pT2 (Fig. 3c), there was no significant difference in this regard between NMIBC and MIBC (p = 0.586; Fig. 3d). In contrast, among high-grade tumors, FES IRS demonstrated consistent increases from stage pTa to pT3/4 (Fig. 3e), and was significantly higher in MIBC than NMIBC (p = 0.002; Fig. 3f).

Regression analysis suggested a positive correlation between FES expression and PI in human cancer tissues when all patients were considered together; however, this relationship was not statistically significant (r = 0.125, p = 0.068; Fig. 4a). While no association was evident between these factors among low-grade tumor samples (r = 0.051, p = 0.607; Fig. 4b), a significant positive correlation was identified in the high-grade tumor group (r = 0.296, p = 0.002; Fig. 4c). The summary of pathological roles of FES expression according to malignant potential is presented in Table 2.

In this study, 143 patients (70.4%) received adjuvant therapy, which did not significantly influence FES expression (p = 0.193). On the basis of the median levels of IRS, patients with over 2 IRS are recognized as “positive” for FES expression. Kaplan–Meier survival curves suggested that FES expression was not associated with metastasis after radical surgery (p = 0.195, Fig. 5a). However, in subgroup analyses based on tumor grade (Fig. 5b, c), elevated FES expression was associated with a lower metastasis-free survival rate for patients with high-grade cancer (p = 0.021; Fig. 5c). On the other hand, among those with low-grade tumors, FES expression exhibited no relationship with subsequent metastasis (p = 0.902, Fig. 5b). In a similar analysis, FES expression was not found to be significantly associated with overall survival in the total patient group (p = 0.508). In addition, no such relationship was established in either the low- (p = 0.763) or high-grade subgroups (p = 0.172). A multivariate model including all clinicopathological features identified muscle invasion (pT2 and 3; HR = 5.4, 95% CI = 2.24–13.03; p < 0.001) but not cancer cell FES expression (HR = 1.3, 95% CI = 0.52–2.97; p = 0.617) as an independent predictor of subsequent metastasis.