Unbiased enrichment of urine exfoliated cells on nanostructured substrates for sensitive detection of urothelial tumor cells

Abstract

Background: Early detection of urothelial carcinoma (UC) by noninvasive diagnostic methods with high accuracy is still underscored. This study aimed to develop a noninvasive assay incorporating both enrichment of urine exfoliated cells and immunoassays for UC detection.

Methods: Polystyrene dishes were exposed to oxygen plasma and modified with 3-aminopropyltriethoxysilane to prepare amine-functionalized nanostructured substrates (NS). Performance characterization of NS was evaluated by atomic force microscope and X-ray photoelectron spectroscopy. Urine exfoliated cells were captured by NS and then immunostained to detect urinary tumor cells (UTCs), which was called UTC assay. The receiver operating characteristic (ROC) curve, area under ROC curve (AUC), and Youden index were used to find the cutoff value of UTC assay. ROC analysis and McNemar test were used to compare the diagnostic accuracy of UTC assay with cytology. Kappa test was used to analyze the agreement of UTC assay and cytology with pathological diagnosis.

Results: Nanostructured substrates had good cell binding yields of nucleated cells and tumor cells. CK20⁺ CD45^- CD11b^- cells were considered as UTCs. UTC number ≥ 1 per sample could be considered as a positive result. By AUC and Kappa analysis, UTC assay showed good performance in UC detection. McNemar test demonstrated that UTC assay had a superior sensitivity even in low-grade subgroup and a similar specificity compared to cytology in UC diagnosis.

Conclusions: Nanostructured substrates could be used to enrich the exfoliated cells from urine samples. UTC assay with NS has the potential to play a role in UC detection. The value of this assay still needs additional validation by large, multi-center studies.

Keywords: cytology; immunocytology; nanostructured substrates; urinary tumor cells; urothelial carcinoma.

Figure 1

Schematic diagram of amine‐functionalized NS preparation and urinary tumor cells enrichment. Tumor cell membranes are negatively charged; the amine‐functionalized NS is positively charged. Aptes = 3‐minopropyltriethoxysilane; NS = nanostructured substrates

Figure 2

Performance characterization of amine‐functionalized NS. (A, B) Representative AFM images (1 × 1μm) of (A) untreated PS and (B) amine‐functionalized NS (PS treated with oxygen plasma and amine‐functionalized by APTES). The roughness of NS is significantly improved compared to PS; (C) XPS elemental analysis of PS (black line) and NS (gray line) samples; (D) Binding yields as a function of incubation time for T24 cells on PS and NS; (E) Binding yields at 1 h as a function of seeding density for T24 cells on NS; (F) Capture yields of T24 cells spiked in urine samples at different concentrations (50, 100, 200 cells spiked in 50‐ml urine, respectively) on NS 1 h after cell seeding. NS = nanostructured substrates; PS = polystyrene; APTES = 3‐aminopropyltriethoxysilane; AFM = atomic force microscope; XPS = X‐ray photoelectron spectroscopy

Figure 3

Efficacy of CK20/CD45/CD11b for discriminating cancer cells from healthy urothelial cells and WBCs captured by NS. CK20 was used as the main marker to detect urothelial cancer cells. CD45 and CD11b were used to mark WBCs and myeloid derivatives, respectively. (A) Graph showing the comparison of CK20 and EpCAM. The immunofluorescence tests were conducted on cell lines (T24, 5637, SV‐HUC‐1) and WBCs. Each cell line was divided into two parts, one part was for CK20 experiment and the other was for EpCAM experiment. CD45 and CD11b were tested in both CK20 experiment and EpCAM experiment. CK20 showed good discriminatory ability. T24 and 5637 cells were CK20⁺CD45⁻CD11b⁻, SV‐HUC‐1 cells were CK20⁻CD45⁻CD11b⁻ and WBCs were CK20⁻CD45⁺CD11b⁺. The combination of CK20, CD45, and CD11b demonstrated excellent ability to identify urothelial cancer cells. (B) Graph showing CK20/CD45/CD11b staining of UTCs and WBCs in urine sample isolated on NS. In the preliminary study, we validated the efficacy of CK20/CD45/CD11b marker combination in a large number of urine samples from bladder cancer patients. Here we show a representative example of such experiments, suggesting good ability of UTC assay in detecting UTCs in urine. UTC = urinary tumor cell

Figure 4

UTC number of the collected clinical urine samples stratified according to pathological outcomes. BL = benign lesion; LUC = low‐grade urothelial carcinoma; HUC = high‐grade urothelial carcinoma; UTC = urinary tumor cell. * represents P < .05

Figure 5

ROC curves for UTC assay and cytology. AUC for UTC assay (0.888) was significantly greater than that for cytology (0.694, P < .001). AUC = area under curve; ROC = receiver operating characteristic