Strategies for Isolating and Propagating Circulating Tumor Cells in Men with Metastatic Prostate Cancer

Abstract

Selecting a well-suited method for isolating/characterizing circulating tumor cells (CTCs) is challenging. Evaluating sensitive and specific markers for prostate cancer (PCa)-specific CTC identification and analysis is crucial. We used the CellCollector EpCAM-functionalized system (CC-EpCAM) and evaluated and developed a PCa-functionalized version (CC-PCa); we then compared CTC isolation techniques that exploit the physical and biological properties of CTCs. We established two cohorts of metastatic PCa patients (mPCa; 15 in cohort 1 and 10 in cohort 2). CTC cultivation experiments were conducted with two capturing methods (Ficoll and ScreenCell). The most sensitive detection rates and highest CTC counts were reached with the CC-PCa and ScreenCell system. Patients with ≥5 CTCs isolated with CC-EpCAM had an overall survival (OS) of 0.93 years, and patients with ≥5 CTCs isolated with CC-PCa had an OS of 1.5 years in cohort 1. Nevertheless, we observed the highest sensitivity and specificity for 24-month survival by the Ficoll with CD45 depletion and ScreenCell system with May-Grunwald Giemsa (MGG) staining. The EpCAM molecule is an essential factor related to OS for CTC isolation based on biological properties in mPCa patients. The best-suited CTC capture system is not limited to one characteristic of cells but adapted to downstream analysis.

Keywords: circulating tumor cells; isolation platforms; prostate cancer.

Figure 1

Overview of CTC isolation approaches in the two independent patient cohorts. Abbreviations: Ficoll *: Ficoll + CD45 depletion.

Figure 2

Immunofluorescence staining of LNCaP cells for (b) PSMA, (e) PSA, (h) PSCA and (c,f,i) Hoechst 33258 was used for nuclear counterstaining. Overlay of nuclear staining and marker-specific staining showing (a) PSMA, (d) PSA and (g) PSCA. Scale bares indicate 20 µm.

Figure 3

Direct comparison of CTC detection: (a) with the CC-EpCAM and CC-PCa systems, and the Wilcoxon matched-pairs signed-rank test was used, p ** 0.002. (b) CC-EpCAM, ScreenCell Cyto device (SC) and Ficoll gradient with CD45 depletion Ficoll *-May-Grunwald Giemsa (Ficoll *-MGG) and Ficoll **-immunofluorescence staining (Ficoll **-IF) systems and Dunn’s multiple comparison test were used. Ficoll **-IF vs. SC-MGG, p = ** 0.002 and Ficoll *-MGG vs. SC, p = ** 0.002.

Figure 4

Representative CTC images (a,e) and negative leukocyte staining (c,g) from selected patients of our cohorts collected by Ficoll ** (a–d) and CC-PCa (e–h). CTCs were defined as cells with positive staining for CK (a,e) but negative staining for CD45 (c,g). Hoechst staining showed the presence of cell nucleoli (b,f). The scale bars indicate 20 µm, and cells were scanned at a magnification of 63.3× (a–d). The scale bars indicate 50 µm, and the cells were scanned at a magnification of 20× (e–h). Merge images are presented in (d,h).

Figure 5

CTCs stained with MGG present in blood samples of PCa patients: (a,b) Patient #7 current treatment enzalutamide after docetaxel chemotherapy with a PSA level of 105 ng/mL. (c) Patient #5 current treatment docetaxel chemotherapy with a PSA level of 5.2 ng/mL. Scale bares indicate 20 µm. Arrows indicate CTCs.

Figure 6

Representative images of cultivated CTCs. The CTCs display positive staining for (a) CK 8, 9 and 18, (b) Hoechst 33258 staining, (c) CD45 negativity and (d) overlay of images (a–c). Scale bar presents 20 µm. Brightfield images of growing CTCs (e,f).

Figure 7

Concentration of PSA in ng/mL of cultivated CTCs in the supernatant displayed as median with range. CTCs were successfully cultivated in different single-cell culture wells of a 24-well plate. CTCs originated from a single patient.

Figure 8

Kaplan–Meier curves of OS according to CTC count determined with (a) CC-EpCAM and with (b) CC-PCa: (a) The patients with <5 CTCs and ≥5 CTCs showed a difference in OS (2.1 years versus 0.93 years (HR 0.53, 95% CI: 0.13–2.1)). (b) The patients with <5 CTCs and ≥5 CTCs showed a difference in OS (4.0 years versus 1.5 years (HR 0.33, 95% CI: 0.11–0.97)).